Application of magnolia sieboldii branch and leaf extract and fluconazole in preparation of antifungal drugs
阅读说明：本技术 天女木兰枝叶提取物协同氟康唑在制备抗真菌药物中的应用 (Application of magnolia sieboldii branch and leaf extract and fluconazole in preparation of antifungal drugs ) 是由 贾琳 金永生 刘金春 章誉艺 黄璜 徐莉萍 于 2021-06-03 设计创作，主要内容包括：本发明涉及一种天女木兰枝叶提取物协同氟康唑在制备抗真菌药物中的应用,其中天女木兰枝叶提取物为天女木兰枝叶75%-85%乙醇提取物,真菌为白念珠菌。本发明提供的天女木兰枝叶75%-85%乙醇提取物可协同氟康唑抗耐药白念珠菌,为拓展天女木兰的临床应用以及治疗氟康唑耐药的白念珠菌提供了新思路,可为联合用药治疗真菌感染以及拓展天女木兰的临床应用提供参考。(The invention relates to an application of magnolia sieboldii branch and leaf extract and fluconazole in preparation of antifungal drugs, wherein the magnolia sieboldii branch and leaf extract is 75-85% ethanol extract of magnolia sieboldii branches and leaves, and fungi are candida albicans. The 75-85% ethanol extract of the branches and leaves of the magnolia sieboldii provided by the invention can cooperate with the fluconazole drug-resistant candida albicans, provides a new idea for expanding the clinical application of the magnolia sieboldii and treating the fluconazole drug-resistant candida albicans, and provides a reference for combined drug treatment of fungal infection and expanding the clinical application of the magnolia sieboldii.)
1. An application of Magnolia sieboldii branch and leaf extract in preparing antifungal medicine in combination with fluconazole is provided.
2. The use of the magnolia sieboldii branch and leaf extract in combination with fluconazole in the preparation of antifungal drugs according to claim 1, is characterized in that: the fungus is Candida albicans.
3. The use of the magnolia sieboldii branch and leaf extract in combination with fluconazole in the preparation of antifungal drugs according to claim 1, is characterized in that: the concentration of the magnolia sieboldii branch and leaf extract in the antifungal medicament is not lower than 7.81 mu g/mL.
4. The use of the magnolia sieboldii branch and leaf extract in combination with fluconazole in the preparation of antifungal drugs according to claim 1, is characterized in that: the magnolia sieboldii branch and leaf extract is an ethanol extract of magnolia sieboldii branches and leaves, preferably a 75-85% ethanol extract of magnolia sieboldii branches and leaves, and further preferably an 80% ethanol extract of magnolia sieboldii branches and leaves.
5. The use of the magnolia sieboldii branch and leaf extract in combination with fluconazole of claim 1 in the preparation of antifungal drugs, wherein the preparation method of the magnolia sieboldii branch and leaf extract comprises the following steps: crushing dried branches and leaves of Magnolia sieboldii, heating and refluxing the crushed branches and leaves with 75-85% ethanol water solution by volume fraction for 1-2 hours, and filtering; heating and refluxing the filter residue by using 75-85% ethanol water solution by volume fraction for 1-2 hours, and filtering; mixing the two filtrates, concentrating under reduced pressure, and recovering ethanol to obtain 75-85% ethanol extract of branches and leaves of Magnolia sieboldii.
6. An antifungal agent, characterized by: the drug takes 75-85% of ethanol extract of magnolia sieboldii branches and leaves and fluconazole as active ingredients, or the drug contains 75-85% of ethanol extract of magnolia sieboldii branches and leaves and fluconazole.
7. The antifungal agent of claim 6, wherein: the medicine also comprises a pharmaceutically acceptable carrier or auxiliary material; the medicine is any pharmaceutically acceptable dosage form, and comprises at least one of tablets, capsules, injections, granules, suspensions and solutions.
8. The antifungal agent of claim 6, wherein: the fungus is Candida albicans.
9. The antifungal agent of claim 6, wherein: the concentration of the magnolia sieboldii branch and leaf extract in the antifungal medicament is not lower than 7.81 mu g/mL; the magnolia sieboldii branch and leaf extract is an ethanol extract of magnolia sieboldii branches and leaves, preferably a 75-85% ethanol extract of magnolia sieboldii branches and leaves, and further preferably an 80% ethanol extract of magnolia sieboldii branches and leaves.
10. The antifungal agent of claim 6, wherein: the preparation method of the magnolia sieboldii branch and leaf extract comprises the following steps: crushing dried branches and leaves of Magnolia sieboldii, heating and refluxing the crushed branches and leaves with 75-85% ethanol water solution by volume fraction for 1-2 hours, and filtering; heating and refluxing the filter residue by using 75-85% ethanol water solution by volume fraction for 1-2 hours, and filtering; mixing the two filtrates, concentrating under reduced pressure, and recovering ethanol to obtain 75-85% ethanol extract of branches and leaves of Magnolia sieboldii.
Technical Field
The invention relates to the technical field of antifungal medicines, and in particular relates to application of magnolia sieboldii branch and leaf extract and fluconazole in preparation of an antifungal medicine.
Background
Fungi are a group of eukaryotes that are widely present in nature and can infect different parts of the human body. We refer to diseases caused by fungi collectively as "mycoses". Mycoses can be classified into superficial mycoses and deep mycoses based on clinical pathogenic conditions. Superficial mycosis is very common in China, and common diseases comprise tinea capitis, tinea corporis, tinea cruris, tinea manus and pedis, tinea versicolor and tinea unguium; deep fungi mainly invade internal organs, bones and nervous systems, and also invade skin and mucosa, and are commonly sporular sporotrichosis, pigmented hyphal disease, nematomycosis and cryptococcosis. Candida albicans invades the skin, mucous membranes, nails and internal organs.
At present, the main clinical treatment medicines for deep fungal infection are polyene (amphotericin B) and azole (fluconazole and the like). Of them, Fluconazole (FLC), an azole, is the most used drug in the treatment of Candida albicans (Candida albicans) infection due to its good bioavailability and reliable safety. However, because the effect on candida albicans is mainly bacteriostasis rather than sterilization, FLC resistance is easily caused in the long-term treatment and repeated administration process, which is a troublesome problem in clinical treatment of deep fungal infection. Restoring the sensitivity of drug-resistant fungi to the treatment drugs and improving the sensitivity of the drug-resistant fungi to FLC, is an effective treatment way for treating deep drug-resistant bacterial infection. The measures for clinically dealing with the drug resistance of the fungi are mainly as follows: increasing the dosage of the medicine, combining the medicine and the like. Therefore, it is necessary to find antifungal drug potentiators or drug resistance reversal agents.
Magnolia sieboldii (Magnolia sieboldii K. Koch) is a plant of Magnoliaceae, and is distributed in Liaoning, Anhui, Zhejiang, Jiangxi, northern part of Fujian, Guangxi, Korea and Japan in China, and grows in mountain land with an elevation of 1600-2000 m. Research has disclosed the use of magnolia sieboldii extract as a bacteriostatic agent: the Magnolia sieboldii extractive solution has effects of inhibiting Staphylococcus aureus, mold and yeast. However, the effect of the branches and leaves of the magnolia sieboldii on the synergistic effect of the fluconazole on the drug-resistant fungi is not reported.
Disclosure of Invention
The invention aims to provide an application of magnolia sieboldii branch and leaf extract and fluconazole in preparation of antifungal drugs, solves the problem of Fluconazole (FLC) resistance easily caused in the processes of long-term treatment of fungal infection and repeated administration in the prior art, and achieves the effects of restoring the sensitivity of drug-resistant fungi to the therapeutic drugs and improving the sensitivity of drug-resistant bacteria to the FLC.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an application of magnolia sieboldii branch and leaf extract and fluconazole in preparation of antifungal drugs. The magnolia sieboldii branch and leaf extract provided by the invention can be used for cooperating with the fluconazole drug-resistant candida albicans, so that a new thought is provided for expanding the clinical application of magnolia sieboldii and treating the fluconazole drug-resistant candida albicans, and a reference is provided for treating fungal infection by using combined drugs and expanding the clinical application of magnolia sieboldii.
In addition, the application of the magnolia sieboldii branch and leaf extract in combination with fluconazole in preparing antifungal medicaments can also have the following additional technical characteristics.
According to the invention, the fungus is Candida albicans.
According to the invention, the concentration of the magnolia sieboldii branch and leaf extract in the antifungal medicament is not lower than 7.81 mu g/mL.
According to the invention, the magnolia sieboldii branch and leaf extract is an ethanol extract of magnolia sieboldii branches and leaves, preferably a 75-85% ethanol extract of magnolia sieboldii branches and leaves, and more preferably an 80% ethanol extract of magnolia sieboldii branches and leaves.
According to the present invention, the preparation method of the magnolia sieboldii branch and leaf extract comprises: crushing dried branches and leaves of the Magnolia sieboldii, heating and refluxing the crushed branches and leaves for 1 to 2 hours by using 75 to 85 percent ethanol water solution by volume, and filtering the mixture; heating and refluxing the filter residue by using 75-85% ethanol water solution by volume fraction for 1-2 hours, and filtering; mixing the two filtrates, concentrating under reduced pressure, and recovering ethanol to obtain 75-85% ethanol extract of branches and leaves of Magnolia sieboldii.
The invention also provides an antifungal medicine which takes 75-85% of ethanol extract of magnolia sieboldii branches and leaves and fluconazole as active ingredients, or the medicine contains 75-85% of ethanol extract of magnolia sieboldii branches and leaves and fluconazole. The drug provided by the invention can restore the sensitivity of drug-resistant fungi to therapeutic drugs and improve the sensitivity of drug-resistant bacteria to FLC.
In addition, the application of the magnolia sieboldii branch and leaf extract in combination with fluconazole in preparing antifungal medicaments can also have the following additional technical characteristics.
According to the invention, the medicament also comprises a pharmaceutically acceptable carrier or auxiliary material; the medicament is any pharmaceutically acceptable dosage form, and comprises at least one of tablets, capsules, injections, granules, suspensions and solutions.
According to the invention, the fungus is Candida albicans.
According to the invention, the concentration of the magnolia sieboldii branch and leaf extract in the antifungal medicament is not lower than 7.81 mu g/mL; the magnolia sieboldii branch and leaf extract is an ethanol extract of magnolia sieboldii branches and leaves, preferably a 75-85% ethanol extract of magnolia sieboldii branches and leaves, and further preferably a 80% ethanol extract of magnolia sieboldii branches and leaves.
According to the present invention, the preparation method of the magnolia sieboldii branch and leaf extract comprises: crushing dried branches and leaves of the Magnolia sieboldii, heating and refluxing the crushed branches and leaves for 1 to 2 hours by using 75 to 85 percent ethanol water solution by volume, and filtering the mixture; heating and refluxing the filter residue by using 75-85% ethanol water solution by volume fraction for 1-2 hours, and filtering; mixing the two filtrates, concentrating under reduced pressure, and recovering ethanol to obtain 75-85% ethanol extract of branches and leaves of Magnolia sieboldii.
One or more technical schemes provided by the invention at least have the following beneficial effects:
(1) according to the invention, the magnolia sieboldii branch and leaf extract is adopted to cooperate with fluconazole, so that the sensitivity of drug-resistant fungi to therapeutic drugs can be recovered, and the sensitivity of drug-resistant bacteria to FLC can be improved.
(2) The invention carries out the research on the synergistic fluconazole antifungal activity of the magnolia sieboldii branches and leaves, inspects the effect of the synergistic fluconazole antifungal activity on drug-resistant candida albicans, provides a method for expanding the clinical application of the magnolia sieboldii branches and leaves and treating the drug-resistant candida albicans of the fluconazole, and provides a reference for the combined drug treatment of drug-resistant fungal infection and the expansion of the clinical application of the magnolia sieboldii.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
In order to better understand the above technical solutions, the following detailed descriptions will be provided with reference to specific embodiments.
The invention also provides an antifungal medicine which takes 75-85% of ethanol extract of magnolia sieboldii branches and leaves and fluconazole as active ingredients or contains 75-85% of ethanol extract of magnolia sieboldii branches and leaves and fluconazole. The magnolia sieboldii branch and leaf extract is cooperated with fluconazole, so that the sensitivity of drug-resistant fungi candida albicans to treatment drugs can be effectively restored, and the sensitivity of drug-resistant fungi to Fluconazole (FLC) is improved. According to the embodiment of the invention, the medicament also comprises a pharmaceutically acceptable carrier or auxiliary material, diluent and the like; the medicament is any pharmaceutically acceptable dosage form, and comprises at least one of tablets, capsules, injections, granules, suspensions and solutions. According to the embodiment of the invention, the fungus is candida albicans, and the extract of the branches and leaves of the magnolia sieboldii cooperates with fluconazole, so that the sensitivity of the drug-resistant fungus candida albicans to a therapeutic drug can be effectively restored, and the sensitivity of the drug-resistant fungus to Fluconazole (FLC) can be improved. According to the embodiment of the invention, in order to obtain more ideal curative effect, the concentration of the magnolia sieboldii branch and leaf extract in the antifungal medicine is not lower than 7.81 mu g/mL. According to an embodiment of the present invention, the magnolia sieboldii branch and leaf extract is an ethanol extract of magnolia sieboldii branches and leaves, preferably an ethanol extract of 75% -85% of magnolia sieboldii branches and leaves, and more preferably an ethanol extract of 80% of magnolia sieboldii branches and leaves. The branches and leaves of the magnolia sieboldii are extracted by using 75-85% of ethanol aqueous solution by volume fraction, so that effective components for synergistic antibiosis can be extracted, the effects of effectively recovering the sensitivity of drug-resistant fungus candida albicans to treatment drugs and improving the sensitivity of drug-resistant fungus to Fluconazole (FLC) are achieved, and the extraction effect is better by using 80% of ethanol aqueous solution by volume fraction. In some embodiments of the invention, the method of preparing the magnolia sieboldii branch and leaf extract comprises: crushing dried branches and leaves of the Magnolia sieboldii, heating and refluxing the crushed branches and leaves for 1 to 2 hours by using 75 to 85 percent ethanol water solution by volume, and filtering the mixture; heating and refluxing the filter residue by using 75-85% ethanol water solution by volume fraction for 1-2 hours, and filtering; and combining the two filtrates, then carrying out reduced pressure concentration, and recovering ethanol to obtain 75-85% ethanol extract of branches and leaves of Magnolia sieboldii. The apparatus used in the present invention for the concentration under reduced pressure is not particularly limited, and may be a rotary evaporator. By adopting a mode of twice reflux extraction, the invention can more fully extract effective components for synergistic antibiosis, thereby achieving the effects of more effectively recovering the sensitivity of drug-resistant fungus candida albicans to treatment drugs and improving the sensitivity of drug-resistant bacteria to Fluconazole (FLC).
In order to better understand the above technical solutions, the following detailed descriptions will be provided with reference to specific embodiments.
Examples
First, experimental materials and methods
(I) test materials
1. Bacterial strains
Candida albicans 103(Candida albicans 103) is provided by the Long-sea hospital, fungi Room, university of navy military medical. The strain used in this example was a clinically isolated drug-resistant Candida albicans strain donated from the Changhai (this strain has been published in CN105503788A, time 20160420).
The strain culture conditions are as follows: all experimental strains are subjected to scratching activation on a Saburg glucose agar (SDA) culture medium, Candida albicans 103 is cultured at 30 ℃ for 2 weeks, then monoclonals are respectively picked and scratched and activated again, the monoclonals obtained in the second time are taken and placed on an SDA inclined plane, and the monoclonals are cultured by the method and then stored at 4 ℃ for later use.
2. Test drug
Name: extract of branches and leaves of Magnolia sieboldii.
3. Reagent
(1) RPMI 1640 liquid culture solution
RPMI 1640(Gibco BRL)10g，NaHCO32.0g, morpholine propanesulfonic acid (MOPS) (Sigma)34.5g (0.165M), adding 900ml of triple distilled water for dissolution, adjusting the pH to 7.0(25 ℃) by 1.0mol/L NaOH, fixing the volume of the triple distilled water to 1000ml, filtering and sterilizing by a 0.22 mu M microporous filter membrane, subpackaging and storing at 4 ℃ for later use.
(2) Sambo glucose agar solid medium (SDA)
10g of peptone, 40g of glucose and 18g of agar, adding 900ml of triple distilled water for dissolving, adding 50ml of 2mg/ml chloramphenicol aqueous solution, adjusting the pH to 7.0, fixing the volume to 1000ml with the triple distilled water, sterilizing under high pressure (121 ℃, 15min), and storing at 4 ℃ for later use.
(3) YEPD culture solution
10g of yeast extract, 20g of peptone and 20g of glucose, adding 900ml of triple distilled water for dissolving, adding 50ml of 2mg/ml chloramphenicol aqueous solution, fixing the volume to 1000ml of triple distilled water, and storing at 4 ℃ for later use after autoclaving (121 ℃, 15 min).
(4) PBS buffer
NaCl 8.0g，KCl 0.4g，Na2HPO4 0.133g，KH2PO40.06g，NaHCO30.35g, adding triple distilled water to a constant volume of 1000ml, sterilizing under high pressure, and storing at 4 ℃.
(5) Antifungal medicine
Fluconazole injection, shanghai xin yi jinzhu pharmaceutical limited. Baicalein (Baicalein, BE), shanghai calendar tripod biotechnology limited.
(6) Solvent(s)
Dimethyl sulfoxide (DMSO), shanghai chemical company, china pharmaceutical group.
(7) Preparing a medicine mother solution:
the fluconazole is an aqueous solution.
4. Instrumentation and equipment
Multiskan MK3 model enzyme standard detector (Labsystems);
a water-proof electric heating constant temperature incubator (jumping into a medical instrument factory from Shanghai);
model MJX Intelligent mold incubator (Ningbo Jiangnan Instrument works);
THZ-82A desk type constant temperature oscillator (jumping into medical instrument factory in Shanghai);
model sw.ct-IF ultra clean bench (suzhou altai air technologies, ltd);
inverted microscope (Amersham Pharmacia);
microsyrinths (Finnpette);
96-well cell culture plates (Nunclon);
LightCycler Real Time PCR instrument (Roche Diagnostics);
eppendorf5417R high speed refrigerated centrifuge (Eppendorf);
biofuge grates high speed refrigerated centrifuge (Heraeus);
laser confocal microscopy (Leica TCS sp 2);
microcon YM-30 filter (Millipore, Bedford, Mass.).
(II) Experimental method
1. Preparation method of Magnolia sieboldii branch and leaf extract
80% ethanol extract: pulverizing dried branches and leaves of Magnolia sieboldii, weighing 20g, extracting with 300ml of 80% ethanol water solution by heating and refluxing for 1 hr, and filtering; and heating and refluxing the filter residue by 300ml of 80% ethanol water solution for 1 hour, and filtering. Mixing the two filtrates, concentrating the filtrate under reduced pressure with a rotary evaporator, and recovering ethanol to obtain 80% ethanol extract 4.2 g. The extraction rate was 25.7%.
2. Preparation of fungal suspensions
Before the experiment, candida albicans 103 is picked from an SDA culture medium stored at 4 ℃ by using an inoculation ring, inoculated into 1ml of YEPD culture solution, subjected to shaking culture at 30 ℃ and 200rpm, and activated for 16 hours, so that the fungus is in the later period of exponential growth. Adding the bacterial solution into 1ml YEPD culture solution, activating again by the above method, counting with blood cell counting plate after 16h, adjusting bacterial solution concentration to 1 × 10 with RPMI 1640 culture solution3-5×103CFU/ml. Inoculating filamentous fungi to SDA slant, wherein subcutaneous tissue fungus and systemic fungus (Sporothrix schenckii) are cultured at 30 deg.C for one week(ii) a Superficial fungi (microsporum lanosum) were cultured at 30 ℃ for two weeks. Activating the bacteria twice, adding proper amount of RPMI 1640 culture solution on SDA slant, blowing and beating the bacterial colony with a straw to make the fungal spore free in the RPMI 1640 culture solution, and filtering with four layers of sterile gauze. Counting the culture solution by a blood cell counting plate, adding RPMI 1640 culture solution to adjust the spore concentration to 1 × 103-5×103CFU/ml。
3. Preparation of drug sensitive reaction plate
Taking a sterile 96-well plate, and adding 100 mu l of RPMI 1640 liquid culture medium into each row of No. 1 wells as a blank control; adding 100 mul of freshly prepared bacterial liquid into each of the 3-12 holes; 160 mul of bacteria liquid and 40 mul of tested compound solution are respectively added into a No. 2 hole; no. 12 wells contained no drug, and 100. mu.l of the bacterial suspension was added as a positive growth control. Wells 2-11 were diluted in duplicate to give final drug concentrations of 250.0, 125.0, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0.49 μ g/ml in each well, with DMSO levels below 1%. A quality control bacteria drug sensitive plate is prepared while preparing the drug sensitive plate each time, and each drug sensitive plate is cultured in a constant temperature box at 30 ℃.
4. Determination of minimum inhibitory concentration (MIC value)
After 2 weeks of Candida culture in a 30 ℃ incubator, the OD of each well was measured at 620nm using an enzyme-labeled analyzer. The OD value of the positive control hole is controlled to be about 0.2, and compared with the positive control hole, the MIC is the concentration of the medicine in the hole with the lowest concentration and the OD value of the medicine is reduced by more than 80 percent80(drug concentration at which fungal growth was 80% inhibited). MIC of drug80When the value exceeds the measured concentration range, counting according to the following method: MIC80A value higher than the maximum concentration of 64. mu.g/ml, is counted as ">64μg/ml”；MIC80When the concentration is the lowest concentration or below, the concentration is calculated to be less than or equal to 0.125 mu g/ml without distinction. All the above experiments were performed in parallel 2 to 3 times when MIC is80Values can be accurately repeated or accepted only at one concentration difference, and higher concentrations are taken as MICs80A value; when MIC80If the values differ by more than two concentrations, the experiment needs to be repeated until the values meet the requirements.
Reference to 1997 U.S. national clinical test standardsStandard J (M27-a protocol) by the chemical commission (NCCLS): determining the MIC of the AmB, comparing with a growth control hole, completely inhibiting the growth by 100 percent, and clearing the culture medium to obtain the MIC of the AmB corresponding to the lowest drug concentration; the lowest drug concentration corresponding to the growth inhibition of more than or equal to 80 percent compared with the growth control hole is the MIC of fluconazole80。
5. Chessboard type microdilution method
The checkerboard microdilution method is to use two kinds of medicines which are combined to be diluted by two times in the longitudinal (A to H) and transverse (2 to 11) directions of a two-dimensional chessboard on a 96-well plate. The test drug (80% ethanol extract of branches and leaves of Magnolia sieboldii) and the positive drug (scutellarin) were combined with the antifungal drug fluconazole respectively, so that the final concentrations of fluconazole and positive drug (scutellarin) were 128.0, 64.0, 32.0, 16.0, 8.0, 4.0, 2.0, 1.0, 0.5 and 0.25. mu.g/ml, and the final concentrations of test drug (80% ethanol extract of branches and leaves of Magnolia sieboldii) and positive drug (scutellarin) were 250.0, 125.0, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0.49. mu.g/ml, respectively. The reagents, medicines and experimental operation steps used in the experiment are the same as above.
Culturing 96-well plate in 30 deg.C constant temperature incubator for 24 hr, taking out, and reading MIC80The value is obtained. The results of the antibacterial activity are shown in Table 1.
TABLE 1 MIC of in vitro anti-Candida Activity of Magnolia sieboldii Branch and leaf extract in combination with Fluconazole80
(ii) use in combination with 8 μ g/ml fluconazole; FICI value is less than or equal to 0.5: synergistic effect, > 0.5: has no relevant effect.
As can be seen from the results in Table 1, 80% ethanol extract of Magnolia sieboldii has a strong synergistic antifungal activity against fluconazole.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the inventive content of the present invention.