Preparation method and application of triterpenoids with fungicidal activity

Abstract

The invention relates to a fermentation preparation method and application of a triterpenoid compound with fungicidal activity, and belongs to the technical field of medicines. According to the invention, a marine fungus Penicillium sp. OUCMDZ-1435 is subjected to solid fermentation, separation and purification, such that two triterpenoid compounds 1 and 2 with fungicidal activity are obtained. Researches find that the compound has the activity of inhibiting candida albicans and candida glabrata and has a bactericidal effect on the candida glabrata; meanwhile, when the compound 1 is combined with the market antifungal drug for use, a low-dose fungicidal effect can be achieved, and the compound 1 has the development value of drug combination. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to a fermentation preparation method of a triterpenoid compound with fungicidal activity and use of the triterpenoid compound in preparing antifungal drugs. Background Art

[0002] At present, the trend of fungal infections around the world is becoming increasingly severe. According to statistics so far, fungal infections directly cause 1.3 million deaths each year and are associated with the deaths of 5 million people. In 2022, the World Health Organization announced a list of "priority fungal pathogens". The 19 invasive fungi on its statistical report were divided into three categories according to priority, including extremely important, highly important, and moderately important. Among them, Candida infections are extremely important and highly important. Candida albicans in the genus Candida is an important conditional pathogen, a type of deep infection fungus that can cause acute or chronic inflammation of the skin or internal organs. At present, there are three main types of antifungal drugs used clinically to treat Candida infections: azoles, polyenes, and echinocandins. However, existing drugs have many disadvantages, such as severe drug resistance, narrow antibacterial spectrum, many adverse reactions, and high prices. Therefore, the development of new antifungal drugs is urgent.

[0003] In 2021, the U.S. FDA approved the launch of Brexafemme, the only antifungal drug in nearly 20 years. This compound is a triterpene nucleus structure compound obtained by semi-synthetic modification of Enfumafungin obtained by fermentation of Aspergillus fumigatus. The fourth-generation antifungal drug triterpenoids act on glucan synthase, and mammals, including humans, do not have cell walls, so they have strong biosafety, and their development and research value is huge.

[0004] Triterpenoid 1 was first isolated and identified in 1998 from the fungus Preussia aemulans (Rehm) v. Arx by liquid fermentation extraction; in 2004, in the structural study of other new compounds, it was mentioned that the compound had antifungal activity, but only the half inhibitory concentration (IC50) against Candida albicans (FP578) was given. 50 1.6 μg / mL)(Arioka M., J. Antibiot, 1998, Fujii T., J Antibiot, 2004). Triterpenoid 2 was isolated from Phomopsis sp. YM355364 by liquid fermentation in 2014, and its minimum inhibitory concentration (MIC 512 μg / mL) against Candida albicans (YM2005) was only reported (Heb J., Nat Prod Commun, 2014).

[0005]

[0006] At present, there are few studies and applications on the antifungal activity of triterpenoids. Although the above two documents mentioned the antifungal activity, there has never been any literature reporting the fungicidal effect and synergistic drug effect of these two triterpenoids. Most of the current first-line antifungal drugs can only achieve fungal inhibition and can only be used for superficial infections. However, deep infection is the main reason for the high mortality rate of fungal infections. Therefore, compounds with fungicidal activity are more likely to be developed as drugs for the treatment of deep fungal infections. At the same time, solving the drug resistance problem of marketed drugs and enhancing drug activity are also effective measures for antifungal treatment. For example, combined drug use can be used to achieve this problem. However, there is no literature reporting the combined use of triterpenoids and marketed antifungal drugs to kill fungi, thereby achieving the treatment of deep fungal infections. Summary of the invention

[0007] In view of the above problems, the first object of the present invention is to provide a method for preparing triterpenoid compounds 1 and 2 having fungicidal activity.

[0008] The second object of the present invention is to provide a use of the above triterpenoid compound having fungicidal activity in the preparation of fungicidal drugs.

[0009] The present invention uses marine fungus Penicillium sp.OUCMDZ-1435 to obtain two triterpenoid compounds 1 and 2 with fungicidal activity through solid fermentation, separation and purification. Studies have found that they have the activity of inhibiting Candida albicans and Candida glabrata, and have a bactericidal effect on Candida glabrata. At the same time, when compound 1 is used in combination with an antifungal drug already on the market, a low-dose fungicidal effect can be achieved, and the compound has the development value of combined medication.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0011] The first aspect of the present invention provides a method for preparing triterpenoid compounds 1 and 2 having fungicidal activity, characterized in that the method comprises the following steps:

[0012] (1) Strain activation: Penicillium sp.OUCMDZ-1435 was inoculated into PDA solid culture medium and incubated at 28°C for 3 days. An appropriate amount of spores was picked up with an inoculation loop and inoculated into 150 mL of liquid seed culture medium for activation. The culture was shaken at 28°C and 180 rpm for 2 days to obtain seed solution.

[0013] The Penicillium sp.OUCMDZ-1435 strain was deposited in the China General Microbiological Culture Collection Center (CGMCC) on October 26, 2022, with the deposit number CGMCC No.40345.

[0014] The PDA solid culture medium is prepared by adding 200 g of potato blocks to 1 liter of water, boiling for 15 minutes, filtering to obtain potato juice, making up the filtrate to 1 liter with water, adding 20 g of glucose and 20 g of agar, and sterilizing by high pressure at 121° C. for 20 minutes.

[0015] The liquid seed culture medium is prepared by adding 200 g of potato cubes into 1 liter of water, boiling for 15 minutes, filtering to obtain potato juice, making up the filtrate to 1 liter with water, adding 20 g of glucose, and sterilizing by high pressure at 121° C. for 20 minutes.

[0016] (2) Strain fermentation: About 10 mL of seed liquid was inoculated into a fermentation bag containing fermentation medium, and the mixture was allowed to ferment at 28° C. for 15 days to obtain a fermentation product.

[0017] The fermentation medium is: 100 g rice, 80 ml tap water, natural pH.

[0018] (3) Extraction of fermentation products: The solid fermentation product was transferred to a container, an equal volume of ethyl acetate was added, and extraction was performed three times. The ethyl acetate phase was concentrated to obtain the fermentation product.

[0019] (4) Product separation and purification: The crude extract was separated and purified by normal phase silica gel column chromatography, reverse phase silica gel column chromatography, gel column chromatography, and semi-preparative HPLC to obtain triterpenoid compounds 1 and 2.

[0020] The structures of the triterpenoid compounds 1 and 2 are as follows:

[0021]

[0022] The second aspect of the present invention provides a use of the triterpenoid compounds 1 and 2 or their pharmaceutically acceptable salts in the preparation of antifungal drugs or fungicides.

[0023] Furthermore, provided is the use of the triterpenoid compounds 1 and 2 or their pharmaceutically acceptable salts in the preparation of drugs against Candida albicans (Candida albicans 10231) or Candida glabrata.

[0024] The present invention also provides the use of the triterpenoid compounds 1 and 2 or their pharmaceutically acceptable salts in combination with other commercially available antifungal drugs in the preparation of antifungal drugs or fungicides.

[0025] Furthermore, provided is the use of the triterpenoid compounds 1 and 2 or their pharmaceutically acceptable salts in combination with other commercially available antifungal drugs in the preparation of drugs against Candida albicans 10231 or Candida glabrata 2001 or drugs that kill Candida albicans 10231 or Candida glabrata 2001.

[0026] The commercially available antifungal drug is selected from ketoconazole, fluconazole, itraconazole, and amphotericin B.

[0027] Due to the adoption of the above technical solution, the present invention has the following advantages:

[0028] (1) The present invention provides a method for obtaining compounds 1 and 2 by static fermentation of marine-derived Penicillium sp.OUCMDZ-1435 using rice solid culture medium. The raw materials are cheap and readily available, the steps are simple, and the preparation conditions are mild.

[0029] (2) The present invention carried out antifungal activity test on the above two compounds, and the results showed that the MIC values ​​of compound 1 against Candida albicans and Candida glabrata were 0.5 μg / mL and 0.25 μg / mL, respectively, and the MFC (minimum fungicidal concentration) value against Candida glabrata was 2 μg / mL; the MIC values ​​of compound 1 against Candida albicans and Candida glabrata were 0.5 μg / mL and 0.25 μg / mL, respectively, and the MFC value against Candida glabrata was 2 μg / mL; the MIC values ​​of compound 2 against Candida albicans and Candida glabrata were 4 μg / mL and 8 μg / mL, respectively, and the MFC value against Candida glabrata was 64 μg / mL. When the fungicidal effect was determined, high concentrations of ketoconazole alone did not kill Candida albicans, while the results of the synergistic antifungal activity determination showed that when 64 μg / mL or 32 μg / mL of ketoconazole was used in combination with 2 μg / mL of compound 1, the effect of completely killing Candida albicans that could not be achieved by using ketoconazole alone could be achieved. Therefore, the results of the present invention show that triterpenoid compounds 1 and 2 have significant antifungal activity, especially the lethal killing effect of the compounds on Candida glabrata, which has not been reported in the literature. At the same time, the drug can synergistically exert an antifungal effect with commercially available antifungal drugs, greatly reduce the concentration of drug use, solve the problem of drug resistance, and be used to prepare a new antifungal drug for combating deep infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the strain morphology of Penicillium sp.OUCMDZ-1435.

[0031] Figure 2 The compound 1 of the present invention 1 H NMR spectrum.

[0032] Figure 3 The compound 1 of the present invention 13 C NMR spectrum.

[0033] Figure 4 The compound 2 of the present invention 1 H NMR spectrum.

[0034] Figure 5 The compound 2 of the present invention 13 C NMR spectrum.

[0035] Figure 6 The synergistic antifungal activity test results of compound 1 of the present invention are shown in FIG. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0037] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available.

[0038] Example 1: Preparation of Compounds 1 and 2

[0039] 1. Isolation and preservation of Penicillium sp.OUCMDZ-1435 strain:

[0040] The compounds 1 and 2 of the present invention are obtained by fermentation, extraction and separation of Penicillium sp.OUCMDZ-1435 strain, and the strain is separated and purified from root mud samples of Wenchang Mangrove Nature Reserve in Hainan.

[0041] (1) Sample collection

[0042] The samples were 14 soil samples collected from the roots of plants such as Bruguiera gymnorrhiza, Aconitum carmichaelii, Prunus elata, Acanthus sinensis and Sonneratia ovalifolia in the Wenchang Mangrove Nature Reserve in Hainan. After collection, they were immediately placed in sterile sealed bags, frozen and transported to the laboratory, and stored in a -20°C refrigerator.

[0043] (2) Sample processing

[0044] The soil samples were separated by gradient dilution method. 1 g of sample was weighed in a sterile environment and surface sterilized with 75% ethanol. The sample was then rinsed with sterile seawater for multiple times and ground into a homogenate in a mortar. The supernatant was diluted with sterile seawater into three gradients of 0.1 g / mL, 0.01 g / mL, and 0.001 g / mL.

[0045] (3) Isolation and purification of strains

[0046] Use PDA medium (natural pH value) to isolate and culture microorganisms. First, use a sterile pipette to absorb 0.2mL of sample solution with different dilution ratios, add it dropwise to the PDA medium, and then use a sterilized coating rod to spread it evenly. After the bacterial solution penetrates into the medium, place it in a 28℃ constant temperature incubator and culture it upside down for 4-7 days.

[0047] Use the plate streak method to purify the strain, observe the growth of the plate bacteria, and when a single colony appears, you can start streak culture. The specific method of plate streak: Use the outer flame of the alcohol lamp to burn the inoculation loop. After sufficient cooling, scrape a single colony from the plate, and use the inoculation loop to make a zigzag streak on the agar plate. This operation needs to be performed in a sterile area near the flame of the alcohol lamp. After the inoculation is completed, the strain number, name and date are marked on the bottom of the culture dish, and then it is placed in a 28℃ constant temperature incubator for inverted culture. If the strain cannot be purified by streaking once, a second streak operation is required until the strain is completely purified.

[0048] (4) Preservation of strains

[0049] Slant preservation: Use the zigzag streaking method to inoculate the purified strain into the slant PDA medium. After the strain grows to maturity, store it in a 4°C refrigerator. Set up 3 parallels for each strain.

[0050] Glycerol tube cryopreservation: Use PDA liquid culture medium (add 20% glycerol) as cryopreservation liquid to cryopreserve the purified strain. Take 1mL of cryopreservation liquid into a cryopreservation tube and sterilize it. After cooling, scrape an appropriate amount of mycelium of the strain on the agar plate into a cryopreservation tube and store it in a -80℃ refrigerator.

[0051] (5) Identification and preservation of strains

[0052] The strains were identified by morphological methods, as shown in the photo. Figure 1As shown. The strain has the following morphological characteristics: the aerial hyphae are short, and the hyphae are white or green; the microscopic morphology of the insert under a 400x microscope shows that the aerial hyphae are broken into spores, which are spherical and smooth. The strain was sent to the China General Microbiological Culture Collection Center (CGMCC) for preservation, named Penicillium sp.OUCMDZ-1435, the preservation number is CGMCC No.40345, and the preservation time is October 26, 2022.

[0053] 2. Preparation of compounds 1 and 2 using strains:

[0054] (1) Strain activation: Penicillium sp.OUCMDZ-1435 was inoculated into PDA solid culture medium and incubated at 28°C for 3 days. An appropriate amount of spores was picked up with an inoculation loop and inoculated into 150 mL of liquid seed culture medium for activation. The culture was shaken at 28°C and 180 rpm for 2 days to obtain seed solution.

[0055] The PDA solid culture medium is prepared by adding 200 g of potato blocks to 1 liter of water, boiling for 15 minutes, filtering to obtain potato juice, making up the filtrate to 1 liter with water, adding 20 g of glucose and 20 g of agar, and sterilizing by high pressure at 121° C. for 20 minutes.

[0056] The liquid seed culture medium is prepared by adding 200 g of potato cubes to 1 liter of water, boiling for 15 minutes, filtering to obtain potato juice, making up the filtrate to 1 liter with water, adding 20 g of glucose, and sterilizing under high pressure at 121°C for 20 minutes.

[0057] (2) Strain fermentation: About 10 mL of seed liquid was inoculated into a fermentation bag containing fermentation medium, and the mixture was allowed to ferment at 28° C. for 15 days to obtain a fermentation product.

[0058] The fermentation medium is: 100 g rice, 80 ml tap water, natural pH.

[0059] (3) Extraction of fermentation product: The solid fermentation product was transferred to a container, and an equal volume of ethyl acetate was added. The extraction was performed three times, and the ethyl acetate phase was concentrated to obtain 70 g of fermentation product.

[0060] (4) Product separation and purification: The crude extract was separated by normal phase silica gel column chromatography with a mobile phase of (CH2Cl2-CH3OH 100:1-0:100). Each 1 L of the collected fraction was combined into one fraction, resulting in a total of 10 fractions. Fr.7 was selected for subsequent reverse phase silica gel column chromatography with a mobile phase of (CH3OH:H2O 10:90-100:0). A total of 16 fractions were obtained. Fr.7-16 was separated by gel column chromatography. The target compound was found in Fr.7-16-5 and separated and purified by semi-preparative HPLC (43% MeCN-0.5‰ TFA-H2O, C18). It was identified as triterpenoid compounds 1 and 2.

[0061] (5) Structural identification:

[0062] Compound 1: white solid, low resolution mass spectrum gave [M+H] at m / z 485.322 + The molecular ion peak indicates a molecular weight of 484. 1 H and 13 C NMR spectrum confirmed the molecular formula to be C 30 H 44 O5, unsaturation degree is 9. Further comparison of NMR and mass spectrometry data with literature data revealed that the structure of this compound is basically consistent with that of compound S19159 reported in the literature. Combined with the optical rotation [α] of compound 1 33 Optical rotation of D-89.5 (c 1 mg / mL, MeOH) and the compound S19159 in the literature [α] 23 The optical rotation comparison of D-74 (c 0.54 mg / mL, pyridine) further confirmed the structure of compound 1. The NMR data are as follows: 1 HNMR (400MHz, CD3OD)δ H: 0.68 (s, 3H, 18-H), 0.98 (d, 3H, 21-H), 1.02 (d, 3H, J = 2.33 Hz, 26-H), 1.04 (d, 3H, J = 2.63 Hz, 27-H), 1.16 (s, 3H, 29-H), 1.21 (m, 1H, 22-H), 1.31 (m, 1H, 16-H), 1.41 (m, 1H, 19-H), 1.42 (m, 1H, 15-H, 20-H), 1.46 (m, 1H, 17-H), 1.51 (m, 1H, 19-H), 1.61 (m, 1H, 22-H), 1.71 (m, 1H, 15-H), 1.88 (m, 2H, 6-H), 1.92 (m, 1H, 23-H), 1.95 (d, 1H, J = 15.2 Hz, 12-H), 1.98 (dd, 1H, J1 = 15.3 Hz, J2 = 6.7 Hz, 12-H), 1.99 (m, 1H, 16-H), 2.14 (m, 1H, 23-H), 2,25 (m, 1H, 25-H), 2.43 (dd, 1H, J1 = 12.5 Hz, J2 = 5.4 Hz 5-H), 2.50 (dd, J1 = 16.5 Hz, J2 = 9.2 Hz, 1H, 2-H), 2.89 (dd, J1 = 15.9 Hz, J2 = 6.7 Hz, 1H, 2-H), 3.06 (m, 1H, 14-H), 4.39 (d, 1H, 11-H), 4.56 (dd, 1H, J1 = 9.6 Hz, J2 = 7.3 Hz, 3-H), 4.66 (s, 1H, 3-OH), 4.73 (s, 2H, 30-H), 5.25 (m, 1H, 7-H), see Figure 2 。

[0063] 13 C NMR (125 MHz, CD3OD) δ C:210.9(C-1),179.4(C-28),157.7(C-24),142.7(C-8),117.5(C-7),106.9(C-30),73.1(C-3),69.8( C-11),58.7(C-17),53.4(C-4),49.7(C-14),47.78(C-2),46.9(C-12),42.9(C-13),39.1(C-10),38. 8(C-5),37.8(C-9),37.2(C-20),35.6(C-22),34.9(C-25),32.1(C-23),29.7(C-16),28.6(C-19),24 .2(C-6),24.2(C-15),22.4(C-26),22.2(C-27),18.9(C-21),17.2(C-18),9.4(C-29),22.4(C-26), see Figure 3 .

[0064] Combining the above characterization data, its structure is determined as follows:

[0065]

[0066] Compound 2: white solid, low resolution mass spectrum gave [M+H] at m / z 445.327 + The molecular ion peak indicates a molecular weight of 444. 1 H and 13 C NMR spectrum confirmed the molecular formula to be C 28 H 44 O4, unsaturation degree is 7. Further comparison of NMR and mass spectrometry data with literature data revealed that the structure of this compound is basically consistent with that of the compound 3β,5α,9α-trihydroxyergosta-7,22-diene-6-one reported in the literature. Combined with the optical rotation of compound 2 [α] 33 Optical rotation of D-70.2 (c 1 mg / mL, MeOH) and 3β,5α,9α-trihydroxyergosta-7,22-diene-6-one in the literature [α] 20 The optical rotation comparison of D-60 (c 0.14 mg / mL, CHCl3) further identified compound 2 as 3β,5α,9α-trihydroxyergosta-7,22-diene-6-one.

[0067] The NMR data are characterized as follows: 1 H NMR (400MHz, CD3OD)δ H:0.67(s,1H,19-H),0.85(d,3H,J=6.9Hz,27-H),0.87(d,3H,J=7.3Hz,26-H), 0.95(d,3H,J=6.9Hz,28-H),0.99(s,1H,18-H),1.05(d,1H,J=6.8Hz,21-H),1. 44(m,1H,17-H),1.46(m,1H,2-H,16-H),1.49(m,1H,1-H,24-H),1.52(m,1H,1 5-H),1.62(m,1H,4-H,15-H),1.71(m,1H,12-H),1.74(m,1H,11-H),1.80(m,1H ,11-H),1.86(m,1H,24-H),1.88(m,1H,2-H),1.90(m,1H,12-H),1.99(m,1H,1 6-H),2.02(m,1H,4-H),2.07(m,1H,20-H),2.30(td,J1=13.9Hz,J2=3.9Hz,1H, 1-H),2.76(m,1H,14-H),3.93(m,1H,3-H),5.21(dd,J1=15.2Hz,J2=7.8Hz,1H, 22-H), 5.27 (dd, J1=15.1Hz, J2=7.1Hz, 1H, 23-H), 5.59 (d, 1H, J=2.1Hz, 7-H), see Figure 4 .

[0068] 13 C NMR (125MHz, CD3OD)δ C :200.1(C-6),165.1(C-8),136.6(C-23),133.6(C-22),120.9(C-7),80.1(C-5),76.1(C-9),6 7.8(C-3),57.4(C-17),52.8(C-14),46.2(C-13),44.3(C-24),42.7(C-10),41.6(C-20),37.1 (C-4), 34.4 (C-25), 30.9 (C-2), 29.3 (C-16), 29.1 (C-11), 26.6 (C-1), 26.1 (C-12), 23.4 (C-15), 21.5 (C-21), 20.5 (C-18), 20.4 (C-26), 20.1 (C-27), 18.2 (C-28), 12.5 (C-19), 20.4 (C-26), see Figure 5 .

[0069] Combining the above characterization data, its structure is determined as follows:

[0070]

[0071] Example 2: Determination of antifungal activity of compounds 1 and 2

[0072] (1) Preparation of bacterial solution

[0073] Use the inoculation circle to pick the newly passaged Candida albicans on the SDA plate and transfer it to the YPD medium. Incubate it at 28℃ and 180r / min for 12h to fully activate the fungus. Count the bacterial solution with a hemocytometer and adjust the concentration of the bacterial solution to 1×10 5 cfu / mL.

[0074] The SDA solid culture medium includes: 10 g of peptone, 40 g of glucose, 20 g of agar, 1 liter of water, and natural pH.

[0075] YPD medium includes 10 g yeast extract, 20 g peptone, 20 g glucose, 8 g agar, 1 liter water, natural pH.

[0076] (2) Preparation of drug solution

[0077] The test compound and positive drug ketoconazole were prepared with cell-grade DMSO to an initial concentration of 2.56 mg / mL.

[0078] (3) MIC value determination

[0079] In the 96-well plate, 200 μL of culture medium was added to the blank control well, and 100 μL of culture medium was added to the positive control well, growth control well, and remaining wells. The positive control was ketoconazole, and 5 μL of ketoconazole solution with an initial concentration of 2.56 mg / mL was added to the positive control well. The test wells were diluted by the two-fold dilution method, that is, 5 μL of the initial compound solution (initial concentration of 2.56 mg / mL) was added to the first well, and 100 μL was transferred to the second well after mixing, and then added to the remaining wells by analogy. No test compound was added to the growth control well and the blank control well, and then 100 μL of the prepared solution diluted to 1×10 was added to the test well, the positive control well, and the growth control well. 5 -1×10 6 cfu / mL of Candida albicans or Candida glabrata suspension, place the 96-well plate in a 37°C incubator and culture it, and read the MIC value (visual observation) after 24 hours.

[0080] (4) MFC value determination

[0081] Based on the MIC determination, 50 μL was aspirated from each well of the 96-well plate after 12 hours of culture and inoculated onto a YPD solid plate (mixing was required before aspiration). The solid plate was placed in a 37°C incubator for 12 hours and then observed to see if there were any colonies growing on the solid plate. The minimum fungicidal concentration (MFC) was determined if the count was less than 5 colonies.

[0082] Table 1 MIC values ​​of compound 1 and compound 2

[0083]

[0084] As can be seen from Table 1, the MIC values ​​of compound 1 against Candida albicans and Candida glabrata were 0.5 μg / mL and 0.25 μg / mL, respectively; the MIC values ​​of compound 2 against Candida albicans and Candida glabrata were 4 μg / mL and 8 μg / mL, respectively. The results showed that the MIC values ​​of compounds 1 and 2 were significantly lower than that of commercially available ketoconazole (positive control), and compound 1 in particular showed a better antibacterial effect.

[0085] Table 2 MFC values ​​of compound 1 and compound 2

[0086]

[0087] As can be seen from Table 2, the positive control ketoconazole has no bactericidal effect on both pathogenic fungi, and compounds 1 and 2 have no bactericidal effect on Candida albicans, but compounds 1 and 2 have bactericidal effects on Candida glabrata, among which the MFC value of compound 1 on Candida glabrata is 2 μg / mL, and the MFC value of compound 2 on Candida glabrata is 64 μg / mL, which is not achievable by ketoconazole (positive control).

[0088] (5) Determination of synergistic antifungal activity

[0089] Pick the newly passaged Candida albicans on the SDA plate and add it to the YPD medium. Culture it at 28°C and 180 rpm for 12 h. Use YPD medium for culture. Filter and sterilize again before adding the medium. Use a hemocytometer to count and record it as 1×10 5cfu / mL. Add 190 μL of YPD medium to the first well of a 96-well plate, 200 μL of medium to the blank control well, and 100 μL of medium to the growth control well and the remaining wells. Then, 10 μL of the initial compound solution (initial concentration was 2 mg / mL) was added to the first well, and the solution was diluted by the two-fold dilution method, that is, 100 μL was transferred to the second well after mixing, and then added to the remaining wells in turn. After the addition, 5 μL of the prepared 2 μg / mL compound 1, 0.25 μg / mL compound 1, 32 μg / mL compound 2, and 2 μg / mL compound 2 were added to each test well, respectively, to form 4 different series, namely, different concentrations of ketoconazole and 2 μg / mL compound 1, different concentrations of ketoconazole and 0.25 μg / mL compound 1, different concentrations of ketoconazole and 32 μg / mL compound 2, and different concentrations of ketoconazole and 2 μg / mL compound 2. No compound was added to the growth control wells and the blank control wells, and then 100 μL of the solution diluted to 1×10 in advance was added to the test wells and the growth control wells. 5 -1×10 6 cfu / mL of bacterial suspension, place the 96-well plate in a 37°C incubator for static culture, and read the minimum value of fungal growth inhibition after 12 hours of culture (visual observation). On this basis, draw 50 μL from each well of the 96-well plate cultured for 12 hours and inoculate it on a YPD solid plate (mix before drawing), place the solid plate in a 37°C incubator for 12 hours, and observe whether there is colony growth on the solid plate. If the count is less than 5 colonies, it is fungicidal.

[0090] Its antibacterial effect is Figure 6 .in, Figure 6 A: Colony growth of Candida albicans under the action of 64μg / mL ketoconazole; Figure 6 B: Colony growth of Candida albicans under the action of 32 μg / mL ketoconazole; Figure 6 C: Colony growth of Candida albicans under the action of 2 μg / mL compound 1; Figure 6 D: Colony growth of Candida albicans under the synergistic effect of 64 μg / mL ketoconazole and 2 μg / mL compound 1; Figure 6 E: Colony growth of Candida albicans under the synergistic action of 32 μg / mL ketoconazole and 2 μg / mL compound 1.

[0091] As can be seen from Table 2, although compounds 1 and 2 have bactericidal effects on Candida glabrata, even when the concentration of ketoconazole, compound 1 and compound 2 reaches 64 μg / mL, they still have no bactericidal effect on Candida albicans (see the results). Figure 6A, 6B, 6C), so the combined drug evaluation was further carried out. First, the above-mentioned concentrations of ketoconazole and compound 1 were selected for bactericidal effect determination. The results showed that when 64μg / mL ketoconazole was used in combination with 2μg / mL compound 1, no Candida albicans grew. Therefore, when 64μg / mL ketoconazole was used in combination with 2μg / mL compound 1, a bactericidal effect could be achieved; and then a lower concentration of ketoconazole and compound 1 was used for combined drug evaluation. The results showed that when 32μg / mL ketoconazole was used in combination with 2μg / mL compound 1, a bactericidal effect could still be achieved. Therefore, when 64μg / mL or 32μg / mL ketoconazole was used in combination with 2μg / mL compound 1, the effect of killing Candida albicans that could not be achieved by using a single drug could be achieved (see the results). Figure 6 D, 6E). The above experimental results indicate that compound 1 is expected to become a lead compound for a new antifungal method for treating deep Candida albicans infection through combined drug therapy.

Claims

1. A method for preparing a triterpenoid compound having fungicidal activity, characterized in that: The following steps are involved: (1) Strain activation: Penicillium sp.OUCMDZ-1435 was inoculated into PDA solid medium and incubated at 28°C for 3 days. An appropriate amount of spores was picked up with an inoculation loop and inoculated into 150 mL of liquid seed medium for activation. The spores were incubated at 28°C and 180 rpm for 2 days to obtain seed solution. (2) Strain fermentation: About 10 mL of the seed solution was inoculated into a fermentation bag containing a fermentation medium, and the fermentation was allowed to stand at 28° C. for 15 days to obtain a fermentation product; (3) Extraction of fermentation product: The solid fermentation product was transferred to a container, an equal volume of ethyl acetate was added, and extraction was performed three times. The ethyl acetate phase was concentrated to obtain the fermentation product; (4) Product separation and purification: The crude extract was separated and purified by normal phase silica gel column chromatography, reverse phase silica gel column chromatography, gel column chromatography, and semi-preparative HPLC to obtain triterpenoid compounds 1 and 2; The structures of the triterpenoid compounds 1 and 2 are as follows: The Penicillium sp.OUCMDZ-1435 strain described in step (1) was deposited in the China General Microbiological Culture Collection Center (CGMCC) on October 26, 2022, with the deposit number CGMCC No.40345.

2. The method for preparing triterpenoid compounds according to claim 1, characterized in that: In step (1), the PDA solid culture medium is prepared by adding 200 g of block potatoes to 1 liter of water, boiling for 15 minutes, filtering to obtain potato juice, and if the filtrate is insufficient, adding water to 1 liter, adding 20 g of glucose and 20 g of agar, and sterilizing by high pressure at 121° C. for 20 minutes; the liquid seed culture medium is prepared by adding 200 g of block potatoes to 1 liter of water, boiling for 15 minutes, filtering to obtain potato juice, and if the filtrate is insufficient, adding water to 1 liter, adding 20 g of glucose, and sterilizing by high pressure at 121° C. for 20 minutes.

3. The method for preparing triterpenoid compounds according to claim 1, characterized in that: In step (2), the fermentation medium is: 100 g rice, 80 ml tap water, natural pH.

4. Use of the triterpenoid compound 1 or 2 or a pharmaceutically acceptable salt thereof having fungicidal activity according to claim 1 in the preparation of an antifungal drug.

5. Use of the triterpenoid compound 1 or 2 or a pharmaceutically acceptable salt thereof having fungicidal activity according to claim 1 in the preparation of an anti-Candida albicans or anti-Candida glabrata drug.

6. Use of the triterpenoid compound 1 or 2 or a pharmaceutically acceptable salt thereof having fungicidal activity according to claim 1 in the preparation of a drug for killing Candida glabrata.

7. Use of the triterpenoid compound 1 or 2 having antifungal activity or a pharmaceutically acceptable salt thereof according to claim 1 in combination with other commercially available antifungal drugs in the preparation of antifungal drugs.

8. Use of the triterpenoid compound 1 or 2 having antifungal activity or a pharmaceutically acceptable salt thereof according to claim 1 in combination with other commercially available antifungal drugs in the preparation of antifungal drugs.

9. Use of the triterpenoid compound 1 or 2 having antifungal activity or a pharmaceutically acceptable salt thereof according to claim 1 in combination with other commercially available antifungal drugs in the preparation of drugs for killing Candida albicans or Candida glabrata.

10. The use according to claim 6, 7 or 8, characterized in that The commercially available antifungal drug is selected from ketoconazole, fluconazole, itraconazole or amphotericin B.

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