CN113952373A - Application of gamboge extract in preparing antibacterial drugs - Google Patents

Application of gamboge extract in preparing antibacterial drugs Download PDF

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CN113952373A
CN113952373A CN202111493469.XA CN202111493469A CN113952373A CN 113952373 A CN113952373 A CN 113952373A CN 202111493469 A CN202111493469 A CN 202111493469A CN 113952373 A CN113952373 A CN 113952373A
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acid
staphylococcus aureus
gamboge
upp
neogambogic
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CN113952373B (en
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林炜
陈培英
李明珠
张超
佟书娟
史婧
陈园
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Nanjing University of Chinese Medicine
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Abstract

The invention discloses antibacterial pharmacological activity of gamboge extract, which is developed by a large number of experimental screens, wherein the gamboge extract containing neogambogic acid and gambogic acid shows good antibacterial activity on gram-positive bacteria such as staphylococcus aureus, enterococcus faecalis, methicillin-resistant staphylococcus aureus and the like by combining with UPP synthetase and reducing the enzymatic activity of the UPP synthetase. Is expected to be developed into a new antibacterial natural product and has important application value.

Description

Application of gamboge extract in preparing antibacterial drugs
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of active ingredients, namely neogambogic acid and gambogic acid, in gamboge extracts as inhibitors targeting gram-positive bacteria UPP synthetase.
Background
In recent years, the emergence of drug-resistant or multi-drug-resistant strains of various pathogenic bacteria such as staphylococcus aureus, enterococcus faecalis and the like seriously endangers the health of people, and the development of novel drug-resistant bacteria infection-resistant drugs is an urgent requirement in the field of public health. The traditional Chinese medicine is a unique resource in China, and is worth of being well inherited, well developed and well utilized. The Chinese medicinal material gamboge is gambogic plant secretory resin, has the effects of breaking blood and resolving hard mass, attacking toxin and corroding sores, is applied to the aspects of removing putrefaction, astringing sores, attacking toxin and diminishing swelling, stopping bleeding, killing insects and the like, and is mainly used for clinically treating ulcers, eczema, tumors, carbuncle, cellulitis and pyogenic infections, stubborn dermatitis, traumatic injury, traumatic bleeding and scalds. Researches report that gambogic acid (gambogic acid) and neogambogic acid (neogambogic acid), which are main components in gamboge, have remarkable effects of resisting tumor cell differentiation and apoptosis induced by tumor, resisting inflammation and resisting virus, and have the characteristics of wide antitumor spectrum and low toxicity.
The bacterial cell wall plays an important biological function in the aspects of maintaining the cell shape, controlling the cell reproduction, resisting external force damage and the like. Peptidoglycan is a major component of bacterial cell walls. In the early stage of peptidoglycan synthesis, the peptidoglycan precursor C55 isoprenoid pyrophosphate synthesis is dependent on UPP synthase (uncaprenyl pyrophosphate synthase). UPP synthase catalyzes the condensation of 8 molecules of isopentenyl pyrophosphate (IPP) containing a five-carbon unit and 1 molecule of farnesyl pyrophosphate (FPP) containing a fifteen-carbon unit to form C55 isoprenoid pyrophosphate. The C55 isoprenoid pyrophosphoric acid is involved in the synthesis and transportation of cell wall peptidoglycan and the synthesis of other cell wall polysaccharides, and plays an important role in the growth and the propagation of bacteria. Thus, UPP synthase is essential for bacterial growth. Because the genome of human eukaryotes and the like does not contain UPP synthetase homologues, the UPP synthetase homologues are potential drug targets.
At present, no report about the application of the neogambogic acid and the gambogic acid to gram-positive bacteria with UPP enzyme activity inhibition effect exists, and particularly the report about the antibacterial effect on methicillin-resistant staphylococcus aureus with drug resistance exists.
Disclosure of Invention
The invention aims to screen out new clinical application of gamboge through a large number of experiments on the basis of the existing pharmacological activity of the gamboge, and develops that gamboge extracts including neogambogic acid and gambogic acid show good antibacterial activity on gram-positive bacteria such as staphylococcus aureus, enterococcus faecalis, methicillin-resistant staphylococcus aureus and the like through combining with UPP synthetase and reducing the enzymatic activity of the UPP synthetase.
It has been discovered herein that neogambogic acid and gambogic acid bind to and effectively inhibit the activity of UPP synthase, which has important biological functions on bacterial cell wall integrity. Therefore, the present application provides two gamboge extracts which combine with UPP synthetase and effectively inhibit the activity thereof so as to exert antibacterial activity, therefore, the invention screens out neogambogic acid, gambogic acid and derivatives thereof which can be used for preparing UPP enzyme activity inhibitors.
Preferably, the gamboge extract or the neogambogic acid and the gambogic acid and the pharmaceutically acceptable carrier are prepared into tablets, granules, capsules, pills, powder, paste, oral liquid and injection.
The research of the invention finds that the minimum inhibitory concentrations MIC of the neogambogic acid to staphylococcus aureus, enterococcus faecalis and methicillin-resistant staphylococcus aureus are respectively 4 mug/ml, 2 mug/ml and 4 mug/ml. The minimum inhibitory concentrations MIC of the gambogic acid on staphylococcus aureus, enterococcus faecalis and methicillin-resistant staphylococcus aureus are respectively 2 mug/ml, 2 mug/ml and 4 mug/ml. Shows that the neogambogic acid and the gambogic acid have good antibacterial effect, and especially have effective antibacterial activity on drug-resistant staphylococcus aureus. Staphylococcus aureus, enterococcus faecalis and methicillin-resistant Staphylococcus aureus are important pathogenic bacteria for causing clinical bacterial infection, and the invention has important clinical significance.
Drawings
FIG. 1 is the IC of Neogambogic acid and gambogic acid on the UPP synthase from enterococcus faecalis50。
FIG. 2 is the interaction of neogambogic acid and gambogic acid with the enzyme enterococcus faecalis UPP synthase.
Detailed Description
Through a large number of experimental screens, the invention discovers that the neogambogic acid and the gambogic acid can inhibit the growth of staphylococcus aureus, enterococcus faecalis and methicillin-resistant staphylococcus aureus in vitro. The invention is further illustrated below with reference to specific examples, which should not be construed as limiting the invention.
Example 1 in vitro antibacterial Activity of Neogambogic acid and gambogic acid against gram-Positive pathogenic bacteria
The compounds tested: neogambogic acid and gambogic acid.
MIC is the minimum inhibitory concentration, is an index for measuring the antibacterial activity of the antibacterial drug, and is the minimum drug concentration capable of inhibiting the growth of pathogenic bacteria in a culture medium after bacteria are cultured in vitro for 18 to 24 hours.
MIC determination method: the bacteria selected for determination include Staphylococcus aureus, enterococcus faecalis, and methicillin-resistant Staphylococcus aureus. Coli as negative control bacteria, and tetracycline and ampicillin as positive control drugs. The test strain is from China center for culture Collection of microorganisms, and the drug is from bioengineering, Inc. The antibacterial activity and the minimum inhibitory concentration of the neogambogic acid and the gambogic acid are measured by adopting a trace broth dilution method. And (3) selecting the monoclonal colony in 5ml of LB culture medium, shaking at 200rpm for 12 hours, and continuously diluting by 10000 times to obtain experimental bacteria liquid for later use. Adopting a 96-well plate, wherein the highest concentration of the compounds to be detected (neogambogic acid or gambogic acid) in the first well is 16 mug/ml, the concentrations are sequentially diluted by half, the lowest concentration of the compounds to be detected in the 6 th well is 0.5 mug/ml, the bacterial liquid in the 7 th well is used as a positive control, the LB culture medium is added into the 8 th well as a blank negative control, the culture is carried out at 37 ℃ for 18h, and the lowest compound concentration of the completely clarified bacterial liquid can be considered as the lowest bacteriostatic concentration. The minimum inhibitory concentrations MIC of the neogambogic acid to staphylococcus aureus, enterococcus faecalis and methicillin-resistant staphylococcus aureus are respectively 4 mug/ml, 2 mug/ml and 4 mug/ml. The minimum inhibitory concentrations MIC of the gambogic acid on staphylococcus aureus, enterococcus faecalis and methicillin-resistant staphylococcus aureus are respectively 2 mug/ml, 2 mug/ml and 4 mug/ml. The results of the specific experiments are shown in tables 1 and 2.
TABLE 1 antibacterial Activity of Neogambogic acid
Figure BDA0003400117150000031
TABLE 2 antibacterial Activity of gambogic acid
Figure BDA0003400117150000032
Example 2 IC on enterococcus faecalis UPP synthase for Neogambogic acid and Gambogic acid50。
The determination of the minimum inhibitory concentration shows that the neogambogic acid and the gambogic acid both have good antibacterial activity. Thus, their inhibition of the activity of the target enzyme UPP synthetase IC was further analyzed50。
The experimental principle equation is as follows:
Figure BDA0003400117150000041
the upper part is the reaction formula of UPP synthetase, acting on two substrates of isopentyl diphosphate and farnesyl diphosphate, the combination of an inhibitor and UPP synthetase can influence the generation of pyrophosphoric acid, and the inhibition capability can be seen by calculating the percentage of fluorescence.
The specific method comprises the following steps:
the UPP synthetase reaction was performed on black 96-well microtiter plates (Corning, NY, USA). The purified UPP synthetase was added to a mixture containing 50. mu.l of reaction mixture (100mM Tris-HCl (pH 7.5), 0.5mM MgCl2,50mM KCl,35 its isopentyl diphosphate (IPP), 5. mu.M farnesyl diphosphate (FPP) and 0.005% (w/v) Triton X-100), and 9mM, 8mM, 6mM, 5mM, 4mM, 2.5mM, 2mM, 1.5mM, 1.25mM, 0.4mM, 0.25mM, 0.2mM of neogambogic acid or gambogic acid, tetracycline (as a negative control). The reaction was carried out at 37 ℃ and stopped after 30min by adding 10. mu.l of 0.5M EDTA solution. Mu.l of the reaction mixture was transferred to a new 96-well microplate well and quenched with 50. mu.l of Master reaction Mix from the pyrophosphate detection kit (Sigma-Aldrich, St Louis, USA). After incubation for 30min at room temperature, the fluorescence of the reaction mixture at 316 and 456nm was measured with FlexStation 3(MD, usa). The calculated percent fluorescence was plotted against mixture concentration using GraphPad Prism (La Jolla, usa). The experiment was repeated at least 3 times independently. The results of the experiment are shown in FIG. 1.
Example 3 interaction of Neogambogic acid and gambogic acid on the enzyme enterococcus faecalis UPP synthase.
Use of Monolith NTTMProtein labeling kit MonolithTMHis-Tag Labeling Kit RED-tris-NTA2nd Generation (NanoTemper Technologies) the His-labeled UPP was labeled with the dye RED-tris-NTA2nd Generation dye. Serial dilutions of unlabeled compound and 10nM labeled UPP were mixed in binding buffer (1.8mM KH2PO4,10mM Na2HPO4,137mM NaCl,2.7mM KCl, 0.05% Tween-20, pH 7.8) to a final volume of 10. mu.L. Subsequently, the samples were loaded into nt.115 quality coated capillaries (NanoTemper Technologies). The binding experiments were carried out using a Monolith NT.115Pico apparatus (NanoTemper Technologies) with the following parameters: LED power 5%, MST power high. The results were obtained using MO Control software version 1.6. MO affinity analysis software version 2.3 was used to determine the fraction of complex formed. Using GraphPad Prism 7 software, assuming a specific binding site, the apparent dissociation constant (Kd) was calculated using nonlinear fitting, as follows, Y BMax X/Kd + X (where BMax is the largest theoretical specific binding, where BMax is 1). The results of the experiment are shown in FIG. 2.

Claims (6)

1. Application of resina Garciniae extract in preparing antibacterial agent is provided.
2. The use of claim 1, wherein said gamboge extract comprises neogambogic acid and gambogic acid of gamboge.
3. The use of claim 2, wherein said bacteria comprise gram positive bacteria.
4. The use of claim 3, wherein said bacteria comprise Staphylococcus aureus, enterococcus faecalis, methicillin-resistant Staphylococcus aureus.
5. Application of neogambogic acid, gambogic acid and derivatives thereof in preparing UPP enzyme activity inhibitor is provided.
6. The use according to any one of claims 1 to 6, wherein the gamboge extract or the neogambogic acid and the gambogic acid are prepared into tablets, granules, capsules, pills, powders, ointments, oral liquids, injections with a pharmaceutically acceptable carrier.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107648217A (en) * 2017-09-29 2018-02-02 中国农业科学院哈尔滨兽医研究所 Neo-garcinolic acid or derivatives thereof is being prepared for preventing and/or treating by the purposes in the medicine of bacterial relevant disease
US20210113615A1 (en) * 2018-04-13 2021-04-22 Ludwig Institute For Cancer Research Ltd. Heterodimeric inactivatable chimeric antigen receptors
WO2021168168A1 (en) * 2020-02-20 2021-08-26 University Of Florida Research Foundation, Incorporated Identification of host-targeting modulators of bacterial uptake and assay for quantifying intracellular bacteria

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107648217A (en) * 2017-09-29 2018-02-02 中国农业科学院哈尔滨兽医研究所 Neo-garcinolic acid or derivatives thereof is being prepared for preventing and/or treating by the purposes in the medicine of bacterial relevant disease
US20210113615A1 (en) * 2018-04-13 2021-04-22 Ludwig Institute For Cancer Research Ltd. Heterodimeric inactivatable chimeric antigen receptors
WO2021168168A1 (en) * 2020-02-20 2021-08-26 University Of Florida Research Foundation, Incorporated Identification of host-targeting modulators of bacterial uptake and assay for quantifying intracellular bacteria

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
XIN HUA ET AL: "Transcriptional Analysis of the Effects of Gambogic Acid and Neogambogic Acid on Methicillin-Resistant Staphylococcus aureus", 《FRONTIERS IN PHARMACOLOGY》 *
付秀英主编: "《特殊管理类中药实用集录》", 30 November 2007, 人民军医出版社 *
王丹等: "藤黄酸衍生物的合成与抗菌活性研究及其对拓扑异构酶的影响", 《辽宁大学学报(自然科学版)》 *
陈馥衡等: "天然活性化合物的研究——藤黄中杀菌活性成分的研究", 《北京农业大学学报》 *

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