Chloro-spiro-gentianol derivative as well as preparation method and application thereof

Abstract

The invention discloses a chlorinated spiro-gentianol derivative as well as a preparation method and application thereof, and belongs to the technical field of active ingredient analysis of marine fungi. The chlorinated spiro-gentianol derivative is a derivative with a chlorogentisyl alcohol and gbosine heterozygous skeleton, the chlorinated spiro-gentianol derivative with a novel structure is separated from a fermentation culture of marine fungi Epicoccum sorghine.GST-5 by utilizing the polarity difference of the chlorogentisyl alcohol and the gbosine derivative, and through an in-vitro anti-beta-glucuronidase test, the chlorinated spiro-gentianol derivative with the novel structure can be used for preparing the anti-beta-glucuronidase derivative with the novel structure and the application of the anti-beta-glucuronidase derivative in the marine fungi Epicoccum sorghine.GST-5, so that the anti-beta-glucuronidase derivative with the novel structure is obtained. It is shown that the chloro-spiro-gentianol derivative provided by the invention has good beta-glucuronidase inhibitory activity, is low in cytotoxicity, and has development prospects in the aspect of preparation of anti-beta-glucuronidase drugs or functional foods.

Description

[0001] This application is a divisional application of the application with the application number 202210955910.X, the application date of August 10, 2022, and the invention title "A Class of Polyhydroxycyclohexane Derivatives and Their Preparation Methods and Applications". Technical Field

[0002] The present invention relates to the technical field of analysis of active components of marine fungi, and specifically relates to derivatives with a gabosine and chlorogentisyl alcohol hybrid skeleton extracted from the fungus Epicoccum sorghinum GST-5 and their applications. Background Art

[0003] Compared with terrestrial microorganisms, marine microorganisms can tolerate extreme conditions unique to the ocean, such as high salt, high pressure, low oxygen, and low light. The specificity of the living environment leads to the diversity of marine microorganisms in terms of species, gene composition, and ecological functions. The particularity of the marine environment combined with the progress of marine microorganism resource acquisition technology has brought unprecedented opportunities to the research of natural drug source compounds from marine microorganisms.

[0004] Marine fungi among marine microorganisms are a rich source of active secondary metabolites. 70-80% of the secondary metabolites of marine fungi have biological activities, including small molecule lactone compounds; mycotoxins; new substances with inhibitory activity on the central nervous system; 1-dodecanol, unsaturated hydrocarbons, acids, esters; lipopeptide antibiotics that can inhibit fungal viruses in plants and humans and act on new targets for fungal cell wall synthesis. Discovering natural products with specific structural types from marine fungi is of great significance for the research and development of marine drugs.

[0005] β-glucuronidase (GUS) is an important class of hydrolases produced by gut microbiota, which can catalyze the hydrolysis of glucuronide conjugates to generate the corresponding aglycones. In the human body, GUS distributed in the intestine can also catalyze the hydrolysis of glucuronides of various drugs (such as indomethacin, diclofenac, and the active metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin) and endogenous sex hormones. The released aglycones can be reabsorbed by the intestine, thus forming a bimodal phenomenon in pharmacokinetics. It is worth noting that this process may cause serious gastrointestinal adverse reactions. For example, GUS can catalyze the hydrolysis of 7-ethyl-10-hydroxycamptothecin-O-glucuronide (SN-38G) to generate SN-38, a product with strong cytotoxic effects. The accumulation of the latter in the intestine can cause intestinal mucosal shedding and lead to serious adverse reactions such as severe delayed diarrhea. Therefore, the development of highly efficient and safe GUS inhibitors is of great significance for alleviating lethal diarrhea caused by drugs such as irinotecan and indomethacin (Weng Zimiao, Lv Shanshan, Ge Guangbo, Wang Ping, Hou Jie, Progress in Pharmaceutical Sciences, Research Progress of β-Glucuronidase Inhibitors Produced by Intestinal Bacteria, 2018, 42(03)).

[0006] Currently, there is little research on the chemical constituents of the genus Epicoccum. Hao Baocong et al. isolated (4R*,5R*,6S*)-4,5-dihydroxy-6-(6'-methylsalicyloyloxy)-2-methoxymethyl-2-cyclohexen-1-one, (4R*,5R*,6S*)-4,5-dihydroxy-6-(6'-methylsalicyloyloxy)-2-methyl-2-cyclohexen-1-one, (4R,5R,6S)-4,5-dihydroxy-6-(6'-methylsalicyloyloxy)-2-hydroxymethyl-2-cyclohexen-1-one, (-)-gabosine E, theobroxide, 3-chlorogentisyl alcohol, and 3-hydroxybenzyl alcohol from an endophytic fungus Epicoccum sorghinum of Taraxacum mongolicum (Hao Baocong, Zheng Yaoyao, Chen Xu, Chen Qiuxia, Ji Ruonan, Chen Min, Acta Pharmaceutica Sinica, Study on Secondary Metabolites of an Endophytic Fungus Epicoccum sorghinum 1-2 of Taraxacum mongolicum, 2022, 57(07)).

[0007] Yuan Chao et al. studied the chemical constituents of an endophytic fungus Epicoccum nigrum 14one 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-methylisobenzofuran-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 them, the alkaloid fusaricide and epicoccolide compounds have strong antibacterial activities. Among them, the alkaloid fusaricide has good in vitro inhibitory activity against lung cancer cell M109 (Yuan Chao, Guo Yuhua, Zhang Yingbo, Hu Xuan, Wang Dan, Yu Fulai, Li Gang, China Journal of Chinese Materia Medica, Study on Secondary Metabolites of an Endophytic Fungus Epicoccum nigrum 14one, 2019, 44(18), 4021-4025).

[0008] However, so far, no literature has reported the isolation of chlorogentisyl alcohol and gabosine compounds with anti-GUS enzyme from fungi of the genus Epicoccum. Summary of the Invention

[0009] The object of the present invention is to extract natural active substances with medicinal value from marine fungi of the genus Epicoccum.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] Twenty-five compounds were isolated from the fermentation products of the fungus Epicoccum sorghinum GST-5. Through structure identification, the 25 compounds are polyhydroxycyclohexane derivatives, belonging to chlorogentisylalcohol derivatives (with the structure of 2-chloro-6-(hydroxymethyl)benzene-1,4-diol), gabosine derivatives (with the structure of 4,5,6-trihydroxy-2-(hydroxymethyl)cyclohex-2-en-1-one), and derivatives with a hybrid skeleton of chlorogentisyl alcohol and gabosine. The specific structural formulas are as shown in Figure 1 and are numbered 1 to 25 respectively.

[0012] The fungus Epicoccum sorghinum GST-5 was isolated from a large marine plant in the hydrothermal vent sediment of Guishan Island, Taiwan Province, China, and the preservation number is: CGMCC 3.20238.

[0013] By consulting databases and literature, the present invention identified 19 of the above 25 compounds as new compounds. Therefore, the present invention provides a new class of chlorospirogentianol derivatives, namely derivatives with a hybrid skeleton of chlorogentisyl alcohol and gabosine, and the structural formulas are as shown in any one of Formula (9), (16), (17), corresponding to Compounds 9, 10, 11, 12, 16, 17 respectively.

[0014]

[0015] The present invention also provides a method for isolating and extracting the above new compounds from the fermentation products of the fungus Epicoccum sorghinum GST-5, but the preparation method of the above compounds in the present invention is not limited thereto.

[0016] The method for extracting the derivative with a hybrid skeleton of chlorogentisyl alcohol and gabosine from the fermentation products includes the following steps:

[0017] (1) Activate the fungus Epicoccum sorghinum GST-5 and inoculate it into a liquid medium for fermentation culture;

[0018] (2) After the fermentation culture is completed, the mycelium and the fermentation broth are separated. The mycelium is extracted in an organic solvent, and the extract is separated. The extract is concentrated and suspended in distilled water to obtain an aqueous suspension. Then, the aqueous suspension is extracted with ethyl acetate or n-butanol to obtain extract A; after the fermentation broth is stirred with diatomaceous earth, it is extracted by refluxing with ethyl acetate or n-butanol to obtain extract B;

[0019] (3) The extract A or extract B is concentrated and then separated by normal-phase silica gel column chromatography. Gradient elution is carried out successively with dichloromethane / methanol mixed solutions with volume ratios of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, and 10:1. The fractions eluted with the dichloromethane / methanol mixed solution with a volume ratio of 10:1 are collected, and then reverse-phase silica gel column chromatography and high-performance liquid chromatography separation are carried out to obtain the derivative.

[0020] In step (1), the fungus Epicoccum sorghinum GST-5 is fermented and cultured.

[0021] The fungus Epicoccum sorghinum GST-5 is a fungus, and a conventional PDA liquid medium or wort medium can be used for fermentation and culture. To increase the yield and provide sufficient nutrients for the growth and metabolism of microorganisms, preferably, based on 1 L of volume, the liquid medium includes the following raw materials: 1-5 g of starch, 10-20 g of bran, 3-15 g of yeast extract, KH 2 PO 4 1-8 g, 0.1-0.8 g of MgSO 4 ·7H 2 O, and the balance is water;

[0022] Alternatively, based on 1 L of volume, the liquid medium includes 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 solution is 6.0-7.0;

[0023] Alternatively, based on 1 L of volume, the liquid medium includes the following raw materials: 10-40 g of sucrose, 5-20 g of corn flour, 1-4 g of NaNO 3 1, 1-4 g of yeast extract, 0.2-0.8 g of KH 2 PO 4 0.2-1 g of MgSO 4 ·7H 2 O, 0.2-1 g of KCl, 0.001-0.005 g of FeSO 4 0.001-0.005 g, and the balance is water;

[0024] Alternatively, per liter of volume, the liquid culture medium comprises the following raw materials: 20 - 30 g of malt extract powder, 15 - 20 g of glucose, 1 - 2 g of casein peptone, and the balance is water.

[0025] The conditions for fermentation culture are static culture at 20 - 30 °C for 10 - 40 days. The static culture method means no shake flask culture.

[0026] Preferably, the temperature for fermentation culture is 22 - 26 °C. More preferably, it is cultured at 25 °C for 20 days, and the yield of the derivative with a chlorogentisyl alcohol and gabosine hybrid skeleton is the highest under this condition.

[0027] In step (2), the mycelium and the fermentation broth are separated, and the derivative with a chlorogentisyl alcohol and gabosine hybrid skeleton can be extracted and separated from both the mycelium and the fermentation broth.

[0028] Among them, when obtaining the derivative with a chlorogentisyl alcohol and gabosine hybrid skeleton from the mycelium, the mycelium is soaked in an organic solvent for 7 - 14 days, and the mycelium is fully broken walled to effectively dissolve the intracellular substances. The organic solvent is one or two of methanol, ethanol, ethyl acetate, and acetone.

[0029] In step (3), the separation and purification method is: subjecting the extract to normal-phase silica gel column chromatography separation, and then subjecting the obtained fractions to recrystallization, reverse-phase silica gel column chromatography, and high-performance liquid chromatography separation. Through multiple steps of separation and purification, a derivative with a chlorogentisyl alcohol and gabosine hybrid skeleton with a relatively high purity can be obtained.

[0030] Preferably, the fraction is subjected to reverse-phase silica gel column chromatography and gradient eluted successively with methanol / water mixtures at volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. Each gradient is eluted 5 times and numbered 1-45 successively. The fractions numbered 1-3 are combined into sub-component 1, fractions 4-5 are combined into sub-component 2, fractions 14-17 are combined into sub-component 6, fractions 18-25 are combined into sub-component 7, fractions 26-28 are combined into sub-component 8, and fractions 38-39 are combined into sub-component 12. Subsequently, separation is performed by high-performance liquid chromatography. Sub-component 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 with the structure shown in formula (9). Sub-component 8 is eluted with an acetonitrile / water mixture at a volume ratio of 18%. The peaks with retention times of 23.3 minutes and 26.3 minutes are compounds 16 and 17 with the structures shown in formula (16) and formula (17), respectively.

[0031] The research of the present invention shows that the chlorogentisyl alcohol and gabosine hybrid skeleton derivatives isolated from the fermentation culture of the fungus Epicoccum sorghinum. GST-5 by the above method have good anti-β-glucuronidase (GUS) activity. Further, the present invention uses the inhibitory activity of the compound on cells as its cytotoxicity data. The results show that except for compound 12, other compounds have low cytotoxicity to human cells. Therefore, the present invention provides the application of the chlorogentisyl alcohol and gabosine hybrid skeleton derivatives in the preparation of drugs or functional foods for anti-β-glucuronidase.

[0032] Further, the anti-β-glucuronidase drug is an inhibitor of intestinal bacteria β-glucuronidase. Research shows that β-glucuronidase distributed in the intestine can cause gastrointestinal adverse reactions during the catalytic hydrolysis of drugs (such as indomethacin, irinotecan, etc.). The compounds provided by the present invention can relieve the gastrointestinal adverse reactions caused by drugs by inhibiting the activity of GUS enzyme.

[0033] The present invention provides the application of the chlorogentisyl alcohol and gabosine hybrid skeleton derivatives in the preparation of drugs or functional foods for relieving the gastrointestinal toxic and side reactions caused by irinotecan and indomethacin drugs.

[0034] Furthermore, the research of the present invention has found that compound 12 has a significant inhibitory effect on Namalwa cell line and U266 cell line. Therefore, the present invention provides the use of the compound shown in the structural formula (12) in the preparation of anti-tumor drugs. Specifically, the tumor is myeloma or lymphoma.

[0035]

[0036] The drug takes the derivative with a hybrid skeleton of chlorogentisyl alcohol and gabosine of the present invention as the main active ingredient, and is made by adding pharmaceutically acceptable excipients, and can be made into a preparation according to the preparation methods of preparations recorded in pharmacy. The said preparation can be injection, drip infusion, powder for injection, granule, tablet, infusion, powder, oral liquid, sugar-coated tablet, film-coated tablet, enteric-coated tablet, buccal tablet, pill, paste, bolus, spray, drop pill, disintegrant, orally disintegrating tablet, pellet, etc.

[0037] The said functional food takes the derivative with a hybrid skeleton of chlorogentisyl alcohol and gabosine of the present invention as the main active ingredient, and is made by adding acceptable food excipients.

[0038] The beneficial effects of the present invention are as follows:

[0039] (1) The present invention utilizes the polarity difference of the derivative with a hybrid skeleton of chlorogentisyl alcohol and gabosine to extract and isolate a derivative with a novel structure and a hybrid skeleton of chlorogentisyl alcohol and gabosine from the fermentation culture of marine fungi. This method is simple in operation, high in extraction yield, and high in product purity, and is suitable for large-scale production.

[0040] (2) Through the in vitro anti-GUS enzyme test, it is shown that the derivative with a hybrid skeleton of chlorogentisyl alcohol and gabosine provided by the present invention has good anti-GUS enzyme activity, and the IC 50 value of the most active compound 17 is 0.24 μM. Further cytotoxicity tests show that the carbon sugar compounds provided by the present invention have low cytotoxicity and have good development prospects in the preparation of anti-GUS enzyme drugs and preventive health foods.

[0041] (3) Through the in vitro tumor cell test, the tumor cell lines used are Namalwa cells and U266 cells. Among them, the survival rate of compound 12 against Namalwa cells is -0.33%, and it has good development prospects in the preparation of anti-tumor drugs and preventive health foods. Description of the Drawings

[0042] Figure 1 These are the structural formulas of 25 chlorogentisyl alcohol and gabosine derivatives of the present invention.

[0043] Figure 2 For compound 9 1 1H NMR data (in DMSO-d6, 600 MHz) and 13 13C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 1H NMR data, (B) is 13 13C NMR data.

[0044] Figure 3 For compound 9 1 1H NMR data (in CD 3 OD, 600 MHz) and 13 13C NMR data (in CD 3 OD, 150 MHz), where (A) is 1 1H NMR data, (B) is 13 13C NMR data.

[0045] Figure 4 For compound 10 1 1H NMR data (in CD 3 OD, 600 MHz) and 13 13C NMR data (in CD 3 OD, 150 MHz), where (A) is 1 1H NMR data, (B) is 13 13C NMR data.

[0046] Figure 5 For compound 11 1 1H NMR data (in CD 3 OD, 600 MHz) and 13 13C NMR data (in CD 3 OD, 150 MHz), where (A) is 1 1H NMR data, (B) is 13 13C NMR data.

[0047] Figure 6 For compound 12 1 1H NMR data (in CD 3 OD, 600 MHz) and 13 13C NMR data (in CD 3OD, 150 MHz), where (A) is 1 1H NMR data, and (B) is 13 13C NMR data.

[0048] Figure 7 For compound 16, the 1 1H NMR data (in DMSO-d6, 600 MHz) and 13 13C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 1H NMR data, and (B) is 13 13C NMR data.

[0049] Figure 8 For compound 17, the 1 1H NMR data (in DMSO-d6, 600 MHz) and 13 13C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 1H NMR data, and (B) is 13 13C NMR data. Detailed implementation manners

[0050] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0051] Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0052] Example 1: Fungal isolation

[0053] Large marine plants were collected from the hydrothermal vent seafloor sediments of Guishan Island, Taiwan Province, China. After the samples were brought back to the laboratory, they were first rinsed 3 times with sterile seawater to remove non-attached microorganisms; the plants were placed in centrifuge tubes, then a small amount of seawater was added, and they were oscillated for 10 min with a vortex oscillator. Then, the suspension after removing the algal bodies was centrifuged for 20 min (5000 r / min), and the supernatant was poured off; the precipitate was suspended with a small amount of sterile seawater, and 0.1 mL was taken and spread on a Martin's medium (containing 8 U / L gentamicin) plate; after culturing at room temperature of 20 °C for 10 d, single colonies were picked, purified by streaking, and transferred to a slant and stored at 4 °C for later use.

[0054] Example 2: Identification of Epichloë fungi

[0055] The isolated fungus was cultured on PDA, and the 18S rDNA gene sequence of the strain was determined. The 18S rDNA sequence of this strain is shown as SEQ ID No.1.

[0056] Based on the morphological characteristics of the strain and the results of 18S rDNA sequence analysis, this strain was identified as Epicoccum sp., and named Epicoccum sorghinum.GST-5. It was deposited in the China General Microbiological Culture Collection Center (CGMCC) on July 12, 2021. The deposit was identified as viable. Deposit address: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. Deposit number: CGMCC 3.20238.

[0057] Example 3: Fermentation culture of Epicoccum sp.

[0058] The activated Epicoccum sorghinum.GST-5 was made into a spore suspension, and then inoculated into the culture medium, followed by static fermentation culture at 25°C for 20 days.

[0059] Among them, the formula of the culture medium is: 3 g of starch, 14 g of bran, 6 g of yeast extract, 5 g of KH 2 PO 4 5, 0.4 g of MgSO 4 ·7H 2 O, and 1000 mL of water.

[0060] Example 4: Fermentation culture of Epicoccum sp.

[0061] The activated Epicoccum sorghinum.GST-5 was made into a spore suspension, and then inoculated into the culture medium, followed by static fermentation culture at 24°C for 20 days.

[0062] Among them, the formula of the culture medium is: 400 g of potato, 6 g of peptone, 2 g of yeast extract, 10 g of glucose, and 1000 mL of water; the initial pH value of the culture medium is 6.5.

[0063] Example 5: Fermentation culture of Epicoccum sp.

[0064] The activated Epicoccum sorghinum.GST-5 was made into a spore suspension, and then inoculated into the culture medium, followed by static fermentation culture at 25°C for 20 days.

[0065] Among them, the formula of the culture medium is: 25 g of sucrose, 10 g of corn flour, 2 g of NaNO 3 2, 2 g of yeast extract, 0.5 g of KH 2 PO 4 0, 0.5 g of MgSO 4 ·7H 2O 0.5 g, KCl 0.5 g, FeSO 4 0.001 g, water 1000 mL.

[0066] Example 6: Fermentation culture of Epichloe fungi

[0067] The activated Epichloe fungi GST-5 was made into a spore suspension, and then inoculated into the culture medium, and statically fermented at 25 °C for 20 days.

[0068] Among them, the culture medium formula was: malt extract powder 25 g, glucose 15 g, casein peptone 1.5 g, water 1000 mL.

[0069] Example 7: Preparation of chlorogentisyl alcohol and gabosine derivatives

[0070] After the fermentation culture of Epichloe fungi GST-5, 5 L of the fermentation culture broth was taken, and the precipitate was obtained by centrifugation to get mycelia; the mycelia were soaked in methanol for 1 week, the soaking solution was concentrated and then suspended in 1 L of distilled water, the aqueous suspension was combined, extracted with 6 L of n-butanol, and the n-butanol extract was concentrated to obtain 10 g of extract; it was mixed with silica gel (100 mesh, 100 g) and subjected to normal-phase silica gel column chromatography separation (200 - 300 mesh, 100 g; silica gel column size L 50 mm, ), successively eluted with dichloromethane / methanol mixed solutions with volume ratios of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1 and methanol in a gradient manner, 300 mL each time; TLC was used to detect the fractions; the fractions eluted with dichloromethane / methanol mixed solutions with volume ratios of 35:1 to 10:1 were collected and recrystallized with methanol.

[0071] Example 8: Preparation of chlorogentisyl alcohol and gabosine derivatives

[0072] After the fermentation culture of Epichloe fungi, 5 L of the fermentation culture broth was taken, and the supernatant was obtained by centrifugation to get the fermentation liquid; the fermentation liquid was concentrated, mixed with 10 g of diatomaceous earth, refluxed with 1 L of n-butanol, and subjected to normal-phase silica gel column chromatography separation (200 - 300 mesh, 1 kg; silica gel column size L50 mm, ) It was successively eluted with dichloromethane / methanol mixtures with volume ratios of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1 and methanol in a gradient manner. 400 mL of fractions were collected each time for each eluent, and each eluent was repeated 5 times. A total of 30 fractions eluted with dichloromethane / methanol mixtures with volume ratios from 35:1 to 10:1 were collected and numbered 1 - 30 in sequence. The fractions were detected by TLC and similar components were combined. Fractions 1 - 7 were combined into component 1, fractions 8 - 11 were combined into component 2, fractions 12 - 15 were combined into component 3, fractions 16 - 25 were combined into component 4, and fractions 26 - 30 were combined into component 5.

[0073] Component 5 was subjected to reverse-phase silica gel column chromatography. The eluent was methanol / water (1:9 - 9:1). It was successively eluted with methanol / water mixtures with volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 in a gradient manner. 400 mL of fractions were collected each time for each eluent, and each eluent was repeated 5 times. A total of 45 fractions eluted with each eluent were collected and numbered 1 - 45 in sequence. The fractions were detected and similar components were combined. Fractions 1 - 3 were combined into sub-component 5-1, fractions 4 - 5 were combined into sub-component 5-2, fractions 6 - 8 were combined into sub-component 5-3, fractions 9 - 10 were combined into sub-component 5-4, fractions 11 - 13 were combined into sub-component 5-5, fractions 14 - 17 were combined into sub-component 5-6, fractions 18 - 25 were combined into sub-component 5-7, fractions 26 - 28 were combined into sub-component 5-8, fractions 29 - 30 were combined into sub-component 5-9, fractions 31 - 33 were combined into sub-component 5-10, fractions 34 - 37 were combined into sub-component 5-11, fractions 38 - 39 were combined into sub-component 5-12, fractions 40 - 41 were combined into sub-component 5-13, fractions 42 - 43 were combined into sub-component 5-14, and fractions 44 - 45 were combined into sub-component 5-15.

[0074] Subsequently, separation was performed by high performance liquid chromatography. The mobile phase was: methanol / water or acetonitrile / water, the flow rate was 10 mL / min, the detection wavelength of the high performance liquid chromatography was 254 nm. For sub-component 5-1, under an eluent of 28% (methanol-water), the peaks with retention times of 13.1 minutes, 15.1 minutes, 18.3 minutes, 20.8 minutes, and 25.6 minutes were recrystallized with cyclohexane to obtain compounds 1, 2, 3, 19, and 20; for sub-component 5-2, under an eluent of 35% (methanol-water), the peaks with retention times of 29.3 minutes, 24.5 minutes, 35.6 minutes, 36.9 minutes, and 27.3 minutes were recrystallized with cyclohexane to obtain compounds 4, 5, 21, 22, and 23; for sub-component 5-6, under an eluent of 36% (methanol-water), the peaks with retention times of 23.3 minutes, 25.5 minutes, 30.6 minutes, and 33.3 minutes were recrystallized with cyclohexane to obtain compounds 6, 7, 8, and 18; for sub-component 5-7, under an eluent of 42% (methanol-water), the peaks with retention times of 18.2 minutes, 24.4 minutes, 26.9 minutes, and 22.3 minutes were recrystallized with cyclohexane to obtain compounds 9, 11, 12, and 10; for sub-component 5-8, under an eluent of 18% (acetonitrile-water), the peaks with retention times of 15.2 minutes, 17.5 minutes, 19.6 minutes, 23.3 minutes, and 26.3 minutes were recrystallized with cyclohexane to obtain compounds 13, 14, 15, 16, and 17; for sub-component 5-12, under an eluent of 25% (acetonitrile-water), the peaks with retention times of 14.3 minutes and 18.3 minutes were recrystallized with cyclohexane to obtain compounds 24 and 25.

[0075] Example 9: Structure Identification of Chlorogentisyl Alcohol and Gabosine Derivatives

[0076] The purity of the prepared compounds was identified by HPLC. Samples with a purity greater than 98% were subjected to structure identification using mass spectrometry and nuclear magnetic resonance techniques. Nuclear magnetic resonance was measured using a Bruker AVANCE DRX-600 NMR Sectrometer with TMS as the internal standard; high-resolution mass spectrometry FTICRMS was measured using a Bruker Apex Spectrometer; electrospray mass spectrometry ESI-MS was measured using a Bruker Esquire3000 plus Spectrometer. The structural formulas of the above 25 compounds are shown as Figure 1 follows. The structure identification of derivatives with a chlorogentisyl alcohol and gabosine hybrid skeleton is as follows:

[0077] According to the one-dimensional NMR and mass spectrometry analysis results of compound 9 (see Table 1), it can be known that the molecular formula of compound 9 is C 14 H12 Cl 2 O 5 , with an unsaturation degree of 8. By comparing the 1 H NMR and 13 C NMR data of the comparative compounds, it was found that Compound 9 was very similar to Compound 1, both containing a 2-chloro-6-(hydroxymethyl)benzene-1,4-diol structure. Through the results of two-dimensional NMR analysis, Compound 9 was identified as a new compound. The structure is as Figure 1 shown:

[0078] Table 1. NMR data of Compound 9

[0079]

[0080] According to the results of one-dimensional NMR and mass spectrometry analysis of Compound 10 (see Table 2), it was known that the molecular formula of Compound 10 was C 15 H 14 Cl 2 O 5 , with an unsaturation degree of 8. By comparing the 1 H NMR and 13 C NMR data of the comparative compounds, it was found that Compound 10 was very similar to Compound 9, with only one more methoxy signal. Through the results of two-dimensional NMR analysis, Compound 10 was identified as a new compound. The structure is as Figure 1 shown:

[0081] Table 2. NMR data of Compound 10

[0082]

[0083]

[0084] According to the results of one-dimensional NMR and mass spectrometry analysis of Compound 11 (see Table 3), it was known that the molecular formula of Compound 9 was C 14 H 12 Cl 2 O 5 , with an unsaturation degree of 8. By comparing the 1 H NMR and 13 C NMR data of the comparative compounds, it was found that Compound 11 was very similar to Compound 9, with only minor changes in the chiral carbon region. Through the results of two-dimensional NMR analysis, Compound 11 was identified as a new compound. The structure is as Figure 1 shown:

[0085] Table 3. NMR data of Compound 11

[0086]

[0087] Based on the results of one-dimensional NMR and mass spectrometry analysis of compound 12 (see Table 4), it can be seen that the molecular formula of compound 12 is C 14 H 12 Cl 2 O 5 , and the degree of unsaturation is 8. By comparing the 1 H NMR and 13 C NMR data of the compounds, it was found that compound 12 was very similar to compound 9, with only minor changes in the chiral carbon region. Through the results of two-dimensional NMR analysis, compound 12 was identified as a new compound. The structure is as Figure 1 shown:

[0088] Table 4. NMR data of compound 12

[0089]

[0090] Based on the results of one-dimensional NMR and mass spectrometry analysis of compound 16 (see Table 5), it can be seen that the molecular formula of compound 16 is C 21 H 24 Cl 2 O 7 , and the degree of unsaturation is 9. Combining the 1 H and 13 C-NMR data, it was found that this compound was similar to compound 10, with only its methoxy group replaced by an ampelomin group. Through the results of two-dimensional NMR analysis, compound 16 was identified as a new compound. The structure is as Figure 1 shown:

[0091] Table 5. NMR data of compound 16

[0092]

[0093]

[0094] Based on the results of one-dimensional NMR and mass spectrometry analysis of compound 17 (see Table 6), it can be seen that the molecular formula of compound 17 is C 21 H 24 Cl 2 O 7 , and the degree of unsaturation is 9. Combining the 1 H and 13 C-NMR data, it was found that this compound was similar to compound 9, with only the chlorine substituent on its gabosine group replaced by a 2-chloro-6-(hydroxymethyl)benzene-1,4-diol group. Through the results of two-dimensional NMR analysis, compound 17 was identified as a new compound. The structure is as Figure 1 shown:

[0095] Table 6. NMR data of compound 17

[0096]

[0097]

[0098] Example 10: Anti-GUS Activity Analysis of Chlorogentisyl Alcohol and Gabosine Derivatives

[0099] (1) Preparation of Solutions

[0100] Preparation of samples: 1.0 mg of the sample and the reference substance D-glucaro-1,4-lactone (DSL) were dissolved in DMSO to prepare 10.0 mM sample and reference substance solutions;

[0101] Preparation of the substrate PNPG solution: 3.0 mg of PNPG was accurately weighed and dissolved in PBS buffer (pH 7.4) to make its concentration 2.5 mM;

[0102] Preparation of 10% DMSO solution: 10.0 μL of DMSO was accurately measured and added to 90.0 μL of PBS buffer (pH 7.4) to prepare 10% DMSO solution;

[0103] Preparation of the liquid sample mother liquor: 100.0 μL of the above sample was accurately measured and mixed with DMSO to prepare a 10.0 mM sample solution (final concentration: 0.1 mM).

[0104] (2) Preliminary Screening of GUS Inhibitors

[0105] Using DMSO as the blank control and DSL as the positive control, screening of GUS inhibitors was carried out. Among them, the blank control: the total volume of the mixed system was 100.0 μL, containing 10.0 μL of GUS (final concentration: 2.0 μg / mL), 79.0 μL of PBS buffer (pH 7.4), 1.0 μL of 10% DMSO solution (final concentration: 100.0 μM), and 10.0 μL of PNPG (final concentration: 250.0 μM).

[0106] Inhibitor and positive control: the total volume of the mixed system was 100.0 μL, containing 10.0 μL of Escherichia coli β-glucuronidase (final concentration: 2.0 μg / mL), 70.0 μL of PBS buffer (pH 7.4), 10.0 μL of the inhibitor, DSL mother liquor (final concentration: 100.0 μM), and 10.0 μL of PNPG (final concentration: 250.0 μM).

[0107] After sample addition, immediately measure the absorbance at 405 nm using an enzyme-linked immunosorbent assay (ELISA) reader. After incubating for 30 min in a 37 °C constant temperature incubator, repeat the measurement. Perform three parallel groups and calculate their relative activities. The relative activity is the percentage of the PNP content within 30 min in the presence of an inhibitor or DSL compared to the blank control.

[0108] (3) Determination of the IC 50 value of the GUS inhibitor

[0109] Perform the IC 50 value determination on the GUS inhibitor with stronger inhibitory effect screened in (2).

[0110] The reaction conditions are as follows: The total volume of the reaction mixture is 100.0 μL, containing 10.0 μL of GUS (final concentration: 2.0 μg / mL), 70.0 μL of PBS buffer (pH 7.4), 10.0 μL of PNPG (final concentration: 250.0 μM), and 10.0 μL of inhibitor solution (final concentration: 1.0 nM - 100.0 μM).

[0111] After sample addition, immediately measure the absorbance at 405 nm using an ELISA reader. After incubating for 30 min in a 37 °C constant temperature incubator, repeat the measurement. Perform three parallel groups and calculate their relative activities. Finally, use Prism 6.0 (GraphPad Software, La Jolla, CA) to plot a concentration-response curve to calculate the IC 50 value.

[0112] (4) Study on the inhibition kinetics of the GUS inhibitor

[0113] Study the inhibition kinetics type (competitive, non-competitive, mixed) of the GUS inhibitor selected in (3) to explore the interactions between the substrate, inhibitor, and GUS. To determine the inhibition kinetics type of the inhibitor, use multiple concentrations of PNPG and multiple concentrations of the inhibitor to determine the corresponding reaction rates and evaluate them through the intersection position of the Lineweaver - Burk plot.

[0114] (5) Experimental results

[0115] Table 7. GUS inhibition rate and IC 50 value of the compound

[0116]

[0117] a ND indicates not detected

[0118] The preliminary screening data of the compounds in the above table showed that the enzyme inhibition rates of compounds 9, 10, 12, and 17 at a concentration of 10 μM were 85% or above. Further rescreening of the above compounds showed that the half-maximal inhibitory concentration (IC50) of these compounds was between 0.24 μM and 2.83 μM, which had a significant inhibitory effect compared with the positive drug DSL. The Ki inhibition constant was between 1.09 μM and 4.43 μM. In the analysis of the enzyme-substrate binding type, they were all mixed inhibitors.

[0119] Example 11: Antitumor Activity Analysis of Chlorogentisyl Alcohol and Gabosine Derivatives

[0120] (1) Reagent Preparation

[0121] 0.4% SRB solution: Weigh 0.8 g of SRB and dissolve it in 200 mL of 1% acetic acid, and store it at room temperature.

[0122] 50% TCA solution: Weigh 50 g of TCA, add water to make up to 100 mL, and store it at 4°C.

[0123] 10 mM Tris-base solution: Weigh 0.6057 g of Tris-base, add water to make up to 500 mL, pH 10.5, and store it at 4°C.

[0124] (2) Experimental Instruments

[0125] CO 2 Incubator (Thermo), Mini shaker (Kylin-Bell Lab instruments), Microplate reader (MD company, model M5)

[0126] (3) Method

[0127] Logarithmic growth phase cells were selected. After trypsin digestion, the cell concentration was adjusted to 2×104 cells / mL with RPMI 1640 medium containing 10% fetal bovine serum. 190 μl of cell suspension was inoculated into a 96-well culture plate per well, and cultured at 37°C, 5% CO 2 for 24 h. 10 μl of sample solution (final concentration: 5 μg / mL for the compound; 50 μg / mL for the mixture) was added to the drug treatment wells, and 5-FU with a final concentration of 5 μg / mL was added to the positive drug wells for treatment; the control wells were added with the medium containing the same volume of solvent, and cultured at 37°C, 5% CO 2Culture 3D. Discard the culture medium, and gently add 100 μl of 50% TCA pre-cooled at 4°C to fix the cells. Let it stand for 5 min first, and then transfer it to 4°C for 1 h. Pour out the fixing solution, wash it 5 times with distilled water to remove TCA, and air dry for 1 h. Add 80 μl of 0.4% SRB solution to each well and stain at room temperature for 30 min. Discard the staining solution, wash it 5 times with 1% acetic acid to fully remove the unbound SRB, and air dry. Add 150 μl of 10 mM Tris-base (pH 10.5) to dissolve, and oscillate on a micro oscillator for 5 min. Measure the OD510nm value with an M5 microplate reader.

[0128] (4) Result calculation

[0129] Tumor cell survival rate (%) = 100% - (OD 对照 - OD 药物 ) / (OD 对照 - OD 空白 ) × 100%

[0130] (5) Experimental results

[0131] Table 8. Survival rates of Namalwa and U266 cells of the compounds

[0132]

[0133] a ND indicates not detected

[0134] Using Namalwa and U266 tumor cell lines to further determine the cytotoxicity of compounds 9, 10, 12, and 17 at a concentration of 20 μM. The data show that except for compound 12, the other compounds have low toxicity to cells. The survival rate of compound 12 for the Namalwa cell line is -0.33%, which is close to the survival rate of 11.28% of the positive drug 5-Fu for the Namalwa cell line. The positive drug As 2 O 3 has a survival rate of 2.59% for the U266 cell line, indicating that compound 12 has dual-functional pharmacodynamic effects of anti-tumor while inhibiting GUS.

Claims

1. A chlorinated spirogentiopicrin derivative, characterized in that, the chlorinated spirogentiopicrin derivative is a derivative with a hybrid skeleton of chlorogentisyl alcohol and gabosine, and the structural formula is shown as any one of formula (9), (16), and (17), 2. A preparation method of the chlorinated spirogentiopicrin derivative according to claim 1, characterized in that, comprises the following steps: (1) Activate the Epicoccum sorghinum GST-5 with the preservation number of CGMCC 3.20238, inoculate it into a liquid medium, and carry out fermentation culture; (2) After the fermentation culture is completed, separate the mycelium and the fermentation broth. Add an organic solvent to the mycelium for extraction, separate to obtain an extract, concentrate the extract, suspend it with distilled water to obtain a water suspension, and then extract the water suspension with ethyl acetate or n-butanol to obtain an extraction solution A; after stirring the fermentation broth with diatomaceous earth, carry out extraction by refluxing with ethyl acetate or n-butanol to obtain an extraction solution B; (3) Concentrate the extraction solution A or extraction solution B and then carry out normal-phase silica gel column chromatography separation. Gradient elution is carried out successively with dichloromethane / methanol mixed solutions with volume ratios of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:

1. Collect the fractions eluted by the dichloromethane / methanol mixed solution with a volume ratio of 10:1, and then carry out reverse-phase silica gel column chromatography and high-performance liquid chromatography separation to obtain the derivative.

3. The preparation method according to claim 2, characterized in that, In step (1), based on 1 L of volume, the liquid medium comprises the following raw materials: 1 - 5 g of starch, 10 - 20 g of wheat bran, 3 - 15 g of yeast extract, KH 2 PO 4 1 - 8 g, MgSO 4 ·7H 2 O 0.1 - 0.8 g, and the balance is water; alternatively, based on a volume of 1 L, the liquid 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 solution is 6.0 - 7.0; Alternatively, per liter of volume, the liquid culture medium comprises the following raw materials: 10 - 40 g of sucrose, 5 - 20 g of corn flour, 1 - 4 g of NaNO 3 1 - 4 g, 1 - 4 g of yeast extract, 0.2 - 0.8 g of KH 2 PO 4 4, 0.2 - 1 g of MgSO 4 ·7H 2 O, 0.2 - 1 g of KCl, 0.001 - 0.005 g of FeSO 4 4, with the balance being water; alternatively, based on a volume of 1 L, the liquid medium comprises the following raw materials: 20 - 30 g of malt extract powder, 15 - 20 g of glucose, 1 - 2 g of casein peptone, and the balance is water.

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

5. The preparation method according to claim 2, characterized in that, In step (3), the fraction is subjected to reverse-phase silica gel column chromatography and gradient eluted successively with methanol / water mixtures with volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, with each gradient eluted 5 times and numbered 1-45 in sequence. Fractions 18-25 are combined into sub-component 7, and fractions 26-28 are combined into sub-component 8. Subsequently, separation is performed by high-performance liquid chromatography. For sub-component 7, eluted with a methanol / water mixture with 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 with the structure shown in formula (9). For sub-component 8, eluted with an acetonitrile / water mixture with a volume ratio of 18%, the peaks with retention times of 23.3 minutes and 26.3 minutes are compounds 16 and 17, with the structures shown in formula (16) and formula (17) respectively.

6. Use of the chlorospirocyclic gentianol derivative according to claim 1 in the preparation of a drug or functional food for resisting β-glucuronidase.

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

8. Use of the chlorospirocyclic gentianol derivative according to claim 1 in the preparation of a drug or functional food for alleviating gastrointestinal toxic and side reactions caused by drugs such as irinotecan and indomethacin.

9. Use of the compound with the structure shown in formula (12) in the preparation of an antitumor drug, characterized in that, the tumor is myeloma or lymphoma.

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