阅读说明：本技术 一种桔梗汤提取物及其制备方法和应用 (Platycodon grandiflorum decoction extract and preparation method and application thereof ) 是由 王书芳 钱景 王毅 于 2019-12-03 设计创作，主要内容包括：本发明公开了一种桔梗汤提取物及其制备方法和应用,属于中药化学领域,该制备方法,包括：将桔梗与甘草混合后粉碎、浸泡,得到混合药物；将混合药物的水溶液加热回流,过滤,收集滤液；利用非极性大孔吸附树脂对滤液进行吸附后,洗脱剂洗脱,得到洗脱液；将乙腈与水作为流动相,在液相色谱中对浓缩后的洗脱液进行梯度洗脱分离,收集30％-33％乙腈水溶液,浓缩、冷冻干燥,得到桔梗汤提取物。桔梗汤提取物包括甘草查尔酮B、甘草皂苷G2与乌拉尔甘草A；该桔梗汤提取物具有抑制金黄色葡萄球菌活性的作用。本发明采用水提、非极性大孔树脂纯化、不同醇浓度分级洗脱等操作,操作方法简单,实现性强,得到的桔梗汤提取物可应用于抗金黄色葡萄球菌药物中。(The invention discloses a platycodon grandiflorum decoction extract and a preparation method and application thereof, belonging to the field of traditional Chinese medicine chemistry, wherein the preparation method comprises the following steps: mixing radix Platycodi and Glycyrrhrizae radix, pulverizing, and soaking to obtain mixed medicine; heating and refluxing the water solution of the mixed medicine, filtering, and collecting filtrate; adsorbing the filtrate by using nonpolar macroporous adsorption resin, and eluting by using an eluant to obtain an eluent; taking acetonitrile and water as mobile phases, carrying out gradient elution separation on the concentrated eluent in liquid chromatography, collecting 30-33% acetonitrile water solution, concentrating, and freeze-drying to obtain the platycodon grandiflorum decoction extract. The radix Platycodi decoction extract comprises licochalcone B, glycyrrhizin G2 and Ural Glycyrrhrizae radix A; the radix Platycodi decoction extract has effect in inhibiting Staphylococcus aureus activity. The invention adopts the operations of water extraction, nonpolar macroporous resin purification, different alcohol concentration graded elution and the like, has simple operation method and strong realizability, and the obtained platycodon grandiflorum decoction extract can be applied to the anti-staphylococcus aureus medicines.)

1. A preparation method of a platycodon grandiflorum decoction extract is characterized by comprising the following steps:

(1) mixing radix Platycodi and Glycyrrhrizae radix, pulverizing, and soaking to obtain mixed medicine;

(2) heating and refluxing the water solution of the mixed medicine, filtering, and collecting filtrate;

(3) adsorbing the filtrate obtained in the step (2) by using nonpolar macroporous adsorption resin, and eluting by using an eluant to obtain an eluent;

(4) taking acetonitrile and water as mobile phases, carrying out gradient elution separation on the concentrated eluent in liquid chromatography, collecting 30-33% acetonitrile water solution, concentrating, and freeze-drying to obtain the platycodon grandiflorum decoction extract.

2. The method for preparing a platycodon grandiflorum decoction extract according to claim 1, wherein in the step (1), the mass ratio of platycodon grandiflorum to liquorice is 1: 1-5.

3. The method for preparing the platycodon grandiflorum soup extract as defined in claim 1, wherein in the step (2), the temperature of the heating reflux is 110-150 ℃, and the time of the heating reflux is 1-12 h.

4. The method for preparing Platycodon grandiflorum decoction extract according to claim 1, wherein in step (3), the nonpolar macroporous adsorbent resin is D101, XDA-5 or HP-10.

5. The method for preparing Platycodon grandiflorum decoction extract according to claim 1, wherein the adsorption time in step (3) is 2-5 h.

6. The method for preparing platycodon grandiflorum soup extract according to claim 1, wherein in the step (3), the eluent is 20-40% ethanol water solution.

7. A Platycodon grandiflorum decoction extract prepared by the method of any one of claims 1 to 6.

8. Use of the platycodon grandiflorum soup extract according to claim 7 for inhibiting the activity of staphylococcus aureus.

Technical Field

The invention relates to the field of traditional Chinese medicine chemistry, and in particular relates to a platycodon grandiflorum decoction extract, a preparation method thereof and application thereof in inhibiting activity of staphylococcus aureus.

Background

Acute Lung Injury (ALI) refers to acute hypoxic respiratory insufficiency syndrome caused by diffuse diffusion of pulmonary interstitium and pulmonary edema due to the destruction of the lung endothelial and epithelial cell barrier caused by infection or trauma to the body.

Bacterial infection is a common cause of acute lung injury, and the acute lung injury caused by bacterial infection mainly has two forms, one is gram-negative bacillus infection, such as Klebsiella pneumoniae, an ALI animal model is usually induced by LPS in experiments to simulate the gram-negative bacillus infection, the other is gram-positive coccus infection, such as staphylococcus aureus, which is an important pathogen causing infectious pneumonia, with the increasing drug resistance of the staphylococcus aureus, the separation rate of methicillin-resistant staphylococcus aureus (MRSA) is over 60 percent, the separation rate of community acquired MRSA (CA-MRSA) is gradually increased in recent years, and most MRSA strains are multi-drug resistant, so that the MRSA strains are not only resistant to almost all β -lactam antibiotics, but also highly resistant to other antibacterial drugs, such as fluoroquinolones, aminoglycosides and the like, and are one of the clinical anti-infection treatment problems at present.

Infectious diseases belong to the category of warm diseases and febrile diseases in the traditional Chinese medicine theory, the traditional Chinese medicine for treating the infectious diseases has better curative effect, a large number of modern researches show that the chemical components of the traditional Chinese medicine have good antibacterial activity, and because the traditional Chinese medicine has the advantages of difficult generation of drug resistance, reversible antibiotic drug resistance combined with antibiotic, low adverse reaction and the like, the search for natural antibiotic becomes a new way for solving the drug resistance of bacteria.

The platycodon root decoction consists of two traditional Chinese medicines, namely platycodon root and liquorice, and is originally invented from Zhang Zhongjing, Shanghai Lun of typhoid, Shaoyin: for shaoyin disease, two or three days, pharyngalgia, it can be combined with gan Cao Tang; for patients without recovery, add Jie Geng Tang. The record in "jin Kui Yao L ü e Lung fistula pulmonary abscess cough" is also: cough with fullness in chest, rapid pulse due to cold, dry throat without thirst, turbid saliva with fishy smell, and pus discharge for a long time like rice gruel are the main symptoms of lung abscess and platycodon root decoction. Platycodon grandiflorum, pungent and bitter with mild flavor, can ventilate lung and relieve sore throat, dispel phlegm and expel pus; the liquorice is sweet and neutral, and can tonify spleen and qi, clear heat and remove toxicity, eliminate phlegm and relieve cough, and relieve spasm and pain. They are combined for treating swollen sore throat, pulmonary abscess and cough.

In order to return to the essence of the traditional medicine for treating diseases, the publication No. CN101396425A discloses a preparation method of a platycodon grandiflorum decoction preparation, wherein a classic formula of the platycodon grandiflorum decoction is extracted by water according to the guiding thought of following the ancient way, and the preparation process parameters and applicable auxiliary materials which are most matched with the platycodon grandiflorum decoction preparation are summarized by combining the modern concentration and granulation processes; the preparation method of the platycodon grandiflorum decoction is described, but the application of the platycodon grandiflorum decoction in inhibiting the activity of staphylococcus aureus is not involved.

The publication No. 101396425A discloses a traditional Chinese medicine for treating chronic pharyngolaryngitis, and the traditional Chinese medicine is prepared into an oral buccal tablet, wherein platycodon grandiflorum and liquorice are both traditional Chinese medicines with homology of medicine and food (traditional Chinese medicine for health care), and researches show that the platycodon grandiflorum decoction prepared into the buccal tablet has obvious effects of clearing heat and removing toxicity, relieving sore throat and relieving pain, and has good prevention and treatment effects on symptoms of lesser yin diseases and pharyngalgia; the platycodon grandiflorum decoction is mainly used for treating sphagitis, and does not have a protection effect on acute lung injury induced by LPS.

Disclosure of Invention

The invention provides a preparation method of a platycodon grandiflorum decoction extract, which adopts the operations of water extraction, nonpolar macroporous resin purification, different alcohol concentration fractional elution and the like to obtain the platycodon grandiflorum decoction extract with strong activity of functional components.

A method for preparing radix Platycodi soup extract comprises:

(1) mixing radix Platycodi and Glycyrrhrizae radix, pulverizing, and soaking to obtain mixed medicine;

(2) heating and refluxing the water solution of the mixed medicine, filtering, and collecting filtrate;

(3) adsorbing the filtrate obtained in the step (2) by using nonpolar macroporous adsorption resin, and then eluting by using an eluant to obtain an eluent;

(4) and (3) taking acetonitrile and water as mobile phases, carrying out gradient elution separation on the concentrated eluent in a liquid chromatogram, collecting separated liquid, and concentrating to obtain the platycodon grandiflorum decoction extract.

In the step (1), the mass ratio of the platycodon grandiflorum to the liquorice is 1: 1-5.

In order to better dissolve medicinal components in the platycodon grandiflorum and the liquorice in the solvent, the soaking solvent is water, the soaking time is 2-24h, and the soaking temperature is 25-50 ℃.

In the step (2), the heating reflux temperature is 110-.

In order to fully dissolve the medicinal components in the platycodon grandiflorum and the liquorice in the water, in the step (2), the mixed medicine is filtered, the dregs of a decoction are added into the water again, and the heating and refluxing are carried out for 2-4 times.

In the step (3), the nonpolar macroporous adsorption resin is one of D101, XDA-5 or HP-10; because the D101 macroporous resin has a relatively large specific surface area and a proper pore size, the D101 macroporous resin has better selectivity on saponin substances in plants, and preferably, the macroporous resin is D101.

The macroporous adsorption resin can adsorb the filtrate for 2-5h, and can completely absorb the medicinal components of radix Platycodi and Glycyrrhrizae radix in the filtrate.

The water solution of ethanol has larger polarity, so that the adsorbed drug can be completely eluted on the premise of not damaging macroporous resin, and the eluent is 20-40% ethanol water solution, preferably 40% ethanol water solution.

And (4) performing gradient elution separation on the concentrated eluent in liquid chromatography, and collecting a separation liquid, wherein the separation liquid is a 30-33% acetonitrile aqueous solution.

The invention also provides a platycodon grandiflorum decoction extract prepared by the preparation method, wherein the platycodon grandiflorum decoction extract comprises licochalcone B and glycyrrhizin G ₂And Ural licorice A; wherein licochalcone B (licochalcone B) is used as main ingredient of radix Platycodi decoction extract, and has content of 50-60%.

The invention also provides application of the platycodon grandiflorum decoction extract in inhibiting the activity of staphylococcus aureus.

The invention has the beneficial effects that:

(1) the preparation method of the platycodon grandiflorum decoction extract adopts the operations of water extraction, nonpolar macroporous resin purification, different alcohol concentration graded elution and the like, is simple to operate and strong in realizability, and obtains the platycodon grandiflorum decoction extract with strong activity of functional components.

(2) The platycodon grandiflorum decoction extract comprises licochalcone B, glycyrrhizin G2 and Ural liquorice A; wherein licochalcone B is used as main ingredient of radix Platycodi decoction extract, and has content of 50-60%.

(3) Animal test research and antibacterial activity test research of the platycodon grandiflorum decoction extract prove that the platycodon grandiflorum decoction extract has the effect of inhibiting the activity of staphylococcus aureus.

Drawings

FIG. 1 is an HPLC analysis chromatogram of Platycodon grandiflorum decoction extract D1 in example 1 of the present invention, wherein 1 is licochalcone B, 2 is glycyrrhizin G2, and 3 is Glycyrrhiza uralensis A.

FIG. 2 is a mass spectrum of licochalcone B in the Platycodon grandiflorum decoction extract D1 in example 1 of the present invention.

FIG. 3 is a mass spectrum of glycyrrhizin G2 in Platycodon grandiflorum decoction extract D1 in example 1 of the present invention.

FIG. 4 is a mass spectrum of Ural licorice A in Platycodon grandiflorum decoction extract D1 in example 1 of the present invention.

FIG. 5 is a graph showing the effect of Platycodon grandiflorum decoction on pathological morphology of lung tissue; wherein A is a lung tissue section of a male mouse in a blank control group; b is a lung tissue section picture of a male rat in the model group; c is a lung tissue section diagram of a male mouse in a platycodon grandiflorum decoction non-infected group, and D is a lung tissue section diagram of a male mouse in a platycodon grandiflorum decoction infected group.

FIG. 6 is a statistical chart of the lung bacterial load of the male mouse in the invention; a is a CFU result statistical chart of lung homogenate of a male rat; panel B is a statistical chart of CFU results for alveolar lavage fluid.

Detailed Description

The invention will be further described in detail with reference to the following examples and drawings, which are given by way of illustration and are not intended to be limiting.