LU502206B1 - Lactarazulene, preparation method and its application - Google Patents

Abstract

The invention relates to the technical field of natural extracts, in particular to lactarazulene, its preparation method and application. It is extracted from Lactarius hatsudake of Russulaceae family, and has excellent anti-inflammatory activity, which can be used to prepare anti-inflammatory drugs.

Description

DESCRIPTION LU502206 Lactarazulene, preparation method and its application

TECHNICAL FIELD The invention relates to the technical field of natural extracts, in particular to lactarazulene, its preparation method and application.

BACKGROUND Lactarius hatsudake is a kind of edible and medicinal fungus belonging to Russulaceae and Lactarius, which is widely distributed in Hunan, Anhui, Fujian, Sichuan, Hubei and Guangxi province. Lactarius hatsudake contains polysaccharides, terpenes, sterols, polyphenols, fatty acids and other bioactive components. Terpenoids are hydrocarbons and their derivatives with a multiple of isoprene in nature. Terpenoids are named and classified according to the number of isoprene, such as monoterpenes (10 carbons), sesquiterpenes (15 carbons), diterpenes (20 carbons), disesquiterpenes (25 carbons), triterpenes (30 carbons) and tetraterpenes (35 carbons). Common terpenoids include monoterpene borneol and camphor in Lauraceae, sesquiterpene artemisinin in Compositae, diterpene vitamin A in animal liver and triterpene ganoderic acid in edible fungi. Terpenoids have anti-inflammatory, anti-tumor, anti-oxidation and antibacterial activities, which have great development and utilization value in the pharmaceutical industry.

Inflammation is a response to damaged local tissues, characterized by dysfunction, pain, fever, redness and swelling, etc. Usually related to pathogen diseases, such as diabetes, arthritis, obesity, metabolic syndrome, cancer and cardiovascular diseases. Inflammation iNOS caused by inflammatory factors that stimulate cells (such as macrophages and monocytes) to secrete a large number of inflammatory mediators such as interleukin (IL-1P, IL-6 and IL-8), tumor necrosis factor (TNF-0), nuclear factor-kB (NF-kB), intercellular adhesion molecule -1(ICAM-1), and cyclooxygenase-2 (Cox-2). Common inflammatory factors include biological factors such as bacteria, viruses and fungi, physical factors such as high temperature and ultraviolet rays, chemical factors such as acid, alkali and toxins, and foreign bodies entering the body and tissue necrosis, which can induce the occurrence of inflammatory reaction.

Clinical anti-inflammatory drugs include steroid anti-inflammatory drugs and non-steroidal anti-inflammatory drugs. Steroid anti-inflammatory drugs have strong anti-inflammatory effect, but they have side effects such as water and sodium retention, puffiness, infection risk and osteoporosis. Non-steroidal anti-inflammatory drugs (NSAIDs) are mainly used to treat chronké/502206 inflammation, but they can also damage gastrointestinal tract, kidney, liver and cardiovascular system, and may also cause rash and asthma. Therefore, anti-inflammatory drugs with good anti-inflammatory effect, low side effects and low price should be developed to meet the market and clinical needs. Studying the biological activity of terpenoids in Lactarius hatsudake can not only increase the added value of Lactarius hatsudake products and promote local economic development, but also have great significance for the development of medicines and health products.

In view of this, the present invention is proposed.

SUMMARY The purpose of the invention is to provide lactarazulene, its preparation method and application. The embodiment of the invention provides lactarazulene, which is extracted from the genus Lactarius (family Russulaceae, class Basidiomycotina) , has excellent anti-inflammatory activity, and can be used for preparing anti-inflammatory drugs.

The invention is realized as follows: In the first aspect, the invention provides a lactarazulene, the structural formula of which is as follows: In the second aspect, the present invention provides a preparation method of lactarazulene according to the aforementioned embodiment, which comprises: extracting Lactarius hatsudake containing the lactarazulene, to form the lactarazulene,.

In the third aspect, the present invention provides the application of the Lactarius hatsudake in the preparation of anti-inflammatory drugs.

In the fourth aspect, the invention provides the application of the lactarazulene described in the previous embodiment in preparing an inhibitor for inhibiting or reducing any of the following conditions: (1) the mRNA expression of 11-6, IL-1, TNF-a and iNOS;

(2) The expression of Cox-2, IL-1, IL-6, iNOS and TNF-a protein; LU502206 (3) Phosphorylation levels of P44/42, p38 and JNK; (4) Activation of MAPKs inflammatory pathway.

The invention has the following beneficial effects: the embodiment of the invention provides a novel structure of lactarazulene extract, which is extracted from Lactarius hatsudake and has excellent anti-inflammatory activity, and then can be used for preparing anti-inflammatory drugs, further expanding the types of anti-inflammatory drugs in natural extracts and expanding the application of Lactarius hatsudake.

BRIEF DESCRIPTION OF THE FIGURES In order to explain the technical scheme of the embodiments of the present invention more clearly, the following drawings that need to be used in the embodiments will be briefly introduced. It should be understood that the following drawings only show some embodiments of the present invention, so they should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained according to these drawings without any creative effort.

Fig. 1 is the preparation liquid chromatogram (a) and purity analysis chromatogram (b) of lactarazulene provided by the embodiment of the present invention; Fig. 2 is the UV-visible spectrum of lactarazulene provided by the embodiment of the present invention; Fig. 3 is the infrared spectrum of lactarazulene provided by the embodiment of the present invention; Fig. 4 is the mass spectrum of lactarazulene provided by the embodiment of the present invention; Fig. 5 is the nuclear magnetic resonance spectrum of lactarazulene provided by the embodiment of the invention; Fig. 6 is a combined carbon spectrum of lactarazulene provided by the embodiment of the present invention; Fig. 7 is the HPLC analysis diagram provided by the comparative example of the present invention;

Fig. 8 is a graph showing the effect of lactarazulene on macrophage activity provided 11502206 Experimental Example 1 of the present invention; Fig. 9 is a graph showing the effect of lactarazulene provided in Experimental Example 2 of the present invention on LPS-induced macrophage inflammatory factor mRNA expression, in which (a) IL-6 mRNA; (B) IL-IB mRNA; (C) TNF-a mRNA; (D) iNOS mRNA; Fig. 10 is a graph showing the effect of lactarazulene provided in Experimental Example 3 of the present invention on the expression level of inflammatory factor protein in macrophages induced by LPS; Among them, (A)COX-2 protein; (B)IL-1ß protein; (C)INOS protein; (D)TNF-a protein; (E)IL-6 protein; Fig. 11 is a graph showing the effect of lactarazulene provided in Experimental Example 4 of the present invention on LPS-induced phosphorylation of MAPKs in macrophages; Among them, (A)p-JNK protein; (B)p-p38 protein; (C)p-p44/42 protein.

DESCRIPTION OF THE INVENTION In order to make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below. If the specific conditions are not indicated in the examples, the conventional conditions or the conditions suggested by the manufacturer shall be followed. The reagents or instruments used, if the manufacturer is not indicated, are conventional products that can be purchased on the market.

The invention provides a lactarazulene, the molecular formula of which is CisHis, and the structural formula of which is as follows: The present invention provides a preparation method of lactarazulene in the previous embodiment, which comprises the following steps: extracting Lactarius hatsudake containing the lactarazulene and to form the lactarazulene.

Specifically, the Lactarius hatsudake containing the lactucazulene is a damaged Lactarius hatsudake of Russulaceae, and the inventor guessed that the lactucazulene is a compound that can only be formed after the Lactarius hatsudake is damaged in order to repair the damaged part/502206 or a stress reaction is generated after the damage. In a word, whatever the reason, it is necessary for Lactarius hatsudake of Russulaceae family to be damaged or damaged before it can be extracted. If Lactarius hatsudake of Russulaceae family is not damaged, the lactucazulene can't be detected, and then it can't be extracted. Wherein, the Lactarius of Russulaceae can be Lactarius hatsudake or Lactarius aurantiacus.

Of course, it can be understood that if the damaged fruiting bodies of Lactarius or fungi of other families and genera can contain lactucazulene, the damaged fruiting bodies of Lactarius or fungi of other families and genera can also be extracted, and if the fruiting bodies of Lactarius or fungi of other families and genera directly contain lactucazulene, they can also be directly extracted.

Specifically, the steps of preparing Lactarius hatsudake containing the lactucazulene include: crushing freshly picked Lactarius hatsudake of Russulaceae, and standing for more than 2 hours; the Lactarius hatsudake of Russulaceae family needs to be freshly picked, and the freshly picked Lactarius hatsudake of Russulaceae family needs to be crushed, that is, the Lactarius hatsudake of Russulaceae family is damaged, and then the lactarazulene can be formed, so that the substance can be extracted in the subsequent extraction process. However, standing for more than 2 hours can make the crushed or damaged Lactarius hatsudake of Russulaceae family fully form lactarazulene, which is beneficial to improve the yield of lactarazulene.

The standing time is preferably 3-4 hours, although the standing time can be more than 2 hours, even up to 2 days, such as 2 hours, 3 hours, 4 hours, 10 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, etc. However, if the standing time is too long, the components in the fruiting body of Lactarius hatsudake of Russulaceae family may change, which will affect the subsequent extraction effect.

After standing, the Lactarius hatsudake of Russulaceae was further pulverized, and the main purpose of the pulverization here was to improve the dissolution of Lactarius hatsudake extract, and then improve its extraction efficiency.

Then extracting with organic solvent to obtain extract, wherein the organic solvent is ktJ502206 least one of ketone solvent (such as acetone), alcohol solvent (such as methanol) and halogen substituted methane solvent (such as chloroform).

It can be understood that the above acetone, methanol and chloroform are only examples of corresponding types of solvents. As long as the lactarazulene can be extracted, other solvents of the same type, such as ethanol and dichloromethane, and even other types of solvents, such as cyclohexane, can be used.

Specifically, acetone is mixed with the Lactarius hatsudake containing the lactarazulene for multiple times of extraction; Wherein the temperature of each extraction is room temperature (25-30°C) and the extraction time is 20-36 hours, for example, any value between 20-36 hours such as 20 hours, 24 hours, 30 hours, 32 hours, 35 hours and 36 hours.

Mixing the mixed solvent formed by methanol and chloroform with the Lactarius hatsudake containing the lactarazulene extract for multiple times; the extraction temperature is room temperature (25-30°C) and the extraction time is 20-36 hours, for example, any value between 20-36 hours such as 20 hours, 24 hours, 30 hours, 35 hours and 36 hours, and the volume ratio of methanol to chloroform is 1:1-1:2, for example, 1:1, 1:1.2.

It should be noted that: (1) The extracted Lactarius hatsutak containing the lactarazulene extract can be a new Lactarius hatsutak which has not been extracted, or the above Lactarius hatsutak which has been extracted with acetone.

(2) The times of multiple extractions and extractions can be 1, 2 or even 3 times.

(3) The above-mentioned extraction by acetone extraction, mixed solution of methanol and chloroform is only an example, and is not limited to the extraction solvent or method.

Then, mix the extract obtained by multiple extraction, that is, an extraction mixed solution. It can be understood that it is also possible to combine the leaching solutions obtained by each extraction, combine the extraction solutions obtained by each extraction, and then combine the two solutions to form an extraction mixture.

Then, concentrate the extract and remove the solvent in the extract to form a crude extract; then, the crude extract is extracted, and the extracted organic solvent part is collected.

Specifically, the extraction steps include: extracting with the mixed solvent of ester solveht/502206 and water, and collecting the ester part, that is, the organic solvent part; Wherein the ester solvent comprises any one of ethyl acetate, methyl acetate, ethyl formate and methyl formate; the volume ratio of the solvent and water is 1: 1-1: 2; For example, 1:1, 1:1.2, 1:1.5, 1:1.9, 1:2, etc. are any values between 1: 1 and 1: 2.

Then, the ester part is separated by column chromatography, in which ether solvent (such as petroleum ether) is used for elution, and the eluent is collected, and then the eluent is separated and purified by liquid chromatography, wherein the conditions of liquid chromatography separation and purification preparation include: the mobile phase includes alcohol solvent (such as pure methanol solution of analytical grade).

The embodiment of the invention also provides an application of Lactarius hatsudake which can inhibit the mRNA expression of IL-6, IL-1B, TNF-a and iNOS, reduce the expression of COX-2, IL-1B, IL-6, iNOS and TNF-a protein, reduce the phosphorylation level of p44/42, p38 and JNK, and then inhibit the inflammatory pathway of MAPKs.

The features and performances of the present invention will be further described in detail with reference to the following examples.

Example 1 The embodiment of the invention provides a preparation method of lactarazulene , which comprises the following steps: (1) 1.3 kg of fresh Lactarius hatsudake fruiting body was ground with a tissue crusher, and then stood at room temperature for 3 hours; (2) Further crushing the ground fruiting body of Lactarius hatsudake in the above step (1), then extracting with acetone (0.5 L) at room temperature for 3 times, each time for 24 hours, combining the extractive solutions of each acetone extraction, and continuously extracting the extracted fruiting body of Lactarius hatsudake with the mixed solution of methanol and chloroform with the volume ratio of 1: 1 (0.5 L) for 2 times, each time for 24 hours, collecting and combining each extraction. Then, the above extracts and extractive solutions are combined to form an extract, then, organic solvents such as acetone, methanol and chloroform are removed by rotary evaporation to obtain a crude extract, the crude extract is completely dissolved in ethyl acetate, the same volume of water is added into the ethyl acetate solution in which the crude extract is dissolved to extract for three times, the water phase is discarded, the ethyl acetate part/502206 is collected, and the ethyl acetate part is evaporated in vacuum to obtain an ethyl acetate extract.

(3) Weigh 40 times the loading amount of 200-300 mesh silica gel (150 g), add petroleum ether (0.5 L), stir into bubble-free homogenate, add the silica gel homogenate into the glass chromatographic column with the piston opened, after the silica gel settles, continue to wash with petroleum ether until the liquid level at the top of the column is stable and the chromatographic column has no bubbles, and then load the sample. Dissolve the ethyl acetate extract (5 g) obtained in the above step (2) with petroleum ether (25 mL), drop it onto the top of silica gel column with a dropper, elute with petroleum ether, and collect the blue fraction.

(4) Dissolve the blue fraction in methanol, and prepare and purify it by HPLC with pure methanol as mobile phase. The preparation conditions of liquid phase are as follows: the mobile phase is pure methanol solution, the flow rate is 10 mL/min, the injection concentration is 10 mg/mL, the injection volume is 2 mL, and the detection wavelength is 289 nm. The blue target compound was obtained, see A in Figure 1.

Purity detection: pure methanol as mobile phase, ODS C18 column (4.6 mmx250 mm, 5 um) as analytical column, wavelength of 289 nm, flow rate of 1 mL/min. The detection result is shown in Figure 1, B, and the purity is 96.7%.

The results of the characterization of lactarazulene provided by the embodiment of the invention are shown in Figure 2- Figure 6, wherein Figure 2 is an ultraviolet-visible spectrum; Figure 3 shows the infrared spectrum; Fig. 4 is a mass spectrum diagram; Fig. 5 is a nuclear magnetic resonance spectrum; Figure 6 shows the NMR spectrum. According to Fig. 2- Fig. 6, the characterization data are as follows: there are infrared characteristic absorption peaks at 1523 and 1421 cm-1, indicating that the compound contains C=C double bond, and there is an absorption peak at 2924 cm-1, which contains -CH3; M/Z: 197 [M+H]+,with a molecular weight of 196. It is speculated that its molecular formula is: C15H16;1H-NMR (DMSO-d6, 400 MHz):

8.36 (1H, d, J = 2.0 Hz, H-8), 7.69 (1H, dd, J = 10.9, 2.0 Hz, H-6), 7.64 (1H, d, J = 3.8 Hz, H-2),

7.30 (1H, d, J = 3.8 Hz, H-3), 7.07 (1H, d, J = 10.9 Hz, H-5), 5.37 (1H, br s, H-13a), 5.20 (1H, br s, H-13b), 2.79 (3H, s, H-14), 2.62 (3H, s, H-15), 2.26 (3H, br s, H-12);13C-NMR (DMSO-d6, 100 MHz): 146.4 (s, C-4), 145.1 (s, C-11), 136.9 (s, C-7), 136.5 (d, C-8), 135.0 (s, C-10), 134.0

(d, C-6), 132.8 (s, C-9), 131.7 (d, C-5), 127.1 (s, C-1), 124.5 (d, C-2), 114.4 (t, C-13), 113.8 (4502206 C-3), 23.5 (q, C-14), 22.9 (q, C-12), 12.8 (q, C-15).

Based on all the above spectral results, it was finally determined that the blue compound was guaiacane sesquiterpene, and the monomer was a blue solid compound, which was easily decomposed by light, and its structural formula was as follows: .

Comparative Example 1: The preparation method of Example 1 was used for extraction and separation and purification, with the only difference: the step (1) was not carried out, that is, fresh Lactarius hatsudake fruiting body was ground and then further ground, and then acetone was directly used for extraction. However, the steps and operations were the same as those of Example 1.

In step (3), when petroleum ether is used for column chromatography elution, the collected fractions do not contain the lactarazulene of the embodiment of the invention. Specifically, the results of high performance liquid chromatography are shown in Figure 7, in which the undamaged samples of Sample 1 (damaged) and Sample 2 (undamaged) show that the extraction of undamaged fresh Lactarius hatsudake fruiting body cannot be separated to obtain the lactarazulene and the damaged fresh Lactarius hatsudake fruiting body needs to be extracted to obtain the required one.

Example 1 MTS experiment The MTS experiment was used to determine the effect of different concentrations of Lactarius hatsudake on the activity of macrophage RAW264.7. The specific experimental methods are as follows: Macrophages RAW264.7 in logarithmic phase were made into uniform suspension with trypsin, and the concentration was adjusted to 6 x104 cells /mL. 100 pL of cell suspension was inoculated into 96-well plate, and cultured in 5% CO2 incubator at 37°C for 24 hours. The control group was added with solvent control, and the experimental group was added with Example 1 with final concentrations of 25 pg/mL, 50 ug/mL and 100 pg/mL. In each group, five wells were used for repeated 3 times. After 24 hours of culture, 20 uL MTS was added. After 2 hours of incubation in the dark, 20 uL 10% SDS was added to stop the reaction. The OD value 681502206 each well was determined by microplate reader.

The experimental results are shown in Fig. 8. According to Fig. 8, it can be seen that different concentrations of Lactarius hatsudake have no obvious toxicity to macrophage RAW264.7, so follow-up experiments can be carried out.

Example 2 The expression of inflammatory factor mRNA was detected by PCR.

The specific experimental method is as follows: Macrophages with a concentration of 106 cells /mL are cultured in an incubator with 5% CO2 at 37°C for 24 hours, while the control group is not treated. The experimental group is added with LPS and lactarazulene (25 pg/mL, 50 ug/mL and 100 pg/mL), while the LPS model group is added with LPS with a final concentration of 1 pg/mL. Continue reverse transcription of RNA and then carry out PCR reaction. The reaction conditions are pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 60°C for 40 s, extension at 72°C for 1 min, and finally circulation for 40 minutes. According to 2-CT (RQ), the mRNA expression of inflammatory factors was calculated.

The results are shown in Fig. 9. According to Fig. 9, different concentrations of lactarazulene (25 pg/mL, 50 pg/mL and 100 pg/mL) can significantly reduce the expression of inflammatory factors IL-6, IL-1ß, TNF-à and iNOS mRNA, and all of them are concentration-dependent.

Example 3 Western blot was used to detect the expression of protein.

The specific experimental method is as follows: Macrophages with a concentration of 106 cells /mL were cultured in an incubator with 5% CO2 at 37°C for 24 hours, while the control group was not treated. LPS and lactarazulene (25 ng/mL, 50 ng/mL and 100 ug/mL) were added to the experimental group, and LPS with a final concentration of 1 ng/mL was added to the LPS model group.

The results are shown in Fig. 10. According to Fig. 10, different concentrations of lactarazulene (25 pg/mL, 50 pg/mL and 100 pg/mL) have obvious inhibitory effects on protein expression of inflammatory factors COX-2, TNF-a and iNOS, and they are concentration-dependent, while 100 pg/mL lactarazulene has an effect on IL.

Example 4 Effect of lactarazulene on MAPKs signaling pathway of LPS-induced macrophages

Specific experimental methods: LU502206 Take macrophages in good growth state and in logarithmic growth phase, wash them twice with D-Hanks buffer, and after sucking up the D-Hanks solution, digest the macrophages with

0.25 mL trypsin digestive solution with a concentration of 0.25% to make them a uniform and stable single cell suspension. Adjust the concentration of cell suspension to about 106 cells /mL with a micro spectrophotometer, and inoculate the cell suspension into each culture dish. Each culture dish contains 8 mL of mixed solution of cells and culture medium, and the mixed solution is placed in a 37°C, 5%CO2 incubator for 12 -16 hours. After the cells adhere to the wall, the old culture medium is dumped and fresh culture medium is added. The blank control group did not add any drugs, while the experimental group first added different concentrations of lactarazulene to the final concentration of 25 pg/mL, 50 pg/mL and 100 ug/mL. After 4 hours of culture, LPS was added to the final concentration of 1 pg/mL, while LPS model group only contained lipopolysaccharide with a concentration of 1 pg/mL. 1 mL of phosphatase inhibitor and RIPA lysate were mixed and completely dissolved to prepare inhibitor working solution with concentration of 10 mmol/L.. Remove the old culture medium from the culture dish, wash the old culture medium with phosphate buffer twice, add 0.3 mL of pre-cooled phosphorylated protein extract RIPA protein lysate (final concentration: 1 mmol/l of phospholipase inhibitor, 1 mmol/L of PMSF and 10% of protease inhibitor Cocktail) into each culture dish, transfer all cells into a 1.5 mL centrifuge tube on ice, You can use vortex oscillation or pipetting gun to blow the cells repeatedly to help them crack. After the cells are cracked on ice for 30 min, centrifuge (rotating speed is 13000 r/min, centrifuging at 4°C for 20 min), and the supernatant obtained is the phosphorylated protein of the cells. The concentration of phosphorylated protein was measured and the expression of protein was analyzed by Western blot.

The results are shown in Fig. 11. According to Fig. 11, different concentrations of lactarazulene (25 pg/mL, 50 pg/mL and 100 pg/mL) can affect the expression of JNK phosphorylated protein, and when the concentration of lactarazulene reaches 100 ug/mL, the expression of JNK phosphorylated protein decreases obviously. The inhibitory effect of different concentrations of lactarazulene on p-p38 protein expression was almost the same. The concentration of 50 and 100 pg/mL lactarazulene can obviously regulate the decrease of p44/42 phosphorylated protein in macrophages stimulated by LPS.

To sum up, the lactarazulene provided by the embodiment of the invention MAPKs n&/502206 toxicity to normal macrophages, and can inhibit the mRNA expression of IL-6, IL-1, TNF-a and iNOS, reduce the expression of COX-2, IL-1, IL-6, iNOS and TNF-a protein, and reduce the phosphorylation level of p44/42, p38 and JNK. It shows that it has obvious inhibitory effect on LPS-induced inflammatory reaction of macrophage RAW264.7, which proves that lactarazulene has good anti-inflammatory activity and has the development potential as an anti-inflammatory drug.

The above is only the preferred embodiment of the present invention, and it is not intended to limit the present invention. For those skilled in the art, the present invention can be modified and varied. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of protection of the present invention.

Claims (10)

1. A lactarazulene, characterized in that its structural formula is as follows:

2. The preparation method of lactarazulene according to claim 1, which is characterized by comprising: extracting Lactarius hatsudake containing the lactarazulene to form the lactarazulene.

3. The preparation method according to claim 2, characterized in that the Lactarius hatsudake containing the lactarazulene belongs to Russulaceae, preferably, the Lactarius of Russulaceae includes Lactarius aurantium or Lactarius hatsudake; preferably, the Lactarius hatsudake containing the lactarazulene is damaged Lactarius hatsudake of Russulaceae.

4. The preparation method according to claim 2, characterized in that the step of preparing Lactarius hatsudake containing the lactarazulene extract comprises: crushing freshly picked Lactarius hatsudake of Russulaceae, and standing for more than 2 hours; Preferably, the standing time is 3-4 hours.

5. The preparation method according to claim 2, characterized in that the extraction step comprises: extracting the Lactarius hatsudake containing the lactarazulene with an organic solvent, and then sequentially concentrating, extracting, separating and purifying the obtained extract by column chromatography and liquid chromatography.

6. The preparation method according to claim 5, characterized in that the organic solvent is selected from at least one of ketone solvent, alcohol solvent and halogen substituted methane solvent; preferably, the step of obtaining the extract comprises: extracting the Lactarius hatsudake containing the lactarazulene with at least one solvent selected from acetone, methanol and chloroform;

preferably, the step of obtaining the extract comprises: mixing acetone with Lactariks)502206 hatsudake containing the lactarazulene for multiple times; Mixing the mixed solvent formed by methanol and chloroform with the extracted lactarazulene, and extracting for several times; then combining the extractive solution obtained by the extraction and the extractive solution obtained by leaching; preferably, the volume ratio of methanol to chloroform is 1:1-1:2.

7. The preparation method according to claim 5, characterized in that the extraction step comprises: extracting with a mixed solvent of ester solvent and water, and collecting the ester part, preferably, the ester solvent includes any one of ethyl acetate, methyl acetate, ethyl formate and methyl formate; preferably, the volume ratio of ester solvent to water is 1: 1-1: 2; preferably, the steps of column chromatography include: eluting with ether solvent; preferably, the ether solvent comprises petroleum ether; preferably, the conditions of liquid chromatography separation and purification preparation include: the mobile phase includes alcohol solvent, preferably methanol solvent.

8. Application of Lactarius hatsudake extract according to claim 1 in preparation of anti-inflammatory drugs.

9. The application according to claim 8, characterized in that the drug is a drug that inhibits the expression of mRNA of IL-6, IL-1, TNF-a and iNOS; preferably, the drug is a drug that reduces the expression of COX-2, IL-1, IL-6, iNOS and TNF-a protein, preferably, the drug is a drug that reduces the phosphorylation level of p44/42, p38 and JNK; preferably, the drug is a drug that inhibits the activation of MAPKs inflammatory pathway.

10. The application of lactarazulene according to claim 1 in preparing an inhibitor for inhibiting or reducing any of the following conditions: (1) the mRNA expression of 11-6, IL-1, TNF-a and iNOS; (2) the expression of Cox-2, IL-1, IL-6, iNOS and TNF-a protein; (3) phosphorylation levels of P44/42, p38 and JNK;

(4) activation of MAPKs inflammatory pathway.

LU502206