Terpenoid as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to terpenoids as well as a preparation method and application thereof. The compound is derived from a secondary metabolite of Irpex lacteus, the chemical name of the compound is determined as (6S, 6aS, 9aR, Z)-6, 8, 8-trimetyl-6, 6a, 7, 8, 9, 9a-hexahydrozoulene [5, 4-c] furan-3, 5 (1H, 4H)-dione through a nuclear magnetic resonance hydrogen spectrum, a carbon spectrum, a two-dimensional spectrum, a high-resolution mass spectrum, an infrared spectrogram and the like, and the chemical structure of the compound is shown as follows: # imgabs 0 # database screening shows that the compound is a novel terpenoid compound, and the compound has a wide application prospect. The strain is separated from Irpex lacteus leavening for the first time. A biological activity test shows that the compound has relatively strong triglyceride reduction activity, and the effect of the compound is superior to that of a common drug lovastatin. Therefore, the novel terpenoid has a wide application prospect in the aspects of medicinal or non-medicinal lipid reduction and blood fat reduction.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a terpenoid compound and a preparation method and application thereof. Technical Background

[0002] Irpex lacteus (scientific name: Irpex lacteus), also known as white-sac Irpex and white-sac Irpex, belongs to the genus Irpex, family Irpexaceae, order Polyporaceae, order Agaricales, phylum Basidiomycetes. This species contains a variety of secondary metabolites, including terpenoids, alkaloids, sugars, furopyridine derivatives, and fatty acids. Terpenoids are particularly abundant in its secondary metabolites and exhibit significant lipid-lowering activity.

[0003] Hyperlipidemia is a common lipid metabolism disorder characterized by abnormally elevated levels of lipids and lipoproteins (such as total cholesterol, triglycerides, chylomicrons, low-density lipoprotein cholesterol, and very low-density lipoprotein cholesterol) in plasma. Furthermore, decreased levels of high-density lipoprotein cholesterol (HDL-C) are also a contributing factor to hyperlipidemia.

[0004] Given the diversity of secondary metabolites of Psoralea corylifolia and its good biological activity, it is worthwhile to extract compounds with novel structures and stronger lipid-lowering activity from its fermentation broth. Summary of the Invention

[0005] The present invention aims to provide a novel terpenoid compound, a preparation method and application thereof, in particular a novel terpenoid compound isolated from the fermentation broth of Irpex lacteus, and its application in lowering blood lipids.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] In one aspect, a terpenoid compound is compound 1, and the chemical structural formula of compound 1 is shown below:

[0008]

[0009] Its chemical name is: (6S,6aS,9aR,Z)-6,8,8-trimethyl-6,6a,7,8,9,9a-hexahydroazuleno[5,4-c]furan-3,5(1H,4H)-dione

[0010] On the other hand, the preparation method of the above-mentioned compound 1 is as follows:

[0011] (1) Strain activation: Select the part of the parent strain of the white rake tooth fungus that is not contaminated and has uniform mycelium, inoculate it on the PDA agar plate, and then place it in a constant temperature incubator at 28°C for 7 days to observe the growth status.

[0012] (2) Strain fermentation: The activated mycelium was inoculated into the fungus liquid culture medium No. 2 and cultured at room temperature for 25 days.

[0013] (3) Preparation of crude extract: After the fermentation is completed, the mycelium is crushed and combined with the fermentation liquid, extracted with 2 times the volume of ethyl acetate 3-5 times, and concentrated under reduced pressure to obtain the crude extract of white rake tooth fungus.

[0014] (4) Normal phase column chromatography: elution was performed using a petroleum ether-ethyl acetate mixed solvent with different gradients (volume ratios of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, and 0:1). The fractions (F4) with a petroleum ether-ethyl acetate volume ratio of 4:1 were collected. The F4 fractions were divided into fractions F4.1, F4.2, F4.3, F4.4, and F4.5 in the order of elution, with each liter being a fraction.

[0015] (5) Semi-preparative liquid chromatography separation: The F4.5 fraction was separated to obtain compound 1. The preparation conditions were: detection wavelength 225 nm, flow rate 4 mL / min, and methanol to water volume ratio 29:21.

[0016] (6) Structural identification: The structure of compound 1 was determined by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry.

[0017] After database comparison, compound 1 was determined to be a new terpenoid compound, which was isolated from the extract of the white rake fungus for the first time and had not been reported before.

[0018] The study also found that compound 1 has potential medicinal value in the treatment and prevention of hyperlipidemia and high triglycerides. Lipid-lowering activity experiments showed that compound 1 significantly reduced triglyceride levels (P < 0.01), with an effect superior to that of the positive drug group, particularly in the high- and medium-dose groups. Therefore, this terpenoid compound has the potential to become an active ingredient in drugs or health supplements for the treatment and prevention of hyperlipidemia and high triglycerides. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of compound 1 of the present invention ( 1 H-NMR).

[0020] Figure 2 is the carbon NMR spectrum of compound 1 of the present invention ( 13 C-NMR).

[0021] Figure 3It is the nuclear magnetic resonance HMQC pattern of compound 1 of the present invention.

[0022] Figure 4 This is the COSY nuclear magnetic resonance pattern of compound 1 of the present invention.

[0023] Figure 5 It is the nuclear magnetic resonance NOESY pattern of compound 1 of the present invention.

[0024] Figure 6 It is the ECD curve diagram of compound 1 of the present invention.

[0025] Figure 7 This is a high-resolution mass spectrum of compound 1 of the present invention.

[0026] Figure 8 is the infrared spectrum of compound 1. DETAILED DESCRIPTION

[0027] In the following examples, in order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with specific examples. The content described below is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0028] Example 1

[0029] 1. Preparation of a terpenoid compound 1

[0030] Provided is a method for preparing a terpenoid compound 1, comprising the following steps:

[0031] (1) Strain activation: Select the part of the parent strain of the white rake tooth fungus that is not contaminated and has uniform mycelium, inoculate it on the PDA agar plate, and then place it in a constant temperature incubator at 28°C for 7 days to observe the growth status.

[0032] (2) Strain fermentation: The activated mycelium was inoculated into the fungus liquid culture medium No. 2 and cultured at room temperature for 25 days.

[0033] (3) Preparation of crude extract: After the fermentation is completed, the mycelium is crushed and combined with the fermentation liquid, extracted with 2 times the volume of ethyl acetate 3-5 times, and concentrated under reduced pressure to obtain the crude extract of white rake tooth fungus.

[0034] (4) Normal phase column chromatography: elution was performed using a petroleum ether-ethyl acetate mixed solvent with different gradients (volume ratios of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, and 0:1). The fractions (F4) with a petroleum ether-ethyl acetate volume ratio of 4:1 were collected. The F4 fractions were divided into fractions F4.1, F4.2, F4.3, F4.4, and F4.5 in the order of elution, with each liter being a fraction.

[0035] (5) Semi-preparative liquid chromatography separation: The F4.5 fraction was separated to obtain compound 1, which was a bright yellow oily liquid. The preparation conditions were a detection wavelength of 225 nm, a flow rate of 4 mL / min, and a methanol to water volume ratio of 29:21.

[0036] 2. Structure determination of compound 1

[0037] Provided is a method for structural characterization of a terpenoid compound 1.

[0038] Weigh about 3 mg of compound 1, dissolve it in deuterated chloroform, filter it into an NMR tube, and perform hydrogen and carbon spectrum detection using NMR technology; 1 H NMR ( Figure 1 )and 13 C NMR ( Figure 2 ) data (400MHz, Chloroform-d) are shown in Table 1. Molecular formula of compound 1 C 15 H 20 O3, the degree of unsaturation of the molecule is 6. The molecular mass of compound 1 was determined by high-resolution mass spectrometry to be m / z = 249.1481 ([M+H]+, calcd 249.1485). 13 C NMR data, the signal at δC 209.30 should be a ketone carbonyl group on a ring. According to HSQC ( Figure 3 ), the hydroxyl group at C-12 (δC 41.79) was replaced by a methylene group (δC 41.79, δH 1.66 & δH 1.27), and the position of the keto carbonyl group was determined to be at C-4 based on the signals of the methylene groups on the ring. Figure 4 ) was also verified. Thus, the planar structure of the compound was obtained. According to NOESY ( Figure 5 ), H3-15 (δH 0.99) and H1-5, H1-7 (δH 3.11overlap) and H1-6 (δH 2.75) all have signals, and H1-7 (δH 3.11overlap) and H1-6 (δH 2.75) are coplanar, and are not in the same plane with the methyl group (δC 28.91H 1.04), which gives the relative configuration of compound 4. Experimental ECD curve ( Figure 6 ) and the calculated ECD curve ( Figure 6 ) show that their absolute configurations are 7R, 6S and 5S, respectively.

[0039] Table 1 Compound 1 1 H NMR and 13 C NMR data

[0040]

[0041] The molecular formula of compound 1 is: C 17 H 24 04, the chemical structure of compound 1 is:

[0042]

[0043] Its chemical name is: (6S,6aS,9aR,Z)-6,8,8-trimethyl-6,6a,7,8,9,9a-hexahydroazuleno[5,4-c]furan-3,5(1H,4H)-dione

[0044] Example 2

[0045] Determination of the lipid-lowering and triglyceride-lowering activity of compound 1.

[0046] 1. Experimental methods

[0047] 1.1 Cell culture

[0048] HepG2 cells were cultured at 37° C. and 5% CO 2 in a DMEM medium containing 10% FBS and 1% penicillin-streptomycin.

[0049] 1.2 Establishment of a high-lipid cell model and in vitro lipid-lowering experiment

[0050] HepG2 cells in good growth condition, converging 80%-90% of the cell flask, were trypsinized and digested with 5 mL of culture medium. Subsequently, the cells were gently pipetted to evenly suspend them in the culture medium and seeded into 6-well plates, with 2 mL of cell suspension added to each well. After 24 hours of incubation in a cell culture incubator, the old culture medium was removed. A normal control group received only fresh complete culture medium, while the other groups received complete culture medium containing 10% DMEM supplemented with oleic acid to induce a hyperlipidemia model. Simultaneously, the positive drug group received 24.7 μmol / L lovastatin, and the drug-treated groups received high, medium, and low doses (100, 50, and 25 μmol / L) of compound 1. After 24 hours of incubation, the old culture medium was removed, and 200 μL of RIPA lysis buffer was added to each well, and the cells were lysed on ice for 10 minutes. The cell lysate was transferred to a 1.5 mL centrifuge tube, sonicated 2-3 times (5 seconds each), and then centrifuged at 12,000 rpm for 5 minutes. The supernatant was taken for TG content determination.

[0051] TG content determination: Set up a blank group, a calibration group, and a sample group, with triplicate wells in each group. Add 250 μL of the corresponding working solution to each well, 2.5 μL of distilled water to the blank well, an equal amount of calibrator to the calibration well, and an equal amount of sample from each group to the sample well. Mix thoroughly, incubate at 37°C for 10 min, measure the absorbance at 510 nm, and calculate the TG content.

[0052] 2. Experimental results

[0053] Table 1 Effect of compound 1 on TG content in HepG2 cells induced by oleic acid

[0054]

[0055] Note: Compared with the normal group, # p<0.05, ## p<0.01; compared with the model group, *p<0.05, **p<0.01;

[0056] The results showed that compound 1 significantly reduced TG levels in HepG2 cells induced by oleic acid. Specifically, TG levels in the high-, medium-, and low-dose groups decreased by 59.32%, 57.63%, and 55.93%, respectively. This data demonstrates that compound 1 has an excellent TG-lowering effect.

[0057] In summary, compound 1, isolated from the extract of the fungus Echinops leucoderma, is a newly discovered terpenoid. As a pure natural ingredient, it has excellent bioactivity in lowering triglyceride levels. Therefore, compound 1 has the potential to be developed as a new drug or health product for the treatment of hyperlipidemia and hypertriglyceridemia.

Claims

1. A terpene compound 1, characterized in that: Its chemical structure is shown in the figure below: The chemical name is: (6S,6aS,9aR,Z)-6,8,8-trimethyl-6,6a,7,8,9,9a-hexahydroazuleno[5,4-c]furan-3,5(1H,4H)-dione.

2. A terpene compound according to claim 1, characterized in that: Prepared by the following steps: <1> Strain activation: Select the part of the Irpex lacteus parent strain plate with uniform and clear mycelium without obvious bacterial contamination, and place it on the PDA agar plate. After inoculation, place the daughter plate with mycelium in a constant temperature incubator and observe the growth status of the strain after culturing at 28℃ for 7 days. The mycelium is clear, without any different color or odor. <2> Strain fermentation: Take the activated mycelium and inoculate it into Fungus No. 2 liquid culture medium, and culture it at room temperature for 25 days. The formula of Fungus No. 2 culture medium is: 20g / L maltose, 10g / L monosodium glutamate, 0.5g / L potassium dihydrogen phosphate, 0.3g / L magnesium sulfate, 10g / L glucose, 3g / L yeast extract, 1g / L corn flour, and 20g / L mannitol. <3> Preparation of crude extract of white rake tooth fungus: after the fermentation of white rake tooth fungus is completed, the mycelium is crushed and combined with the fermentation liquid, extracted with 2 volumes of ethyl acetate for 3-5 times, and the extract is concentrated under reduced pressure to obtain the crude extract of white rake tooth fungus. <4> Normal phase column chromatography separation of the crude extract of white rake tooth fungus: The crude extract of white rake tooth fungus was separated by column chromatography using a normal phase column chromatography method through the following gradient elution program, that is, a petroleum ether-ethyl acetate mixture with a volume ratio of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, and 0:1 was used for elution, and the elution volume of each gradient was 5 L. The elution position of the mixed solvent with a volume ratio of petroleum ether to ethyl acetate of 4:1 was fraction F4. <5> Normal phase column chromatography separation of the crude extract of Albizia odorifera: the F4 fractions were separated in the order of elution, with each 1 L being one fraction, namely F4.1, F4.2, F4.3, F4.4 and F4.

5. <6> Semi-preparative liquid chromatography separation and preparation of compounds: The compounds in fraction F4.5 were separated by semi-preparative liquid chromatography to obtain compound 1.

3. A terpene compound 1 according to claim 1, characterized in that: The liquid phase preparation conditions were as follows: detection wavelength was 225 nm, flow rate was 4 mL / min, and volume ratio of methanol to water was 29:

21.

4. A terpene compound according to claim 1, characterized in that: The compound 1 is used in the preparation of therapeutic and preventive drugs or health products for lowering blood lipids and triglycerides.

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