Gemmarane type sesquiterpene as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of phytochemistry, and particularly relates to gemmarane type sesquiterpene as well as a preparation method and application thereof. The gemmarane type sesquiterpenoids disclosed by the invention are prepared from a fermentation solution of an endophytic fungus Aspergillus aculeatus Aa-1 (Aspergillus aculeatus Aa-1) derived from enteromorpha prolifera, and the gemmarane type sesquiterpenoids disclosed by the invention are prepared from the fermentation solution of the endophytic fungus Aspergillus aculeatus Aa-1 (Aspergillus aculeatus Aa-1) derived from the enteromorpha prolifera. According to the present invention, through the modern spectroscopy technology, such as high resolution mass spectrum HRESIMS, superconductive nuclear magnetic resonance spectrum NMR and X-ray single crystal diffraction technology, the gemmarane type sesquiterpene with the new structure is named as asperiunoid A. The invention further discloses a preparation method of the gemmarane type sesquiterpene with the new structure, the gemmarane type sesquiterpene asperiunoid A with the new structure has relatively good inhibitory activity on retinoblastoma HXO-Rb44 and human gastric cancer cells MKN-45, and a new lead compound entity is provided for research and development of anti-tumor drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of plant chemistry, and specifically relates to a germarane-type sesquiterpene and a preparation method and application thereof. Background Art

[0002] Germane-type sesquiterpenes are widely present in plants and fungi, and their main structural feature is a typical ten-membered ring skeleton. Germane-type sesquiterpenes are an important natural active substance, which has been proven to have anti-tumor, anti-platelet aggregation, anti-neuritis, acetylcholinesterase inhibition and other activities. Therefore, this type of compound has important application value in the field of biomedicine. Chinese invention patent CN115197238B discloses achigermalide D or achigermalide E, a sesquiterpenes with hypoglycemic activity isolated from yarrow, which provides a new lead molecule for the research and development of hypoglycemic drugs. Chinese invention patent CN113896629B discloses a preparation method of a sesquiterpenoid compound derived from Valeriana officinalis and its application in anti-inflammatory drugs. Chinese invention patent CN114933602B discloses 14 new highly oxidized sesquiterpenoid lactone compounds extracted and separated from the genus Valeriana of the Asteraceae family. This class of compounds has the same mother core of the germarane sesquiterpene lactone and has good anti-liver cancer activity. Based on the wide biological activity of the germarane sesquiterpene, the new germarane sesquiterpene compounds excavated from nature have potential application value in the creation and development of biomedicine. Summary of the invention

[0003] In view of this, the present invention discloses a germarane-type sesquiterpenoid and its preparation method and application. The present invention first discovered and reported a novel germarane-type sesquiterpenoid compound and its preparation method from a fungus, and found that the compound has significant anti-tumor activity against retinoblastoma and has the potential to be developed into a new biomedicine.

[0004] In order to achieve the above object, the present invention adopts the following technical solution:

[0005] The first technical purpose of the present invention is to provide a novel germarane-type sesquiterpenoid compound, which is prepared from the fermentation broth of an endophytic fungus Aspergillus aculeatus Aa-1 derived from Enteromorpha prolifera. By using modern spectroscopy techniques, including high-resolution mass spectrometry HRESIMS, superconducting nuclear magnetic resonance spectroscopy NMR and X-ray single crystal diffraction technology, the novel structure of the germarane-type sesquiterpenoid is named asperacunoid A, and the molecular formula of asperacunoid A is C 15 H24 O2, the structural formula is as follows:

[0006]

[0007] The second technical purpose of the present invention is to provide a method for preparing the germarane-type sesquiterpenoid compound.

[0008] The present invention accurately separates and prepares the germarane-type sesquiterpenoid compound by culturing and fermenting the fungus and extracting and purifying the target compound. The specific process of separating and preparing the germarane-type sesquiterpenoid asperacunoid A in the present invention is as follows:

[0009] (1) Under sterile conditions, the Aspergillus aculeatus Aa-1 was inoculated in 1 L conical flasks, each of which was filled with sterilized potato dextrose broth medium (Beijing Solebow Technology Co., Ltd.);

[0010] (2) placing the conical flask in a constant temperature environment at 28° C. for 30 days for static fermentation, extracting the culture medium of the Aspergillus aculeatus Aa-1 strain after fermentation with 300 mL of ethyl acetate by ultrasonic extraction, and concentrating under vacuum to a non-flowing extract to obtain a fermentation crude extract (vacuum degree -0.1 MPa);

[0011] (3) The crude fermentation extract was added to 100-200 mesh silica gel and mixed, with the mass ratio of the crude fermentation extract to the silica gel being 1:1; then, the components were separated by reduced pressure vacuum silica gel column chromatography, and gradient elution was performed using dichloromethane-methanol (volume ratios of 50:1, 40:1, 30:1, 20:1, 10:1 and 5:1, respectively) as gradient eluents, to obtain 6 components Fr.1 to Fr.6 with increasing polarity;

[0012] (4) The component Fr.2 (dichloromethane-methanol volume ratio 40:1 component) obtained in step (3) was subjected to C18 reverse phase silica gel column chromatography (Lobar LiChroprep RP-18, Merck, Germany), and methanol-water (volume ratio 10% to 100%) was selected as the eluent for gradient elution to obtain 10 sub-components Fr.2.1 to Fr.2.10;

[0013] (5) The subfraction Fr.2.5 obtained in step (4) (the eluent is methanol-water with a volume ratio of 50%) is subjected to silica gel column chromatography (200-300 mesh, the eluent is dichloromethane-methanol volume ratio of 30:1), and the second column volume component is collected (the total elution time is 3.5h, the flow rate is 0.1mL / s, and the elution time during collection is 2h) and vacuum reduced pressure concentration (vacuum degree -0.1Mpa, concentrated to a non-flowing extract) to purify the jermane-type sesquiterpene compound asperacunoid A.

[0014] The third technical purpose of the present invention is to provide the use of the germarane-type sesquiterpenes in the preparation of anti-tumor drugs.

[0015] The germarane-type sesquiterpenoid compounds of the present invention have good inhibitory activity on retinoblastoma HXO-Rb44 and human gastric cancer cell MKN-45, and provide a new lead compound entity for the research and development of anti-tumor drugs.

[0016] Specifically, the novel structured germane-type sesquiterpenoid asperacunoid A has good inhibitory activity against retinoblastoma HXO-Rb44 and human gastric cancer cell MKN-45, wherein the inhibitory activity of the compound against retinoblastoma HXO-Rb44 is better than that of the positive drug doxorubicin.

[0017] The specific process of using the fungus-derived new structure germarane-type sesquiterpene asperacunoid A to test the anti-tumor activity is as follows:

[0018] The MTT method was used to detect the cytotoxic activity of the compound asperacunoid A on human non-small cell lung cancer A549 cells, human cervical cancer HeLa cells, retinoblastoma HXO-Rb44 cells and human gastric cancer MKN-45 cells.

[0019] Take the cells that have been cultured for more than the third generation and have grown to 90% and add 5×10 3 The cells were evenly inoculated in a 96-well plate, 100 μL per well, and cultured in a 37°C, 5% CO2 incubator for 24 hours. They were randomly divided into a blank control group and a drug-treated group (drug concentrations were 3.125, 6.25, 12.5, 25, 50 and 100 μM, respectively), with 6 replicates for each concentration. After the cells adhered to the wall, the corresponding reagents were added according to the groups. After incubation for 24 hours, the supernatant was aspirated, 10 μL of MTT was added to each well in the dark, and the incubation continued for 4 hours. After the incubation was completed, the supernatant was aspirated, 150 μL of DMSO was added, the plate was slowly vibrated for 10 minutes using an enzyme reader, the absorbance was measured at 490 nm and the cell survival rate was calculated, and the drug half-inhibitory concentration IC was calculated using SPSS software. 50 .

[0020] It should be noted that the fungus Aspergillus aculeatus Aa-1 is deposited by the General Microbiology Center of the China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.40424 and a classification name of Aspergillus aculeatus. Deposit date: November 14, 2022, deposit location: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The germarane-type sesquiterpenoid asperacunoid A separated and prepared by the present invention is a new structural compound that has not been reported before, as determined by searching and comparing the natural product SciFinder database.

[0023] (2) The method provided by the present invention can accurately separate the gemmane-type sesquiterpenes.

[0024] (3) The inhibitory activity of the germane-type sesquiterpenoid asperacunoid A on retinoblastoma HXO-Rb44 is superior to that of the positive drug doxorubicin. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Figure 1 The figure is a chemical structure diagram of the germarane-type sesquiterpene asperacunoid A of the present invention.

[0027] Figure 2 This is a high-resolution mass spectrum of the germarane-type sesquiterpene asperacunoid A described in the present invention.

[0028] Figure 3 This is the hydrogen spectrum (500 MHz, DMSO-d6) of the germarane-type sesquiterpene asperacunoid A of the present invention.

[0029] Figure 4 This is the carbon spectrum (125MHz, DMSO-d6) of the germarane-type sesquiterpene asperacunoid A described in the present invention.

[0030] Figure 5This is a single crystal diffraction pattern of the gemmane-type sesquiterpene asperacunoid A described in the present invention. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0033] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0034] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0035] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0036] The invention discloses a germarane-type sesquiterpene, a preparation method and anti-tumor activity against retinoblastoma.

[0037] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0038] Example 1

[0039] A method for preparing a germarane-type sesquiterpenoid compound asperacunoid A, which accurately separates and prepares the germarane-type sesquiterpenoid compound through fungal culture and fermentation, extraction and purification technology of the target compound. The specific process of separating and preparing the germarane-type sesquiterpenoid asperacunoid A of the present invention is as follows:

[0040] (1) Under sterile conditions, the Aspergillus aculeatus Aa-1 was inoculated in 1 L conical flasks, each of which was filled with sterilized potato dextrose broth medium (Beijing Solebow Technology Co., Ltd.);

[0041] (2) placing the conical flask in a constant temperature environment at 28° C. for 30 days for static fermentation, extracting the culture medium of the Aspergillus aculeatus Aa-1 strain after fermentation with 300 mL of ethyl acetate by ultrasonic extraction, and concentrating under vacuum to a non-flowing extract to obtain a fermentation crude extract (vacuum degree -0.1 MPa);

[0042] (3) The crude fermentation extract was added to 100-200 mesh silica gel and mixed, with the mass ratio of the crude fermentation extract to the silica gel being 1:1; then, the components were separated by reduced pressure vacuum silica gel column chromatography, and gradient elution was performed using dichloromethane-methanol (volume ratios of 50:1, 40:1, 30:1, 20:1, 10:1 and 5:1, respectively) as gradient eluents, to obtain 6 components Fr.1 to Fr.6 with increasing polarity;

[0043] (4) The component Fr.2 (dichloromethane-methanol volume ratio 40:1 component) obtained in step (3) was subjected to C18 reverse phase silica gel column chromatography (Lobar LiChroprep RP-18, Merck, Germany), and methanol-water (volume ratio 10% to 100%) was selected as the eluent for gradient elution to obtain 10 sub-components Fr.2.1 to Fr.2.10;

[0044] (5) The subfraction Fr.2.5 obtained in step (4) (the eluent is methanol-water with a volume ratio of 50%) is subjected to silica gel column chromatography (200-300 mesh, the eluent is dichloromethane-methanol volume ratio of 30:1), and the second column volume component is collected (total elution time 3.5h, flow rate 0.1mL / s, elution time during collection 2h) and vacuum reduced pressure concentration (vacuum degree -0.1Mpa, concentrated to a non-flowing extract) to purify the jermane-type sesquiterpene compound asperacunoid A.

[0045] It should be noted that the fungus Aspergillus aculeatus Aa-1 is deposited by the General Microbiology Center of the China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.40424 and a classification name of Aspergillus aculeatus. Deposit date: November 14, 2022, deposit location: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0046] In order to further demonstrate the beneficial effects of the present invention and to better understand the present invention, the following experimental examples are used to further illustrate the technical features disclosed in the present invention, but they should not be understood as limiting the present invention. Other improvements made by those skilled in the art based on the above invention content without creative work are also considered to fall within the scope of protection of the present invention.

[0047] It should be noted that the preparation of the germarane-type sesquiterpene asperacunoid A described in the following experimental examples is as described in Example 1.

[0048] Experimental Example 1:

[0049] In this experimental example, the chemical structure of the germarane-type sesquiterpenoid asperacunoid A was determined by high-resolution mass spectrometry HRESIMS, superconducting nuclear magnetic resonance spectroscopy NMR and X-ray single crystal diffraction technology. The physicochemical properties of the germarane-type sesquiterpenoid asperacunoid A are as follows:

[0050] Properties: colorless crystals; Melting point: 125-128°C; Solubility: easily soluble in methanol and acetone, slightly soluble in dichloromethane; Molecular formula: C 15 H 24 O2; optical rotation: [α] 20 D +56.6 (methanol); UV absorption spectrum λ max (Methanol, logε) 200 (4.01) nm; High resolution mass spectrometry (HRESIMS): m / z 235.1695 [MH] - (Theoretical value C 15 H 23 O2 - , 235.1693); the H NMR spectrum data and C NMR spectrum data are shown in Table 1.

[0051] Table 1: NMR data of the germarane-type sesquiterpenoid asperacunoid A

[0052]

[0053] Experimental Example 2:

[0054] In this experimental example, the MTT method was used to detect the cytotoxic activity of the compound asperacunoid A on human non-small cell lung cancer A549 cells, human cervical cancer HeLa cells, retinoblastoma HXO-Rb44 cells and human gastric cancer MKN-45 cells. The results are shown in Table 2. The specific process is as follows:

[0055] Take the cells that have been cultured for more than the third generation and have grown to 90% and add 5×10 3 The cells were evenly inoculated in a 96-well plate, 100 μL per well, and cultured in a 37°C, 5% CO2 incubator for 24 hours. They were randomly divided into a blank control group and a drug-treated group (drug concentrations were 3.125, 6.25, 12.5, 25, 50 and 100 μM, respectively), with 6 replicates for each concentration. After the cells adhered to the wall, the corresponding reagents were added according to the groups. After incubation for 24 hours, the supernatant was aspirated, 10 μL of MTT was added to each well in the dark, and the incubation continued for 4 hours. After the incubation was completed, the supernatant was aspirated, 150 μL of DMSO was added, the plate was slowly vibrated for 10 minutes using an enzyme reader, the absorbance was measured at 490 nm and the cell survival rate was calculated, and the drug half-inhibitory concentration IC was calculated using SPSS software. 50 .

[0056] Table 2: Cytotoxicity test results of the germarane-type sesquiterpenoid asperacunoid A (IC 50 , μM)

[0057]

[0058] The results showed that, as shown in Table 2, the novel structure of the germane-type sesquiterpenoid asperacunoid A had good inhibitory activity against retinoblastoma HXO-Rb44 and human gastric cancer cell MKN-45, IC 50 The values ​​were 10.9 and 23.8 μM, respectively. Among them, the compound has the best inhibitory activity against retinoblastoma HXO-Rb44, which is better than the positive drug doxorubicin (IC 50 =11.2 μM). The compound had no significant inhibitory activity on human non-small cell lung cancer A549 cells and human cervical cancer HeLa cells.

[0059] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A germarane-type sesquiterpene, characterized in that: The germarane-type sesquiterpenoid A is prepared from the fermentation broth of the endophytic fungus Aspergillus aculeatus Aa-1 from Enteromorpha prolifera. The molecular formula of asperacunoid A is C 15 H 24 O2, the structural formula is as follows:

2. A method for preparing a germarane-type sesquiterpene as claimed in claim 1, characterized in that: The specific preparation process of the germarane-type sesquiterpene asperacunoid A is as follows: (1) Under sterile conditions, the Aspergillus aculeatus Aa-1 is inoculated in conical flasks, each of which is filled with sterilized potato dextrose broth medium; (2) placing the conical flask in a constant temperature environment of 28° C. for static fermentation culture for 30 days, ultrasonically extracting the culture medium of the Aspergillus aculeatus Aa-1 strain after fermentation with ethyl acetate, and concentrating under vacuum to a non-flowing extract to obtain a fermentation crude extract; (3) adding 100-200 mesh silica gel to the crude fermentation extract obtained in step (2) and mixing the sample, and then separating the components by reduced pressure vacuum silica gel column chromatography, and gradient eluting with dichloromethane-methanol solutions with volume ratios of 50:1, 40:1, 30:1, 20:1, 10:1 and 5:1 as gradient eluents, to obtain 6 components Fr.1 to Fr.6 in increasing polarity; (4) subjecting the component Fr.2 obtained in step (3) to C18 reverse phase silica gel column chromatography, selecting a gradient eluent of 10% to 100% methanol-water solution by volume to perform gradient elution, and obtaining a total of 10 sub-components Fr.2.1 to Fr.2.10; (5) The subfraction Fr.2.5 obtained in step (4) is subjected to silica gel column chromatography, eluted with dichloromethane-methanol in a volume ratio of 30:1, the second column volume component is collected and concentrated under vacuum to a non-flowing extract, and the gemmane-type sesquiterpene asperacunoid A is purified.

3. The method for preparing the germarane-type sesquiterpenes according to claim 2, characterized in that: In step (2), the vacuum degree of vacuum decompression concentration is -0.1 MPa.

4. The method for preparing the germarane-type sesquiterpenes according to claim 2, characterized in that: In step (3), the mass ratio of the fermentation crude extract to the silica gel is 1:

1.

5. The method for preparing the germarane-type sesquiterpenes according to claim 2, characterized in that: In step (5), the vacuum degree of vacuum decompression concentration is -0.1 MPa.

6. The method for preparing the germarane-type sesquiterpenes according to claim 2 or 5, characterized in that: The total elution time was 3.5 h, the flow rate was 0.1 mL / s, and the elution time during collection was 2 h.

7. Use of the germarane sesquiterpene as claimed in claim 1 or the germarane sesquiterpene prepared by the method as claimed in claim 2 in the preparation of antitumor drugs.

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