CN115252662B - Plum slice extract, preparation method and application thereof in preparation of antibacterial preparation - Google Patents
Plum slice extract, preparation method and application thereof in preparation of antibacterial preparation Download PDFInfo
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Abstract
The invention relates to the technical field of natural pharmaceutical chemistry, and particularly discloses a plum slice extract, a preparation method thereof and application thereof in preparation of antibacterial preparations. The preparation method of the plum slice extract comprises the following steps: mixing fresh plum leaves and branches, cutting, and adding supercritical CO 2 Extracting in an extraction instrument to obtain supercritical extract of plum slice leaves and branches, namely the plum slice extract. Researches show that the plum slice extract prepared by the method has the effect of resisting methicillin-resistant staphylococcus aureus; therefore, the plum slice extract provided by the invention has important application prospect in preparing an antibacterial preparation with the effect of resisting methicillin-resistant staphylococcus aureus.
Description
Technical Field
The invention relates to the technical field of natural pharmaceutical chemistry, in particular to a plum slice extract, a preparation method thereof and application thereof in preparation of antibacterial preparations.
Background
Nanjiao Mei tablet (natural borneol), the original name borneol, also called dextrorotatory borneol, is a special south medicine resource in the state of Mei Yudong. The Chinese pharmacopoeia records that the plum slice tree is rich in natural borneol and has the effects of preventing corrosion, promoting tissue regeneration, diminishing inflammation, resisting bacteria, promoting skin absorption and the like. Natural borneol is a precious medicinal material and a high-grade spice, is widely applied to the fields of medicine, chemical industry, food health care and the like, and has the reputation of 'plant gold'. The Chinese herbal medicine extract components are characterized by unique natural green, safe and low-sensitivity, environment friendliness and the like, and the Chinese herbal medicine extract components are also widely applied to the formula of Chinese herbal medicine preparation products.
Methicillin-resistant staphylococcus aureus is a clinically common bacterium with stronger toxicity, and is resistant to all other beta-lactams and cephalosporins with the same structure as methicillin except for methicillin; meanwhile, the composition has different degrees of drug resistance to aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides and rifampicin. Therefore, the antibacterial preparation for resisting the methicillin-resistant staphylococcus aureus has important application value.
However, natural borneol rich in the plum tree has been reported to have antibacterial effect; however, the prior art does not report that the prune branches and leaves contain active ingredients for resisting methicillin-resistant staphylococcus aureus; there is no report in the prior art that an effective component or an extract for resisting methicillin-resistant staphylococcus aureus is prepared from plum branches and leaves.
Disclosure of Invention
In order to overcome at least one technical problem existing in the prior art, the invention firstly provides a preparation method of a plum slice extract. Researches show that the plum slice extract prepared by the method has the effect of resisting methicillin-resistant staphylococcus aureus.
The invention aims to solve the technical problems, and is realized by the following technical scheme:
a preparation method of a plum slice extract, which comprises the following steps:
mixing fresh plum leaves and branches, cutting, and adding supercritical CO 2 Extracting in an extraction instrument to obtain supercritical extract of the leaves and the branches of the plum slice, namely the plum slice extract.
The inventors have surprisingly found that mixing the leaves and branches of the plum leaves with supercritical CO 2 Plum slice and leaf obtained by extraction with extraction instrumentAnd branch supercritical extract, which has certain inhibition effect on methicillin-resistant staphylococcus aureus.
Preferably, the weight ratio of fresh plum leaves to branches is 1:1-5.
Most preferably, the weight ratio of fresh plum leaves to branches is 1:2-3.
Preferably, supercritical CO 2 The extraction conditions of the extractor are as follows: the extraction temperature is 50-60 ℃, the extraction pressure is 30-35 MPa, the flow rate is 10-20L/h, and the extraction time is 2-3 h.
Preferably, supercritical CO 2 The extraction conditions of the extractor are as follows: the extraction temperature is 55 ℃, the extraction pressure is 35MPa, the flow rate is 15L/h, and the extraction time is 2.5h.
Preferably, in the use of supercritical CO 2 An entrainer is also adopted in the extraction process of the extraction instrument, and the entrainer is acetone.
Preferably, the weight of the entrainer is 5-15% of the total weight of fresh plum leaves and branches.
Most preferably, the entrainer is used in an amount of 10% by weight based on the total weight of fresh prune leaves and branches.
The inventors found in further studies that, in the use of supercritical CO 2 The supercritical extract of the plum slice leaves and the branches, which is obtained by adding the acetone entrainer in the extraction process of the extraction instrument, has a remarkable improvement on the inhibition effect on the methicillin-resistant staphylococcus aureus compared with the supercritical extract of the plum slice leaves and the branches, which is prepared by not adding the acetone entrainer.
Preferably, further, the supercritical extract of the leaves and branches of the plum slice is enriched with the active ingredients through a silica gel column;
the specific method for enriching the active ingredients through the silica gel column comprises the following steps: loading supercritical extract of plum slice leaves and branches onto silica gel column, eluting with 6-10 times of organic solvent composed of cyclohexane and acetone, collecting eluate, concentrating, and drying to obtain the extract.
Preferably, the volume ratio of cyclohexane to acetone in the organic solvent is 9-10:1.
Most preferably, the volume ratio of cyclohexane to acetone in the organic solvent is 9:1.
In order to further improve the inhibition effect of supercritical extract of plum leaves and branches on methicillin-resistant staphylococcus aureus; the inventor further adopts a silica gel column to enrich the active ingredients. In the process of enriching the active ingredients by taking supercritical extracts of the leaves and the branches of the plum slices as objects, the composition of the organic solvent of the eluting silica gel column plays a decisive role in further preparing the plum slice extract with more excellent inhibition effect on methicillin-resistant staphylococcus aureus. Not eluting with organic solvent of arbitrary composition can obtain extract of Borneolum with excellent inhibiting effect.
The inventor has found that when the organic solvent consisting of cyclohexane and acetone in the volume ratio of 8-10:1 is adopted for elution, the prepared plum slice extract has very excellent effect of resisting methicillin-resistant staphylococcus aureus, and the inhibition effect of the plum slice extract on the methicillin-resistant staphylococcus aureus is greatly improved compared with that of supercritical extracts of plum slice leaves and branches. However, the inhibitory effect of the extract of the plum slice prepared by eluting with other or other organic solvents on methicillin-resistant staphylococcus aureus can not be improved greatly, or even can not be improved, compared with the supercritical extracts of the leaves and branches of the plum slice.
The invention also provides a plum slice extract prepared by the preparation method.
The invention also provides an application of the plum slice extract in preparing an antibacterial preparation.
Preferably, the antibacterial preparation is an antibacterial preparation with an effect of resisting methicillin-resistant staphylococcus aureus.
The beneficial effects are that: the invention provides a plum slice extract prepared by a brand new method; researches show that the plum slice extract prepared by the method has the effect of resisting methicillin-resistant staphylococcus aureus; therefore, the plum slice extract provided by the invention has important application prospect in preparing an antibacterial preparation with the effect of resisting methicillin-resistant staphylococcus aureus.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way.
Example 1 preparation of Mei pian extract
Mixing fresh plum leaves and branches at a weight ratio of 1:3, cutting, and adding supercritical CO 2 Extracting in an extraction instrument to obtain supercritical extract of plum slice leaves and branches (namely the plum slice extract);
wherein, supercritical CO 2 The extraction conditions of the extractor are as follows: the extraction temperature is 55 ℃, the extraction pressure is 35MPa, the flow rate is 15L/h, and the extraction time is 2.5h.
EXAMPLE 2 preparation of Mei pian extract
Mixing fresh plum leaves and branches at a weight ratio of 1:3, cutting, and adding supercritical CO 2 Extracting in an extraction instrument to obtain supercritical extract of plum slice leaves and branches (namely the plum slice extract);
wherein, supercritical CO 2 The extraction conditions of the extractor are as follows: acetone is adopted as an entrainer, and the weight dosage of the entrainer is 10% of the total weight of fresh plum leaves and branches; the extraction temperature is 55 ℃, the extraction pressure is 35MPa, the flow rate is 15L/h, and the extraction time is 2.5h.
EXAMPLE 3 preparation of Mei pian extract
(1) Mixing fresh plum leaves and branches at a weight ratio of 1:3, cutting, and adding supercritical CO 2 Extracting in an extraction instrument to obtain supercritical extracts of the leaves and the branches of the plum slices;
(2) Loading supercritical extract of folium Pruni mume and branch onto silica gel column, eluting with 8 times of organic solvent composed of cyclohexane and acetone, collecting eluate, concentrating, and drying to obtain extract;
supercritical CO in step (1) 2 The extraction conditions of the extractor are as follows: acetone is adopted as entrainer for entrainingThe weight of the agent is 10% of the total weight of fresh plum leaves and branches; the extraction temperature is 55 ℃, the extraction pressure is 35MPa, the flow rate is 15L/h, and the extraction time is 2.5h;
in the step (2), the weight of the silica gel in the silica gel column is 30 times of the weight of the supercritical extract of the plum leaves and the branches; the silica gel is 200-300 mesh silica gel; the volume ratio of cyclohexane to acetone is 9:1.
EXAMPLE 4 preparation of Mei pian extract
(1) Mixing fresh plum leaves and branches at a weight ratio of 1:1, cutting, and adding supercritical CO 2 Extracting in an extraction instrument to obtain supercritical extracts of the leaves and the branches of the plum slices;
(2) Loading supercritical extract of folium Pruni mume and branch onto silica gel column, eluting with organic solvent composed of cyclohexane and acetone with volume 10 times of that of the column, collecting eluate, concentrating, and drying to obtain extract;
supercritical CO in step (1) 2 The extraction conditions of the extractor are as follows: acetone is adopted as an entrainer, and the weight dosage of the entrainer is 15% of the total weight of fresh plum leaves and branches; the extraction temperature is 50 ℃, the extraction pressure is 30MPa, the flow rate is 20L/h, and the extraction time is 2h;
in the step (2), the weight of the silica gel in the silica gel column is 30 times of the weight of the supercritical extract of the plum leaves and the branches; the silica gel is 200-300 mesh silica gel; the volume ratio of cyclohexane to acetone is 10:1.
EXAMPLE 5 preparation of Mei pian extract
(1) Mixing fresh plum leaves and branches at a weight ratio of 1:5, cutting, and adding supercritical CO 2 Extracting in an extraction instrument to obtain supercritical extracts of the leaves and the branches of the plum slices;
(2) Loading supercritical extract of folium Pruni mume and branch onto silica gel column, eluting with 6 times of organic solvent composed of cyclohexane and acetone, collecting eluate, concentrating, and drying to obtain extract;
supercritical CO in step (1) 2 Extraction instrumentThe extraction conditions of (2) are as follows: acetone is adopted as an entrainer, and the weight dosage of the entrainer is 5% of the total weight of fresh plum leaves and branches; the extraction temperature is 60 ℃, the extraction pressure is 35MPa, the flow rate is 10L/h, and the extraction time is 3h;
in the step (2), the weight of the silica gel in the silica gel column is 30 times of the weight of the supercritical extract of the plum leaves and the branches; the silica gel is 200-300 mesh silica gel; the volume ratio of cyclohexane to acetone is 10:1.
Comparative example 1 preparation of Mei pian extract
(1) Mixing fresh plum leaves and branches at a weight ratio of 1:3, cutting, and adding supercritical CO 2 Extracting in an extraction instrument to obtain supercritical extracts of the leaves and the branches of the plum slices;
(2) Loading supercritical extract of folium Pruni mume and branch onto silica gel column, eluting with cyclohexane with volume of 8 times of column volume, collecting eluate, concentrating, and drying to obtain extract;
supercritical CO in step (1) 2 The extraction conditions of the extractor are as follows: acetone is adopted as an entrainer, and the weight dosage of the entrainer is 10% of the total weight of fresh plum leaves and branches; the extraction temperature is 55 ℃, the extraction pressure is 35MPa, the flow rate is 15L/h, and the extraction time is 2.5h;
in the step (2), the weight of the silica gel in the silica gel column is 30 times of the weight of the supercritical extract of the plum leaves and the branches; the silica gel adopts 200-300 mesh silica gel.
Comparative example 1 differs from example 3 in that the organic solvent used in the silica gel column elution step is different; comparative example 1 elution with cyclohexane; in example 3, the elution was carried out using an organic solvent consisting of cyclohexane and acetone in a volume ratio of 9:1.
Comparative example 2 preparation of Mei pian extract
(1) Mixing fresh plum leaves and branches at a weight ratio of 1:3, cutting, and adding supercritical CO 2 Extracting in an extraction instrument to obtain supercritical extracts of the leaves and the branches of the plum slices;
(2) Loading supercritical extract of folium Pruni mume and branch onto silica gel column, eluting with 8 times of acetone, collecting eluate, concentrating, and drying to obtain extract;
supercritical CO in step (1) 2 The extraction conditions of the extractor are as follows: acetone is adopted as an entrainer, and the weight dosage of the entrainer is 10% of the total weight of fresh plum leaves and branches; the extraction temperature is 55 ℃, the extraction pressure is 35MPa, the flow rate is 15L/h, and the extraction time is 2.5h;
in the step (2), the weight of the silica gel in the silica gel column is 30 times of the weight of the supercritical extract of the plum leaves and the branches; the silica gel adopts 200-300 mesh silica gel.
Comparative example 2 differs from example 3 in that the organic solvent used in the silica gel column elution step is different; comparative example 2 elution with acetone; in example 3, the elution was carried out using an organic solvent consisting of cyclohexane and acetone in a volume ratio of 9:1.
Comparative example 3 preparation of extract of Mei pian
(1) Mixing fresh plum leaves and branches at a weight ratio of 1:3, cutting, and adding supercritical CO 2 Extracting in an extraction instrument to obtain supercritical extracts of the leaves and the branches of the plum slices;
(2) Loading supercritical extract of folium Pruni mume and branch onto silica gel column, eluting with 8 times of organic solvent composed of cyclohexane and ethyl acetate, collecting eluate, concentrating, and drying to obtain extract;
supercritical CO in step (1) 2 The extraction conditions of the extractor are as follows: acetone is adopted as an entrainer, and the weight dosage of the entrainer is 10% of the total weight of fresh plum leaves and branches; the extraction temperature is 55 ℃, the extraction pressure is 35MPa, the flow rate is 15L/h, and the extraction time is 2.5h;
in the step (2), the weight of the silica gel in the silica gel column is 30 times of the weight of the supercritical extract of the plum leaves and the branches; the silica gel is 200-300 mesh silica gel; the volume ratio of cyclohexane to ethyl acetate is 9:1.
Comparative example 3 differs from example 3 in that the composition of the organic solvent is different in the silica gel column elution step; comparative example 3 elution was performed with an organic solvent consisting of cyclohexane and ethyl acetate in a volume ratio of 9:1; in example 3, the elution was carried out using an organic solvent consisting of cyclohexane and acetone in a volume ratio of 9:1.
Comparative example 4 preparation of Mei pian extract
(1) Mixing fresh plum leaves and branches at a weight ratio of 1:3, cutting, and adding supercritical CO 2 Extracting in an extraction instrument to obtain supercritical extracts of the leaves and the branches of the plum slices;
(2) Loading supercritical extract of folium Pruni mume and branch onto silica gel column, eluting with 8 times of organic solvent composed of cyclohexane and acetone, collecting eluate, concentrating, and drying to obtain extract;
supercritical CO in step (1) 2 The extraction conditions of the extractor are as follows: acetone is adopted as an entrainer, and the weight dosage of the entrainer is 10% of the total weight of fresh plum leaves and branches; the extraction temperature is 55 ℃, the extraction pressure is 35MPa, the flow rate is 15L/h, and the extraction time is 2.5h;
in the step (2), the weight of the silica gel in the silica gel column is 30 times of the weight of the supercritical extract of the plum leaves and the branches; the silica gel is 200-300 mesh silica gel; the volume ratio of cyclohexane to acetone is 8:1.
Comparative example 4 is different from example 3 in that the volume ratio of cyclohexane and acetone in the organic solvent is different in the silica gel column elution step; comparative example 4 elution was performed with an organic solvent consisting of cyclohexane and acetone in a volume ratio of 8:1; in example 3, the elution was carried out using an organic solvent consisting of cyclohexane and acetone in a volume ratio of 9:1.
Experimental example methicillin-resistant Staphylococcus aureus antibacterial test
Experimental drugs: the extract of Mei pian prepared in examples 1 to 5 and comparative examples 1 to 4; in the test process, each extract is prepared into 20mg/mL of liquid medicine to be tested.
The testing method comprises the following steps: taking 0.2mL of bacterial liquid containing methicillin-resistant staphylococcus aureus (concentration is 1×10) 7 Adding the components per mL) into a sterile culture medium, smearing uniformly, placing an oxford cup on the culture medium after drying, and adding 300 mu L of each liquid medicine to be tested into the oxford cup; after the addition of the liquid medicine is finished, the liquid medicine is put into an incubator at 37 ℃ for culturing for 24 hours; finally, measuring the diameter of a bacteriostasis ring of each liquid medicine to be measured on methicillin-resistant staphylococcus aureus; each test liquid was repeatedly tested 10 times, and the average value was obtained, and the results are shown in table 1.
TABLE 1 diameter of inhibition zone of each Mexicillin-resistant Staphylococcus aureus by each Mexicillin extract
In the experimental data of Table 1, the diameter of the inhibition zone of the extract of the plum slice of example 1 against methicillin-resistant staphylococcus aureus is 11.3mm; this shows that the supercritical CO described in this method is used as raw material of plum leaves and branches 2 The supercritical extract (namely the plum slice extract) of the leaves and the branches of the plum slice extracted by the extraction method has a certain inhibition effect on methicillin-resistant staphylococcus aureus.
In the experimental data of Table 1, the diameter of the inhibition zone of the plum slice extract of example 2 against methicillin-resistant staphylococcus aureus is 14.4mm; significantly greater than example 1; this indicates that, in the case of supercritical CO 2 The supercritical extracts of the plum slice leaves and the branches (namely the plum slice extracts) obtained by adding the acetone entrainer in the extraction process of the extraction instrument have remarkable improvement on the inhibition effect of the methicillin-resistant staphylococcus aureus compared with the supercritical extracts of the plum slice leaves and the branches prepared by not adding the acetone entrainer.
In the experimental data of table 1, the diameter of the antibacterial zone of the plum slice extract prepared in examples 3 to 5 on methicillin-resistant staphylococcus aureus is greatly larger than 14.4mm of example 2; this indicates that: the supercritical extract of the plum leaves and the branches is enriched with active ingredients by adopting a silica gel column, and organic solvent consisting of cyclohexane and acetone with the volume ratio of 8-10:1 is adopted for eluting, so that the prepared plum leaves extract has very excellent effect of resisting methicillin-resistant staphylococcus aureus, and the inhibition effect of the plum leaves and the branches on the methicillin-resistant staphylococcus aureus is greatly improved compared with the supercritical extract of the plum leaves and the branches.
In the experimental data in table 1, the diameter of the antibacterial zone of the plum slice extract prepared in comparative examples 1-2 on methicillin-resistant staphylococcus aureus is not obviously increased, even is reduced compared with that in example 2; the diameter of the antibacterial zone of the plum slice extract prepared in the comparative examples 3 and 4 on methicillin-resistant staphylococcus aureus is not greatly increased compared with that of the plum slice extract prepared in the example 2; are far smaller than the extract of the plum slice prepared in the example 3. This indicates that: in the process of taking supercritical extracts of plum leaves and branches as objects to enrich active ingredients, the composition of organic solvent of the eluting silica gel column plays a decisive role in further preparing the plum slice extract with more excellent inhibition effect on methicillin-resistant staphylococcus aureus. Not eluting with organic solvent of arbitrary composition can obtain extract of Borneolum with excellent inhibiting effect. Only when the organic solvent consisting of cyclohexane and acetone with the volume ratio of 8-10:1 is adopted for elution, the prepared plum slice extract has very excellent effect of resisting methicillin-resistant staphylococcus aureus, and the inhibition effect of the plum slice extract on the methicillin-resistant staphylococcus aureus is greatly improved compared with that of supercritical extracts of plum slice leaves and branches. However, the inhibitory effect of the extract on methicillin-resistant staphylococcus aureus, which is prepared by eluting with other organic solvents or other organic solvents with other compositions or other volume ratios, cannot be further improved or greatly improved compared with the supercritical extracts of the leaves and branches of the plum slice.
Claims (5)
1. Application of Borneolum Syntheticum extract in preparing preparation for resisting methicillin-resistant Staphylococcus aureus is provided; which is a kind of
Is characterized in that the preparation method of the plum slice extract comprises the following steps:
mixing fresh plum leaves and branches, cutting, and adding supercritical CO 2 In an extraction apparatus
Extracting to obtain supercritical extract of plum leaves and branches;
loading supercritical extracts of the leaves and the branches of the plum slices onto a silica gel column, eluting with 6-10 times of organic solvent consisting of cyclohexane and acetone, collecting eluate, concentrating and drying to obtain the plum slice extract;
wherein the weight ratio of fresh plum leaves to branches is 1:1-5; supercritical CO 2 The extraction conditions of the extractor are as follows: the extraction temperature is 50-60 ℃, the extraction pressure is 30-35 MPa, the flow rate is 10-20L/h, and the extraction time is 2-3 h; in the use of supercritical CO 2 An entrainer is also adopted in the extraction process of the extraction instrument, and acetone is adopted as the entrainer; the weight dosage of the entrainer is 5% -15% of the total weight of fresh plum leaves and branches;
the volume ratio of cyclohexane to acetone in the organic solvent is 9-10:1.
2. The use of a prune leaf extract according to claim 1, characterized in that the weight ratio of fresh prune leaf to branch is 1:2-3.
3. Use of a prune tablet extract according to claim 1, characterized in that supercritical CO 2 The extraction conditions of the extractor are as follows: the extraction temperature is 55 ℃, the extraction pressure is 35MPa, the flow rate is 15L/h, and the extraction time is 2.5h.
4. The use according to claim 1, wherein the entraining agent is used in an amount of 10% by weight based on the total weight of fresh prune leaves and branches.
5. Use according to claim 1, characterized in that the volume ratio of cyclohexane to acetone in the organic solvent is 9:1.
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| Durable Antibacterial Cotton Fabrics Based on Natural Borneol-Derived Anti-MRSA Agents;Liu Yang，等;Advanced Healthcare Materials;09(11);第1-8页 * |
| 梅州香樟挥发油抗炎活性试验;叶小玲;赵莹;张声源;张伟楠;饶颂华;聂华;;广东化工(08);第49-50页 * |
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