A sesquiterpenoid compound and its preparation method and application

Abstract

The present invention belongs to the field of medical technology, and specifically relates to a sesquiterpenoid compound and a preparation method and application thereof. The present invention provides a sesquiterpenoid compound having a structure shown in Formula 1 or Formula 2. The sesquiterpenoid compound having a structure shown in Formula 1 or Formula 2 provided by the present invention is a sesquiterpenoid compound containing a 2-(trihydro-2H-pyran-2-yl)propanol unit, which enriches the diversity of sesquiterpenoid compounds. At the same time, the immunostimulating activity screening test of the sesquiterpenoid compound provided by the present invention shows that the sesquiterpenoid compound provided by the present invention has a certain effect of promoting immune enhancement in inducing mouse spleen lymphocyte proliferation by concanavalin (ConA) or lipopolysaccharide (LPS), and can be used to prepare immunostimulating drugs.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a sesquiterpenoid compound and a preparation method and application thereof. Background Art

[0002] Sesquiterpenes are natural terpenoid compounds with 15 carbon atoms in one molecule. They contain three isoprene units and are an important group of natural products with diverse structures.

[0003] According to the literature, a variety of sesquiterpenoids with different skeletons have stimulated the study of drug analogs. Among them, the sesquiterpenoid unit containing 2-(trihydro-2H-pyran-2-yl)propan-2-ol has been reported as an important subunit of hexapeptide-terpenoids, as well as indole diterpenoids. However, sesquiterpenoids with 2-(trihydro-2H-pyran-2-yl)propanol units remain an extremely rare class of terpenoids. So far, in the studies on the structure determination and biosynthesis of hexaketide-terpenoids in 1971 and 1973, only three artificial products containing lactone or lactone ring-opening units have been reported, and no NMR data and activity evaluation are available. Summary of the invention

[0004] The object of the present invention is to provide a sesquiterpenoid compound and a preparation method and application thereof. The sesquiterpenoid compound provided by the present invention has immunostimulating activity, can be used to prepare immunostimulating drugs, and enriches the diversity of sesquiterpenoid compounds.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a sesquiterpene compound having a structure shown in Formula 1 or Formula 2:

[0007]

[0008] The present invention provides a method for preparing the sesquiterpenoid compound described in the above technical solution, comprising the following steps:

[0009] inoculating the Cynodon dactylon spore fungus into a fermentation medium for fermentation to obtain a Cynodon dactylon spore fungus fermentation product;

[0010] The fermented product of Cynodon dactylon fungus is mixed with an alcohol solvent and subjected to ultrasonic extraction to obtain a crude extract of sesquiterpenoid compounds;

[0011] The crude extract of the sesquiterpenoid compound and silica gel are mixed, and the obtained mixture is loaded into a column, and a first column chromatography separation is performed, wherein the first column chromatography separation is performed according to a volume ratio from large to small, and a chloroform-methanol system with a volume ratio of chloroform to methanol of 100:0 to 10:1 is used for gradient elution, and the same fractions are combined to obtain five fractions, which are named as Fr.1 to Fr.5 fractions respectively;

[0012] The Fr.2 fraction is subjected to a second column chromatography separation to obtain an eluent containing a sesquiterpenoid compound having a structure shown in Formula 1; the eluent for the second column chromatography separation is a petroleum ether-acetone system or a petroleum ether-ethyl acetate system;

[0013] The Fr.4 fraction is subjected to a third column chromatography separation to obtain an eluent containing a sesquiterpenoid compound having a structure shown in Formula 2; the eluent for the third column chromatography separation is a petroleum ether-acetone system or a petroleum ether-ethyl acetate system;

[0014] The eluate containing the sesquiterpenoid compound having the structure shown in Formula 1 is subjected to a first gel chromatography purification to obtain a pure product of the sesquiterpenoid compound having the structure shown in Formula 1;

[0015] The eluate containing the sesquiterpenoid compound having the structure shown in Formula 2 is subjected to a second gel chromatography purification to obtain a pure product of the sesquiterpenoid compound having the structure shown in Formula 2.

[0016] Preferably, the raw materials of the fermentation medium include potatoes and / or rice; and the fermentation is solid fermentation.

[0017] Preferably, the fermentation temperature is 20-25° C., and the fermentation time is 30-35 days.

[0018] Preferably, the alcohol solvent includes one or more of methanol, ethanol, propanol and isopropanol; and the ratio of the mass of the fermentation product of Cynodon dactylon to the volume of the alcohol solvent is (50-70) g: (80-130) mL.

[0019] Preferably, the first column chromatography separation is: gradient elution using a chloroform-methanol system with volume ratios of 100:0, 100:1, 50:1, 30:1 and 10:1 in sequence.

[0020] Preferably, the mass ratio of the dry weight of the crude extract of the sesquiterpenoid compound to the silica gel is 1:(1-1.2);

[0021] The mesh number of the silica gel is 300 to 400 meshes.

[0022] Preferably, the eluent for the second column chromatography separation is a petroleum ether-acetone system; the volume ratio of petroleum ether to acetone is 30:1.

[0023] Preferably, the eluent for the third column chromatography separation is a petroleum ether-acetone system; the volume ratio of petroleum ether to acetone is 15:1.

[0024] The present invention provides the use of the sesquiterpenoid compound of the above technical solution or the sesquiterpenoid compound prepared by the preparation method of the above technical solution in the preparation of immunostimulating drugs.

[0025] The present invention provides a sesquiterpenoid compound having a structure shown in Formula 1 or Formula 2. The sesquiterpenoid compound having a structure shown in Formula 1 or Formula 2 provided by the present invention is a sesquiterpenoid compound containing a 2-(trihydro-2H-pyran-2-yl)propanol (2-(tetrahydro-2H-pyran-2-yl)propan-2-ol) unit, which enriches the diversity of sesquiterpenoid compounds. At the same time, the immunostimulating activity screening test of the sesquiterpenoid compound provided by the present invention shows that the sesquiterpenoid compound provided by the present invention has a certain immunostimulating effect on the proliferation of mouse spleen lymphocytes induced by concanavalin (ConA) or lipopolysaccharide (LPS), and can be used to prepare immunostimulating drugs.

[0026] The present invention provides a method for preparing the sesquiterpenoid compounds described in the above technical solution. The preparation method provided by the present invention is based on microbial fermentation, has a short preparation cycle, mild culture conditions, few by-products, and low cost, thus meeting the requirements of environmental protection and low carbon, and is easy to realize industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The immunostimulating active compound bipodonine E of the present invention 1 H-NMR spectrum;

[0028] Figure 2 The immunostimulating active compound bipodonine E of the present invention 13 C-NMR and DEPT spectra;

[0029] Figure 3 The immunostimulating active compound bipodonine E of the present invention 1 H- 1 H COSY spectrum;

[0030] Figure 4 is the HMBC spectrum of bipodonine E, an immunomodulatory compound of the present invention;

[0031] Figure 5 is the HSQC spectrum of bipodonine E, an immunomodulatory compound of the present invention;

[0032] Figure 6 is the NOESY spectrum of bipodonine E, an immunomodulatory compound of the present invention;

[0033] Figure 7 is the HR-ESI-MS spectrum of bipodonine E, an immunomodulatory compound of the present invention;

[0034] Figure 8 The ECD experimental and calculated graphs of bipodonine E, an immunomodulatory compound of the present invention;

[0035] Fig. 9 The immunostimulating active compound bipodonine G of the present invention 1 H-NMR spectrum;

[0036] Fig.10 The immunostimulating active compound bipodonine G of the present invention 13 C-NMR and DEPT spectra;

[0037] Fig.11 The immunostimulating active compound bipodonine G of the present invention 1 H- 1 H COSY spectrum;

[0038] Fig.12 is the HMBC spectrum of bipodonine G, an immunomodulatory compound of the present invention;

[0039] Fig.13 is the HSQC spectrum of bipodonine G, an immunomodulatory compound of the present invention;

[0040] Fig.14 is the ROESY spectrum of bipodonine G, an immunomodulatory compound of the present invention;

[0041] Fig.15 is the HR-ESI-MS spectrum of bipodonine G, an immunomodulatory compound of the present invention;

[0042] Fig.16 The ECD experimental and calculated graphs of bipodonine G, an immunomodulatory compound of the present invention. DETAILED DESCRIPTION

[0043] The present invention provides a sesquiterpene compound having a structure shown in Formula 1 or Formula 2:

[0044]

[0045] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0046] The present invention identifies the structures of the sesquiterpenoid compounds of Formula 1 and Formula 2 by combining one-dimensional nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy (1D / 2D NMR), HR-ESI-MS and quantum chemical ECD calculation. 1 H. 13 C. 1 H- 1 H COSY, HMBC, HSQC, ROESY NMR data, HR-ESI-MS and ECD calculated spectra are shown in the appendix of the instruction manual Figures 1 to 16 .

[0047] The present invention provides a method for preparing the sesquiterpenoid compound described in the above technical solution, comprising the following steps:

[0048] inoculating the Cynodon dactylon spore fungus into a fermentation medium for fermentation to obtain a Cynodon dactylon spore fungus fermentation product;

[0049] The fermented product of Cynodon dactylon fungus is mixed with an alcohol solvent and subjected to ultrasonic extraction to obtain a crude extract of sesquiterpenoid compounds;

[0050] The crude extract of the sesquiterpenoid compound and silica gel are mixed, and the obtained mixture is loaded into a column, and a first column chromatography separation is performed, wherein the first column chromatography separation is performed according to a volume ratio from large to small, and a chloroform-methanol system with a volume ratio of chloroform to methanol of 100:0 to 10:1 is used for gradient elution, and the same fractions are combined to obtain five fractions, which are named as Fr.1 to Fr.5 fractions respectively;

[0051] The Fr.2 fraction is subjected to a second column chromatography separation to obtain an eluent containing a sesquiterpenoid compound having a structure shown in Formula 1; the eluent for the second column chromatography separation is a first petroleum ether-acetone system;

[0052] The Fr.4 fraction is subjected to a third column chromatography separation to obtain an eluent containing a sesquiterpenoid compound having a structure shown in Formula 2; the eluent for the third column chromatography separation is a second petroleum ether-acetone system;

[0053] The eluate containing the sesquiterpenoid compound having the structure shown in Formula 1 is subjected to a first gel chromatography purification to obtain a pure product of the sesquiterpenoid compound having the structure shown in Formula 1;

[0054] The eluate containing the sesquiterpenoid compound having the structure shown in Formula 2 is subjected to a second gel chromatography purification to obtain a pure product of the sesquiterpenoid compound having the structure shown in Formula 2.

[0055] The invention inoculates the Cynodon dactylon fungus into a fermentation medium for fermentation to obtain a Cynodon dactylon fungus fermentation product.

[0056] In the present invention, the Cynodon dactylon helminthus fungus is an endophytic fungus of Aconitum brevis, and is preferably isolated from the root of Aconitum brevis. The present invention has no special requirements for the separation method, and conventional technical means in the art may be used. Before the fermentation, the present invention preferably activates the Cynodon dactylon helminthus fungus; the activation is preferably: inoculating the Cynodon dactylon helminthus fungus onto a PDA slant culture medium, culturing at a constant temperature, and obtaining activated Cynodon dactylon helminthus fungus. In the present invention, the temperature of the constant temperature culture is preferably 25 to 30°C, more preferably 28°C, and the time is preferably 3 to 7 days, more preferably 5 days. The present invention preferably stores the obtained activated Cynodon dactylon helminthus fungus in an environment of 5°C for standby use.

[0057] In the present invention, the fermentation is preferably solid fermentation.

[0058] In the present invention, the raw materials of the fermentation medium preferably include potatoes and / or rice. In the present invention, the method for preparing the fermentation medium used in the fermentation preferably includes the following steps:

[0059] The raw materials of the culture medium are sterilized at high temperature and cooled in sequence to obtain the fermentation medium.

[0060] In the present invention, when the raw material of the culture medium is preferably potato, before the high temperature sterilization, the potato is preferably peeled and crushed, and the crushing is preferably to cut the potato into pieces, and the volume of the pieces obtained by cutting is preferably 1.0 cm 3 . In the present invention, the preparation process of the fermentation medium is preferably carried out in a tissue culture flask; the ratio of the mass of the raw material of the fermentation medium to the volume of the tissue culture flask is preferably 45-55g:75-85mL, more preferably 50g:80mL. In the present invention, the temperature of the high-temperature sterilization is preferably 120-150°C, more preferably 121-130°C; the time is preferably 30-50min, more preferably 35-40min. In the present invention, the tissue culture flask is preferably capped when performing the high-temperature sterilization. The present invention has no special limitation on the cooling method, as long as it can be cooled to room temperature.

[0061] In the present invention, the fermentation is preferably: the activated Cynodon dactylon fungus is inoculated in a fermentation medium, and the inoculation amount (the mass ratio of Cynodon dactylon fungus to the fermentation medium) is preferably 0.5%. The present invention has no special requirements for the conditions of the inoculation, and the inoculation can be carried out in a manner familiar to those skilled in the art. In the present invention, the fermentation temperature is preferably 20 to 30°C, more preferably 20 to 25°C; the fermentation time is preferably 30 to 40 days, more preferably 35 days. The solid fermentation method used in the present invention can effectively shorten the fermentation time.

[0062] After obtaining the fermented product of Cynodon dactylon fungus, the present invention mixes the fermented product of Cynodon dactylon fungus with an alcohol solvent and performs ultrasonic extraction to obtain a crude extract of sesquiterpenoid compounds.

[0063] In the present invention, the alcohol solvent preferably includes one or more of methanol, ethanol, propanol and isopropanol, more preferably includes methanol, ethanol, propanol or isopropanol, and further preferably is methanol. In the present invention, the ratio of the mass of the Cynodon dactylon fungus fermentation product to the volume of the alcohol solvent is preferably (50-70) g: (80-130) mL, more preferably 60 g: 120 mL. In the present invention, the alcohol solvent has good solubility for the structural sesquiterpenoid compounds shown in Formula 1 and Formula 2, and can well separate the structural sesquiterpenoid compounds shown in Formula 1 and Formula 2 from the Cynodon dactylon fungus fermentation product. The present invention has no special requirements for the mixing method as long as it can be mixed evenly.

[0064] In the present invention, the power of the ultrasonic extraction is preferably 250-350 W, more preferably 300 W, and the time is preferably 20-40 min, more preferably 30 min.

[0065] In the present invention, after the ultrasonic extraction, the present invention also preferably includes filtering the system after the ultrasonic extraction; the present invention has no special limitation on the filtration, and a conventional filtration method can be used. After the filtration, the present invention preferably performs reduced pressure distillation on the obtained filtrate to remove the solvent in the filtrate to obtain a crude extract of sesquiterpenoid compounds. In the present invention, the vacuum degree of the reduced pressure distillation is preferably 10 to 15 kPa, more preferably 12 kPa; the temperature of the reduced pressure distillation is preferably 45 to 55°C, more preferably 50°C. The present invention has no special limitation on the time of reduced pressure distillation as long as the solvent in the filtrate can be removed.

[0066] After obtaining the crude extract of sesquiterpenoid compounds, the present invention mixes the crude extract of sesquiterpenoid compounds with silica gel, and the obtained mixture is loaded into a column for first column chromatography separation. The first column chromatography separation is carried out according to a volume ratio from large to small, using a chloroform-methanol system with a volume ratio of chloroform to methanol of 100:0 to 10:1 for gradient elution, and the same fractions are combined to obtain five fractions, which are named Fr.1 to Fr.5 fractions respectively.

[0067] In the present invention, the crude extract of the sesquiterpenoid compound is preferably mixed with the silica gel in the form of a crude extract solution. In the present invention, the solvent in the crude extract solution is preferably dichloromethane. The present invention preferably uses dichloromethane to obtain the crude extract of the sesquiterpenoid compound to obtain the crude extract solution.

[0068] In the present invention, the mass ratio of the dry weight of the crude extract of the sesquiterpenoid compound to the silica gel is preferably 1:(1-1.2). In the present invention, the mesh number of the silica gel is 300-400 mesh. In the present invention, the mixture obtained after removing the solvent is preferably loaded into a column.

[0069] In the present invention, the first column chromatography separation is preferably: gradient elution is performed using a chloroform-methanol system with a volume ratio of 100:0, 100:1, 50:1, 30:1 and 10:1 in sequence. In the present invention, three column volumes are eluted in each ratio, and then the eluents of each ratio are combined. The obtained eluent is subjected to thin layer chromatography (TLC), and the same fractions are combined to obtain five fractions, which are named as fractions Fr.1, Fr.2, Fr.3, Fr.4 and Fr.5 respectively; the first column chromatography separation is preferably performed at room temperature.

[0070] The present invention subjects the Fr.2 fraction to a second column chromatography separation to obtain an eluent containing a sesquiterpenoid compound having a structure shown in Formula 1. In the present invention, the second column chromatography separation is preferably a silica gel column chromatography separation; the eluent for the second column chromatography separation is preferably a petroleum ether-acetone system or a petroleum ether-ethyl acetate system, more preferably a petroleum ether-acetone system. In the present invention, in the petroleum ether-acetone system used in the second column chromatography separation, the volume ratio of petroleum ether to acetone is preferably 30:1. In the present invention, the flow rate of the eluent for the second column chromatography separation is preferably 2 to 5 mL / min, more preferably 3.5 mL / min.

[0071] The present invention performs a third column chromatography separation on the Fr.4 fraction to obtain an eluent containing a sesquiterpenoid compound having a structure shown in Formula 2. In the present invention, the third column chromatography separation is preferably a silica gel column chromatography separation; the eluent for the third column chromatography separation is preferably a petroleum ether-acetone system or a petroleum ether-ethyl acetate system, more preferably a petroleum ether-acetone system. In the present invention, in the petroleum ether-acetone system used in the second column chromatography separation, the volume ratio of petroleum ether to acetone is preferably 15:1. In the present invention, the flow rate of the eluent for the third column chromatography separation is preferably 2 to 5 mL / min, more preferably 3.5 mL / min.

[0072] The present invention performs a first gel chromatography purification on the eluate containing the sesquiterpenoid compound having the structure shown in Formula 1 to obtain a pure product of the sesquiterpenoid compound having the structure shown in Formula 1. In the present invention, the filler for the first gel chromatography purification is preferably dextran gel, and the solvent for the first gel chromatography purification is preferably methanol or a methanol-chloroform system, more preferably methanol; in the methanol-chloroform system, the volume ratio of methanol to chloroform is preferably 2:1. The flow rate of the solvent for the first gel chromatography purification is preferably 0.5 to 0.7 mL / min, more preferably 0.6 mL / min. In the present invention, the first gel chromatography purification obtains a first purified solution, and the present invention preferably removes the solvent from the first purified solution to obtain a pure product of the sesquiterpenoid compound having the structure shown in Formula 1. In the present invention, the method for removing the solvent is preferably reduced pressure distillation; the temperature for the reduced pressure distillation is preferably 48 to 55° C., more preferably 50° C.

[0073] The present invention performs a second gel chromatography purification on the eluate containing the sesquiterpenoid compound of the structure shown in Formula 2 to obtain a pure product of the sesquiterpenoid compound of the structure shown in Formula 2. In the present invention, the filler of the second gel chromatography purification is preferably dextran gel, and the solvent of the second gel chromatography purification is preferably methanol or a methanol-chloroform system, more preferably methanol; in the methanol-chloroform system, the volume ratio of methanol to chloroform is preferably 2:1. The flow rate of the solvent for the second gel chromatography purification is preferably 0.5 to 0.7 mL / min, more preferably 0.6 mL / min. In the present invention, the second gel chromatography purification obtains a second purified solution, and the present invention preferably removes the solvent from the second purified solution to obtain a pure product of the sesquiterpenoid compound of the structure shown in Formula 2. In the present invention, the method for removing the solvent is preferably reduced pressure distillation; the temperature of the reduced pressure distillation is preferably 48 to 55°C, more preferably 50°C.

[0074] The present invention provides the use of the sesquiterpenoid compound of the above technical solution or the sesquiterpenoid compound prepared by the preparation method of the above technical solution in the preparation of immunostimulating drugs.

[0075] The present invention provides an immunostimulatory drug, comprising the sesquiterpenoid compound of the above technical solution or the sesquiterpenoid compound prepared by the preparation method described in the above technical solution and pharmaceutical excipients. In the present invention, in the immunostimulatory drug, the mass percentage of the sesquiterpenoid compound of the above technical solution or the sesquiterpenoid compound prepared by the preparation method described in the above technical solution is preferably 1 to 99%, more preferably 55 to 90%. The present invention has no special limitation on the pharmaceutical excipients, and conventional pharmaceutical excipients in the art can be selected. The present invention has no special limitation on the dosage form and preparation method of the immunostimulatory drug, and the preparation method well known in the art can be used to prepare tablets, granules or injections and other dosage forms.

[0076] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0077] Example 1

[0078] The Cynodon dactylon helminthes fungus was inoculated onto a PDA slant medium, and after constant temperature culture at 28° C. for 5 days, the activated Cynodon dactylon helminthes fungus was obtained, and the culture was placed in a 5° C. environment for storage for future use;

[0079] 5.0 kg of potatoes were peeled, washed and cut into 1 cm 3 The potato pieces were divided into 100 tissue culture bottles with a volume of 350 mL (50 g / bottle), and then the tissue culture bottles were capped and sterilized at 121° C. for 30 min, and cooled to obtain a fermentation medium;

[0080] Inoculating the activated Cynodon dactylon fungus into the fermentation medium at an inoculation rate of 0.5%, covering the medium and culturing the medium at room temperature for 35 days to obtain a Cynodon dactylon fungus fermentation product;

[0081] The obtained Cynodon dactylon spp. fermentation product was mixed with 120 mL of methanol in a mass volume ratio (50 g / 120 mL), and then ultrasonicated for 30 min at 40 kHz, filtered, and the filtrate was subjected to reduced pressure distillation (vacuum degree of 12 kPa, 50° C.) until there was no alcohol taste, to obtain 83.0 g of crude extract;

[0082] The 83.0 g crude extract was dissolved in 100 mL of dichloromethane, and then mixed with 83.0 g of silica gel (300-400 mesh), and then concentrated under reduced pressure (vacuum degree of 12 kPa, 50° C.) to remove the solvent, and loaded into a column; gradient elution was performed using solutions of chloroform and methanol with a volume ratio of 100:0, 100:1, 50:1, 30:1, and 10:1 to obtain five fractions, namely, Fr.1 fraction, Fr.2 fraction, Fr.3 fraction, Fr.4 fraction, and Fr.5 fraction; the Fr.3 fraction was eluted by silica gel column chromatography using petroleum ether-acetone with a volume ratio of 30:1 as an eluent to obtain a sesquiterpenoid compound (denoted as bipodonine E) eluate, the Fr.4 fraction was subjected to silica gel column chromatography using petroleum ether-acetone with a volume ratio of 15:1 as an eluent to obtain an eluate of a sesquiterpenoid compound (denoted as bipodonine G) having a structure shown in Formula 1;

[0083] The eluate of bipodonine E and the eluate of bipodonine G were purified by dextran gel using methanol as solvent to obtain pure bipodonine E and pure bipodonine G.

[0084] Example 2 Identification of the structure

[0085] The bipodonine E and bipodonine G prepared in Example 1 were detected by nuclear magnetic resonance spectrometer to obtain 1D / 2D NMR (one-dimensional nuclear magnetic resonance spectrum and two-dimensional nuclear magnetic resonance spectrum), HR-ESI-MS (high-resolution electrospray ionization mass spectrometry) and quantum chemical ECD calculation (such as Figures 1 to 16 ) The obtained compounds bipodonine E and bipodonine G and their structures were identified.

[0086] The chemical shift δ assignments of H and the C connected to bipodonine E and bipodonine G can be obtained by combining HSQC spectrum with carbon spectrum as shown in Table 1.

[0087] Table 1 Bipodonine E and bipodonine G 13 C and 1 H NMR data.

[0088] No. <![CDATA[1 a ]]> <![CDATA[2 b ]]> <![CDATA[δ H (JinHz)]]> <![CDATA[δ C ]]> <![CDATA[δ H (JinHz)]]> <![CDATA[δ C ]]> 1 1.76dd(6.9,1.2) 44.6 10.01d 207.2 1.10overlap 2 4.14td(11.2,4.7) 66.5 2.07s 64.8 3 211.5 71.4 4 2.43m 57.3 1.41m 39.1 1.75dt(14.3,3.1) 5 1.50m 29.0 1.45m 21.4 1.96dt(12.7,4.1) 1.66m 6 3.20overlap 82.1 3.16dd(11.9,3.5) 83.7 7 33.7 37.6 8 1.31m 38.5 1.57dd(12.6,3.9) 37.6 1.60m 1.91dt(11.5,3.8) 9 1.47m 21.6 1.50m 24.0 1.60m 1.84m 10 3.23oerlap 85.4 3.25dd(11.7,2.8) 85.1 11 72.0 72.0 12 1.16s3H 26.1 1.18s3H 26.1 13 1.14s3H 23.9 1.17s3H 24.0 14 0.91s3H 16.5 1.23s3H 13.8 15 2.19s3H 29.1 1.21s3H 31.1

[0089] a Measured in CDCl 3 (600 MHz for 1 H NMR and 150 MHz for13 C NMR)

[0090] b Measured in CDCl 3 (400 MHz for 1 H NMR and 100 MHz for 13 C NMR)

[0091] Compound 1 is a white powder. The high-resolution electrosprayionization mass spectrum (HR-ESI-MS) of compound 1 has an m / z of 293.1723 [M+Na] + (calcd.for C 15 H 26 O 4 Na + ,293.1723) indicates that its molecular formula is C 15 H 26 O 4 , the unsaturation degree is 3. One of the carbonyl groups (-CO-) occupies one unsaturation, and the remaining two unsaturations indicate that compound 1 has a two-ring skeleton. 1 H, 13 C, DEPT and HSQCNMR spectra show that compound 1 has 15 carbon atoms, including three methyl groups [δ H 1.16(H 3 -12),1.14(H 3 -13),0.91(H 3 -14),2.19(H 3 -15); δ C 26.1 (C-12), 23.9 (C-13), 16.5 (C-14), 29.1 (C-15)], four methylene groups [δ H 1.76(H-1a),1.10(H-1b),1.50(H-5a),1.96(H-5b),1.31(H-8a),1.60(H-8b),1.47(H-9a),1.60(H-9b); δ C 44.6 (C-1), 29.0 (C-5), 38.5 (C-8), 21.6 (C-9)], four methines [δ H 4.14(H-2,oxygenated),2.43(H-4),3.20(H-6,oxygenated),3.23(H-10,oxygenated); δ C66.5 (C-2, oxygenated), 57.3 (C-4), 82.1 (C-6, oxygenated), 85.4 (C-10, oxygenated)], three quaternary carbons [δ C 33.7 (C-7), 72.0 (C-11, oxygenated)], and a carbonyl group [δ C 211.5(C-3)].

[0092] Combining the 1D and 2D NMR spectra of compound 1, it can be observed from the HMBC spectrum that H-10 and H 2 -8 are respectively related to C-6, H 2 -8 and H 2 -9 is associated with C-7, and then combined with H-8 / H-9 / H-10 1 H- 1 H COSY and CH-6(δ H 3.20,δ C 82.1) and CH-10(δ H 3.23,δ C 85.4) indicates the presence of an oxypentane ring (ring A). 3 -12 is related to C-13, C-10 and C-11 respectively; H 3 -13 are related to C-12, C-11 and C-10 respectively; combining their chemical shifts indicates that the 2-propanol of compound 1 is 10 -C 11 The bond is located at C-10, thus constructing the 2-(tetrahydro-2H-pyran-2-yl)propan-2-ol unit. The presence of cyclohexane (B ring) is due to the presence of H 2 -4 / H 2 -5 / H-6 1 H- 1 The results were confirmed by H COSY correlation and HMBC correlation between H-2 and C-7. H 4.14,δ C 66.5) indicates that the hydroxyl group (-OH) is located at C-2. 3 -15(δ H 2.19) are related to C-3 and C-4, respectively, indicating that the methyl ketone is located at the C-4 position.

[0093] The relative configuration of compound 1 was determined by NOESY experiment. 3 -14(δ H0.91) and H-8β(δ H 1.60) and H-2(δ H 4.14), confirming that the relative configuration of C-7 is R*. H 1.31) and H-6 and H-10 (δ H 3.21), and the NOEs of H-6 and H-4 are correlated, and H 3 -14(δ H 0.91) and H-4(δ H 4.14) NOE-related missing, indicating that the relative configuration of compound 1 is 2S*, 4S*, 6R*, 7R*, 10R*. Using time-dependent density functional theory calculation method (TDDFT) calculation at B3LYP / 6-311+G(d,p) / / B3LYP / 6-31G(d,p) level, by comparing with the ECD experimental spectrum, the absolute configuration of compound 1 was determined to be 2S, 4S, 6R, 7R, 10R, and it was named bipodonine E.

[0094] Compound 2, light yellow powder. Its HR-ESI-MS m / z 293.1720[M+Na] + (calcdfor C 15 H 26 O 4 Na + ,293.1723) and 13 C and DEPTNMR data show that its molecular formula is C 15 H 26 O 4 , corresponding to 3 unsaturations. A careful comparison and analysis with compound 1 revealed that compound 2 has similar A ring - oxypentane ring and B ring - cyclohexane as 1. Remove two unsaturations, and the other unsaturation is the aldehyde group (-CHO). The chemical shift of the combined C-3 is δ C 71.4ppm and through H 3 -15(δ H 1.21) are related to the HMBC of C-2, C-3, and C-4, respectively, and it is speculated that there is a methyl group in addition to the hydroxyl (-OH) substitution at C-3. H 10.01) and C-2(δ C 64.8), it was inferred that the aldehyde group was located at the C-2 position.

[0095] The relative configuration of compound 2 was determined based on the NOESY experimental spectrum. 3 -14 and H 3 -15 and H-8β(δH 1.91) NOE correlation, it is inferred that H 3 -15 is β-configuration. H-6 and H-8α(δ H 1.57) and H-2, as well as H-8α and H-10 (δ H 3.25), it is speculated that they are all α-configured. Density functional theory (DFT) was used to perform ECD calculations on compound 2 ((2R,3R,6R,7R,10R)-2) using Gauss 09 at the B3LYP / 6-311+G(d,p) / / B3LYP / 6-31G(d,p) level, confirming that the configuration of compound 2 is 2R,3R,6R,7R,10R and it was named bipodonine G.

[0096] In summary, it can be determined that the compound bipodonine E (Compound 1) prepared in Example 1 is a sesquiterpene compound with a structure shown in Formula 1, and the compound bipodonine G (Compound 2) is a sesquiterpene compound with a structure shown in Formula 2:

[0097]

[0098] Example 3 Screening of the immunostimulatory activity of compounds bipodonine E and bipodonine G

[0099] Preparation of mouse spleen lymphocyte suspension: ICR mice were killed by cervical dislocation, soaked in 75% ethanol for 5 minutes, and the mice were taken out and placed on a clean bench for aseptic separation of the spleen. The spleen was rinsed with phosphate buffer (PBS) and the surrounding connective tissue and fat components were removed. The spleen tissue was placed on a stainless steel mesh (200 mesh), lightly pressed with a glass syringe needle, rinsed with 3 mL of PBS, and the rinse fluid was collected and transferred to a centrifuge tube. Centrifuged at 4°C for 15 minutes (1000 rpm / min), and the supernatant was discarded. After adding 3 mL of red blood cell lysis solution, it was placed at room temperature for 2 minutes, and 7 mL of PBS was added to balance the osmotic pressure of spleen cells. 7 mL of PBS was added repeatedly and centrifuged and washed twice (1000 rpm / min, 5 minutes), and the supernatant was discarded to obtain a gray-white spleen cell precipitate. The cells were suspended in RPMI-1640 medium to make a single cell suspension of a certain concentration. The cells were counted by trypan blue staining. The number of live cells was greater than 95%, and the cell concentration was adjusted to 1×10 5 Pieces / mL.

[0100] MTS method to detect mouse spleen lymphocyte proliferation: Take the prepared mouse spleen lymphocyte suspension and add it to a 96-well cell culture plate, 180μL / well. The experiment was divided into a cell blank control group (40μL RPMI-1640 was added to each well); an inducer ConA or LPS stimulation group (10μL inducer and 10μL RPMI-1640 were added to each well); a sample synergistic ConA or LPS stimulation group, 10μL of sample solution (final sample concentration of 20μM), 10μL of inducer ConA or LPS (final concentration of 10μg / mL) were added to each well, and 3 replicates were added to each group. Incubate at 37°C, 5% CO 2 After culturing in an incubator for 72 h, 20 μL MTS was added, cultured for 3 h, shaken for 10 min, and the absorbance was measured at 490 nm.

[0101] The experimental results showed that the compounds bipodonine E and bipodonine G showed a certain immune-enhancing effect on the proliferation of mouse spleen lymphocytes induced by concanavalin (ConA) or lipopolysaccharide (LPS). (Table 2ConA / LPS is the positive control, and Control is the blank control). Therefore, bipodonine E and bipodonine G have research value as lead compounds for the development of immune-enhancing drugs. The method of producing large amounts of simple immune-enhancing compounds bipodonineE and bipodonine G based on microbial fermentation can not only test the needs of environmental protection and low carbon, but also realize a new way of mass production, providing a new option for the development of immune-enhancing drugs.

[0102] Table 2 Effects of bipodonine E and bipodonine G on (A) concanavalin A (ConA) and (B) lipopolysaccharide (LPS)-induced cell proliferation rates

[0103] compd Cell proliferation (%) compd Cell proliferation (%) Compound 1 - Compound 1 28.8 Compound 2 37.2 Compound 2 40.3 Control 12.3 Control 11.0 ConA 28.9 LPS 21.3

[0104] Example 4

[0105] The Cynodon dactylon helminthes fungus was inoculated onto a PDA slant medium, and after constant temperature culture at 28° C. for 5 days, the activated Cynodon dactylon helminthes fungus was obtained, and the culture was placed in a 5° C. environment for storage for future use;

[0106] 50 g of rice was soaked in water overnight, and the soaked rice was placed in a 350 mL tissue culture bottle, and then the tissue culture bottle was covered and sterilized at 121° C. for 30 min, and cooled to obtain a fermentation medium;

[0107] Inoculating the activated Cynodon dactylon fungus into the fermentation medium at an inoculation rate of 0.5%, covering the medium and culturing the medium at room temperature for 35 days to obtain a Cynodon dactylon fungus fermentation product;

[0108] The obtained Cynodon dactylon spp. fermentation product was mixed with 120 mL of methanol in a mass volume ratio (50 g / 120 mL), and then ultrasonicated for 30 min at 40 kHz, filtered, and the filtrate was subjected to reduced pressure distillation (vacuum degree of 12 kPa, 50° C.) until there was no alcohol taste, to obtain 14.0 g of crude extract;

[0109] The 14.0 g crude extract was dissolved in 40 mL of dichloromethane, and then mixed with 14.0 g of silica gel (300-400 mesh), and then concentrated under reduced pressure (vacuum degree of 12 kPa, 50° C.) to remove the solvent, and loaded into a column; gradient elution was performed using solutions of chloroform and methanol with a volume ratio of 100:0, 100:1, 50:1, 30:1, and 10:1 to obtain five fractions, namely, Fr.1 fraction, Fr.2 fraction, Fr.3 fraction, Fr.4 fraction, and Fr.5 fraction; the Fr.3 fraction was eluted by silica gel column chromatography using petroleum ether-acetone with a volume ratio of 30:1 as an eluent to obtain the eluent of bipodonine E, and the Fr.4 fraction was eluted by silica gel column chromatography using petroleum ether-acetone with a volume ratio of 15:1 as an eluent to obtain bipodonine The above eluates were purified by dextran gel using methanol as solvent to obtain compounds bipodonine E and bipodonine G.

[0110] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A sesquiterpenoid compound, It is characterized in that It has a structure shown in Formula 1 or Formula 2:

2. The method for preparing the sesquiterpenoid compound according to claim 1, It is characterized in that The following steps are involved: inoculating the Cynodon dactylon spore fungus into a fermentation medium for fermentation to obtain a Cynodon dactylon spore fungus fermentation product; The fermented product of Cynodon dactylon fungus is mixed with an alcohol solvent and subjected to ultrasonic extraction to obtain a crude extract of sesquiterpenoid compounds; The crude extract of the sesquiterpenoid compound and silica gel are mixed, and the obtained mixture is loaded into a column, and a first column chromatography separation is performed, wherein the first column chromatography separation is performed according to a volume ratio from large to small, and a chloroform-methanol system with a volume ratio of chloroform to methanol of 100:0 to 10:1 is used for gradient elution, and the same fractions are combined to obtain five fractions, which are named as Fr.1 to Fr.5 fractions respectively; The Fr.2 fraction is subjected to a second column chromatography separation to obtain an eluent containing a sesquiterpenoid compound having a structure shown in Formula 1; the eluent for the second column chromatography separation is a petroleum ether-acetone system or a petroleum ether-ethyl acetate system; The Fr.4 fraction is subjected to a third column chromatography separation to obtain an eluent containing a sesquiterpenoid compound having a structure shown in Formula 2; the eluent for the third column chromatography separation is a petroleum ether-acetone system or a petroleum ether-ethyl acetate system; The eluate containing the sesquiterpenoid compound having the structure shown in Formula 1 is subjected to a first gel chromatography purification to obtain a pure product of the sesquiterpenoid compound having the structure shown in Formula 1; The eluate containing the sesquiterpenoid compound having the structure shown in Formula 2 is subjected to a second gel chromatography purification to obtain a pure product of the sesquiterpenoid compound having the structure shown in Formula 2.

3. The preparation method according to claim 2, It is characterized in that The raw materials of the fermentation medium include potato and / or rice; and the fermentation is solid fermentation.

4. The preparation method according to claim 2 or 3, It is characterized in that The fermentation temperature is 20-25° C., and the fermentation time is 30-35 days.

5. The preparation method according to claim 2, It is characterized in that The alcohol solvent includes one or more of methanol, ethanol, propanol and isopropanol; the ratio of the mass of the fermentation product of Cynodon dactylon to the volume of the alcohol solvent is (50-70) g: (80-130) mL.

6. The preparation method according to claim 2, It is characterized in that The first column chromatography separation is: gradient elution using a chloroform-methanol system with volume ratios of 100:0, 100:1, 50:1, 30:1 and 10:1 in sequence.

7. The preparation method according to claim 2 or 6, It is characterized in that The mass ratio of the dry weight of the crude extract of the sesquiterpenoid compound to the silica gel is 1:(1-1.2); The mesh number of the silica gel is 300 to 400 meshes.

8. The preparation method according to claim 2, It is characterized in that The eluent for the second column chromatography separation is a petroleum ether-acetone system; the volume ratio of petroleum ether to acetone is 30:

1.

9. The preparation method according to claim 2, It is characterized in that The eluent for the third column chromatography separation is a petroleum ether-acetone system, and the volume ratio of petroleum ether to acetone is 15:

1.

10. Use of the sesquiterpenoid compound according to claim 1 or the sesquiterpenoid compound prepared by the preparation method according to claims 2 to 9 in the preparation of immunostimulatory drugs.

Images