A mixed-source terpene compound and its application in the preparation of a drug with antifungal activity

Abstract

The present invention relates to the technical field of medicinal chemistry, and specifically discloses a meroterpenoid compound bipolaricochlioquinones B isolated from endophytic fungi in potatoes Bipolaris eleusines and its potential application in the preparation of antifungal drugs. More specifically, it describes the application in the preparation of drugs with significant inhibitory activity against Trichophyton floccosum and Microsporum gypseum. The meroterpenoid compound of the present invention can strongly inhibit Trichophyton floccosum and Microsporum gypseum, and has the potential to be used in the preparation of novel antifungal drugs.

Description

Technical Field

[0001] The present invention relates to the fields of medicine and chemical technology, and particularly relates to a mixed-source terpene compound derived from the endophytic fungus Bipolaris eleusines of Solanum tuberosum and its application in the preparation of a drug with antifungal activity. Background Art

[0002] In the past half century, due to the increase in the number of immunocompromised hosts, the clinical manifestations of fungal diseases have increased significantly. Fungi include many species related to human diseases, usually including the genera Coccus, Aspergillus, and Candida. They are inhaled through the lungs or penetrate and spread throughout many organs of the body through the gastrointestinal tract, thus causing fungal infections. Human fungal infections can be roughly divided into superficial and deep infections of the skin and mucous membranes, systemic or disseminated infections. Superficial infections do not necessarily require systemic predisposing factors, while deep and systemic infections are more common in immunocompromised hosts. The incidence of fungal infections is increasing at an alarming rate, posing a huge challenge to healthcare professionals. Systemic infections are the most difficult-to-treat diseases among fungal infections. Systemic fungal infections usually originate in the lungs or endogenous flora and can spread to many other organs, which is an urgent situation that needs to be overcome currently. In the case of delayed treatment, the mortality rate is extremely high. It is reported that up to 95% of bone marrow transplant recipients infected with Aspergillus, and the mortality rate of opportunistic fungal infections exceeds 50%. Therefore, it is necessary to discover antifungal compounds with enhanced efficacy and better compatibility, and the research on antifungal substances remains a hot topic.

[0003] Potato (Solanum tuberosum L.) is an annual plant of the Solanaceae family, native to the Andes Mountains in South America. It is one of the world's major food crops, with the effects of regulating the stomach, strengthening the spleen, and replenishing qi, and can prevent and treat various diseases. It also has the effects of detoxification and anti-inflammation. Endophytic fungi refer to fungi, yeasts, and bacteria that invade or live within plant tissues without causing any diseases or harm to them. They also promote the growth of host plants and the formation of secondary metabolites related to plant defense. Usually, several to hundreds of endophytic fungal species can be isolated from a single plant, and at least one of them is host-specific. Compared with isolates of fungal plant pathogens and fungal soils, fewer secondary metabolites are isolated from endophytic fungi. The rich and diverse secondary metabolites of endophytic fungi make them a broad source of unexplored and uncharacterized microorganisms capable of producing new metabolites. Screening for bioactive lead compounds from the secondary metabolites of endophytic fungi is one of the effective ways to study innovative drugs.

[0004] Bipolaris is an important plant pathogen distributed worldwide, capable of causing leaf spot and leaf rust in corn, wheat, and rice. So far, a total of 47 species of Bipolaris have been identified, and their taxonomy, geographical distribution, and species synonyms have been clarified. Due to the important role of Bipolaris in plant pathogens, some have been widely used in biotechnology and genetic manipulation. Meroterpenoids are a class of hybrid natural products derived from terpenoids, with complex and diverse skeletons and various biological activities such as antifungal, anti-inflammatory, and antitumor effects. They have received extensive attention in the fields of food, daily chemicals, and medicine, showing great application potential and broad market prospects. The applicant has studied the antifungal activity of a meroterpenoid compound isolated from the endophytic fungus Bipolaris eleusines of potato. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide a meroterpenoid compound and its application in the preparation of a drug with antifungal activity.

[0006] A meroterpenoid with antifungal activity has the following structural formula:

[0007]

[0008] Molecular formula: C 29 H 41 NO 6 S

[0009] The application of the meroterpenoid compound in the preparation of a drug that can inhibit Trichophyton floccosum and Microsporum gypseum also belongs to the protection scope of the present invention.

[0010] In the present invention, the specific manifestation of the antifungal activity is as follows: it can strongly inhibit Trichophyton floccosum (the inhibition rate is 65.06% ± 0%), and can also strongly inhibit Microsporum gypseum (the inhibition rate is 51.07% ± 5.954%). The experiments of the present invention prove that the meroterpenoid compound can effectively inhibit some fungi and has the potential use for the preparation of a new antifungal drug.

[0011] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0012] 1. The present invention discloses a meroterpenoid compound derived from the endophytic fungus Bipolaris eleusines of potato, which can effectively inhibit Trichophyton floccosum and Microsporum gypseum.

[0013] 2. The present invention provides a mixed-source terpenoid compound with antifungal activity, which can effectively inhibit Trichophyton concentricum and Microsporum gypseum, and has the potential use for preparing a novel antifungal drug.

[0014] 3. The mixed-source terpenoid compound whose use is intended to be protected by the present invention can be obtained by extraction and purification from endophytic fungi of plants, and has the advantages of short culture cycle, high operation feasibility, no chemical pollution, environmental friendliness, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 1H NMR spectrum (600 MHz, CDCl 3 ) of the compound prepared in Example 1 in the specific implementation manner;

[0016] Figure 2 13C NMR spectrum (150 MHz, CDCl 3 ) and DEPT spectrum of the compound prepared in Example 1 in the specific implementation manner;

[0017] Figure 3 HMQC spectrum of the compound prepared in Example 1 in the specific implementation manner;

[0018] Figure 4 HMBC spectrum of the compound prepared in Example 1 in the specific implementation manner;

[0019] Figure 5 H- 1 H COSY spectrum of the compound prepared in Example 1 in the specific implementation manner; 1 H COSY spectrum;

[0020] Figure 6 ROESY spectrum of the compound prepared in Example 1 in the specific implementation manner;

[0021] Figure 7 C calculation of the compound prepared in Example 1 in the specific implementation manner 13 C calculation;

[0022] Figure 8 DP4+ analysis of the compound prepared in Example 1 in the specific implementation manner;

[0023] Figure 9 ECD calculation of the compound prepared in Example 1 in the specific implementation manner. SPECIFIC IMPLEMENTATION MANNER

[0024] To make the invention purpose and content of the present application presented more clearly, the applicant will clearly and completely describe the technical solution of the present invention below in combination with specific examples.

[0025] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0026] Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0027] Example 1:

[0028] The raw material potato endophytic fungus was isolated from the stem of a fresh potato plant in Lincang. The self-number of the strain is E-19. Through the comparison of the ITS sequence determination results, it is known that the maximum similarity of this sequence with Bipolaris eleusines is as high as 99%. The gene bank accession number is KY909768.1. Therefore, it was identified as Bipolaris eleusines, and this strain was preserved in the Microbial Strain Bank of the School of Pharmacy, South-Central University for Nationalities (already publicly available, see: Yang Mansi, Wang Wenxiang, Li Longgen, etc. Isolation, identification and secondary metabolite research of potato endophytic antagonistic fungus E-19 [J]. Natural Product Research and Development, 2015, 27(10): 1728-1731, 1736).

[0029] The specific isolation process of the above-mentioned potato endophytic fungus E-19 in Lincang is as follows: Wash the stem of a healthy potato plant clean under tap water, dry it, and cut the stem into 3-cm segments. Then, under sterile conditions, rinse it with 75% ethanol for 1 min and wash it 3 times with sterile water; disinfect it with 2% sodium hypochlorite for 2 min and rinse it 3 times with sterile water; then place it on sterile filter paper to absorb the moisture. Cut off the two ends of the surface-sterilized stem, split the remaining segments in the middle, and cut them into 0.5-cm 3 small pieces, and perform surface sterilization using a vertical pressure steam sterilizer (YXQ-LS-100S1, Shanghai Boxun). Then, under sterile conditions, transfer the small pieces onto a PDA medium and place them in an incubator at 25 °C for cultivation. Using the apical mycelium purification method, regularly observe the growth of endophytic fungi every day, pick the tip part of the newly grown mycelium at the incision and transfer it onto a PDA medium, and purify and culture it until a pure strain is obtained. Transfer the purified pure strain into a test tube containing PDA medium and place it in a 4 °C refrigerator for storage.

[0030] The strain E-19 was amplified by wheat solid fermentation: Take the test tube out of the refrigerator, place it in a sterile environment at 25 °C for one night, and then take a 0.5-cm mycelium block from the test tube under sterile conditions and inoculate it onto a plate PDA medium. After it grows for about 7 days and the colony covers the PDA, it is reserved. Wheat medium ratio: 50 g of wheat per bottle, 50 mL of distilled water per bottle, placed in a 500-mL culture bottle, sterilized at 120 °C for 30 min, and after cooling, pick 0.5-cm 3The mycelium blocks were inoculated onto wheat culture media, with a total of 224 bottles. Culture conditions: Dark culture at 25 °C for 40 d.

[0031] The separation and purification process of the mixed-source terpenoid compounds described in the claims and the invention content of the specification: Mash all the fermented wheat solid culture media described above and soak them 5 times with a strongly polar mixed solvent (dichloromethane: methanol = 1:1, v / v), 12 h each time. After each soaking, centrifuge, combine the extracts after 5 centrifugations, evaporate the solvent under reduced pressure until dry, dissolve with a small amount of water and ethyl acetate (water: ethyl acetate = 1:1, v / v), and extract 5 times with ethyl acetate. After combining the ethyl acetate parts, concentrate under reduced pressure to obtain 166.7 g of a crude extract paste. Perform gradient elution using a normal-phase silica gel chromatography column with 80–100 mesh (the eluent is dichloromethane: methanol = 100:0 - 0:100, v / v), and detect and develop color using thin-layer chromatography (the developing agent is dichloromethane: methanol = 20:1, v / v; thin-layer chromatography silica gel plate, Qingdao Ocean Chemical Factory) to roughly divide into six components A–F.

[0032] Component D (eluent: dichloromethane:methanol = 20:1, v / v, eluent volume: 10 column volumes, 31.6 g) was subjected to medium-pressure liquid chromatography (Biotage SP1, reverse-phase packing material: RP-18, 20 - 45 μm, Fuji Silysia Chemical Ltd., Japan). Methanol and water were used as eluents (methanol / water = 40:60, 60:40, 70:30, 80:20, 100:0, v / v, eluted in sequence), gradient elution was carried out at a flow rate of 20 mL / min, the elution time for each ratio was 40 min, thin-layer chromatography (developing agent: chloroform:methanol = 10:1, v / v; thin-layer chromatography silica gel plate, Qingdao Marine Chemical Factory) was used for detection and color development. The same or similar components were combined to obtain 10 sub-components, which were labeled D1–D10 in ascending order of polarity. Fraction D9 (methanol:water = 70:30, v / v, 903.5 mg) was chromatographed on a Sephadex LH-20 gel column (Pharmacia Fine Chemical Co., Ltd., Sweden) with methanol elution (methanol volume: 4 column volumes), thin-layer chromatography (developing agent: chloroform:methanol = 10:1, v / v; thin-layer chromatography silica gel plate, Qingdao Marine Chemical Factory) was used for detection and color development. The same or similar components were combined and separated into six sub-components, which were labeled D9-1–D9-6 in ascending order of the polarity of the obtained components. Fraction D9-3 (obtained from the second column volume during methanol elution, 113.6 mg) was subjected to preparative high-performance liquid chromatography (Agilent 1260; chromatographic column: Agilent Zorbax SB-C18, specification: 9.4 mm × 150 mm, 5 μm; acetonitrile-water, 50:50–60:40, v / v; flow rate: 4 mL / min) for gradient elution for 30 min, and 4.1 mg of the mixed-source terpenoid compound of the present invention (retention time: 20.5 min) was prepared.

[0033] Structure identification of the compound: The mixed-source terpenoid compound prepared in Example 1 was dissolved in 0.5 mL of deuterated chloroform, transferred to a nuclear magnetic resonance tube with a 200 μL pipette, and the hydrogen spectrum, carbon spectrum, two-dimensional spectrum, and 13 C quantum chemical calculation, ECD, and DP4+ probability analysis (as Figures 1-9 ) were detected on a nuclear magnetic resonance spectrometer (Bruker Avance III 600 MHz, Germany). The structure and configuration of the compound were solved by integrating various physical and chemical data and named bipolaricochlioquinones B.

[0034] Nuclear magnetic resonance data of the obtained mixed-source terpenoids:

[0035]

[0036]

[0037] Other physicochemical data of this mixed-source terpene:

[0038] Appearance: colorless solid; UV(MeOH)λ max (logε) = 235(3.61); IR(KBr): 3414, 2970, 2939, 2874, 1713, 1655, 1458, 1431, 1381, 1342, 1308, 1246, 1146, 1092, 1026, 833 cm -1 ; +361(c 0.5, MeOH); HRESIMS m / z 532.27271[M+H] + (calcd for C 29 H 42 NO 6 S + , 532.27274).

[0039] According to the results of the above tests, it is confirmed that the structural formula of the compound obtained in this example is:

[0040]

[0041] Molecular formula: C 29 H 41 NO 6 S

[0042] Example 2: Determination of antifungal activity

[0043] Principle: Inhibition rate = (absorbance value of blank bacterial solution - absorbance value of sample bacterial solution) × 100% / absorbance value of blank bacterial solution. The national standard stipulates that a product that can kill > 50% of fungi can be labeled as having an antibacterial effect; a product that can kill > 80% of fungi can be labeled as having a bactericidal effect. Both the bactericidal rate and the inhibition rate are indicators of bactericidal ability. The modes of action of bactericidal and antibacterial are different. Antibacterial is to inhibit the growth of fungi and prevent them from continuing to reproduce, while bactericidal is to destroy the structure of fungi and cause their death.

[0044] Experimental steps:

[0045] (1) Preparation of the compound to be tested: The compound obtained in Example 1, amphotericin B, and terbinafine hydrochloride were respectively prepared into 20 mM stock solutions with DMSO (terbinafine hydrochloride, amphotericin B, and DMSO were purchased from Sigma Corporation.

[0046] (2) Activation of the test strains: Take out the cryopreserved test strains (Candida albicans ATCC10231 purchased from Microbiologics, USA; Epidermophyton floccosum CBS 566.94, Trichophyton rubrum ATCC4438, Microsporum gypseum CBS118893 purchased from the Medical Fungal Culture Collection Center of the Chinese Academy of Medical Sciences) from the -80°C low-temperature storage box. Pipette 10 μl of the bacterial solution into a glass shaking culture tube containing 1 ml of YEPD culture medium, and place it in a 30°C air-bath constant temperature shaking incubator for shaking culture at 200 rpm. After 24 h, pipette 10 μl from the YEPD bacterial suspension into a new 1 ml of YEPD culture medium and continue shaking culture at 30°C for 16 h. The activation is completed, and the fungus at this time is in the late exponential growth phase.

[0047] (3) Determination of antifungal activity (inhibition rate): Dilute the mother liquor of the compound obtained in Example 1 in step (1) with DMSO (20 mM, 1 mM, 500 μM, 200 μM, 100 μM, 40 μM, 1 μM) to obtain the test compound solution; Take a 96-well culture plate, add the test compound solution and the fungal bacterial solution (dilute the bacterial solution activated in step (2) to a certain concentration with serum-free DMEM medium) to each well, and the final concentration is 1×10 5 CFU / mL (Candida albicans) and 5×10 5 CFU / mL (filamentous fungi: Epidermophyton floccosum, Trichophyton rubrum, and Microsporum gypseum). Incubate Candida albicans at 37°C for 24 hours and filamentous fungi at 25°C for 5 days, and then detect the absorbance value at 625 nm of each well with an enzyme-linked immunosorbent assay (ELISA) reader. At the same time, set up a medium blank control (only add 100 μL of serum-free DMEM medium), a blank bacterial solution (only add the fungal bacterial solution, 100 μM for all the above four fungi), amphotericin B (dilute the mother liquor in step (1) with DMSO to 0.5 μM), and terbinafine hydrochloride (dilute the mother liquor in step (1) with DMSO to 3 μM and 15 μM) as positive drug controls. Inhibition rate = (absorbance value of the blank bacterial solution - absorbance value of the sample bacterial solution) × 100% / absorbance value of the blank bacterial solution.

[0048] Experimental results:

[0049] In this experiment, the antifungal activity of the mixed-source terpenoid compounds obtained in Example 1 was screened against Candida albicans, Epidermophyton floccosum, Trichophyton rubrum, and Microsporum gypseum. The experimental results showed that at a concentration of 100 μM, the compound had a strong inhibitory effect on Epidermophyton floccosum and Microsporum gypseum (inhibition rate > 50%, see the following table).

[0050] Table 1: Detection results of inhibition rate

[0051]

[0052] Example 3: Determination of the sensitivity of yeast-like fungi to antifungal agents by microdilution method

[0053] Principle: Dilute the stock solution of the compound obtained in Example 1 in step (1) of Example 2 with DMSO (20 mM, 1 mM, 500 μM, 200 μM, 100 μM, 40 μM, 1 μM) to obtain the test compound solution. Take a 96-well plate and add 100 μl of the above-mentioned test compound solutions with different concentrations to wells 1-7 in each row. Wrap the prepared 96-well plate with plastic wrap and place it for 6 months; then take out the 96-well culture plate and add fungal broth (dilute the activated broth in step (2) of Example 2 to a certain concentration with serum-free DMEM medium) to wells 1-7. The final concentration is 1×10 5 CFU / mL (Candida albicans) and 5×10 5 CFU / mL (filamentous fungi: Trichophyton floccosum, Trichophyton rubrum, and Microsporum gypsum) are cultured. Candida albicans is cultured at 37 °C for 24 hours, and filamentous fungi are cultured at 25 °C for 5 days. Then, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at 625 nm in each well. Set a medium blank control in well 8 (add only 100 μL of serum-free DMEM medium), and a blank broth control (add only fungal broth, 100 μM for all four above-mentioned fungi). It is concluded that Candida albicans, Trichophyton floccosum, Trichophyton rubrum, and Microsporum gypsum are sensitive to this compound.

Claims

1. A mixed-source terpene compound, whose structural formula is:

2. Use of the mixed-source terpene compound according to claim 1 in the preparation of a drug for inhibiting Trichophyton floccosum and / or Microsporum gypseum.

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