阅读说明：本技术 一个倍半萜类化合物的制备及其协同氟康唑抗耐药真菌应用 (Preparation of sesquiterpenoids and application of sesquiterpenoids in synergism of fluconazole in resisting drug-resistant fungi ) 是由 刘娜 孙钰琳 吴聪 宋美娜 杨凤英 葛迪 于 2021-01-29 设计创作，主要内容包括：本发明提供了本发明提供了一个倍半萜类化合物,该化合物从中药毛梗豨莶中提取：毛梗豨莶地上部位干粉先用95％乙醇浸提、浓缩得到粗浸膏,再用乙酸乙酯萃取,萃取物以甲醇-水体系过MCI树脂柱层析,其中组分分别以氯仿-甲醇、石油醚-乙酸乙酯体系过正相硅胶柱层析,最后通过高效液相色谱ODS柱和手性柱进行分离纯化后得到。评价白色念珠菌对这个化合物的体外敏感性,并观察与氟康唑合用对白色念珠菌耐药菌株(28A)的联合抗真菌作用。本发明为有效的控制真菌感染,研发新型的抗真菌药物奠定了物质基础,有利于毛梗豨莶药用价值的进一步的开发。(The invention provides a sesquiterpenoids compound, which is extracted from siegesbeckia pubescens Makino as a traditional Chinese medicine: extracting dry powder of the overground part of siegesbeckia Makino with 95% ethanol, concentrating to obtain a crude extract, extracting with ethyl acetate, performing MCI resin column chromatography on the extract with a methanol-water system, performing normal phase silica gel column chromatography on the components with chloroform-methanol and petroleum ether-ethyl acetate systems respectively, and finally performing separation and purification by using a high performance liquid chromatography (ODS) column and a chiral column. The in vitro sensitivity of Candida albicans to this compound was evaluated, and the combined antifungal effect on Candida albicans resistant strain (28A) in combination with fluconazole was observed. The invention lays a material foundation for effectively controlling fungal infection and developing novel antifungal drugs, and is beneficial to further development of the medicinal value of siegesbeckia pubescens Makino.)

1. A sesquiterpene compound having the structure of formula (I):

the structural type of the sesquiterpenoids is clofane-2 beta, 9 beta-diol.

2. A method of preparing a sesquiterpene compound of claim 1, wherein: the method comprises the following steps:

(1) drying ground parts of siegesbeckia Makino, crushing, leaching with 95% ethanol, and concentrating the leaching solution to obtain a crude extract;

(2) suspending the crude extract in water, extracting with ethyl acetate, and concentrating the organic phase to obtain ethyl acetate extract;

(3) subjecting the ethyl acetate extract to MCI resin column chromatography, and eluting with 50% -85% v/v methanol-water to obtain main eluting component;

(4) subjecting the MCI column chromatography eluate to normal phase silica gel column chromatography, sequentially eluting with chloroform-methanol at a ratio of 100:1, 50:1, 20:1, 8:1, and 1:1v/v to obtain 5 components Fr.1-Fr.5:

(5) passing the component Fr.4 through normal phase silica gel column chromatography, eluting with petroleum ether-ethyl acetate at a ratio of 8:1v/v to obtain component Fr.4.1-Fr.4.3;

(6) subjecting the fraction Fr.4.2 to high performance liquid chromatography with YMC-pack ODS-A, eluting with 74% v/v methanol-water, detecting wavelength at 210nm, and collecting fraction with retention time of 12min to obtain pure sesquiterpene compound.

3. The method of claim 2, wherein: in the step (1), the material-to-liquid ratio of siegesbeckia Makino to 95% ethanol is 1: 2-3 (w/v).

4. The method of claim 2, wherein: in the step (1), the siegesbeckia Makino is crushed to a particle size diameter of less than 3 mm.

5. The method of claim 2, wherein: in the step (1), the leaching times are 3 times, and each time is 7 days.

6. The method of claim 2, wherein: in the step (1), the mixture is concentrated to 1/100-1/150 in original volume.

7. The method of claim 2, wherein: in the step (2), the ratio of the crude extract to water is 1: 3-4 (w/v).

8. The method of claim 2, wherein: in the step (2), the volume ratio of the ethyl acetate to the water is 1: 3-4; the extraction times of the ethyl acetate are 1-3 times.

9. Use of a sesquiterpene compound of claim 1 in combination with fluconazole against a drug-resistant fungus.

10. Use according to claim 9, characterized in that: the fungus 28A is Candida albicans separated from human oral cavity with drug resistance to Fluconazole (FLC).

Technical Field

The invention belongs to the field of natural medicinal chemistry, and relates to a preparation method of a sesquiterpenoids and application thereof in synergistic effect of fluconazole in resisting drug-resistant fungi.

Background

Botanical drugs have been the important source of natural drugs and also the main source of human diseases prevention and treatment. The incidence of fungal infection is on the rising trend year by year, and the clinical fungal drug resistance is generated, so people are promoted to hope to ask for novel natural antibacterial drugs in order to find drug lead compounds with better curative effect or new targets. Therefore, the development of new types of safe and efficient drug-resistant fungal drugs is one of the important challenges in the field of drug development today. The structural diversity and the characteristic of easy combination with biological macromolecules of the natural product determine incomparable advantages of the natural product in the process of participating in life physiology, and the natural product has important position irreplaceable in the research and development of new drugs and is an important source for finding drug lead structures and candidate drugs. Herba siegesbeckiae, an important Chinese medicinal resource, has been reported to have various structural types and various biological activities as a secondary metabolite. Therefore, metabolites having antifungal activity mined from siegesbeckia orientalis are of great importance for effective control of fungal infections. The method can lay a foundation for researching and developing novel antifungal medicines, provides strategy guidance and theoretical support for clinically and reasonably using the antifungal medicines in the future and exploring future research trends of the antifungal medicines, and provides a preparation method of the sesquiterpene compound and application of the sesquiterpene compound in cooperation with fluconazole in resisting medicine-resistant fungi.

Disclosure of Invention

In order to further discover the medicinal value of siegesbeckia Makino, the invention provides sesquiterpenes extracted from siegesbeckia Makino, which have synergistic activity of fluconazole against drug-resistant fungi.

Another object of the present invention is to provide a method for preparing the sesquiterpene.

It is still another object of the present invention to provide an antifungal use of the sesquiterpenes described above.

In order to achieve the purpose, the invention adopts the following technical scheme:

a sesquiterpene compound having the structure of formula (I):

the structural type of the sesquiterpenoids is clofane-2 beta, 9 beta-diol.

The preparation method of the sesquiterpenoids comprises the following steps:

(1) drying ground parts of siegesbeckia Makino, crushing, leaching with 95% ethanol, and concentrating the leaching solution to obtain a crude extract;

(2) suspending the crude extract in water, extracting with ethyl acetate, and concentrating the organic phase to obtain ethyl acetate extract;

(3) subjecting the ethyl acetate extract to MCI resin column chromatography, and eluting with 50% -85% v/v methanol-water to obtain main eluting component;

(4) subjecting the MCI column chromatography eluate to normal phase silica gel column chromatography, sequentially eluting with chloroform-methanol at a ratio of 100:1, 50:1, 20:1, 8:1, and 1:1v/v to obtain 5 components Fr.1-Fr.5:

(5) passing the component Fr.4 through normal phase silica gel column chromatography, eluting with petroleum ether-ethyl acetate at a ratio of 8:1v/v to obtain component Fr.4.1-Fr.4.3;

(6) subjecting the fraction Fr.4.2 to high performance liquid chromatography with YMC-pack ODS-A, eluting with 74% v/v methanol-water, detecting wavelength at 210nm, and collecting fraction with retention time of 12min to obtain pure sesquiterpene compound.

Preferably, in the step (1), the ratio of siegesbeckia Makino to 95% ethanol is 1: 2-3 (w/v).

Preferably, in the step (1), the siegesbeckia Makino is crushed to have a particle size diameter of less than 3 mm.

Preferably, in the step (1), the leaching times are 3 times, and each time is 7 days.

Preferably, in step (1), the concentrate is 1/100-1/150 in bulk.

Preferably, in the step (2), the ratio of the crude extract to water is 1: 3-4 (w/v).

Preferably, in the step (2), the volume ratio of the ethyl acetate to the water is 1: 3-4; the extraction times of the ethyl acetate are 1-3 times.

Application of a sesquiterpenoid compound in synergy of fluconazole in resisting drug-resistant fungi.

Preferably, the fungus 28A is candida albicans isolated from the human mouth having resistance to Fluconazole (FLC), but is not limited to candida albicans.

The invention has the beneficial effects that: the invention provides a sesquiterpene compound extracted from siegesbeckia Makino, a preparation method thereof and application thereof in antifungal activity, wherein the sesquiterpene is clofane-2 beta, 9 beta-diol, and the sesquiterpene is combined with fluconazole to show synergistic antifungal activity on candida albicans (28A) and obviously reduce the dosage of the fluconazole.

The active compound extracted by the invention provides a material basis for the research and development of antifungal drugs, and is beneficial to the further development of the medicinal value of siegesbeckia pubescens Makino.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a technical scheme that:

a sesquiterpene compound having the structure of formula (I):

the structural type of the sesquiterpenoids is clofane-2 beta, 9 beta-diol.

The preparation method of the sesquiterpenoids comprises the following steps:

(1) drying ground parts of siegesbeckia Makino, crushing, leaching with 95% ethanol, and concentrating the leaching solution to obtain a crude extract;

(2) suspending the crude extract in water, extracting with ethyl acetate, and concentrating the organic phase to obtain ethyl acetate extract;

(3) subjecting the ethyl acetate extract to MCI resin column chromatography, and eluting with 50% -85% v/v methanol-water to obtain main eluting component;

(4) subjecting the MCI column chromatography eluate to normal phase silica gel column chromatography, sequentially eluting with chloroform-methanol at a ratio of 100:1, 50:1, 20:1, 8:1, and 1:1v/v to obtain 5 components Fr.1-Fr.5:

(5) passing the component Fr.4 through normal phase silica gel column chromatography, eluting with petroleum ether-ethyl acetate at a ratio of 8:1v/v to obtain component Fr.4.1-Fr.4.3;

(6) subjecting the fraction Fr.4.2 to high performance liquid chromatography with YMC-pack ODS-A, eluting with 74% v/v methanol-water, detecting wavelength at 210nm, and collecting fraction with retention time of 12min to obtain pure sesquiterpene compound.

Preferably, in the step (1), the ratio of siegesbeckia Makino to 95% ethanol is 1: 2-3 (w/v).

Preferably, in the step (1), the siegesbeckia Makino is crushed to have a particle size diameter of less than 3 mm.

Preferably, in the step (1), the leaching times are 3 times, and each time is 7 days.

Preferably, in step (1), the concentrate is 1/100-1/150 in bulk.

Preferably, in the step (2), the ratio of the crude extract to water is 1: 3-4 (w/v).

Preferably, in the step (2), the volume ratio of the ethyl acetate to the water is 1: 3-4; the extraction times of the ethyl acetate are 1-3 times.

Application of a sesquiterpenoid compound in synergy of fluconazole in resisting drug-resistant fungi.

Preferably, the fungus 28A is candida albicans isolated from the human mouth having resistance to Fluconazole (FLC), but is not limited to candida albicans.

EXAMPLE 1 preparation of a sesquiterpene Compound

5kg of dried overground part of siegesbeckia Makino, pulverizing to particle size less than 3mm, soaking in 95% ethanol, and extracting for 3 times (10L each time) for 7 days. Mixing the ethanol extractive solutions, and concentrating under reduced pressure to obtain 302g crude extract;

the crude extract was suspended in 1.0L of water and then extracted three times with 0.5L of ethyl acetate each time. Mixing ethyl acetate extract phases, and concentrating under reduced pressure to obtain 83g of ethyl acetate extract;

subjecting the extract to MCI resin column chromatography, performing gradient elution with methanol-water (v/v, 50% -80%), detecting by thin layer chromatography, and mixing to obtain main elution sample 32 g;

subjecting the MCI column chromatography eluate to normal phase silica gel column chromatography, eluting with chloroform-methanol system (v/v, 100:1, 50:1, 20:1, 8:1, 1:1), analyzing eluate components by thin layer chromatography, and collecting to obtain components Fr.1-Fr.5;

performing normal phase silica gel column chromatography on the component Fr.4, isocratically eluting with a petroleum ether-ethyl acetate system (v/v, 8:1), analyzing the components of the eluent according to thin layer chromatography, and collecting to obtain a component Fr.4.1-Fr.4.3;

the fraction Fr.4.2 was purified by high performance liquid chromatography (column: YMC-pack ODS-A, 10 x 250mm, flow rate 3mL/min, detection wavelength 210nm), isocratically eluted with methanol-water (v/v, 74%), and the fraction with retention time of 12min was collected to obtain clofrane-2 β,9 β -diol.

Example 2 antifungal Activity of a sesquiterpene Compound

According to the proposal of M27-A recommended by the national committee for standardization of clinical trials (NCCLS), the sensitivity of the experimental strain to volatile oil is determined by microdilution, the bacterial content in each well is about (1-5) x 103cfu/mL, a sterile 96-micro-well plate is taken, 12 wells are arranged in each row, 1024 mu g/mL of drug stock solution is added into the first micro-well, 100 mu L of drug stock solution is filled from the second micro-well to the tenth micro-well by adopting a method of 10-level multiple dilution, the drug stock solution is diluted into 1024 mu g/mL, 512 mu g/mL, 256 mu g/mL, 128 mu g/mL, 64 mu g/mL, 32 mu g/mL, 16 mu g/mL, 8 mu g/mL, 4 mu g/mL and 2 mu g/mL by using liquid culture medium, no liquid is added into the eleventh micro-well, only 100 mu L of the liquid culture medium is added as positive control, no. twelve well only adds 200 mul of liquid culture medium as negative control, incubates for 12h in an incubator at 35 ℃, and uses a microplate reader to determine MIC, namely, the concentration of the compound or fluconazole is the minimum inhibitory concentration when the growth of Candida albicans (28A) is inhibited by 80%.

The minimum inhibitory concentration value of the volatile oil and the fluconazole after serial double-proportion is subjected to serial mixing by adopting a chessboard microdilution method to determine the minimum inhibitory concentration value when the volatile oil and the fluconazole are jointly applied, the experimental result of the joint application of the volatile oil and the fluconazole is evaluated by calculating the inhibitory concentration fraction index FIC, and the FIC is (MIC compound, combined application)/(MIC compound, single application) + (MIC fluconazole, combined application)/(MIC fluconazole, single application), and the evaluation standard is as follows: FIC is less than or equal to 0.5, and shows a synergistic effect; 0.5< FIC < 1, showing additive effect; 1< FIC <2, as an unrelated effect; the FIC is more than or equal to 2, and the smaller the FIC index is, the stronger the synergistic effect of the two medicines is.

The experimental result shows that the MIC of the sesquiterpene shown in the formula (I) and the fluconazole to 28A is more than 256 mu g/mL, when the sesquiterpene and the fluconazole are combined for use, the MIC value of the sesquiterpene and the fluconazole to the drug-resistant strain 28A is remarkably reduced, the FIC value is less than 0.09375, and the sesquiterpene compound shown in the formula (I) shows remarkable synergistic antifungal activity, as shown in the table 1, so that the sesquiterpene compound shown in the formula (I) has the application of synergistic fluconazole in resisting drug-resistant fungi.

TABLE 1 sesquiterpene synergy Fluconazole Activity against drug-resistant fungus 28A (MIC, μ M)

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.