Isoprenylated flavanones, methods of making and uses thereof

Abstract

The application relates to the technical field of medicines, and discloses a kind of isoprenyl-santonin compounds and preparation method and application thereof.The isoprenyl-santonin compound is any one of compounds shown in formula 1 to formula 9, the isoprenyl-santonin compound is isolated and extracted from the secondary metabolites of Aspergillus stellatus, and has excellent antitumor activity and anti-inflammatory activity, can be applied to the preparation of antitumor drugs and anti-inflammatory drugs, and has great practical value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an isopentenyl xanthones compound, its preparation method, and its application. Background Technology

[0002] Fungi are an important source of bioactive compounds. Due to their short lifespan and strong adaptability to external media, they can produce a rich variety of biologically significant secondary metabolites. *Aspergillus* is a typical filamentous fungus whose secondary metabolites include alkaloids, terpenes, anthraquinones, and xanthones. *Aspergillus asteroides* belongs to the genus *Aspergillus*, and research on its secondary metabolites is still lacking. Existing reports mainly focus on various types of xanthones. Further exploration of the secondary metabolites of this species is needed.

[0003] Xanthones are a class of oxyheterocyclic molecules containing dibenzo-γ-pyrrolidones. Their derivatives exhibit a variety of biological activities, and compounds with a xanthone skeleton often have good potential for therapeutic applications. The most numerous and diverse subclass obtained from plant and fungal sources is isopentenylated xanthones. Therefore, systematic research on Aspergillus asteroides to identify bioactive isopentenylated xanthones from its secondary metabolites is of great significance. Summary of the Invention

[0004] The purpose of this invention is to overcome the current limitations of the limited variety of isopentenyl xanthones and the need for further enhancement of their antitumor and anti-inflammatory activity. This invention provides an isopentenyl xanthone compound, its preparation method, and its applications. The isopentenyl xanthone compound described in this invention is isolated and extracted from the secondary metabolites of Aspergillus asteroides and possesses excellent antitumor and anti-inflammatory activity. It can be used in the preparation of antitumor and anti-inflammatory drugs, demonstrating significant practical value.

[0005] To achieve the above objectives, the present invention provides an isopentenyl xanthones, wherein the isopentenyl xanthones is any one of the compounds shown in Formula 1 to Formula 9;

[0006]

[0007] A second aspect of the present invention provides a method for preparing the isopentenyl xanthones, the method comprising the following steps:

[0008] (1) Inoculate the mycelium of Aspergillus stellaria onto potato dextrose agar medium and then incubate at a constant temperature to obtain seed culture medium;

[0009] (2) Cut the seed culture medium containing mycelium into pieces and inoculate it into sterilized rice culture medium for fermentation culture;

[0010] (3) The fermentation product obtained in step (2) is extracted with ethanol and then concentrated under reduced pressure to obtain the total extract;

[0011] (4) The total extract is suspended in water, then extracted with ethyl acetate, and then concentrated under reduced pressure to obtain ethyl acetate extract;

[0012] (5) The ethyl acetate extract was subjected to normal phase column chromatography to obtain five fractions Fr.1 to Fr.5;

[0013] (6) The fraction Fr.3 was subjected to reversed-phase column chromatography to obtain 10 fractions Fr.3.1 to Fr.3.10;

[0014] (7) The component Fr.3.4 was subjected to normal phase column chromatography, followed by high performance liquid chromatography to obtain the compound shown in Formula 6;

[0015] (8) The fraction Fr.3.7 was subjected to normal phase column chromatography to obtain seven fractions Fr.3.7.1 to Fr.3.7.7;

[0016] (9) The component Fr.3.7.3 was purified by high performance liquid chromatography to obtain the compound shown in Formula 1;

[0017] (10) The component Fr.3.7.4 was purified by high performance liquid chromatography to obtain the compounds shown in Formula 2 and Formula 3;

[0018] (11) The component Fr.3.7.5 was purified by high performance liquid chromatography to obtain the compounds shown in Formula 4 and Formula 5;

[0019] (12) The fraction Fr.3.8 was subjected to normal phase column chromatography to obtain nine fractions Fr.3.8.1 to Fr.3.8.9;

[0020] (13) The fraction Fr.3.8.6 was separated by gel column chromatography to obtain three fractions Fr.3.8.6.1 to Fr.3.8.6.3;

[0021] (14) The components Fr.3.8.6.2 were purified by high performance liquid chromatography to obtain the compounds shown in Formula 7, Formula 8 and Formula 9.

[0022] Preferably, the conditions for constant temperature incubation include: a temperature of 20-30℃ and a time of 5-12 days.

[0023] Preferably, the fermentation conditions include a temperature of 20-30℃ and a time of 40-50 days.

[0024] Preferably, in step (5), the eluent for the normal phase column chromatography is a petroleum ether-ethyl acetate-methanol mixed solution with a volume ratio of 10:1:0-10:10:0-10:10:1.

[0025] Preferably, in step (6), the eluent for the reversed-phase column chromatography is a methanol-water mixture with a volume ratio of 20:80-80:20.

[0026] Preferably, in step (7), the eluent for the normal phase column chromatography is a petroleum ether-ethyl acetate mixture with a volume ratio of 8:1 to 0:1.

[0027] Preferably, in step (8), the eluent for the normal phase column chromatography is a petroleum ether-ethyl acetate mixture with a volume ratio of 8:1 to 0:1.

[0028] Preferably, in step (12), the eluent for the normal phase column chromatography is a petroleum ether-ethyl acetate mixture with a volume ratio of 10:1 to 0:1.

[0029] A third aspect of the present invention provides the use of the above-mentioned isopentenyl shankone compounds in the preparation of antitumor drugs.

[0030] A fourth aspect of the present invention provides the use of the above-mentioned isopentenyl shankone compounds in the preparation of anti-inflammatory drugs.

[0031] In this invention, nine isopentenyl xanthones (compounds shown in Formulas 1 to 9) were obtained by separating and purifying the ethyl acetate extract of the fungus *Aspergillus stellaria*. Furthermore, the specific structures of these nine isopentenyl xanthones, as shown in Formulas 1 to 9, were determined using spectral analysis techniques such as nuclear magnetic resonance spectroscopy. Further evaluation of the antitumor and anti-inflammatory activities of the isopentenyl xanthones of this invention demonstrated that all of them possess certain antitumor and anti-inflammatory activities. Among them, the compounds shown in Formula 4, Formula 8, and Formula 9 exhibited significant antitumor activity, with the compound shown in Formula 9 showing a significant antitumor effect. Additionally, the compounds shown in Formula 1, Formula 4, Formula 7, and Formula 8 exhibited significant anti-inflammatory activity. Detailed Implementation

[0032] The following detailed description of specific embodiments of the present invention is provided. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0033] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0034] The isopentenyl shankone compounds described in this invention are any one of the compounds shown in Formulas 1 to 9;

[0035]

[0036] In this invention, the compound shown in Formula 1 is named Compound 1, the compound shown in Formula 2 is named Compound 2, the compound shown in Formula 3 is named Compound 3, the compound shown in Formula 4 is named Compound 4, the compound shown in Formula 5 is named Compound 5, the compound shown in Formula 6 is named Compound 6, the compound shown in Formula 7 is named Compound 7, the compound shown in Formula 8 is named Compound 8, and the compound shown in Formula 9 is named Compound 9.

[0037] In this invention, compounds 1 to 9 are extracted from the secondary metabolites (fermentation products) of the fungus Aspergillus stellaria.

[0038] The present invention further provides a method for preparing the isopentenyl xanthones, the method comprising the following steps:

[0039] (1) Inoculate the mycelium of Aspergillus stellaria onto potato dextrose agar medium and then incubate at a constant temperature to obtain seed culture medium;

[0040] (2) Cut the seed culture medium containing mycelium into pieces and inoculate it into sterilized rice culture medium for fermentation culture;

[0041] (3) The fermentation product obtained in step (2) is extracted with ethanol and then concentrated under reduced pressure to obtain the total extract;

[0042] (4) The total extract is suspended in water, then extracted with ethyl acetate, and then concentrated under reduced pressure to obtain ethyl acetate extract;

[0043] (5) The ethyl acetate extract was subjected to normal phase column chromatography to obtain five fractions Fr.1 to Fr.5;

[0044] (6) The fraction Fr.3 was subjected to reversed-phase column chromatography to obtain 10 fractions Fr.3.1 to Fr.3.10;

[0045] (7) The component Fr.3.4 was subjected to normal phase column chromatography, followed by high performance liquid chromatography to obtain the compound shown in Formula 6;

[0046] (8) The fraction Fr.3.7 was subjected to normal phase column chromatography to obtain seven fractions Fr.3.7.1 to Fr.3.7.7;

[0047] (9) The component Fr.3.7.3 was purified by high performance liquid chromatography to obtain the compound shown in Formula 1;

[0048] (10) The component Fr.3.7.4 was purified by high performance liquid chromatography to obtain the compounds shown in Formula 2 and Formula 3;

[0049] (11) The component Fr.3.7.5 was purified by high performance liquid chromatography to obtain the compounds shown in Formula 4 and Formula 5;

[0050] (12) The fraction Fr.3.8 was subjected to normal phase column chromatography to obtain nine fractions Fr.3.8.1 to Fr.3.8.9;

[0051] (13) The fraction Fr.3.8.6 was separated by gel column chromatography to obtain three fractions Fr.3.8.6.1 to Fr.3.8.6.3;

[0052] (14) The components Fr.3.8.6.2 were purified by high performance liquid chromatography to obtain the compounds shown in Formula 7, Formula 8 and Formula 9.

[0053] In this invention, the Aspergillus stellaria used was purchased from the strain bank of the China General Microbiological Culture Collection Center (CGMCC), and the purchased strain number is CGMCC 3.6292.

[0054] In a preferred embodiment, in step (1), the conditions for the isothermal incubation include: a temperature of 20-30°C and a duration of 5-12 days. Specifically, the isothermal incubation temperature is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C; and the isothermal incubation time is 5 days, 6 days, 7 days, 8 days, 10 days, 11 days, or 12 days.

[0055] In a specific implementation, the specific process of step (1) includes: inoculating the mycelium of Aspergillus stellaria onto potato dextrose agar medium using an inoculation loop, then incubating at a constant temperature, and after three generations of continuous copying, culturing the Aspergillus stellaria strain in large quantities to obtain seed culture medium.

[0056] In this invention, the solid-liquid ratio of rice to water in the rice culture medium is 180-220g:200mL. Specifically, the solid-liquid ratio of rice to water can be 180g:200mL, 190g:200mL, 200g:200mL, 210g:200mL, or 220g:200mL.

[0057] In a preferred embodiment, step (2) includes sterilizing the rice culture medium in an autoclave for 20-40 minutes. Specifically, the sterilization time can be 20 minutes, 30 minutes, or 40 minutes.

[0058] In a preferred embodiment, in step (2), the fermentation conditions include a temperature of 20-30°C and a time of 40-50 days. Specifically, the fermentation temperature can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C; and the fermentation time can be 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, or 50 days.

[0059] In this invention, in step (3), the fermentation product obtained in step (2) refers to the material containing the culture medium obtained after fermentation and mycelium.

[0060] In a preferred embodiment, in step (3), the ethanol extraction is performed 8-15 times, more preferably 9-12 times. Specifically, the number of ethanol extractions can be 8, 9, 10, 11, 12, 13, 14, or 15 times.

[0061] In a specific implementation, in step (3), the solvent ethanol is recovered by vacuum concentration to obtain the total extract.

[0062] In a preferred embodiment, in step (4), the ethyl acetate extraction is performed 8-13 times, more preferably 9-12 times. Specifically, the number of ethyl acetate extractions can be 8, 9, 10, 11, 12, or 13 times.

[0063] In a specific implementation, in step (4), the solvent ethyl acetate is recovered by vacuum concentration to obtain the total extract.

[0064] In a preferred embodiment, in step (5), the eluent for the normal phase column chromatography is a petroleum ether-ethyl acetate-methanol mixed solution with a volume ratio of 10:1:0-10:10:0-10:10:1. Gradient elution is performed using this eluent to obtain five components Fr.1 to Fr.5.

[0065] In a preferred embodiment, in step (6), the eluent for the reversed-phase column chromatography is a methanol-water mixture with a volume ratio of 20:80-80:20. Gradient elution is performed using this eluent to obtain 10 components Fr.3.1 to Fr.3.10.

[0066] In a preferred embodiment, in step (7), the eluent for the normal-phase column chromatography is a petroleum ether-ethyl acetate mixture with a volume ratio of 8:1 to 0:1. Elution is performed using this eluent, and the resulting eluent is then purified by high-performance liquid chromatography to obtain the compound shown in Formula 6.

[0067] In a specific implementation, in step (7), the mobile phase of the high performance liquid chromatography is an acetonitrile-water mixture with a volume ratio of 70:30.

[0068] In a preferred embodiment, in step (8), the eluent for the normal phase column chromatography is a petroleum ether-ethyl acetate mixture with a volume ratio of 8:1 to 0:1.

[0069] In a specific implementation, in step (9), the mobile phase of the high performance liquid chromatography is an acetonitrile-water mixture with a volume ratio of 90:10.

[0070] In a specific implementation, in step (10), the mobile phase of the high performance liquid chromatography is an acetonitrile-water mixture with a volume ratio of 75:25.

[0071] In a specific implementation, in step (11), the mobile phase of the high performance liquid chromatography is an acetonitrile-water mixture with a volume ratio of 80:20.

[0072] In a preferred embodiment, in step (12), the eluent for the normal phase column chromatography is a petroleum ether-ethyl acetate mixture with a volume ratio of 10:1 to 0:1.

[0073] In the method described in this invention, in step (13), the eluent for the gel column chromatography is a methanol-dichloromethane mixture with a volume ratio of 50:50.

[0074] In a specific embodiment, in step (14), the eluent obtained by gel column chromatography in step (13) is purified by high performance liquid chromatography, and an acetonitrile-water mixed solution with a volume ratio of 74:26 is used as the mobile phase to purify the compounds shown in Formula 7, Formula 8 and Formula 9.

[0075] The present invention further provides the application of the isopentenyl shankone compound in the preparation of antitumor drugs.

[0076] This invention provides the use of the isopentenyl xanthones in the preparation of anti-liver cancer drugs, anti-colon cancer drugs, anti-breast cancer drugs, or anti-lung cancer drugs.

[0077] Evaluation of the antitumor activity of the isopentenyl xanthones of this invention reveals that they exhibit certain antitumor activity. Specifically, compounds 4 (Formula 4), 8 (Formula 8), and 9 (Formula 9) demonstrate significant antitumor activity, with compound 9 exhibiting the highest activity. Compound 4 (Formula 4) shows significant activity against liver cancer cells, compound 8 (Formula 8) shows significant activity against liver and colon cancer cells, and compound 9 (Formula 9) shows significant activity against liver cancer cells, colon cancer cells, breast cancer cells, colonic epithelial cells, and lung cancer cells.

[0078] This invention also provides the application of the isopentenyl xanthones in the preparation of anti-inflammatory drugs. Evaluation of the anti-inflammatory activity of the isopentenyl xanthones of this invention shows that all of them possess certain anti-inflammatory activity, with compound 1 (Formula 1), compound 4 (Formula 4), compound 7 (Formula 7), and compound 8 (Formula 8) exhibiting significant anti-inflammatory activity.

[0079] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto.

[0080] The strain *Aspergillus stellatus* used in the following examples is deposited in Room 414, Building 1, School of Pharmacy, Huazhong University of Science and Technology, with the serial number 414JZ-112. This strain was purchased from the China General Microbiological Culture Collection Center (CGMCC) with purchase serial number CGMCC 3.6292. It was identified as *Aspergillus stellatus* by ITS gene sequencing. The ITS sequence number of the strain is:

[0081] .

[0082] Example 1

[0083] (1) The Aspergillus stellaria strain was revived in a sterile operating table. The mycelium of the Aspergillus stellaria strain was inoculated onto potato dextrose agar (PDA) medium with an inoculation loop. The strain was cultured in a constant temperature incubator at 25°C for 7 days. After three generations of continuous copying, the strain was cultured in large quantities to prepare seed culture medium.

[0084] (2) Pour 200g of rice and 200mL of tap water into a 1000mL conical flask, prepare a total of 250 flasks, and sterilize them in an autoclave for 30min to obtain rice culture medium; cut the seed culture medium containing mycelium into small square pieces, inoculate them into the rice culture medium cooled to room temperature (25℃), and ferment them at a constant temperature of 25℃ for 45 days (total fermentation amount is 50kg, calculated by the weight of rice);

[0085] (3) The fermentation product obtained in step (2) was extracted with anhydrous ethanol 10 times, and then the ethanol was recovered by vacuum concentration to obtain a total extract that was brownish-black.

[0086] (4) The total extract obtained in step (3) is suspended in 10L of water, and then extracted 10 times with ethyl acetate of the same volume as water. The solvent is recovered by vacuum concentration to obtain ethyl acetate extract.

[0087] (5) The obtained ethyl acetate extract was subjected to normal phase column chromatography with a gradient elution of a petroleum ether-ethyl acetate-methanol mixed solution with a volume ratio of 10:1:0-10:10:0-10:10:1 to obtain 5 fractions (Fr.1 to Fr.5).

[0088] (6) Fraction Fr.3 was eluted by reversed-phase column chromatography using a methanol-water mixture with a volume ratio of 20:80-80:20 as the eluent to obtain 10 fractions (Fr.3.1 to Fr.3.10);

[0089] (7) The component Fr.3.4 was subjected to normal phase column chromatography and eluted with a mixed solution of petroleum ether-ethyl acetate with a volume ratio of 8:1-0:1. The resulting eluent was then purified by high performance liquid chromatography with a mixed solution of acetonitrile-water with a volume ratio of 70:30 as the mobile phase to obtain compound 6 as shown in Formula 6.

[0090] (8) Fraction Fr.3.7 was subjected to normal phase column chromatography and eluted with a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 8:1-0:1 to obtain 7 fractions (Fr.3.7.1 to Fr.3.7.7);

[0091] (9) The component Fr.3.7.3 was purified by high performance liquid chromatography, using an acetonitrile-water mixture with a volume ratio of 90:10 as the mobile phase, to obtain compound 1 as shown in Formula 1;

[0092] (10) The component Fr.3.7.4 was purified by high performance liquid chromatography, using an acetonitrile-water mixture with a volume ratio of 75:25 as the mobile phase, to obtain compound 2 as shown in formula 2 and compound 3 as shown in formula 3;

[0093] (11) The component Fr.3.7.5 was purified by high performance liquid chromatography, using an acetonitrile-water mixture with a volume ratio of 80:20 as the mobile phase, to obtain compound 4 as shown in formula 4 and compound 5 as shown in formula 5;

[0094] (12) Fraction Fr.3.8 was subjected to normal phase column chromatography and eluted with petroleum ether-ethyl acetate at a volume ratio of 10:1-0:1 to obtain 9 fractions (Fr.3.8.1 to Fr.3.8.9);

[0095] (13) Fraction Fr.3.8.6 was separated by gel column chromatography and eluted with a methanol-dichloromethane mixed solution with a volume ratio of 50:50 to obtain 3 fractions (Fr.3.8.6.1 to Fr.3.8.6.3);

[0096] (14) The component Fr.3.8.6.2 was purified by high performance liquid chromatography using a mixed solution of acetonitrile-water with a volume ratio of 90:10 as the mobile phase to obtain compound 7 shown in formula 7, compound 8 shown in formula 8 and compound 9 shown in formula 9.

[0097] Test case

[0098] Test Example 1

[0099] The structures of the compounds shown in Formulas 1 to 9 were identified.

[0100] High-resolution mass spectrometry (HRESIMS), ultraviolet (UV), infrared (IR), optical rotation (ORD), and nuclear magnetic resonance (NMR) spectrometry were performed on compounds 1 to 9 represented by formulas 1 to 9. 1 H NMR and 13 The structures of compounds 1 to 9 were determined by using C NMR and circular dichroism (ECD) spectroscopy, along with calculations based on ECD, molybdenum salt reaction, and Mosher reaction methods, and by comprehensive analysis of the data. The results are as follows:

[0101] Compound 1: Yellow powder, UV(MeCN)λ max (logε)=247(4.20),271(4.15),292(4.07),382(3.63)nm; ECD(MeCN)λ max (Δε)=206(+4.40),221(+2.82),234(-0.73),252(+1.57),276(-0.48); 1 The H NMR (CDCl3, 600MHz) data are shown in Table 1; 13 The CNMR (CDCl3, 150MHz) data are shown in Table 2; HRESIMS m / z 431.1108 [M+Na] + (The calculated value is C) 23 H 20 O7Na,431.1107);

[0102] Compound 2: Yellow powder, UV(MeCN)λ max(logε)=260(4.24),275(4.36),297(3.82),393(3.64)nm; ECD(MeCN)λ max (Δε)=207(+9.50),226(+1.66),258(-1.32),286(+1.38),334(-0.64); 1 The H NMR (CDCl3, 600MHz) data are shown in Table 1; 13 The CNMR (CDCl3, 150MHz) data are shown in Table 2; HRESIMS m / z 405.1315 [M+Na] + (The calculated value is C) 22 H 22 O6Na,405.1314);

[0103] Compound 3: Yellow powder, UV(MeCN)λ max (logε)=241(4.18),274(4.24),296(3.77),391(3.57)nm; ECD(MeCN)λ max (Δε)=207(+7.88),229(+1.80),257(-0.23),280(+1.06),333(-0.48); 1 The H NMR (CDCl3, 600MHz) data are shown in Table 1; 13 The CNMR (CDCl3, 150MHz) data are shown in Table 2; HRESIMS m / z 461.1566 [M+Na] + (The calculated value is C) 25 H 26 O7Na,461.1576);

[0104] Compound 4: Yellow powder, UV(MeCN)λ max (logε)=241(4.38),273(4.49),387(3.77)nm; ECD(MeCN)λ max (Δε)=221(+3.71),248(-2.57),269(-4.50),296(+0.79),333(-1.22); 1 The H NMR (CDCl3, 600MHz) data are shown in Table 1; 13 The C NMR (CDCl3, 150MHz) data are shown in Table 2; HRESIMS m / z 519.1995 [M+Na] + (The calculated value is C)28 H 32 O8Na,519.1995);

[0105] Compound 5: Yellow powder, UV(MeCN)λ max (logε)=241(4.37),273(4.50),387(3.77)nm; ECD(MeCN)λ max (Δε)=219(+4.95),245(-2.78),260(-1.32),270(-4.38),294(+1.04),335(-1.09); 1 The H NMR (CDCl3, 600MHz) data are shown in Table 3; 13 The CNMR (CDCl3, 150MHz) data are shown in Table 2; HRESIMS m / z 505.1832 [M+Na] + (The calculated value is C) 27 H 30 O8Na, 505.1838);

[0106] Compound 6: Yellow powder, UV(MeCN)λ max (logε)=240(4.08),272(4.18),383(3.47)nm; ECD(MeCN)λ max (Δε)=221(+1.52),250(-1.44),269(-1.77),332(-0.70); 1 The H NMR (CDCl3, 600MHz) data are shown in Table 3; 13 The C NMR (CDCl3, 150MHz) data are shown in Table 2; HRESIMS m / z 535.1940 [M+Na] + (The calculated value is C) 28 H 32 O9Na,535.1944);

[0107] Compound 7: Yellow powder, UV(MeCN)λ max (logε)=241(4.33),275(4.45),393(3.73)nm; 1 The H NMR data (C5D5N, 600MHz) are shown in Table 3; 13 The C10 NMR (C5D5N, 150MHz) data are shown in Table 2; HRESIMS m / z 535.2301 [M+Na] + (The calculated value is C)29 H 36 O8Na, 535.2308);

[0108] Compound 8: Yellow powder, UV(MeCN)λ max (logε)=241(4.42),275(4.54),393(3.51)nm; 1 The H NMR data (C5D5N, 400MHz) are shown in Table 3; 13 The C10 NMR (C5D5N, 100MHz) data are shown in Table 2; HRESIMS m / z 535.2301 [M+Na] + (The calculated value is C) 29 H 36 O8Na, 535.2308);

[0109] Compound 9: Yellow powder. UV(MeCN)λ max (logε)=241(4.40),275(4.51),393(3.79)nm; 1 The H NMR (CDCl3, 600MHz) data are shown in Table 3; 13 The C NMR (CDCl3, 150MHz) data are shown in Table 2; HRESIMS m / z 535.2305 [M+Na] + (The calculated value is C) 29 H 36 O8Na, 535.2308).

[0110] Table 1. Compounds 1 to 4 in deuterated chloroform solvent at 600 MHz 1 H NMR data

[0111]

[0112]

[0113] Table 2 Compounds 1 to 9 13 C NMR data

[0114]

[0115] Note: a 150MHz data in deuterated chloroform solvent; b 100MHz data in deuterated pyridine solvent; c 150MHz data in deuterated pyridine solvent.

[0116] Table 3 Compounds 5 to 91 H NMR data

[0117]

[0118]

[0119] Note: a 150MHz data in deuterated chloroform solvent; b 100MHz data in deuterated pyridine solvent; c 150MHz data in deuterated pyridine solvent.

[0120] Test Example 2

[0121] The antitumor activity of compounds 1 to 9, represented by formulas 1 to 9, was evaluated.

[0122] Test method: The CCK-8 assay was used to screen the activity of five cell types: HepG2 (liver cancer cells), RKO (colon cancer cells), MCF-7 (breast cancer cells), NCM460 (colon epithelial cells), and A549 (lung cancer cells).

[0123] The five cell lines were seeded at a density of 5000 cells / well in 96-well plates and incubated for 24 hours. Then, the cells were treated with different concentrations of compounds 1 to 9 (compound 1 concentrations were 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM, respectively; the concentrations of compounds 2 to 9 were the same as those of compound 1) for 72 hours. Afterward, 10 μL of WST-8 solution was added to each well, and the cells were incubated for another 4 hours. The absorbance at 450 nm was measured using a microplate reader. Each cell line was measured at least three times. The half-maximal inhibitory concentration (IC50) was calculated using IBM SPSS Statistics 25 based on the dose-response curve. 50 The results are shown in Table 4.

[0124] Table 4

[0125] compound <![CDATA[IC 50 (HepG2)]]> <![CDATA[IC 50 (RKO)]]> <![CDATA[IC 50 (MCF-7)]]> <![CDATA[IC 50 (NCM460)]]> <![CDATA[IC 50 (A549)]]> Compound 4 16.65±0.95 >40 >40 >40 >40 Compound 8 18.26±1.80 10.07±1.35 >40 >40 >40 Compound 9 20.81±0.96 8.62±1.18 4.98±0.28 3.35±0.58 5.66±0.86

[0126] As shown in Table 4, compound 9 exhibits significant inhibitory activity against RKO, MCF-7, NCM460, and A549 cell lines, with an IC50 concentration of [missing data]. 50 The values ​​were 8.62 μM, 4.98 μM, 3.35 μM, and 5.66 μM, respectively. For the HepG2 cell line, compounds 4, 8, and 9 exhibited moderate inhibitory activity, with IC50 values ​​of 8.62 μM, 4.98 μM, 3.35 μM, and 5.66 μM. 50 The values ​​were 16.65, 18.26, and 20.81 μM, respectively. Furthermore, compound 8 also exhibited inhibitory activity against the RKO cell line, with an IC50 value of 16.65, 18.26, and 20.81 μM. 50The concentration was 10.07 μM. These results indicate that compound 4 (Formula 4), compound 8 (Formula 8), and compound 9 (Formula 9) exhibit significant antitumor activity, with compound 9 (Formula 9) showing the highest antitumor activity.

[0127] Test Example 3

[0128] The anti-inflammatory activity of compounds 1 to 9, represented by formulas 1 to 9, was evaluated.

[0129] The inhibitory effect of compounds 1 to 9 on NO (nitric oxide) production in LPS (lipopolysaccharide)-induced RAW 264.7 cells (mouse mononuclear macrophage leukemia cells) at non-toxic concentrations was evaluated to simulate an inflammatory response and assess the anti-inflammatory activity of compounds 1 to 9.

[0130] Assay Method: RAW 264.7 cells were cultured at 37°C in a 5% CO2 environment with 10% fetal bovine serum and Penicillium added to the culture medium. RAW 264.7 cells were seeded in 96-well plates. After a 24-hour pre-incubation, RAW 264.7 cells were treated for 2 hours with compounds 1 to 9 at concentrations ranging from 80 to 2.5 μM (compound 1 concentrations were 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, and 2.5 μM respectively; compounds 2 to 9 concentrations were the same as compound 1). The cells were then stimulated with LPS (1 μg / mL) for 12 hours, and the NO content was determined using the Griess method. Cell culture supernatant (50 μL) was mixed with 100 μL of Griess reagent (1% sulfonamide, 0.1% dinaphthyl dihydrochloride, 2% phosphoric acid) in a 96-well plate at room temperature for 10 min. The optical density was measured at 510 nm using a microplate reader. The results are shown in Table 5.

[0131] Table 5

[0132] compound <![CDATA[IC 50 (mean±SD)]]> Compound 1 9.39±0.93 Compound 2 42.12±1.93 Compound 3 45.23±0.93 Compound 4 12.61±1.32 Compound 5 49.78±1.56 Compound 6 46.50±0.88 Compound 7 7.30±0.69 Compound 8 5.16±0.17 Compound 9 43.26±1.78

[0133] As shown in Table 5, compounds 1 through 9 all possess certain anti-inflammatory activities. Among them, compounds 1, 4, 7, and 8 can significantly reduce LPS (lipopolysaccharide)-induced NO (nitric oxide) production, with IC50 values ​​of [missing information]. 50 The concentrations were 9.39 μM, 12.61 μM, 7.30 μM and 5.16 μM, respectively, showing significant anti-inflammatory activity.

[0134] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. An isoprenylsantonin compound, characterized by, The isoprenyl-santonin compound is any one of compounds shown in formula 1 to formula 9.

2. A method for producing the isoprenyl flavanone compound according to claim 1, characterized by, The preparation method comprises the following steps: (1) inoculating mycelia of Aspergillus stellatus on a potato glucose agar medium, and then performing constant temperature culture to obtain a seed culture medium; (2) inoculating the seed culture medium containing the mycelia into a sterilized rice medium to perform fermentation culture; (3) extracting the fermentation product obtained in step (2) with ethanol, and then performing reduced pressure concentration to obtain a total extract; (4) suspending the total extract in water, then performing extraction with ethyl acetate, and then performing reduced pressure concentration to obtain an ethyl acetate extract; (5) performing normal phase column chromatography on the ethyl acetate extract to obtain five components Fr.1 to Fr.5; (6) performing reverse phase column chromatography on the component Fr.3 to obtain ten components Fr.3.1 to Fr.3.10; (7) performing normal phase column chromatography on the component Fr.3.4, and then performing high performance liquid chromatography purification to obtain the compound shown in formula 6; (8) performing normal phase column chromatography on the component Fr.3.7 to obtain seven components Fr.3.7.1 to Fr.3.7.7; (9) performing high performance liquid chromatography purification on the component Fr.3.7.3 to obtain the compound shown in formula 1; (10) performing high performance liquid chromatography purification on the component Fr.3.7.4 to obtain the compound shown in formula 2 and the compound shown in formula 3; (11) performing high performance liquid chromatography purification on the component Fr.3.7.5 to obtain the compound shown in formula 4 and the compound shown in formula 5; (12) performing normal phase column chromatography on the component Fr.3.8 to obtain nine components Fr.3.8.1 to Fr.3.8.9; (13) performing gel column chromatography separation on the component Fr.3.8.6 to obtain three components Fr.3.8.6.1 to Fr.3.8.6.3; (14) performing high performance liquid chromatography purification on the component Fr.3.8.6.2 to obtain the compound shown in formula 7, the compound shown in formula 8 and the compound shown in formula 9.

3. The preparation method according to claim 2, characterized in that, The constant temperature culture comprises the following conditions: the temperature is 20-30 DEG C, and the time is 5-12 days.

4. The preparation method according to claim 2, characterized in that, The fermentation culture comprises the following conditions: the temperature is 20-30 DEG C, and the time is 40-50 days.

5. The preparation method according to claim 2, characterized in that, In step (5), the eluent of the normal phase column chromatography is a mixed solution of petroleum ether-ethyl acetate-methanol with a volume ratio of 10:1:0-10:10:0-10:10:

1.

6. The preparation method according to claim 2, characterized in that, In step (6), the eluent of the reverse phase column chromatography is a mixed solution of methanol-water with a volume ratio of 20:80-80:

20.

7. The preparation method according to claim 2, characterized in that, In step (7), the eluent of the normal phase column chromatography is a mixed solution of petroleum ether-ethyl acetate with a volume ratio of 8:1-0:

1.

8. The preparation method according to claim 2, characterized in that, In step (8), the eluent of the normal phase column chromatography is a mixed solution of petroleum ether-ethyl acetate with a volume ratio of 8:1-0:

1.

9. The isoprenyl-santonin compound of claim 1 is applied to preparation of an antitumor drug.

10. The isoprenyl-santonin compound of claim 1 is applied to preparation of an anti-inflammatory drug.

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