CN115887517A - Application of laggera pterodonta extract in resisting drug-resistant bacteria producing extended-spectrum beta-lactamase - Google Patents
Application of laggera pterodonta extract in resisting drug-resistant bacteria producing extended-spectrum beta-lactamase Download PDFInfo
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention discloses an application of laggera pterodonta extract in resisting drug-resistant bacteria producing extended-spectrum beta-lactamase. The laggera pterodonta extract is obtained by extracting with methanol, ethanol and fat-soluble solvent, and then extracting and performing chromatography. The pterodonta extract is used for measuring the Minimum Inhibitory Concentration (MIC) by a trace broth double dilution method, and experimental data show that the extract has certain antibacterial activity on hyper-broad-spectrum beta-lactamase producing bacteria and non-hyper-broad-spectrum beta-lactamase producing bacteria resistant to escherichia coli, and has the prospect of developing into drugs resistant to bacteria.
Description
Technical Field
The invention belongs to the field of natural medicinal chemistry, and particularly relates to a preparation method of laggera pterodonta extract and application of laggera pterodonta extract in antibiosis, in particular to application of laggera pterodonta extract in resisting drug-resistant bacteria producing extended-spectrum beta-lactamase.
Background
In recent years, with the long-term application and abuse of antibiotics in clinical disease treatment, clinically isolated escherichia coli mostly presents the current situation of multidrug resistance (MDR). Along with the evolution of pathogenic bacteria, a plurality of strains generate drug resistance, the effect is worse and worse when the dosage of antibiotics is larger, even the curative effect is gradually lost, the drug resistance of escherichia coli to cephalosporin drugs is remarkably increased in europe, and the therapeutic effect of the combination of quinolone and aminoglycoside on the escherichia coli is also remarkably reduced. Thus, in order to solve the threat posed by resistant strains, scientists are eagerly exploring new antibiotics and new ways to solve the problem of resistance.
Natural drugs hold an important position in medicinal chemistry in China. The traditional Chinese medicine is a treasure in medical research, wherein a plurality of natural products have good pharmacological and biological effects, have good effects on treating diseases, are a hot spot in modern traditional Chinese medicine research, and have wide application prospects in the aspects of new medicine research and development, traditional Chinese medicine modernization, traditional Chinese medicine resource development and the like.
Laggera Pterodonta (dc.) Benth is a medicinal plant of the genus hexagonia of the family compositae, namely herb of longeperis, smelly leaves, hexagonia and the like, is distributed in Yunnan, sichuan, tibet and other places, is mainly produced in Yunnan province of China, and has rich medicine sources. The writings of Yunnan Chinese herbal medicine, kunming folk herb, yunnan herbal medicine and the like have a large number of records on the laggera pterodonta used in Yunnan folk, and have the efficacy of treating diseases such as cold, sphagitis, bronchitis, malaria and the like. The compounds that have been identified from this genus of plants are mainly eudesmane-type sesquiterpenes and flavonols. The laggera pterodonta has been successfully developed to be used as a medicament for anti-inflammatory treatment of sore throat, cough due to lung heat and the like, but at present, no systematic and clinical research is carried out on drug-resistant escherichia coli, and no report is provided on the application of the laggera pterodonta extract to resisting drug-resistant bacteria producing ultra-broad-spectrum beta-lactamase.
Disclosure of Invention
The invention aims to provide application of laggera pterodonta extract in resisting drug-resistant bacteria producing extended-spectrum beta-lactamase, and aims to solve the problems in the background technology. In order to realize the purpose, the invention adopts the technical scheme that:
the invention obtains active extract by concentrating, extracting and separating the overground part of the laggera pterodonta, subcultures drug-resistant escherichia coli, researches the in-vitro inhibition effect of the laggera pterodonta extract on drug-resistant bacteria by a trace broth two-fold dilution method, fully exerts the biological diversity of natural plant resources in China, and develops and utilizes traditional medicine. The application of the laggera pterodonta extract which has definite curative effect, small side effect, economy, effectiveness and can be widely applied to clinical drug-resistant bacteria resistance is developed by further searching compounds with biological activity to deeply discuss the pharmacological activity of active components.
The active ingredients of the laggera pterodonta extract comprise the following compounds: laggerac acid, 1 beta-hydroxy-laggerac acid, 3 beta-hydroxy-laggerac acid, wintergreen acid, laggera diol, laggera triol A, laggera triol B, 4-O-acetyl cuauthenone 3-O- (2 '-hydroxy-2' -methyl-3 '-acetoxybutyrate), 3- (2', 3 '-diacetyloxy-2' -methylbutylrytyl) -cuauhtemone.
The method for extracting the laggera pterodonta extract comprises the following steps: fully crushing the overground part of the dried pterodonta foetida, adding methanol with the weight 8 times of that of the overground part of the dried pterodonta foetida, carrying out cold soaking or percolation, heating and refluxing, carrying out ultrasonic extraction for multiple times, and collecting a solvent to obtain an initial extract.
The primary extract was further treated as follows: adding 4 times of water, standing, filtering to remove insoluble resin and chlorophyll, concentrating under reduced pressure to obtain concentrated solution, adding 4 times of anhydrous ethanol, standing, filtering to remove insoluble substances, and evaporating to remove solvent to obtain laggera pterodonta extract; or, performing normal phase silica gel column chromatography on the primary extract, removing water-soluble impurities, eluting with 20-95% ethanol, collecting ethanol eluate, and recovering the solvent until the solvent is dried to obtain the laggera pterodonta extract; or passing the primary extract solution through polyamide column or Sephadex column, eluting with water and 95% ethanol in sequence, collecting ethanol eluate, and recovering solvent to dry to obtain Folum inermis extract.
The invention has the beneficial effects that:
the pterodonta foetida extract is used for determining the Minimum Inhibitory Concentration (MIC) by a trace broth double dilution method, and experimental data show that the extract has certain antibacterial activity on Escherichia coli drug-resistant bacteria producing extended-spectrum beta-lactamase and non-extended-spectrum beta-lactamase, and has a prospect of developing into drug-resistant bacteria resistant medicines.
Drawings
FIG. 1 is a structural diagram of pterodontic acid.
FIG. 2 is a structural diagram of 1 β -hydroxy-pterodontic acid.
FIG. 3 is a structural diagram of 3 beta-hydroxy-pterodontic acid.
FIG. 4 is a diagram of the structure of wintergreen.
FIG. 5 is a structural diagram of pterodontolidine.
FIG. 6 is a structural diagram of pteridamol A.
FIG. 7 shows the structure of Laurencide B.
FIG. 8 is a structural diagram of 4-O-acetyl cuauthenone 3-O- (2 ' -hydroxy-2' -methyl-3' -acetoxybutyrate).
FIG. 9 is a structural diagram of 3- (2 ',3' -diacetoxy-2' -methylbutyltyl) -cuauhtemone.
FIG. 10 shows the minimum inhibitory concentration of the laggera pterodonta extract against different bacteria.
Detailed description of the preferred embodiments
To facilitate an understanding of the invention, the invention will now be described more fully hereinafter with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. In contrast, when an element is referred to as being "directly on" another element, there are no intervening elements present. As used herein, the terms "vertical," "horizontal," "left," "right," and the like are for illustrative purposes only and do not represent the only embodiments, and as used herein, the terms "upper," "lower," "left," "right," "front," "rear," and the like are used in a positional relationship with reference to the drawings.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The technical solution of the present patent will be described in further detail with reference to the following embodiments.
As shown in FIGS. 1 to 10, the following examples are explained in detail:
the first embodiment is as follows: a method for preparing laggera pterodonta extract.
Fully crushing the overground part of the dried pterodonta foetida, adding methanol with the weight 8 times of the weight of the ground pterodonta foetida for cold soaking or percolation, heating and refluxing, carrying out ultrasonic extraction for multiple times, collecting and combining solvents to obtain an initial extract.
Adding 4 times of water, standing, filtering to remove insoluble resin and chlorophyll, concentrating under reduced pressure to obtain concentrated solution, adding 4 times of anhydrous ethanol, standing, filtering to remove insoluble substances, and evaporating to remove solvent to obtain laggera pterodonta extract.
Or, performing normal phase silica gel column chromatography on the primary extract, removing water-soluble impurities, eluting with 20-95% ethanol, collecting ethanol eluate, and recovering the solvent until the solvent is dried to obtain the laggera pterodonta extract.
Or passing the primary extract solution through polyamide column or Sephadex column, eluting with water and 95% ethanol in sequence, collecting ethanol eluate, and recovering solvent to dry to obtain Folum inermis extract.
The laggera pterodonta extract comprises laggera pterodonta acid, laggera pterodonta acid (figure 1), 1 beta-hydroxy-laggera pterodonta acid (figure 2), 3 beta-hydroxy-laggera pterodonta acid (figure 3), wintergreen acid (figure 4), laggera pterodonta glycol (figure 5), laggera pteronly (figure 6), laggera pterodonta triol ethyl (figure 7), 4-O-acetyl cuauthenone 3-O- (2 '-hydroxy-2' -methyl-3 '-acetoxybutyrrate) (figure 8), 3- (2', 3 '-diacetyloxy-2' -methoxybutyryl) -cuautemone (figure 9) and other active compounds. Through HPLC quantitative detection, the laggera pterodonta extract contains laggera pterodonta acid 15.2% and wintergreen acid 6.1%.
The above active compounds can be obtained by the following separation methods:
the pterodonta foetida extract obtained by the treatment in the above way is subjected to silica gel column chromatography, and the eluent is petroleum ether-acetone (12 to 100% acetone), so as to obtain 8 parts A to H, wherein the parts A, B and D are washed and recrystallized to obtain the active compound 1,2,3. The remaining mother liquor was sampled with silica gel, subjected to normal phase silica gel column chromatography, and subjected to high performance liquid separation with the addition of an acid (1 drop in 5 ml) to petroleum ether-ethyl acetate (20 to 100% ethyl acetate) to obtain the above-mentioned compounds 4 to 9.
Examples
Antibacterial experiment of laggera pterodonta extract
1 instruments and materials
1.1 Instrument: the enzyme-linked immunosorbent assay device is purchased from Nanjing DeFei laboratory instruments GmbH; the autoclave was purchased from Shanghai Bo News medical Bioinstrumentation, inc.
1.2 materials: broth culture, broth agar culture was purchased from Kyowa Biotech, inc.; the 96-well plate was purchased from Wuxi Kangsi Biotech limited; DMSO is produced by Tianjin Dongtianzheng fine chemical reagent factory.
1.3 strains: the strains used included common and drug resistant strains (ESBL + and ESBL-).
1.3.1 common strains: escherichia coli, staphylococcus aureus, and Bacillus subtilis.
1.3.2 producing extended-spectrum beta-lactamase Escherichia coli drug-resistant bacteria (ESBL +):
USVAST-503；
USVAST-100；
1.3.3 non-extended-spectrum beta-lactamase-producing Escherichia coli drug-resistant bacteria (ESBL-):
USVAST-298；
USVAST-457；
1.3.4 information on drug-resistant bacteria is as follows:
table 1: drug-resistant bacterium information table
1.4 Positive control: the pure berberine is purchased from Shanghai Mecline Biotech limited; cefotaxime sodium powder for injection is purchased from Beijing Yuekang pharmaceutical industry group, inc.
2. Determination of Minimum Inhibitory Concentration (MIC)
All instruments used in the operation experiment are wrapped by newspaper, sterilized in an autoclave (120 ℃,60 min), and irradiated by an ultraviolet sterilizing lamp for more than 30min for later use.
The preparation method of the bacterial liquid concentration comprises the following specific steps:
inoculating bacteria to solid culture medium with inoculating loop, placing in 37 deg.C incubator overnight for culturing, inoculating single colony to solid culture medium, repeating the above operation for three generations, selecting single colony to inoculate MH liquid culture medium, shake culturing in 37 deg.C incubator to logarithmic growth phase, centrifuging for 10min (4200 r/min), discarding upper layer liquid culture medium, washing with sterile normal saline for 3 times, diluting lower layer with sterile normal saline, and precipitating to OD 600nm After 0.10, dilute 1000 times for use.
The minimum inhibitory concentration of the pterodonta foetida extract on the drug-resistant bacteria is determined by a trace broth two-fold dilution method, which comprises the following specific steps:
using 96-well plate, 10% of dmso medium + sterile saline as blank control group; negative control 10% dmso medium + bacterial solution; positive control 10% dmso medium + bacteria solution + positive drug.
The 96-well plate A-E column was used as a sample group, the samples were diluted with 10% DMSO medium for positive drug so that the concentration reached 200 ug/ml, then 200 ul was injected into the 2 nd well of the 2 nd row, 100ul of liquid medium was added to all the wells after the 2 nd well, then 100ul was sucked from the 2 nd well by a pipette gun and injected into the 3 rd well, followed by blow-leveling, then 100ul was injected into the 4 th well, and the above dilution process was repeated until the 11 th well was reached, and 100ul was taken out and discarded.
After the sample is diluted, 100ul of sterile normal saline is added into a blank control group, 100ul of bacterial liquid which is diluted in advance is added into the rest holes, and under the method, the sample concentration from the 2 nd hole to the 11 th holes is as follows: 100. Mu.g/ml, 50. Mu.g/ml, 25. Mu.g/ml, 12.5. Mu.g/ml, 6.25. Mu.g/ml, 3.125. Mu.g/ml, 1.56. Mu.g/ml, 0.79. Mu.g/ml, 0.39. Mu.g/ml, 0.19. Mu.g/ml.
Placing the 96-well plate in an incubator at 37 ℃ for culturing for 16 hours, and then measuring OD (optical density) by using an enzyme-labeling instrument 600nm The value is obtained.
Each experiment was performed 3 times in parallel, with no significant bacterial growth as the minimum inhibitory concentration (MIC, ug/ml) compared to the negative control group, with the following results:
table 2: minimum inhibitory concentration of laggera pterodonta extract on different strains
As shown in Table 2, the pterodonta foetida extract has certain inhibitory action on staphylococcus aureus and bacillus subtilis, and the minimum inhibitory concentration is 100 ug/ml. The MIC to the Escherichia coli is 50 ug/ml, the minimum inhibitory concentration to non-extended spectrum B-lactamase-producing Escherichia coli drug-resistant bacteria is 12.5 ug/ml, the minimum inhibitory concentration to extended spectrum beta-lactamase-producing Escherichia coli drug-resistant bacteria is 25 ug/ml, and the inhibition effect to the drug-resistant bacteria is better than that of natural plant berberine.
The laggera pterodonta extract has stronger bacteriostatic effect on drug-resistant bacteria than a plant antibacterial agent berberine, has broad-spectrum antibacterial action, is effective on drug-resistant bacteria of escherichia coli which produce ultra-broad-spectrum beta-lactamase and non-ultra-broad-spectrum beta-lactamase, and can become a potential active component for inhibiting drug-resistant bacteria.
The above embodiments are only for illustrating the present invention and not for limiting the present invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, therefore all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (5)
1. Application of laggera pterodonta extract in resisting drug-resistant bacteria producing extended-spectrum beta-lactamase.
2. The use of laggera pterodonta extract in resisting extended-spectrum beta-lactamase-producing drug-resistant bacteria according to claim 1, wherein the active ingredients of the laggera pterodonta extract comprise the following compounds: pterodontic acid, 1 beta-hydroxy-pterodontic acid, 3 beta-hydroxy-pterodontic acid, wintergreen, pterodontolene glycol, 4-O-acetyl cuauthenone 3-O- (2 '-hydroxy-2' -methyl-3 '-acetoxybutyrate), and 3- (2', 3'-diacetoxy-2' -methylbutyltyryl) -cuauhtemone.
3. The use of laggera pterodonta extract in resisting extended-spectrum beta-lactamase-producing drug-resistant bacteria according to claim 2, wherein the laggera pterodonta extract has a laggera acid content of not less than 12% and a wintergreen acid content of not less than 5%.
4. The pterodonta foetida extract and the use thereof in resisting extended-spectrum beta-lactamase drug-resistant bacteria according to claim 2, wherein the extended-spectrum beta-lactamase drug-resistant bacteria are extended-spectrum beta-lactamase escherichia coli drug-resistant bacteria and non-extended-spectrum beta-lactamase escherichia coli drug-resistant bacteria.
5. The application of laggera pterodonta extract in resisting extended-spectrum beta-lactamase drug-resistant bacteria according to claim 2, wherein the preparation method of the laggera pterodonta extract comprises the following steps:
s1: pulverizing aerial parts of dried laggera pterodonta, adding organic solvent, cold soaking or percolating, reflux heating, and ultrasonic extracting to recover solvent to obtain primary extract;
s2: adding 1-3 times of water into the primary extract, standing for 4 hours, concentrating the aqueous solution under reduced pressure, adding 1-3 times of absolute ethyl alcohol into the concentrate, and standing for 6 hours; removing water insoluble substance from the obtained alcoholic solution, or evaporating the solvent to obtain Laggera pterodonta extract; or, performing normal phase silica gel column chromatography on the primary extract, removing water-soluble impurities, eluting with 20-95% ethanol, collecting ethanol eluate, and recovering the solvent until the solvent is dried to obtain the laggera pterodonta extract; or passing the primary extract solution through polyamide column or sephadex column, eluting with water and 95% ethanol in sequence, collecting ethanol eluate, and recovering solvent to dry to obtain laggera pterodonta extract.
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| CN105998153A (en) * | 2016-05-10 | 2016-10-12 | 中山大学 | Application of pithecellobium clypearia extract to preparation of drug for resisting generation of extended spectrum beta-lactamase escherichia coli |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105998153A (en) * | 2016-05-10 | 2016-10-12 | 中山大学 | Application of pithecellobium clypearia extract to preparation of drug for resisting generation of extended spectrum beta-lactamase escherichia coli |
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