A class of polyhydroxycyclohexane derivatives and their preparation method and application

Abstract

The present invention discloses a class of polyhydroxycyclohexane derivatives, their preparation methods, and applications, and belongs to the technical field of analysis of active ingredients in marine fungi. The polyhydroxycyclohexane derivatives are chlorogentisyl alcohol derivatives, gabosine derivatives, or derivatives having a hybrid skeleton of chlorogentisyl alcohol and gabosine. The present invention utilizes the polarity differences of chlorogentisyl alcohol and gabosine derivatives to extract and separate novel chlorogentisyl alcohol and gabosine derivatives from the fermentation culture of the marine fungus Epicoccum sorghinum.GST-5. In vitro anti-β-glucuronidase tests show that the chlorogentisyl alcohol and gabosine derivatives provided by the present invention have good β-glucuronidase inhibitory activity and low cytotoxicity, showing development prospects in the preparation of anti-β-glucuronidase drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis of active ingredients of marine fungi, and in particular to gabosine and chlorogentisyl alcohol derivatives extracted from Epicoccum sorghinum GST-5 and applications thereof. Background Art

[0002] Compared to terrestrial microbes, marine microbes are more tolerant of extreme marine conditions, such as high salinity, high pressure, low oxygen, and low light levels. This unique habitat leads to a diverse range of species, genetic composition, and ecological functions. This unique marine environment, coupled with advances in marine microbial resource extraction technologies, has created unprecedented opportunities for the research of natural pharmaceutical compounds derived from marine microbes.

[0003] Marine fungi, a group of marine microorganisms, are a rich source of active secondary metabolites, 70-80% of which are biologically active. These include small molecule lipid compounds; mycotoxins; novel substances with central nervous system inhibitory activity; 1-dodecanol; unsaturated hydrocarbons, acids, and esters; and lipopeptide antibiotics that inhibit plant and human fungal viruses by synthesizing novel targets in the fungal cell wall. The discovery of natural products with specific structural types derived from marine fungi is of great significance for the development of marine pharmaceuticals.

[0004] β-Glucuronidase (GUS) is an important hydrolase produced by intestinal flora, catalyzing the hydrolysis of glucuronide conjugates to produce the corresponding aglycones. In the human body, GUS distributed in the intestine also catalyzes the hydrolysis of glucuronides of various drugs (such as indomethacin, diclofenac, and 7-ethyl-10-hydroxycamptothecin, the active product of irinotecan) and endogenous hormones. The released aglycones can be reabsorbed in the intestine, resulting in a bimodal pharmacokinetic phenomenon. Notably, this process can cause severe gastrointestinal adverse reactions. For example, GUS catalyzes the hydrolysis of 7-ethyl-10-hydroxycamptothecin-O-glucuronide (SN-38G) to produce the highly cytotoxic product SN-38. Accumulation of SN-38 in the intestine can cause intestinal mucosal sloughing and lead to adverse reactions such as severe delayed diarrhea. Therefore, the development of efficient and safe GUS inhibitors is of great significance for alleviating fatal diarrhea caused by drugs such as irinotecan and indomethacin (Weng Zimiao, Lu Shanshan, Ge Guangbo, Wang Ping, Hou Jie, Pharmaceutical Progress, Research Progress of Enteric Bacterial β-glucuronidase Inhibitors, 2018, 42(03)).

[0005] Currently, there are few studies on the chemical components of Epicoccum fungi. Hao Baocong et al. isolated (4R*, 5R*, 6S*)-4,5-dihydroxy-6-(6'-methylsalicylic acid oxy)-2-methoxymethyl-2-cyclohexen-1-one, (4R*, 5R*, 6S*)-4,5-dihydroxy-6-(6'-methylsalicylic acid oxy)-2-methyl-2-cyclohexen-1-one, (4R, 5R, 6S)-4,5-dihydroxy-6-(6'-methylsalicylic acid oxy)-2-hydroxymethyl-2-cyclohexen-1-one, (-)-gabosine E, theobroxide, 3-chlorogentisol, and 3-hydroxybenzyl alcohol from an endophytic fungus of dandelion Epicoccum sorghinum (Hao Baocong, Zheng Yaoyao, Chen Xu, Chen Qiuxia, Ji Ruonan, Chen Min, Acta Pharmaceutica Sinica, 2014). 1-2 Secondary Metabolite Research, 2022, 57(07)).

[0006] Yuan Chao et al. studied the chemical components of an endophytic fungus Epicoccumnigrum14one isolated from the lichen Leptogium masiaticum and identified them as one alkaloid compound fusaricide, seven benzofuran compounds epicoccone B, 4,6-dihydroxy-5-methoxy-7-methyl-1,3-dihydroisobenzofuran, 5-methyl-epicoccone B, 3,6,7-trihydroxy-5-methoxy-4-methylisobenzo furan-1(3H)-one, 3-methoxyepicoccone B, 2,3,4-trihydroxy-6-(hydroxymethyl)-5-methylbenzyl-alcohol, isoochracinic acid and three epicoccolide compounds epicocconigrones A, epicoccolide B, epicocconigrones B, among which alkaloids fusaricide and epicoccolide compounds have strong antibacterial activity, among which alkaloid fusaricide has good in vitro inhibitory activity against lung cancer cells M109 (Yuan Chao, Guo Yuhua, Zhang Yingbo, Hu Xuan, Wang Dan, Yu Fulai, Li Gang, Chinese Journal of Traditional Chinese Medicine, Study on Secondary Metabolites of an Endophytic Fungus Epicoccum nigrum 14one, 2019, 44(18), 4021-4025).

[0007] However, there is no literature report on the isolation of GUS-resistant chlorogentisyl alcohol and gabosine compounds from Epicococcus fungi. Summary of the Invention

[0008] The purpose of the present invention is to extract and obtain natural active substances with medicinal value from the marine Epicoccum fungus (Epicoccum).

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] The present invention isolates 25 compounds from the fermentation product of Epicoccum sorghinum GST-5. Structural identification shows that the 25 compounds are polyhydroxycyclohexane derivatives, which respectively belong to chlorogentisylalcohol derivatives (having a 2-chloro-6-(hydroxymethyl)benzene-1,4-diphenol structure), gabosine derivatives (having a 4,5,6-trihydroxy-2-(hydroxymethyl)cyclohex-2-ene-1-one structure), and derivatives having a hybrid skeleton of chlorogentisyl alcohol and gabosine. The specific structural formula is as follows: Figure 1 As shown, they are numbered 1 to 25 respectively.

[0011] The Epicoccum sorghinum fungus GST-5 is isolated from large marine plants in the hydrothermal vent sediments of Guishan Island, Taiwan Province, and its accession number is CGMCC 3.20238.

[0012] By consulting databases and literature, the present invention identified 19 of the 25 compounds as new compounds. Therefore, the present invention provides a new class of chlorogentisyl alcohol derivatives, gabosine derivatives, or derivatives having a hybrid skeleton of chlorogentisyl alcohol and gabosine, wherein the structural formula of the chlorogentisyl alcohol derivative is shown in any one of formulas (4)-(6) and (13)-(15), corresponding to compounds 4, 5, 6, 13, 14, and 15, respectively.

[0013]

[0014]

[0015] Among them, the structural formula of the gabosine derivative is shown in any one of formulas (8), (20), (24)-(25), corresponding to compounds 8, 20, 21, 22, 24, and 25, respectively.

[0016]

[0017] Among them, the structural formula of the derivative having a hybrid skeleton of chlorogentisyl alcohol and gabosine is shown in any one of formulas (7), (9), (16), and (17), corresponding to compounds 7, 9, 10, 11, 12, 16, and 17, respectively.

[0018]

[0019] The present invention also provides a method for separating and extracting the above-mentioned novel compound from the fermentation product of Epicoccum sorghinum GST-5, but the preparation method of the above-mentioned compound in the present invention is not limited thereto.

[0020] The method for extracting the chlorogentisyl alcohol derivative, the gabosine derivative or the derivative having a hybrid skeleton of chlorogentisyl alcohol and gabosine from a fermentation product comprises the following steps:

[0021] (1) Activating Epicoccum sorghinum GST-5 was inoculated into a liquid culture medium for fermentation;

[0022] (2) After the fermentation culture is completed, mycelium and fermentation liquid are separated, the mycelium is added to an organic solvent for extraction, and the extract is separated to obtain an extract, the extract is concentrated and suspended with distilled water to obtain an aqueous suspension, and then the aqueous suspension is extracted with ethyl acetate or n-butanol to obtain extract A; after the fermentation liquid is stirred with diatomaceous earth, it is extracted with ethyl acetate or n-butanol under reflux to obtain extract B;

[0023] (3) After the extract A or the extract B is concentrated, it is separated by normal phase silica gel column chromatography, and gradient elution is performed with a dichloromethane / methanol mixture with a volume ratio of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, and 10:1, respectively. The fractions eluted with the dichloromethane / methanol mixture with a volume ratio of 10:1 are collected, and then the fractions are separated by reverse phase silica gel column chromatography and high performance liquid chromatography to obtain the derivative.

[0024] In step (1), the fungus Epicoccum sorghinum GST-5 is fermented and cultured.

[0025] Epicococcus GST-5 is a fungus that can be fermented using conventional PDA liquid culture medium or malt extract medium. To increase yield and provide sufficient nutrients for microbial growth and metabolism, the liquid culture medium preferably comprises the following ingredients per liter: 1-5g starch, 10-20g bran, 3-15g yeast extract, 1-8g KH2PO4, 0.1-0.8g MgSO4·7H2O, and the balance water.

[0026] Alternatively, based on a volume of 1 L, the liquid culture medium comprises the following raw materials: 200-600 g of potato, 2-10 g of peptone, 1-5 g of yeast extract, 5-20 g of glucose, and the balance is water; the initial pH value of the culture medium is 6.0-7.0;

[0027] Alternatively, based on a volume of 1 L, the liquid culture medium includes the following raw materials: sucrose 10-40 g, corn flour 5-20 g, NaNO3 1-4 g, yeast extract 1-4 g, KH2PO4 0.2-0.8 g, MgSO4·7H2O 0.2-1 g, KCl 0.2-1 g, FeSO4 0.001-0.005 g, and the balance is water;

[0028] Alternatively, based on a volume of 1 L, the liquid culture medium includes the following raw materials: 20-30 g malt extract powder, 15-20 g glucose, 1-2 g casein peptone, and the balance is water.

[0029] The fermentation culture conditions are static culture at 20-30° C. for 10-40 days. The static culture method does not involve shake flask culture.

[0030] Preferably, the fermentation temperature is 22-26° C., more preferably 25° C. for 20 days, under which conditions the yields of the chlorogentisyl alcohol and gabosine derivatives are the highest.

[0031] In step (2), mycelium and fermentation broth are separated and obtained, and the chlorogentisyl alcohol and gabosine derivatives can be extracted and separated from the mycelium and fermentation broth.

[0032] When using mycelium to obtain chlorogentisyl alcohol and gabosine derivatives, the mycelium is immersed in an organic solvent for 7-14 days to fully break the mycelium wall and effectively dissolve the intracellular substances. The organic solvent is one or two of methanol, ethanol, ethyl acetate and acetone.

[0033] In step (3), the separation and purification method comprises: subjecting the extract to normal-phase silica gel column chromatography, and then subjecting the resulting fraction to recrystallization, reverse-phase silica gel column chromatography, and high-performance liquid chromatography. Through multi-step separation and purification, chlorogentisyl alcohol and gabosine derivatives of high purity can be obtained.

[0034] Preferably, the fractions are subjected to reverse phase silica gel column chromatography, and gradient elution is performed with a methanol / water mixture of volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, and each gradient elution is performed 5 times. The fractions are numbered 1 to 45 in sequence, and the fractions numbered 1 to 3 are combined into subfraction 1, fractions 4 to 5 are combined into subfraction 2, fractions 14 to 17 are combined into subfraction 6, fractions 18 to 25 are combined into subfraction 7, fractions 26 to 28 are combined into subfraction 8, and fractions 38 to 39 are combined into subfraction 12; and then high performance liquid chromatography is used. Spectral separation, subcomponent 1 in the elution of 28% by volume methanol / water mixture, the retention time of the peak of 25.6 minutes is the structural formula shown in formula (20) R1 = Cl, R2 = CH3 compound 20; subcomponent 2 in the elution of 35% by volume methanol / water mixture, the retention time of 29.3 minutes, 24.5 minutes, 35.6 minutes, 36.9 minutes peaks are compounds 4, 5, 21 and 22, the structural formula is shown in formula (4), formula (5), formula (20) R1 = Oxybutyl, R2 = H, formula (20) R1 = OH, R2 = Oxybutyl; when subcomponent 6 is eluted with a methanol / water mixture at a volume ratio of 36%, the peaks with retention times of 23.3 minutes, 25.5 minutes, and 30.6 minutes are compounds 6, 7, and 8, whose structural formulas are shown in Formula (6), Formula (7), and Formula (8), respectively; when subcomponent 7 is eluted with a methanol / water mixture at a volume ratio of 42%, the peaks with retention times of 18.2 minutes, 24.4 minutes, 26.9 minutes, and 22.3 minutes are compounds 9, 11, 12, and 10, whose structural formulas are shown in Formula (9), respectively; when subcomponent 8 is eluted with a methanol / water mixture at a volume ratio of 42%, the peaks with retention times of 18.2 minutes, 24.4 minutes, 26.9 minutes, and 22.3 minutes are compounds 9, 11, 12, and 10, whose structural formulas are shown in Formula (9), respectively. When eluted with an acetonitrile / water mixture with a volume ratio of 18%, the peaks with retention times of 15.2 minutes, 17.5 minutes, 19.6 minutes, 23.3 minutes and 26.3 minutes are compounds 13, 14, 15, 16 and 17, and their structural formulas are shown in Formula (13), Formula (14), Formula (15), Formula (16) and Formula (17), respectively. When eluted with an acetonitrile / water mixture with a volume ratio of 25%, the peaks with retention times of 14.3 minutes and 18.3 minutes are compounds 24 and 25, and their structural formulas are shown in Formula (24) and Formula (25), respectively.

[0035] Research conducted in the present invention demonstrates that chlorogentisyl alcohol and gabosine derivatives isolated from the fermentation culture of the fungus Epicoccumsorghinum GST-5 using the aforementioned method exhibit significant anti-β-glucuronidase (GUS) activity. Furthermore, the present invention uses the inhibitory activity of the compounds against cells as its cytotoxicity data, and the results indicate that, with the exception of compounds 12 and 14, the remaining compounds exhibit low cytotoxicity to human cells. Therefore, the present invention provides the use of the polyhydroxycyclohexane derivatives in the preparation of anti-β-glucuronidase drugs.

[0036] Furthermore, the anti-β-glucuronidase drug is an intestinal bacterial β-glucuronidase inhibitor. Studies have shown that β-glucuronidase distributed in the intestine can cause gastrointestinal adverse reactions during the hydrolysis of catalyzed drugs (such as indomethacin and irinotecan). The compounds provided by the present invention can alleviate drug-induced gastrointestinal adverse reactions by inhibiting GUS enzyme activity.

[0037] The present invention provides the use of the polyhydroxycyclohexane derivatives in preparing a drug for alleviating gastrointestinal toxic and side effects caused by irinotecan and indomethacin.

[0038] Furthermore, the present invention has found that compounds 12 and 14 have significant inhibitory effects on Namalwa cell lines and U266 cell lines. Therefore, the present invention provides the use of compounds represented by the structural formulas (12) and (14) in the preparation of anti-tumor drugs. Specifically, the tumor is myeloma or lymphoma.

[0039]

[0040] Furthermore, among the 25 compounds, the compounds represented by the structural formulae (1), (3), (18), (19), and (23) are known compounds, corresponding to compounds 1, 2, 3, 18, 19, and 23, respectively. Studies conducted in the present invention have shown that the compounds also have anti-GUS enzyme activity. Therefore, the present invention provides the use of the compounds represented by the structural formulae (1), (3), (18), (19), and (23) in the preparation of anti-β-glucuronidase drugs. Furthermore, the anti-β-glucuronidase drugs are intestinal bacterial β-glucuronidase inhibitors.

[0041]

[0042]

[0043] The drug comprises the chlorogentisyl alcohol and gabosine derivatives of the present invention as the main active ingredients, and is supplemented with pharmaceutically acceptable excipients. The drug can be prepared according to pharmaceutically documented preparation methods. The drug can be in the form of injections, drips, powder injections, granules, tablets, granules, powders, oral liquids, sugar-coated tablets, film-coated tablets, enteric-coated tablets, buccal preparations, pills, ointments, pills, sprays, drop pills, disintegrants, orally disintegrating tablets, and pellets.

[0044] The present invention also provides a method for separating and extracting compounds 1, 2, 3, 18, 19, and 23 from the fermentation product of Epicoccum sorghinum GST-5, comprising the following steps: subjecting the above-mentioned subcomponent 1, subcomponent 2, and subcomponent 6 to high performance liquid chromatography separation, specifically: when subcomponent 1 is eluted with a methanol / water mixture with a volume ratio of 28%, the peaks with retention times of 13.1 minutes, 15.1 minutes, 18.3 minutes, and 20.8 minutes are compounds 1, 2, 3, and 19, respectively; when subcomponent 2 is eluted with a methanol / water mixture with a volume ratio of 35%, the peak with a retention time of 27.3 minutes is compound 23; when subcomponent 6 is eluted with a methanol / water mixture with a volume ratio of 36%, the peak with a retention time of 33.3 minutes is compound 18.

[0045] The present invention has the following beneficial effects:

[0046] (1) The present invention utilizes the polarity difference of chlorogentisyl alcohol and gabosine derivatives to extract and separate a novel structure of chlorogentisyl alcohol and gabosine derivatives from the fermentation culture of marine fungi. The method is simple to operate, has a high extraction yield, and has high product purity, and is suitable for large-scale production.

[0047] (2) The in vitro anti-GUS enzyme test showed that the chlorogentisyl alcohol and gabosine derivatives provided by the present invention have good anti-GUS enzyme activity, among which the most active compound 17 has an IC 50 The value was 0.24 μM. Further cytotoxicity tests showed that the carbon sugar compound provided by the present invention had low cytotoxicity and had good development prospects in the preparation of drugs against GUS enzyme.

[0048] (3) In vitro tumor cell tests were conducted using Namalwa cells and U266 cells. The survival rate of compound 12 on Namalwa cells was -0.33%, and the survival rate of compound 14 on U266 cells was 7.86%, which was close to that of the positive drug 5-Fu. This indicates that the compound has good development prospects in the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The following are the structural formulas of 25 chlorogentisyl alcohol and gabosine derivatives of the present invention.

[0050] Figure 2 For compound 1 1 H NMR data (in CD3OD, 600 MHz) and 13 C NMR data (in CD3OD, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0051] Figure 3 For compound 2 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0052] Figure 4 For compound 3 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0053] Figure 5 For compound 4 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0054] Figure 6 For compound 5 1H NMR data (in CD3OD, 600 MHz) and 13 C NMR data (in CD3OD, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0055] Figure 7 For compound 6 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0056] Figure 8 For compound 7 1 H NMR data (in CD3OD, 600 MHz) and 13 C NMR data (in CD3OD, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0057] Figure 9 For compound 8 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0058] Figure 10 For compound 9 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0059] Figure 11 For compound 9 1 H NMR data (in CD3OD, 600 MHz) and 13 C NMR data (in CD3OD, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0060] Figure 12 For compound 10 1 H NMR data (in CD3OD, 600 MHz) and 13 C NMR data (in CD3OD, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0061] Figure 13 For compound 11 1 H NMR data (in CD3OD, 600 MHz) and 13 C NMR data (in CD3OD, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0062] Figure 14 For compound 12 1 H NMR data (in CD3OD, 600 MHz) and 13 C NMR data (in CD3OD, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0063] Figure 15 Compound 13 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0064] Figure 16 Compound 14 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0065] Figure 17 Compound 15 1 H NMR data (in DMSO-d6, 600 MHz) and 13C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0066] Figure 18 For compound 16 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0067] Figure 19 Compound 17 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0068] Figure 20 For compound 18 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0069] Figure 21 Compound 19 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0070] Figure 22 Compound 20 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0071] Figure 23 Compound 21 1 H NMR data (in CD3OD, 600 MHz) and 13 C NMR data (in CD3OD, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0072] Figure 24 Compound 22 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0073] Figure 25 Compound 23 1 H NMR data (in CD3OD, 600 MHz) and 13 C NMR data (in CD3OD, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0074] Figure 26 Compound 24 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data.

[0075] Figure 27 Compound 25 1 H NMR data (in DMSO-d6, 600 MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) 13 C NMR data. DETAILED DESCRIPTION

[0076] The present invention will be further described below in conjunction with specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all fall within the scope of the present invention.

[0077] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0078] Example 1 Fungal Isolation

[0079] Large marine plants were collected from the seabed sediments of the hydrothermal vents on Guishan Island, Taiwan Province. After the samples were brought back to the laboratory, they were first rinsed three times with sterile seawater to remove non-attached microorganisms; the plants were placed in a centrifuge tube, and then a small amount of seawater was added. The suspension was vortexed for 10 minutes, and the suspension after removing the algae was centrifuged for 20 minutes (5000r / min), and the supernatant was poured off; the sediment was suspended in a small amount of sterile seawater, and 0.1mL was spread on a Martin's medium (containing 8U / L gentamicin) plate; after incubation at room temperature of 20°C for 10 days, a single colony was picked, purified by streaking, and transferred to a slant and stored at 4°C for later use.

[0080] Example 2 Identification of Epicococcus fungi

[0081] The isolated fungus was cultured on PDA, and the 18S rDNA gene sequence of the strain was determined. The 18S rDNA sequence of the strain is shown as SEQ ID No. 1.

[0082] Based on the morphological characteristics and 18S rDNA sequence analysis results of the strain, the strain was identified as Epicoccum sp. and named Epicoccum sorghinum.GST-5. It was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms (CGMCC) on July 12, 2021, and was identified as alive. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, and the deposit number is: CGMCC 3.20238.

[0083] Example 3 Fermentation culture of Epicococcus fungi

[0084] The activated Epicococcus fungus GST-5 was prepared into a spore suspension, which was then inoculated into a culture solution and statically fermented at 25°C for 20 days.

[0085] The culture solution formula is: 3g starch, 14g bran, 6g yeast extract, 5g KH2PO4, 0.4g MgSO4·7H2O, and 1000mL water.

[0086] Example 4 Fermentation culture of Epicococcus fungi

[0087] The activated Epicococcus fungus GST-5 was prepared into a spore suspension, which was then inoculated into a culture solution and statically fermented at 24°C for 20 days.

[0088] The culture medium formula is: 400 g potato, 6 g peptone, 2 g yeast extract, 10 g glucose, and 1000 mL water; the initial pH value of the culture medium is 6.5.

[0089] Example 5 Fermentation culture of Epicococcus fungi

[0090] The activated Epicococcus fungus GST-5 was prepared into a spore suspension, which was then inoculated into a culture solution and statically fermented at 25°C for 20 days.

[0091] The culture medium formula is: sucrose 25g, corn flour 10g, NaNO3 2g, yeast extract 2g, KH2PO4 0.5g, MgSO4·7H2O 0.5g, KCl 0.5g, FeSO4 0.001g, water 1000mL.

[0092] Example 6 Fermentation culture of Epicococcus fungi

[0093] The activated Epicococcus fungus GST-5 was prepared into a spore suspension, which was then inoculated into a culture solution and statically fermented at 25°C for 20 days.

[0094] The culture solution formula is: 25g malt extract powder, 15g glucose, 1.5g casein peptone, and 1000mL water.

[0095] Example 7 Preparation of chlorogentisyl alcohol and gabosine derivatives

[0096] After fermentation and cultivation of Ephelococcus fungus GST-5, 5 L of fermentation culture broth was centrifuged to obtain a precipitate to obtain mycelium; the mycelium was soaked in methanol for 1 week, the soaked broth was concentrated and then suspended in 1 L of distilled water. The water suspensions were combined and extracted with 6 L of n-butanol. The n-butanol extract was concentrated to obtain 10 g of extract; the sample was mixed with silica gel (100 mesh, 100 g) and separated by normal phase silica gel column chromatography (200-300 mesh, 100 g; silica gel column size L 50 mm, ), gradient elution with dichloromethane / methanol mixtures and methanol in volume ratios of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, and 10:1, 300 mL each time; fractions were detected by TLC; fractions eluted with dichloromethane / methanol mixtures in volume ratios of 35:1 to 10:1 were collected and recrystallized from methanol.

[0097] Example 8 Preparation of chlorogentisyl alcohol and gabosine derivatives

[0098] After the Epicococcus fungus was fermented, 5 L of the fermentation culture was taken and centrifuged to obtain the supernatant; the fermentation broth was concentrated, mixed with 10 g of diatomaceous earth, refluxed with 1 L of n-butanol, and separated by normal phase silica gel column chromatography (200-300 mesh, 1 kg; silica gel column size L50 mm, ), gradient elution was performed with dichloromethane / methanol mixtures and methanol in volume ratios of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, and 10:1, successively. 400 mL fractions were collected for each elution with each eluent, and each eluent was repeated 5 times. A total of 30 fractions eluted with dichloromethane / methanol mixtures in volume ratios of 35:1 to 10:1 were collected and numbered 1 to 30, and the fractions were detected by TLC and similar components were combined. Fractions 1 to 7 were combined into component 1, fractions 8 to 11 were combined into component 2, fractions 12 to 15 were combined into component 3, fractions 16 to 25 were combined into component 4, and fractions 26 to 30 were combined into component 5.

[0099] Component 5 was subjected to reverse-phase silica gel column chromatography with methanol / water (1:9-9:1) as the eluent. Gradient elution was performed with a methanol / water mixture of volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, collecting 400 mL fractions for each elution with each eluent. The elution was repeated 5 times for each eluent, and a total of 45 fractions eluted with each eluent were collected and numbered 1 to 45 in sequence. The fractions were detected and similar components were merged. Fractions 1 to 3 were merged into subfraction 5-1, fractions 4 to 5 were merged into subfraction 5-2, fractions 6 to 8 were merged into subfraction 5-3, and fractions 9 to 10 were merged. Fractions 11 to 13 are combined into subcomponent 5-5, fractions 14 to 17 are combined into subcomponent 5-6, fractions 18 to 25 are combined into subcomponent 5-7, fractions 26 to 28 are combined into subcomponent 5-8, fractions 29 to 30 are combined into subcomponent 5-9, fractions 31 to 33 are combined into subcomponent 5-10, fractions 34 to 37 are combined into subcomponent 5-11, fractions 38 to 39 are combined into subcomponent 5-12, fractions 40 to 41 are combined into subcomponent 5-13, fractions 42 to 43 are combined into subcomponent 5-14, and fractions 44 to 45 are combined into subcomponent 5-15.

[0100] The product was then separated by high performance liquid chromatography with a mobile phase of methanol / water or acetonitrile / water at a flow rate of 10 mL / min. The detection wavelength of the high performance liquid chromatography was 254 nm. The peaks of subcomponent 5-1 with a retention time of 13.1 minutes, 15.1 minutes, 18.3 minutes, 20.8 minutes and 25.6 minutes, respectively, were recrystallized from cyclohexane to give compounds 1, 2, 3, 19 and 20; the peaks of subcomponent 5-2 with a retention time of 29.3 minutes, 24.5 minutes, 35.6 minutes, 36.9 minutes and 27.3 minutes, respectively, were recrystallized from cyclohexane to give compounds 4, 5, 21, 22 and 23, respectively, and the peaks of subcomponent 5-6 with a retention time of 23.3 minutes, 25.7 minutes, 26.8 minutes, 28.9 minutes, 29.8 minutes, 30.8 minutes, 31.8 minutes, 32.8 minutes, 33.8 minutes, 34.8 minutes, 35.8 minutes, 36.9 minutes, 37.8 minutes, 38.8 minutes, 39.8 minutes, 40.8 minutes, 41.8 minutes, 42.8 minutes, 43.8 minutes, 44.8 minutes, 45.8 minutes, 46.8 minutes, 47.8 minutes, 48.8 minutes, 49.8 minutes, 50.8 minutes, 51.8 minutes, 52.8 minutes, 53.8 minutes, 54.8 minutes, 55.8 minutes, 56.8 minutes, 57.8 minutes, 58.8 minutes, 59.8 minutes, 60.8 minutes, 61.8 minutes, 62. The peaks at 0.5 minutes, 30.6 minutes and 33.3 minutes were recrystallized from cyclohexane to give compounds 6, 7, 8 and 18; the peaks of subcomponent 5-7 with retention times of 18.2 minutes, 24.4 minutes, 26.9 minutes and 22.3 minutes were recrystallized from cyclohexane to give compounds 9, 11, 12 and 10 under the eluent of 42% (methanol-water); the peaks of subcomponent 5-8 with retention times of 15.2 minutes, 17.5 minutes, 19.6 minutes, 23.3 minutes and 26.3 minutes were recrystallized from cyclohexane to give compounds 13, 14, 15, 16 and 17 under the eluent of 18% (acetonitrile-water); the peaks of subcomponent 5-12 with retention times of 14.3 minutes and 18.3 minutes were recrystallized from cyclohexane to give compounds 24 and 25 under the eluent of 25% (acetonitrile-water).

[0101] Example 9 Structural Identification of Chlorogentisyl Alcohol and Gabosine Derivatives

[0102] The purity of the prepared compounds was determined by HPLC. The structures of samples with a purity greater than 98% were identified by mass spectrometry and nuclear magnetic resonance. Nuclear magnetic resonance was determined by a Bruker AVANCE DRX-600 NMR Sectrometer with TMS as the internal standard; high-resolution mass spectrometry (FTICRMS) was determined by a Bruker Apex Spectrometer; and electrospray ionization mass spectrometry (ESI-MS) was determined by a Bruker Esquire 3000. plus Spectrometer determination.

[0103] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 1), the molecular formula of the compound is C7H7ClO3. By consulting the database and literature, compound 1 was identified as the known compound 3-Chlorogentisylalcohol. The structure is as follows Figure 1 As shown:

[0104] Table 1 NMR data of compound 1

[0105]

[0106]

[0107] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 2), the molecular formula of the compound is C8H9ClO3. By consulting the database and literature, compound 2 was identified as the known compound 5-dehydroxy-5S-chlorogabosine C. The structure is as follows Figure 1 As shown:

[0108] Table 2 NMR data of compound 2

[0109]

[0110] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 3), the molecular formula of the compound is C7H5ClO3. By consulting the database and literature, compound 3 was identified as the known compound 1-chloro-3,6-dihydroxybenzaldehyde. The structure is as follows Figure 1 As shown:

[0111] Table 3 NMR data of compound 3

[0112]

[0113]

[0114] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 4), the molecular formula of the compound is C 13 H 10 ClNO5, unsaturation is 9. 1 H and 13 After comparison with C-NMR, it was found that the compound contained the same p-hydroxychlorophenyl group as compound 1. The two-dimensional NMR analysis results identified compound 4 as a new compound. Figure 1 As shown:

[0115] Table 4 NMR data of compound 4

[0116]

[0117] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 5), the molecular formula of the compound is C 13 H 11 ClO5, unsaturation is 8.1 H and 13 The C-NMR data showed that there was an additional α-pyrone fragment. Compound 5 was identified as a new compound through two-dimensional NMR analysis. Figure 1 As shown:

[0118] Table 5 NMR data of compound 5

[0119]

[0120]

[0121] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 6), the molecular formula of the compound is C 14 H 12 Cl2O5, unsaturation is 7. 1 H and 13 After C-NMR comparison, it was found that the compound had a higher C-7 (δ C 67.7) and H-7(δ H 4.52) have slight differences. The two-dimensional NMR analysis results identified compound 6 as a new compound. Figure 1 As shown:

[0122] Table 6 NMR data of compound 6

[0123]

[0124] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 7), the molecular formula of the compound is C 14 H 14 Cl2O5, unsaturation is 7. 1 The H NMR data showed that compound 7 had two more active hydrogen signals than compound 2, and one CH2 hydrogen signal changed from high field to mid-field, containing one p-hydroxychlorobenzene and one gabosinetype I fragment. The two-dimensional NMR analysis results identified compound 7 as a new compound. Figure 1 As shown:

[0125] Table 7 NMR data of compound 7

[0126]

[0127]

[0128] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 8), the molecular formula of the compound is C 13 H 11ClO5, unsaturation degree is 8. 1 H NMR and 13 C NMR data showed that compound 8 was very similar to compound 5, both containing an α-pyrone structure. The two-dimensional NMR analysis results identified compound 8 as a new compound. Figure 1 As shown:

[0129] Table 8 NMR data of compound 8

[0130]

[0131]

[0132] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 9), the molecular formula of the compound is C 14 H 12 Cl2O5, unsaturation is 8. 1 H NMR and 13 C NMR data showed that compound 9 was very similar to compound 1, both containing a 2-chloro-6-(hydroxymethyl)benzene-1,4-diphenol structure. Compound 9 was identified as a new compound by two-dimensional NMR analysis. Figure 1 As shown:

[0133] Table 9 NMR data of compound 9

[0134]

[0135] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 10), the molecular formula of the compound is C 15 H 14 Cl2O5, unsaturation is 8. 1 H NMR and 13 C NMR data showed that compound 10 was very similar to compound 9, with only one more methoxy signal. Based on the two-dimensional NMR analysis results, compound 10 was identified as a new compound. Figure 1 As shown:

[0136] Table 10 NMR data of compound 10

[0137]

[0138]

[0139] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 11), the molecular formula of the compound is C 14 H12 Cl2O5, unsaturation is 8. 1 H NMR and 13 C NMR data showed that compound 11 was very similar to compound 9, with only slight changes in the chiral carbon region. The two-dimensional NMR analysis results identified compound 11 as a new compound. Figure 1 As shown:

[0140] Table 11 NMR data of compound 11

[0141]

[0142]

[0143] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 12), the molecular formula of the compound is C 14 H 12 Cl2O5, unsaturation is 8. 1 H NMR and 13 C NMR data showed that compound 12 was very similar to compound 9, with only slight changes in the chiral carbon region. The two-dimensional NMR analysis results identified compound 12 as a new compound. Figure 1 As shown:

[0144] Table 12 NMR data of compound 12

[0145]

[0146] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 13), the molecular formula of the compound is C 14 H 19 ClO5, unsaturation is 5. Combined 1 H and 13 C-NMR data revealed that this compound had one more ampelomin group than compound 1. Two-dimensional NMR analysis identified compound 13 as a new compound. Figure 1 As shown:

[0147] Table 13 NMR data of compound 13

[0148]

[0149]

[0150] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 14), the molecular formula of the compound is C 14 H 19ClO5, unsaturation is 5. Combined 1 H and 13 C-NMR data showed that the compound had only a slight change in the ampelomin group compared to compound 13. The two-dimensional NMR analysis results identified compound 14 as a new compound. Figure 1 As shown:

[0151] Table 14 NMR data of compound 14

[0152]

[0153]

[0154] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 15), the molecular formula of the compound is C 14 H 19 ClO5, unsaturation is 5. Combined 1 H and 13 C-NMR data showed that the compound had only a slight change in the ampelomin group compared to compound 13. Compound 15 was identified as a new compound by two-dimensional NMR analysis. Figure 1 As shown:

[0155] Table 15 NMR data of compound 15

[0156]

[0157] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 16), the molecular formula of the compound is C 21 H 24 Cl2O7, unsaturation is 9. Combined 1 H and 13 C-NMR data showed that the compound was similar to compound 10, except that its methoxy group was replaced by an ampelomin group. Compound 16 was identified as a new compound by two-dimensional NMR analysis. Figure 1 As shown:

[0158] Table 16 NMR data of compound 16

[0159]

[0160] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 17), the molecular formula of the compound is C 21 H 24 Cl2O7, unsaturation is 9. Combined 1 H and 13C-NMR data showed that the compound was similar to compound 9, except that the chlorine substituent on the gabosine group was replaced by a 2-chloro-6-(hydroxymethyl)benzene-1,4-diphenol group. Compound 17 was identified as a new compound by two-dimensional NMR analysis. Figure 1 As shown:

[0161] Table 17 NMR data of compound 17

[0162]

[0163] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 18), the molecular formula of the compound is C 10 H7NO3. By consulting databases and literature, compound 18 was identified as a known compound, kynurenic acid, with the following structure: Figure 1 As shown:

[0164] Table 18 NMR data of compound 18

[0165]

[0166] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 19), the molecular formula of the compound is C7H9ClO4. By consulting the database and literature, compound 19 was identified as the known compound 5-dehydroxy-5S-chlorogabosine C, with the structure shown in Figure 1 As shown:

[0167] Table 19 NMR data of compound 19

[0168]

[0169] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 20), the molecular formula of the compound is C8H 11 ClO4, unsaturation degree is 3. The NMR data of this compound are very similar to compound 19, with only one more methoxy signal. The two-dimensional NMR analysis results identified compound 20 as a new compound. Figure 1 As shown:

[0170] Table 20 NMR data of compound 20

[0171]

[0172] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 21), the molecular formula of the compound is C8H 11ClO4, unsaturation degree is 3. The NMR data of this compound are very similar to compound 19, with only one butyl signal added. The two-dimensional NMR analysis results identified compound 21 as a new compound. Figure 1 As shown:

[0173] Table 21 NMR data of compound 21

[0174]

[0175] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 22), the molecular formula of the compound is C8H 11 ClO4, the degree of unsaturation is 3. The NMR data of this compound are very similar to those of compound 21, with only slight changes in the carbon-hydrogen signal of the butyl substituent. The two-dimensional NMR analysis results identify compound 22 as a new compound. Figure 1 As shown:

[0176] Table 22 NMR data of compound 22

[0177]

[0178] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 23), the molecular formula of the compound is C 15 H 16 O7. By consulting databases and literature, compound 23 was identified as the known compound 3,4-dihydroxy-5-(11-methylsalicyloxy)-7-hydroxymethyl-2-cyclohexenl-one. Figure 1 As shown:

[0179] Table 23 NMR data of compound 23

[0180]

[0181] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 24), the molecular formula of the compound is C 13 H 14 O8, unsaturation degree is 7. The NMR data of this compound are very similar to those of compound 19, and they belong to the gabosine series of compounds. The two-dimensional NMR analysis results identified compound 24 as a new compound. Figure 1 As shown:

[0182] Table 24 NMR data of compound 24

[0183]

[0184] According to the one-dimensional NMR and mass spectrometry analysis results of the compound (see Table 25), the molecular formula of the compound is C 19 H 18 O9, with an unsaturation degree of 11. The NMR data of this compound are very similar to those of compound 24, with only one more α-pyrone signal. Compound 25 was identified as a new compound by two-dimensional NMR analysis. Figure 1 As shown:

[0185] Table 25 NMR data of compound 25

[0186]

[0187]

[0188] Example 10 Analysis of Anti-GUS Activity of Chlorogentisyl Alcohol and Gabosine Derivatives

[0189] (1) Preparation of solution

[0190] Sample preparation: 1.0 mg of sample and reference substance D-glucaroyl-1,4-lactone (DSL) was dissolved in DMSO to prepare 10.0 mM sample and reference substance solutions;

[0191] Preparation of substrate PNPG solution: Accurately weigh 3.0 mg of PNPG and dissolve it in PBS buffer (pH 7.4) to a concentration of 2.5 mM;

[0192] Preparation of 10% DMSO solution: Accurately measure 10.0 μL of DMSO and add 90.0 μL of PBS buffer (pH 7.4) to prepare a 10% DMSO solution;

[0193] Preparation of liquid sample stock solution: Accurately measure 100.0 μL of the above sample, add DMSO and mix to make a 10.0 mM sample solution (final concentration: 0.1 mM).

[0194] (2) Preliminary screening of GUS inhibitors

[0195] Screening for GUS inhibitors was performed using DMSO as a blank control and DSL as a positive control. The blank control consisted of a 100.0 μL mixture containing 10.0 μL GUS (final concentration: 2.0 μg / mL), 79.0 μL PBS buffer (pH 7.4), 1.0 μL 10% DMSO solution (final concentration: 100.0 μM), and 10.0 μL PNPG (final concentration: 250.0 μM).

[0196] Inhibitor and positive control: The total volume of the mixed system is 100.0 μL, containing 10.0 μL Escherichia coli β-glucuronidase (final concentration: 2.0 μg / mL), 70.0 μL PBS buffer (pH 7.4), 10.0 μL inhibitor, DSL stock solution (final concentration: 100.0 μM), and 10.0 μL PNPG (final concentration: 250.0 μM).

[0197] After sample addition, immediately measure absorbance at 405 nm using a microplate reader. Incubate in a 37°C incubator for 30 minutes and repeat the measurement in triplicate. Calculate relative activity. Relative activity is the percentage of PNP content in the blank control within 30 minutes after addition of inhibitor or DSL.

[0198] (3) GUS inhibitor IC 50 Value determination

[0199] The GUS inhibitors with strong inhibitory effects screened in (2) were subjected to IC 50 Value determination.

[0200] The reaction conditions are as follows: the total volume of the mixed system is 100.0 μL, containing 10.0 μL GUS (final concentration: 2.0 μg / mL), 70.0 μL PBS buffer (pH 7.4), 10.0 μL PNPG (final concentration: 250.0 μM) and 10.0 μL inhibitor solution (final concentration: 1.0 nM-100.0 μM).

[0201] After the sample was added, the absorbance was immediately measured at 405 nm using a microplate reader. The sample was incubated in a 37°C incubator for 30 min and then repeated for three times. The relative activity was calculated. Finally, the concentration response curve was drawn using Prism 6.0 (GraphPad Software, LaJolla, CA) to calculate the IC 50 value.

[0202] (4) Inhibitory kinetics of GUS inhibitors

[0203] The inhibition kinetics of the GUS inhibitors selected in (3) (competitive, non-competitive, mixed) were studied to explore the interactions between substrate, inhibitor, and GUS. To determine the inhibition kinetics of the inhibitors, various concentrations of PNPG and inhibitors were used to determine the corresponding reaction rates and evaluate the intersection points of the Lineweaver-Burk plot.

[0204] (5) Experimental results

[0205] Table 26 GUS inhibition rate and IC of compounds 50 value

[0206]

[0207]

[0208] a ND means not detected

[0209] The initial screening data of the compounds in the above table show that compounds 1, 4, 6, 7, 9, 10, 12, 13, 14, 15, 17, 19, 20, and 21 have enzyme inhibition rates of 85% or above at a concentration of 10 μM. Further rescreening of the above compounds showed that the half-maximal inhibitory concentrations of these compounds ranged from 0.24 μM to 4.61 μM, which are significantly inhibitory compared to the positive drug DSL. The Ki inhibition constants ranged from 0.58 μM to 7.32 μM. In the enzyme-substrate binding type analysis, except for compounds 1 and 20, which were non-competitive inhibition, the others were mixed inhibition.

[0210] Example 11 Analysis of the Antitumor Activity of Chlorogentisyl Alcohol and Gabosine Derivatives

[0211] (1) Reagent preparation

[0212] 0.4% SRB solution: Weigh 0.8 g of SRB, dissolve it in 200 mL of 1% acetic acid, and store at room temperature.

[0213] 50% TCA solution: Weigh 50 g of TCA, add water to make up to 100 mL, and store at 4°C.

[0214] 10mM Tris-base solution: Weigh 0.6057g Tris-base, add water to 500mL, pH 10.5, and store at 4℃.

[0215] (2) Experimental instruments

[0216] CO2 incubator (Thermo), mini shaker (Kylin-Bell Lab instruments), microplate reader (MD company, M5 model)

[0217] (3) Methods

[0218] Logarithmically growing cells were trypsinized and adjusted to a cell concentration of 2×10⁴ cells / mL in RPMI 1640 medium supplemented with 10% fetal bovine serum. 190 μl of the cell suspension was plated per well in a 96-well culture plate and incubated at 37°C, 5% CO₂ for 24 hours. Drug-treated wells were treated with 10 μl of sample solution (final concentration: compound 5 μg / mL; mixture 50 μg / mL). Positive drug wells were treated with 5-FU at a final concentration of 5 μg / mL. Control wells were treated with an equal volume of medium containing vehicle and incubated at 37°C, 5% CO₂ for 3 days. The medium was discarded, and the cells were fixed with 100 μl of 4°C pre-cooled 50% TCA. The cells were allowed to stand for 5 minutes, then transferred to 4°C for 1 hour. The fixative was discarded, the cells were washed five times with distilled water to remove the TCA, and air-dried for 1 hour. 80 μl of 0.4% SRB solution was added to each well and stained at room temperature for 30 minutes. Discard the staining solution, wash with 1% acetic acid five times to remove unbound SRB, and air dry. Dissolve the solution in 150 μl of 10 mM Tris-base (pH 10.5), and shake on a microplate oscillator for 5 minutes. Measure the OD at 10 nm using an M5 microplate reader.

[0219] (4) Result calculation

[0220] Tumor cell survival rate (%) = 100% - (OD 对照 -OD 药物 ) / (OD 对照 -OD 空白 )×100%

[0221] (5) Experimental results

[0222] Table 27 Namalwa and U266 cell viability of compounds

[0223]

[0224] a ND means not detected

[0225] The cytotoxicity of compounds 1, 4, 6, 7, 9, 10, 12, 13, 14, 15, 17, 19, 20, and 21 was further determined at a concentration of 20 μM using Namalwa and U266 tumor cell lines. The data showed that, with the exception of compounds 12 and 14, the other compounds had low cytotoxicity. The survival rate of compound 12 against Namalwa cells was -0.33%, and the survival rate of compound 14 against Namalwa cells was -3.03%, while the survival rate against U266 cells was 7.86%, which was close to the survival rate of the positive drug 5-Fu against Namalwa cells (11.28%) and the survival rate of the positive drug As2O3 against U266 cells (2.59%). This indicates that compounds 12 and 14 have dual anti-tumor efficacy while inhibiting GUS.

Claims

1. A polyhydroxycyclohexane derivative, characterized in that: The polyhydroxycyclohexane derivative is a chlorogentisyl alcohol derivative or a derivative having a hybrid skeleton of chlorogentisyl alcohol and gabosine, and the structural formula of the chlorogentisyl alcohol derivative is shown in any one of formulas (4)-(6) and (13)-(15). The structural formula of the derivative having a hybrid skeleton of chlorogentisyl alcohol and gabosine is shown in formula (7), 2. The method for preparing a polyhydroxycyclohexane derivative according to claim 1, wherein: The following steps are involved: (1) The activated Epicoccum sorghinum GST-5 with the accession number CGMCC 3.20238 was inoculated into a liquid culture medium for fermentation; (2) After the fermentation culture is completed, mycelium and fermentation liquid are separated, an organic solvent is added to the mycelium for extraction, and an extract is separated to obtain the extract, the extract is concentrated and suspended in distilled water to obtain an aqueous suspension, and then the aqueous suspension is extracted with ethyl acetate or n-butanol to obtain extract A; after the fermentation liquid is stirred with diatomaceous earth, ethyl acetate or n-butanol is refluxed for extraction to obtain extract B; (3) After the extract A or the extract B is concentrated, it is separated by normal phase silica gel column chromatography, and gradient elution is performed with a dichloromethane / methanol mixture with a volume ratio of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, and 10:1, respectively. The fractions eluted with the dichloromethane / methanol mixture with a volume ratio of 10:1 are collected, and then the fractions are separated by reverse phase silica gel column chromatography and high performance liquid chromatography to obtain the derivative.

3. The preparation method according to claim 2, wherein In step (1), based on a volume of 1 L, the liquid culture medium comprises the following raw materials: 1-5 g starch, 10-20 g bran, 3-15 g yeast extract, 1-8 g KH2PO4, 0.1-0.8 g MgSO4·7H2O, and the balance water; Alternatively, based on a volume of 1 L, the liquid culture medium comprises the following raw materials: 200-600 g of potato, 2-10 g of peptone, 1-5 g of yeast extract, 5-20 g of glucose, and the balance is water; the initial pH value of the culture medium is 6.0-7.0; Alternatively, based on a volume of 1 L, the liquid culture medium includes the following raw materials: sucrose 10-40 g, corn flour 5-20 g, NaNO3 1-4 g, yeast extract 1-4 g, KH2PO4 0.2-0.8 g, MgSO4·7H2O 0.2-1 g, KCl 0.2-1 g, FeSO4 0.001-0.005 g, and the balance is water; Alternatively, based on a volume of 1 L, the liquid culture medium includes the following raw materials: 20-30 g malt extract powder, 15-20 g glucose, 1-2 g casein peptone, and the balance is water.

4. The preparation method according to claim 2, wherein In step (1), the fermentation culture conditions are static culture at 20-30° C. for 10-40 days.

5. The preparation method according to claim 2, wherein In step (3), the fractions were subjected to reverse phase silica gel column chromatography, and gradient elution was performed with a methanol / water mixture of volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, respectively. Each gradient elution was performed 5 times, and the fractions were numbered 1 to 45 in sequence. Fractions 4 to 5 were combined into subfraction 2, fractions 14 to 17 were combined into subfraction 6, fractions 18 to 25 were combined into subfraction 7, fractions 26 to 28 were combined into subfraction 8, and fractions 38 to 39 were combined into subfraction 12. The fractions were then separated by high performance liquid chromatography, and subfraction 2 was eluted with a methanol / water mixture of volume ratio 35%. The retention time was 2 hours. The peaks with retention times of 29.3 minutes and 24.5 minutes respectively are compounds 4 and 5, and their structural formulas are shown in formula (4) and formula (5) respectively; the peaks with retention times of 23.3 minutes and 25.5 minutes respectively are compounds 6 and 7, and their structural formulas are shown in formula (6) and formula (7) respectively when subcomponent 6 is eluted with a methanol / water mixture with a volume ratio of 36%, and their structural formulas are shown in formula (6) and formula (7) respectively; the peaks with retention times of 15.2 minutes, 17.5 minutes and 19.6 minutes respectively are compounds 13, 14 and 15, and their structural formulas are shown in formula (13), formula (14) and formula (15) respectively when subcomponent 8 is eluted with an acetonitrile / water mixture with a volume ratio of 18%.

6. Use of the polyhydroxycyclohexane derivative according to claim 1 in the preparation of anti-β-glucuronidase drugs.

7. The use according to claim 6, characterized in that The β-glucuronidase is intestinal bacteria β-glucuronidase.

8. Use of the polyhydroxycyclohexane derivative according to claim 1 in the preparation of a drug for alleviating gastrointestinal toxicity and side effects caused by irinotecan and indomethacin.

9. Use of a compound having a structural formula (14) in the preparation of an anti-tumor drug, characterized in that: The tumor is myeloma or lymphoma, 10. Use of compounds represented by formula (1) and (3) in the preparation of drugs for resisting β-glucuronidase.

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