A new sorbicillinoids compound and its extraction and separation method and use

Abstract

The application discloses a new Sorbicillinoids compound and an extraction and separation method and application thereof, relates to the technical fields of natural compound extraction, separation and natural medicine chemistry. The application takes Trichoderma reesei fermentation liquor as raw material, extracts a new Sorbicillinoids compound from Trichoderma reesei metabolites, and names the compound as Trichosorbicil. The molecular formula of the Sorbicillinoids compound Trichosorbicil is C 25 H 28 O7, and a structural formula is as follows: The compound Trichosorbicil has relatively significant in-vitro anti-tumor activity, and provides a basis for further developing in-depth pharmacological and clinical research and developing a new anti-tumor drug with high curative effect and small side effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural compound extraction, separation and natural pharmaceutical chemistry, and particularly relates to a Sorbicillinoids new compound, an extraction and separation method thereof and application. BACKGROUND

[0002] Sorbicillinoids is a kind of natural compound produced by secondary metabolism of fungi. Sorbicillinoids is a family of characteristic yellow hexaketide compounds first isolated from various fungal strains of marine and terrestrial origin, with characteristic absorption at 370 nm. In 1948, Cram and Tishler reported the isolation and structural identification of several Sorbicillinoids compounds. Over the years, more members of the family have been isolated from fungal strains collected in different environments. The C1-C6 sorbyl side chain is a structural feature of this family. Recognizing this structural similarity, and finding that the family has no "official" name, the word "Sorbicillinoids" is used to include the entire family of compounds.

[0003] Sorbicillinoids compounds have complex structures and multiple biological activities, and many Sorbicillinoids compounds have been isolated and identified. Identifying new Sorbicillinoids substances and their potential medical and biotechnological applications can serve as a potential source of new therapeutic agents and new drugs, and have extremely high research value.

[0004] Trichoderma reesei is an important industrial production strain, which meets the GRAS (Generally Regarded as Safe) standard, and its fermentation products have been widely used in food, feed and other industries. Trichoderma reesei is easy to expand culture and has been applied to enzyme preparation production industry in various countries. Literature reports that Trichoderma reesei produces 11% of the world's enzyme preparations. Trichoderma reesei is the only strain in the genus Trichoderma that can produce Sorbicillinoids compounds, with high yield, and the fermentation broth shows characteristic yellow color. Previous studies have found that the crude extract of Trichoderma reesei Sorbicillinoids has antioxidant and anticancer activities, and is particularly significant for inhibiting malignant melanoma. Is there a new type of Sorbicillinoids substance in Trichoderma reesei-derived Sorbicillinoids substance that has never been reported? This is a scientific problem worthy of study. The separation and identification of new Sorbicillinoids substances can discover new synthesis pathways and synthesis enzymes of Sorbicillinoids, and can be a potential source of new therapeutic agents and new drugs, which has very high research and application value. SUMMARY

[0005] The purpose of the present application is to provide a new Sorbicillinoids compound and its extraction and separation method and use, so as to solve the problems existing in the prior art. The new Sorbicillinoids compound provided by the present application has relatively significant in vitro antitumor activity, which provides a basis for further in-depth pharmacological and clinical research and development of new antitumor drugs with strong efficacy and small side effects.

[0006] In order to achieve the above purpose, the present application provides the following solutions:

[0007] The present application provides a Sorbicillinoids compound, named Trichosorbicil, the molecular formula of Trichosorbicil is C 25 H 28 O7, and the chemical name according to the structure is: (S)-3-((1R,2S,E)-2,3-dihydroxy-6-((2E,4E)-1-hydroxyhexa-2,4-dien-1-ylidene)-2,4-dimethyl-5-oxocyclohex-3-en-1-yl)-3-methyl-6-((1E,3E)-penta-1,3-dien-1-yl)-2H-pyran-2,4(3H)-dione, and the structural formula is as follows:

[0008]

[0009] The application further provides an extraction and separation method of the Sorbicillinoids compound, comprising the following steps:

[0010] The mycelium-removed Trichoderma reesei fermentation liquor is extracted by using an extraction solvent to obtain a Sorbicillinoids crude extract;

[0011] The Sorbicillinoids crude extract is separated by using semi-preparative high performance liquid chromatography; the separation condition of the semi-preparative high performance liquid chromatography is that: the chromatographic column is selected as Eclipse XDB-C18, the mobile phase is acetonitrile and water, the volume ratio of acetonitrile and water is: 40-99% acetonitrile for 0-15 min, 99% acetonitrile for 15-20 min, 40% acetonitrile for 20-25 min, and the detection wavelength is 370 nm; the column effluent is collected according to four different times to obtain four fractions, which are respectively recorded as Fr.1-Fr.4, and the corresponding collection times are: Fr.1-11-13 min, Fr.2-13-16 min, Fr.3-16-17.8 min, and Fr.4-17.8-20 min;

[0012] After the fraction Fr.3 is subjected to solvent evaporation treatment, the preparation high performance liquid chromatography is used for separation; the condition of the preparation high performance liquid chromatography is that: the chromatographic column is selected as Sep Basic C18, the mobile phase is acetonitrile and water, the volume ratio of acetonitrile and water is: 55-99% acetonitrile for 0-15 min, 99% acetonitrile for 15-20 min, 55% acetonitrile for 20-25 min, and the detection wavelength is 370 nm; the column effluent of 15.1-15.4 min is collected, and then subjected to solvent evaporation treatment to obtain the Sorbicillinoids compound.

[0013] Further, the extraction solvent is ethyl acetate.

[0014] Further, the Trichoderma reesei fermentation liquor is obtained by inoculating Trichoderma reesei into a fungal culture medium and then performing fermentation culture.

[0015] The fungal culture medium comprises the following components: glucose 100 g / L, tryptone 6 g / L, KH2PO4 0.15 g / L, K2HPO4 0.15 g / L, CaCl2·2H2O 0.10 g / L, MgSO4·7H2O 0.10 g / L, NaCl 0.05 g / L, 1 mL / L trace element solution and 20 g / L calcium carbonate; the trace element solution comprises the following components: 1.6 g / L MnSO4·4H2O, 5 g / L FeSO4·7H2O, 2 g / L CoCl2·6H2O and 1.4 g / L ZnSO4·7H2O.

[0016] Further, the temperature of the fermentation culture is 28℃, the rotation speed is 200rpm, and the time is 10 days.

[0017] The application further provides application of the Sorbicillinoids compound in the preparation of a medicine for treating tumors.

[0018] Further, the tumor is ovarian cancer, brain astrocytoma, colorectal adenocarcinoma, neuroblastoma, breast cancer, malignant melanoma, cervical cancer or liver cancer.

[0019] The application further provides a medicine for treating tumors, wherein the active ingredient comprises the Sorbicillinoids compound.

[0020] Further, the tumor is ovarian cancer, brain astrocytoma, colorectal adenocarcinoma, neuroblastoma, breast cancer, malignant melanoma, cervical cancer or liver cancer.

[0021] Further, the medicine further comprises a pharmaceutically acceptable excipient.

[0022] The application discloses the following technical effects:

[0023] The application takes Trichoderma reesei fermentation liquor as a raw material, extracts a new Sorbicillinoids compound from metabolic products of Trichoderma reesei, and names the compound as Trichosorbicil. The compound Trichosorbicil has relatively significant in-vitro anti-tumor activity, and provides a basis for further in-depth pharmacological and clinical research and development of a new anti-tumor drug with strong curative effect and small side effect.

[0024] The compound Trichosorbicil is obtained from Trichoderma reesei fermentation products for the first time, and provides a basis for research on a new synthesis path of Sorbicillinoids compounds in Trichoderma reesei. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0026] Figure 1 Semi-preparative high performance liquid chromatogram of Sorbicillinoids crude extract;

[0027] Figure 2High-performance liquid chromatography (HPLC) chromatogram for the preparation of fraction Fr.3;

[0028] Figure 3 The HR-ESI-MS spectrum of the positive ion of the compound Trichosorbicil;

[0029] Figure 4 For the compound Trichosorbicil 1 H-NMR spectrum;

[0030] Figure 5 For the compound Trichosorbicil 13 C-NMR spectrum;

[0031] Figure 6 For the compound Trichosorbicil 1 H- 1 H COSY spectrum;

[0032] Figure 7 The HSQC spectrum of the compound Trichosorbicil;

[0033] Figure 8 The HMBC spectrum of the compound Trichosorbicil;

[0034] Figure 9 The NOESY spectrum of the compound Trichosorbicil;

[0035] Figure 10 The ROESY spectrum of the compound Trichosorbicil;

[0036] Figure 11 For the compound Trichosorbicil 1 H- 1 Schematic diagrams related to H COSY and HMBC;

[0037] Figure 12 This is a schematic diagram showing the NOESY and ROESY properties of the compound Trichosorbicil. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, a parameter, an individual value or subrange within that range is also specifically disclosed. Each of these individual values or sub-ranges is also specifically disclosed. The upper and lower limits of these explicitly disclosed ranges of values can independently be included or excluded in the ranges given. These smaller ranges of values are also specifically disclosed.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art or that it is specifically incorporated by reference in the present application.

[0041] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Additional implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The examples in the specification are illustrative only and not restrictive of the application.

[0042] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0043] The experimental methods in the following examples, unless otherwise specified, were generally performed according to routine conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

[0044] The cell culture media and reagents used in the present application are shown in Table 1:

[0045] Table 1 Cell culture media and reagents

[0046]

[0047] Example 1

[0048] 1. Extraction of novel compounds

[0049] Fungal medium components: glucose 100 g / L, tryptone 6 g / L, KH2PO4 0.15 g / L, K2HPO4 0.15 g / L, CaCl2·2H2O 0.10 g / L, MgSO4·7H2O 0.10 g / L, NaCl 0.05 g / L, 1 mL / L trace element solution and 20 g / L calcium carbonate; wherein the components of the trace element solution are as follows: 1.6 g / L MnSO4·4H2O, 5 g / L FeSO4·7H2O, 2 g / L CoCl2·6H2O and 1.4 g / L ZnSO4·7H2O.

[0050] The fungal medium was divided into 20 250 mL volume shake flasks, each containing 50 mL of the medium, which was sterilized, and then the conidiospores of Trichoderma reesei strain QM6a (ATCC 13631) were inoculated into the fungal medium, with an inoculation amount of 10 7 spores per flask. The shake flasks with the inoculated spores were placed in a shaker, and cultured at 200 rpm and 28°C for 10 days to obtain a fermentation broth.

[0051] The mycelia were removed from the fermentation broth by centrifugation (or filtration) to obtain a fermentation supernatant; the supernatant was mixed with ethyl acetate at a volume ratio of 3:1, shaken thoroughly, extracted, and the upper ethyl acetate extract was collected. The extraction was repeated three times, and the upper ethyl acetate extracts from the three times were combined; the upper ethyl acetate extract was concentrated by a rotary evaporator at 40°C under vacuum to obtain a crude Sorbicillinoids extract.

[0052] All of the crude Sorbicillinoids extract was redissolved with 20 mL of methanol, and separated by semi-preparative high performance liquid chromatography (Agilent LC52 semi-preparative liquid chromatography system). The conditions of the semi-preparative high performance liquid chromatography were as follows: the chromatographic column was Eclipse XDB-C18, the mobile phase was acetonitrile and water, and the volume ratio of acetonitrile to water was: 0-15 min, 40-99% acetonitrile; 15-20 min, 99% acetonitrile; 20-25 min, 40% acetonitrile; and the detection wavelength was 370 nm. The column effluent was collected according to four different times (11-13 min, 13-16 min, 16-17.8 min and 17.8-20 min) to obtain four fractions, which were denoted as Fr.1-Fr.4, respectively. Figure 1 The corresponding collection times were: Fr.1—11-13 min, Fr.2—13-16 min, Fr.3—16-17.8 min and Fr.4—17.8-20 min, respectively. Figure 1

[0053] ​The above fraction Fr.3 was concentrated by rotary evaporator at 40°C under vacuum, and then redissolved with 5 mL of methanol. The redissolved fraction Fr.3 was separated by preparative high performance liquid chromatography (Agilent preparative liquid chromatography system). The conditions of the preparative high performance liquid chromatography were as follows: the column was Sep Basic C18, the mobile phase was acetonitrile and water, the volume ratio of acetonitrile to water was 55-99% acetonitrile at 0-15 min, 99% acetonitrile at 15-20 min, 55% acetonitrile at 20-25 min, and the detection wavelength was 370 nm. The column effluent at 15.1-15.4 min was collected (0.5 mL / min) and concentrated by rotary evaporator at 40°C under vacuum to obtain a yellow powder, which was the new compound obtained by extraction, named Trichosorbicil. Figure 2

[0054] 2. Structure identification

[0055] The structure of the new compound Trichosorbicil was identified by high resolution mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance one-dimensional (1H-NMR, 13C-NMR) and two-dimensional (2D) techniques (1H-1H COSY, HSQC, HMBC, NOESY). 1 13 1 1

[0056] The new compound Trichosorbicil was a yellow amorphous powder, and the m / z in HR-EISI-MS was 441.1907. The molecular formula was C 25 28 O7(M = 440.1907), as shown in the following formula. Figure 3

[0057] According to nuclear magnetic resonance technology, the structure of the compound was determined, the nuclear magnetic data are shown in Table 2, the spectrum is shown in Figure 1, and the structural formula is as follows: Figures 4-12

[0058]

[0059] Table 2 Nuclear magnetic data of the compound Trichosorbicil (500 / 150 MHz, DMSO-d6)

[0060]

[0061] Example 2 Test of in vitro anti-tumor activity

[0062] ​​​​​​​​The new compound Trichosorbicil prepared in Example 1 was subjected to in vitro anti-tumor activity test, and the experimental cells were human ovarian cancer cells (SKOV3), human brain astrocytoma cells (U87MG), human colorectal adenocarcinoma cells (HT-29), human neuroblastoma cells (SH-SY5Y), human breast cancer cells (MCF-7), human malignant melanoma cells (A375), human cervical cancer cells (Hela) and human hepatoma cells (Hep G2).

[0063] MTT method was used to perform in vitro cytotoxicity experiment, and the specific implementation method was as follows:

[0064] The new compound Trichosorbicil of Example 1 was dissolved in a small amount of dimethyl sulfoxide to prepare a mother liquor, and the concentration of Trichosorbicil in the mother liquor was 200 mg / mL. Then DMEM high-sugar fetal bovine double-antibiotic medium (45 mL DMEM high-sugar medium, 5 mL fetal bovine serum, 0.5 mL double-antibiotic) was used to prepare test solutions with concentrations of 0, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0, 80.0, 100.0, 150.0 and 200.0 μg / mL, respectively.

[0065] The cancer cells in good logarithmic phase were inoculated in 96-well plates at a density of 3000 cells per well, and the culture medium was DMEM high-sugar fetal bovine double-antibiotic medium; the blank group was not inoculated with cells, and only DMEM high-sugar fetal bovine double-antibiotic medium was added, and the 96-well plate was placed in a 37℃, 5% carbon dioxide incubator for 24 hours.

[0066] Then 100 μL of the above test solution with different concentrations was added to each well of the 96-well plate, and the blank group and the control group were given the test solution with a concentration of 0, and the culture was continued for 48 hours, then 20 μL of tetrazolium blue-phosphate buffer was added to each well, and the 96-well plate was placed in a 37℃, 5% carbon dioxide incubator for 4 hours, and the absorbance OD value of each well was measured at 492 nm on an enzyme marker, repeated 3 times, and the average value was taken.

[0067] The IC 50 values of the compound for inhibiting each kind of tumor cell were calculated using Logit method, and the results are shown in Table 3. The results show that the compound Trichosorbicil has inhibitory activity on multiple tumor cells, and the inhibitory activity on human malignant melanoma cells is the highest, with an IC 50 value of 8.59 μg / mL.

[0068] Table 3 IC 50 values of the compound Trichosorbicil on cancer cells

[0069]

[0070] In summary, the present application has in vitro anti-tumor activity by separating and purifying the prepared novel Sorbicillinoids compound Trichosorbicil, and can be applied to the preparation of drugs for preventing and / or treating tumors. The method for separating and purifying the compound Trichosorbicil provided by the present application provides a research scheme for identifying new compounds. The present application provides a new method and new process for large-scale fermentation production of Trichosorbicil, and also provides a good research idea for developing anticancer drugs.

[0071] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A Sorbicillinoid compound, characterized in that, The structural formula of the Sorbicillinoids compound is as follows:

2. A method for extracting and separating Sorbicillinoids compounds as described in claim 1, characterized in that, Includes the following steps: The fermentation broth of Trichoderma reesei, after removing mycelia, was extracted using an extraction solvent to obtain a crude extract of Sorbicillinoids. The crude extract of Sorbicillinoids was separated using semi-preparative high-performance liquid chromatography (HPLC). The separation conditions for the semi-preparative HPLC were as follows: Eclipse XDB-C18 column; acetonitrile and water as the mobile phase; acetonitrile to water volume ratio: 0-15 min, 40-99% acetonitrile; 15-20 min, 99% acetonitrile; 20-25 min, 40% acetonitrile; detection wavelength: 370 nm; the column effluent was collected at four different times to obtain four fractions, denoted as Fr.1 to Fr.4, with corresponding collection times of: Fr.1—11-13 min, Fr.2—13-16 min, Fr.3—16-17.8 min, and Fr.4—17.8-20 min. After solvent evaporation of fraction Fr.3, preparative high-performance liquid chromatography (HPLC) was used for separation. The preparative HPLC conditions were as follows: Sep Basic C18 column; acetonitrile and water as the mobile phase; acetonitrile to water volume ratio: 0-15 min, 55-99% acetonitrile; 15-20 min, 99% acetonitrile; 20-25 min, 55% acetonitrile; detection wavelength: 370 nm; column effluent was collected for 15.1-15.4 min, and then subjected to solvent evaporation to obtain the Sorbicillinoids compound.

3. The extraction and separation method according to claim 2, characterized in that, The extraction solvent is ethyl acetate.

4. The extraction and separation method according to claim 2, characterized in that, The Trichoderma reesei fermentation broth is obtained by inoculating Trichoderma reesei into a fungal culture medium and then fermenting it. The fungal culture medium comprises the following components: 100 g / L glucose, 6 g / L tryptone, 0.15 g / L KH2PO4, 0.15 g / L K2HPO4, 0.10 g / L CaCl2·2H2O, 0.10 g / L MgSO4·7H2O, 0.05 g / L NaCl, 1 mL / L trace element solution, and 20 g / L calcium carbonate; the trace element solution comprises the following components: 1.6 g / L MnSO4·4H2O, 5 g / L FeSO4·7H2O, 2 g / L CoCl2·6H2O, and 1.4 g / L ZnSO4·7H2O.

5. The extraction and separation method according to claim 4, characterized in that, The fermentation culture was carried out at a temperature of 28°C, a rotation speed of 200 rpm, and a time of 10 days.

6. The use of a Sorbicillinoids compound as described in claim 1 in the preparation of a medicament for treating tumors.

7. The application according to claim 6, characterized in that, The tumors mentioned are ovarian cancer, glioblastoma of the brain, colorectal adenocarcinoma, neuroblastoma, breast cancer, malignant melanoma, cervical cancer, or liver cancer.

8. A drug for treating tumors, characterized in that, The active ingredient includes the Sorbicillinoids compound as described in claim 1.

9. The medicament according to claim 8, characterized in that, The tumors mentioned are ovarian cancer, glioblastoma of the brain, colorectal adenocarcinoma, neuroblastoma, breast cancer, malignant melanoma, cervical cancer, or liver cancer.

10. The medicament according to claim 8, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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