Sesquiterpenoids as well as preparation method and application thereof

Abstract

The invention discloses a sesquiterpenoids compound and a preparation method and application thereof, the sesquiterpenoids compound has a structure as shown in a formula I, and the sesquiterpenoids compound is separated from pogostemon esquirolii of a labiatae thesium plant. The sesquiterpenoids provided by the invention can be used for inhibiting the generation of nitric oxide induced by LPS (Lipopolysaccharide), and also can be used for inhibiting the expression of inflammatory factors IL-6, TNF-alpha, COX-2 and iNOS; the sesquiterpenoids can also inhibit activation of an NF-kappa B pathway and an MAPK pathway induced by LPS, and finally exert an excellent anti-inflammatory effect. The sesquiterpenoids provided by the invention are extracted from a natural plant thesium membranaceum, have small cytotoxicity, can be used for preparing new anti-inflammatory drugs, and provide molecular reserve and scientific basis for research and development of the anti-inflammatory drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a sesquiterpene compound and a preparation method and application thereof. BACKGROUND

[0002] Pogostemon esquirolii, also known as Jiguantoucai (Malipo, Yunnan), Panuapa (Honghe, Yunnan), Yelianjin (Tianlin, Guangxi), is a plant of the genus Pogostemon of the Lamiaceae family. It is mainly distributed in the south and southeast of Yunnan, Guangxi, Guizhou, Guangdong and Hainan. According to records, the branches and leaves of Pogostemon esquirolii can be used as medicine to treat uterine prolapse. At present, there are about 80 species of Pogostemon plants in the world, among which 11 species have been reported, including Pogostemonis, Shui Zhencaicai, Pogostemonis, Pogostemonis, Pogostemonis (P.glaber), P.purpurascens, P.parviflorus, P.benghalense, P.quadrifolius, P.plectranthoides, etc. A variety of natural active ingredients have been identified from them. However, at present, there are few reports on Pogostemon esquirolii, and its chemical composition and biological activity research is not sufficient, and further research is needed.

[0003] Inflammation is a common self-defense mechanism against infection and to avoid tissue damage. Although the inflammatory response is to start the body's immune system to defend against disease, excessive response is also an important cause of many diseases, such as sepsis, systemic reaction syndrome and septic shock, etc. Therefore, anti-inflammation plays an important role in the prevention and treatment of diseases. Sesquiterpenes are derived from active precursors, pyrophosphate acyl, by different position double bond shift or ring closure, which are terpenes composed of 3 isoprene units (15 carbon atoms) and widely distributed in the plant kingdom, such as Magnoliaceae, Rutaceae, Asteraceae, etc. The skeleton structure of sesquiterpenes is complex, with more than 200 types, and there are dozens of common parent carbon skeleton types. This kind of compounds has multiple biological activities, including anti-inflammatory, anti-tumor and immune regulation, etc. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes a sesquiterpene compound, which can inhibit the secretion of inflammatory factors and thus play an anti-inflammatory role.

[0005] The present application also proposes a preparation method of the above-mentioned sesquiterpene compound.

[0006] The present application also proposes a pharmaceutical composition.

[0007] The present application also proposes an application.

[0008] According to a first aspect of the present application, a sesquiterpenoid compound is provided, which has a structure as shown in Formula I:

[0009]

[0010] In some embodiments of the present application, the sesquiterpenoid compound is isolated from Pogostemon esquirolii of Lamiaceae.

[0011] According to a second aspect of the present application, a preparation method of the sesquiterpenoid compound according to the first aspect of the present application is provided, which comprises the following steps:

[0012] S1: taking dried Pogostemon esquirolii whole plant to extract with 85% to 100% ethanol, and combining the extract to obtain extract concentrate; dissolving the extract concentrate in water and sequentially extracting with petroleum ether, ethyl acetate and n-butanol to collect the petroleum ether layer;

[0013] S2: taking the petroleum ether layer to perform column chromatography separation, and performing gradient elution with n-hexane-ethyl acetate as eluent, and collecting 11 components PA1-PA11;

[0014] S3: taking component PA6 obtained in step S2 to perform column chromatography separation, and performing gradient elution with methanol aqueous solution as eluent, and collecting 3 components PA6B1-PA6B3;

[0015] S4: taking component PA6B2 obtained in step S3 to perform column chromatography separation, and performing gradient elution with n-hexane-ethyl acetate as eluent, and collecting 2 components PA6B2C1-PA6B2C2;

[0016] S5: taking component PA6B2C2 obtained in step S4 to perform column chromatography separation, and performing gradient elution with methanol aqueous solution as eluent, and collecting 6 components PA6B2C2D1-PA6B2C2D6;

[0017] S6: taking component PA6B2C2D3 obtained in step S5 to perform liquid chromatography separation, and performing isocratic elution with 20% to 30% acetonitrile aqueous solution, and collecting 3 components PA6B2C2D3E1-PA6B2C2D3E3;

[0018] S7: taking component PA6B2C2D3E1 obtained in step S6 to perform liquid chromatography separation, and performing isocratic elution with 40% to 60% acetonitrile aqueous solution to obtain the sesquiterpenoid compound Pogostesquines E.

[0019] In some embodiments of the present application, the column chromatography separation in step S2 employs normal phase silica gel column chromatography.

[0020] In some embodiments of the present application, the column chromatography separation in step S2 employs dry loading.

[0021] In some embodiments of the present application, the volume ratio of n-hexane-ethyl acetate in step S2 is in turn pure n-hexane, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, pure ethyl acetate.

[0022] In some embodiments of the present application, the column chromatography separation in step S3 employs small pore resin chromatography.

[0023] In some embodiments of the present application, the column chromatography separation in step S3 employs dry loading.

[0024] In some embodiments of the present application, the concentration of methanol aqueous solution in step S3 is in turn 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol, 100% methanol.

[0025] In some embodiments of the present application, the column chromatography separation in step S4 employs normal phase silica gel column chromatography.

[0026] In some embodiments of the present application, the column chromatography separation in step S4 employs dry loading.

[0027] In some embodiments of the present application, the volume ratio of n-hexane-ethyl acetate in step S4 is in turn 20:1, 15:1, 10:1, 8:1, 5:1, 2:1.

[0028] In some embodiments of the present application, the column chromatography separation in step S5 employs C18 reversed phase silica gel column chromatography.

[0029] In some embodiments of the present application, the column chromatography separation in step S5 employs wet loading.

[0030] In some embodiments of the present application, the concentration of methanol aqueous solution in step S5 is in turn 50% methanol, 60% methanol, 70% methanol.

[0031] In some embodiments of the present application, the liquid chromatography in step S6 comprises semi-preparative liquid chromatography.

[0032] In some embodiments of the present application, the liquid chromatography in step S7 comprises semi-preparative liquid chromatography.

[0033] According to a third aspect of the present application, a pharmaceutical composition is provided, which comprises the sesquiterpene compound according to the first aspect of the present application and a pharmaceutically acceptable excipient.

[0034] In some embodiments of the present application, the pharmaceutically acceptable excipient comprises at least one of a binder, a disintegrant, a lubricant, a coating agent, a suspending agent, a viscosity agent and a surfactant.

[0035] In some embodiments of the present application, the binder is at least one of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose or polyvinylpyrrolidone.

[0036] In some embodiments of the present application, the disintegrant is at least one of corn starch, potato starch, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose or alginic acid.

[0037] In some embodiments of the present application, the lubricant is at least one of microfine silica, magnesium stearate, calcium stearate, stearic acid, talc or anhydrous silica.

[0038] In some embodiments of the present application, the coating agent comprises at least one of hydroxypropyl methyl cellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, cellulose acetate phthalate, polyvinyl acetate phthalate, ethyl cellulose, cellulose acetate.

[0039] In some embodiments of the present application, the suspending agent comprises at least one of gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, hydroxymethyl cellulose or aluminum stearate gel.

[0040] In some embodiments of the present application, the surfactant is at least one of lecithin, sorbitan monooleate or glycerol monostearate.

[0041] According to a fourth aspect of the present application, the sesquiterpene compound according to the first aspect of the present application or prepared by the method according to the second aspect of the present application is used for preparing an anti-inflammatory drug.

[0042] In some embodiments of the present application, the anti-inflammatory drug comprises at least one of a NO inhibitor, an IL-6 expression inhibitor, a TNF-α expression inhibitor, a COX-2 expression inhibitor, an iNOS expression inhibitor, an NF-κB pathway inhibitor and a MAPK pathway inhibitor.

[0043] The present application has at least the following beneficial effects:

[0044] The sesquiterpene compound can inhibit LPS-induced nitric oxide production, can also inhibit expression of inflammatory factors IL-6, TNF-alpha, COX-2 and iNOS, can inhibit LPS-induced NF-kappa B pathway and MAPK pathway activation, and finally plays an excellent anti-inflammatory effect. The sesquiterpene compound is extracted from natural plant Memecylon leaf, has small cytotoxicity, can be used for preparing a new anti-inflammatory drug, and provides a molecular reserve and scientific basis for research and development of anti-inflammatory drugs. BRIEF DESCRIPTION OF DRAWINGS DETAILED DESCRIPTION OF THE INVENTION OF THE INVENTION BRIEF DESCRIPTION OF DRAWINGS

[0045] The application will be further described below in combination with the drawings and examples, in which:

[0046] Figure 1 The DEPT spectrum (CDCl3) of the sesquiterpene compound in Example 1 of the application is shown in Figure 6. 1 H NMR spectrum (CDCl3);

[0047] Figure 2 The DEPT spectrum (CDCl3) of the sesquiterpene compound in Example 1 of the application is shown in Figure 6. 13 C NMR spectrum (CDCl3);

[0048] Figure 3 The DEPT spectrum (CDCl3) of the sesquiterpene compound in Example 1 of the application is shown in Figure 6.

[0049] Figure 4 The DEPT spectrum (CDCl3) of the sesquiterpene compound in Example 1 of the application is shown in Figure 6.

[0050] Figure 5 The DEPT spectrum (CDCl3) of the sesquiterpene compound in Example 1 of the application is shown in Figure 6.

[0051] Figure 6 The DEPT spectrum (CDCl3) of the sesquiterpene compound in Example 1 of the application is shown in Figure 6. 1 H- 1 H COSY spectrum (CDCl3);

[0052] Figure 7 The DEPT spectrum (CDCl3) of the sesquiterpene compound in Example 1 of the application is shown in Figure 6. 1 H NMR spectrum (DMSO-d6);

[0053] Figure 8 The DEPT spectrum (CDCl3) of the sesquiterpene compound in Example 1 of the application is shown in Figure 6.

[0054] Figure 9 The DEPT spectrum (CDCl3) of the sesquiterpene compound in Example 1 of the application is shown in Figure 6.

[0055] Figure 10Figure for result of detecting NO production after giving sesquiterpenoids in Example 2 of the present application; wherein, A is for giving 20 μM, B is for giving 40 μM, 50 μM, 60 μM, 70 μM, 80 μM; compared with control group, ### P<0.001; compared with LPS group, * represents p<0.05, ** represents p<0.01, *** represents p<0.001;

[0056] Figure 11 Figure for result of cytotoxicity detection in Example 2 of the present application;

[0057] Figure 12 Figure for result of inflammation factor detection in Example 2 of the present application; wherein, A is for Western blot result, B and C are for statistical result; compared with control group, ## P<0.01, ### P<0.001; compared with LPS group, ** represents p<0.01, *** represents p<0.001;

[0058] Figure 13 Figure for result of inflammation related protein detection in Example 2 of the present application; wherein, A is for Western blot result, B and C are for statistical result; compared with control group, ## P<0.01, ### P<0.001; compared with LPS group, * represents p<0.05, ** represents p<0.01, *** represents p<0.001.

[0059] Figure 14 Figure for result of NF-κB signal path detection in Example 2 of the present application; wherein, A is for Western blot result, B is for statistical result; compared with control group, # P<0.0001; compared with LPS group, * represents p<0.05;

[0060] Figure 15 Figure for result of MAPK signal path detection in Example 2 of the present application; wherein, A is for Western blot result, B is for statistical result; compared with control group, ## P<0.01, ### P<0.001; compared with LPS group, * represents p<0.05, ** represents p<0.01, *** represents p<0.001. DETAILED DESCRIPTION

[0061] The concept and technical effects of the present application will be described clearly and completely in combination with the embodiments below, so as to fully understand the purposes, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0062] Separation, purification and structure identification of sesquiterpenoid Pogostesquines E

[0063] 1. Separation and purification of sesquiterpenoid Pogostesquines E

[0064] 1) Dry the whole grass of Membranous Pogostemone (20 kg) by a pulverizer, and soak it in 20 L of 95% ethanol for extraction for 3 times at room temperature. Collect and combine the extractive liquid, and get the extract 1252 g after spin-drying. Add 3 L of pure water to the extract for stirring and ultrasonic suspension. After sufficient suspension, use petroleum ether, ethyl acetate and n-butanol for 5 times of extraction in sequence, and get 281 g of petroleum ether layer (P layer), 101.5 g of ethyl acetate layer (E layer) and 230 g of n-butanol layer (n-Bu layer). According to the previous research experience and the literature (“F Li, CJ Li, J Ma, et al. Four new sesquiterpenes from the stems of Pogostemon cablin [J]. Fitoterapia, 2013, 86: 183-187.”, “Li Fei. Chemical constituents and biological activity of Pogostemonis Herba and M. hancei [D]. Beijing: Beijing Union Medical College, 2013.”, “Li Xiaohong. Chemical constituents of Pogostemonis Herba and D. japonicus [D]. Sichuan: Chengdu University of Chinese Medicine, 2015.”), it is known that the sesquiterpenoid compounds of Pogostemone plants are mainly concentrated in the petroleum ether part, so the petroleum ether layer of Membranous Pogostemone is taken as the target for separation, purification and identification.

[0065] 2) Use the medium-pressure preparative liquid chromatography method, use 300-400 mesh normal phase silica gel filler to dry column, and then use an air pump to compact the silica gel powder after column packing. Then weigh 420 g of 100-200 mesh silica gel and mix it with the P layer 281 g for dry column loading. Use n-hexane-ethyl acetate for gradient elution, and the mixed volume ratio of the eluent is pure n-hexane, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, pure ethyl acetate in sequence, and a total of 11 components (PA1-PA11) are eluted.

[0066] 3) Dissolve the PA6 component obtained in step 2) in an appropriate amount of dichloromethane and dry load the sample solution. Add the sample solution to an appropriate amount of small pore resin (MCI) packing and stir until homogeneous. Allow the solvent to evaporate to obtain a sample layer. Add the sample layer to a chromatographic column packed with MCI packing. Use methanol-water solution as the eluent for gradient elution. The eluent ratios used are 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol, and 100% methanol, respectively. Elute the sample layer sequentially to obtain components PA6B1 (57.1 mg), PA6B2 (6.38 g), and PA6B3 (7.05 g).

[0067] 4) Take the component PA6B2 obtained in step 3), dissolve it in an appropriate amount of n-hexane-ethyl acetate, and load it onto a chromatographic column packed with 300-400 mesh normal phase silica gel. Use n-hexane-ethyl acetate as the eluent for gradient elution. The volume ratio of the eluent is n-hexane:ethyl acetate = 20:1, 15:1, 10:1, 8:1, 5:1, 2:1, to obtain two components PA6B2C1 to PA6B2C2 (4.80 g).

[0068] 5) Take the component PA6B2C2 obtained in step 4), dissolve it in an appropriate amount of methanol-water, load it onto a C18 SMB 100-20 / 45 reversed-phase column, and elute sequentially with 50%, 60%, and 70% methanol-water solutions to obtain components PA6B2C2D1 to PA6B2C2D6.

[0069] 6) Take 135.4 mg of component PA6B2C2D3 obtained in step 5) and analyze it using a semi-preparative HPLC system (Waters 1500-Series system, including a Waters 2489 UV detector, column type: [missing information]). C18OBD (250×10mm) was separated using liquid chromatography under the following conditions: isocratic elution with 25% acetonitrile aqueous solution for 28 min, and gradient elution with 25%–100% acetonitrile for 8 min. Chromatographic peaks were collected at 20.4–21.6 min, 21.6–22.6 min, and 32.4–34.4 min to obtain components PA6B2C2D3E1–PA6B2C2D3E3.

[0070] 7) Take 28.8 mg of component PA6B2C2D3E1 obtained in step 6) and analyze it using a semi-preparative HPLC system (Waters 1500-Series system, including a Waters 2489 UV detector, column type: [missing information]). The semi-terpenoid compound Pogostesquines E (19.2 mg) was obtained by separation on a C18 OBD (250x10 mm) column using the following liquid chromatography conditions: 50% acetonitrile in water isocratic elution, and collection of the chromatographic peak at 17 min, and the structure of Pogostesquines E is shown in formula I:

[0071]

[0072] 2. Structure analysis of Pogostesquines E:

[0073] Colorless solid, the HR-ESIMS quasi-molecular ion peak m / z 271.1305 [M+Na] + (calcd for C 15 H 20 O3Na 271.1305), the molecular formula of which was deduced as C 15 H 20 O3, with 6 degrees of unsaturation. The UV spectrum showed absorbance at λ max value of 285 nm, and the infrared spectrum showed characteristic absorption of one hydroxyl group (3422 cm -1 ) and two olefinic bonds (1722 cm -1 , 1709 cm -1 ). The nuclear magnetic signals were assigned by 1 H, 13 C, HSQC and DEPT, and the specific information is shown in Table 1, and the spectra are shown in Figures 1-4 .

[0074] Further analysis showed that Pogostesquines E has three methyl groups [δ H 1.06 (d, J = 6.9 Hz), δ C 21.4, 12-CH3; δ H 1.03 (d, J = 6.9 Hz), δ C 22.3, 13-CH3; δ H 0.9 (s), δ C 21.4, 14-CH3], one methylene group [δ H 3.41 (d, J = 18.8 Hz), 2.01 (dd, J = 18.8, 6.4 Hz), δ C 31.2, 9-CH2], one methine group [δ H 2.91 (p, J = 6.8 Hz), δ C 26.8, 11-CH], and one oxygen-containing methine group [δ H 3.65 (d, J = 5.3 Hz), δC 73.3, 1-CH], 1 quaternary carbon [delta C 42.2, 10-C], 1 oxygen-bearing quaternary carbon [delta C 76.6, 5-C], 3 double bonds [delta H 5.91 (dd, J = 9.6, 5.5 Hz), delta C 127.1, 2-CH; delta H 6.12 (d, J = 9.8 Hz), delta C 129.6, 3-CH; delta C 140.7, 4-C; delta H 6.01 (s), 5.35 (s), delta C 121.6, 15-CH2; delta C 143.5, 7-C; delta C 139.5, 8-C], 1 carbonyl [delta C 194.2, 6-C].

[0075] Table 1 Pogostesquines E 1 H NMR and 13 C NMR data (500 MHz, CDC13, delta in ppm)

[0076]

[0077]

[0078] According to Pogostesquines E 1 H- 1 H COSY spectrum can be known: H-8 / H-9, H-3 / H-2, H-1 / H-2, H-11 / H-12, H-11 / H-13, so C-8 and C-9 are connected, C-3 and C-2 are connected, C-1 and C-2 are connected, C-11 and C-12 are connected, C-11 and C-13 are connected.

[0079] Through the HMBC spectrum, Figure 5It can be seen that: H-3 is related to C-1 / C-2 / C-4 / C-5 / C-15; H-2 is related to C-3 / C-4 / C-10; H-1 is related to C-3 / C-14 / C-5 / C-10 / C-9; H-8 is related to C-7 / C-9 / C-6 / C-10 / C-11; H-15 is related to C-4 / C-3 / C-5; and H-11 is related to C-6. The structure of Pogostesquines E is related to C-7 / C-8 / C-12 / C-13, H-9 to C-5 / C-8 / C-7 / C-10 / C-14, H-14 to C-1 / C-5 / C-10 / C-9, H-11 to C-7 / C-6 / C-8 / C-12 / C-13, H-12 to C-7 / C-13, and H-13 to C-7 / C-12. Based on this information, the planar structure of Pogostesquines E is determined.

[0080] Pogostesquines E was obtained by dissolving it in DMSO-d6. 1 H NMR spectrum ( Figure 6 and Figure 7 ) and ROESY spectrum ( Figure 8 From the ROESY spectrum, it can be observed that H3-14 and H-1 have correlated signals, and H2-9α and OH-5 have correlated signals, so they are located in the same plane and are classified as α configuration; while OH-1 and H2-9β have correlated signals, so they are located in the same plane and are classified as β configuration.

[0081] Finally, to confirm the above-mentioned predictions regarding the relative configuration of Pogostesquines E, and to determine the absolute configuration of Pogostesquines E, a comparison was made between calculated and experimental ECD spectra, confirming its absolute configuration as 1S, 5S, 10S (e.g., Figure 9 (As shown).

[0082] Physicochemical properties of Pogostesquines E: colorless solid, [α] 2 D 5 =+67.9(c 0.01Acetone); UV(ACN)λ max (logε)238(4.41)nm; ECD(ACN)λ max (Δε)236(33.44)nm; IR(KBr)ν max 2958, 2972, 1723, 1267cm -1 ;HR-ESIMS m / z 271.1305[M+Na] + (calcd for 271.1305 for C) 15 H20 O3Na).

[0083] Investigation of Anti-inflammatory Activity of Sesquiterpenoid Pogostesquines E

[0084] 1. Preparation of the sample to be tested

[0085] A certain amount of Pogostesquines E was accurately weighed with an analytical balance, and according to its molecular weight, an appropriate amount of biomolecular grade DMSO was added to dissolve it and prepare a sample stock solution with a concentration of 100 mM, which was stored in the refrigerator for standby use.

[0086] 2. Cell culture and treatment

[0087] 1) Cell recovery:

[0088] The mouse microglial BV2 cells were taken out from the liquid nitrogen tank, then placed in a 37°C water bath and quickly shaken to dissolve; then the cell suspension was transferred to a centrifuge tube with an appropriate amount of DMEM medium, and centrifuged at 1000 rpm for 3 minutes; after centrifugation, the supernatant in the centrifuge tube was removed with a pipette; after adding 1 mL of medium, the cell suspension was dispersed by gently blowing the wall of the centrifuge tube, and then the cell suspension was transferred to a cell bottle, and 6 mL of DMEM medium containing 10% FBS + 1% penicillin-streptomycin was added, and the culture bottle was placed in a 37°C CO2 incubator for incubation.

[0089] 2) Cell passage:

[0090] Prepare new cell culture bottles, remove the culture bottle of cells cultured to 80%-90% density, and use a pipette to remove the old culture medium, then wash with PBS buffer for 1-2 times; then add trypsin and digest at 37°C, and shake appropriately to promote detachment; after the cells are partially detached, add DMEM medium containing 10% FBS + 1% penicillin-streptomycin to stop digestion, then gently blow the wall of the culture bottle with a pipette. After mixing the cells and medium, transfer them to a centrifuge tube and centrifuge at 1000 rpm for 3 min; add an appropriate amount of medium, mix well, and then evenly distribute them to the new culture bottles, and place them in a CO2 incubator for incubation.

[0091] 3) Cell plating:

[0092] According to the plating density, calculate the amount of diluted cell suspension, and supplement with medium; after mixing, evenly add the cell suspension to the 96-well plate, and finally place it in the incubator for 24 h.

[0093] 3. Detection of relative content of NO

[0094] The LPS-induced BV2 cell inflammation model was used for anti-inflammatory activity evaluation.

[0095] BV2 cells were used at a rate of 6 × 10 5 Seeds were inoculated at a density of [number] cells / mL in 96-well plates and cultured for 24 h to allow adhesion. Dexamethasone was used as a positive control. 100 μL of culture medium was added to each well of the blank control group (CON); 2 μg / mL LPS was added to each well of the negative control group; a mixed solution of 10 μM dexamethasone (DXMS) and 2 μg / mL LPS was added to each well of the positive control group; 20 μM of the test sample and 2 μg / mL LPS were added to each well of the experimental group. After drug addition, the plates were incubated in a conventional incubator for 24 h. 50 μL of supernatant was aspirated from each well, and 50 μL each of Griess Reagent I and II NO detection kits were added. After shaking for 10 min, the OD value was measured at 540 nm. The results are shown below. Figure 10 As shown in Figure A.

[0096] The effect of five different concentrations of Pogostesquines E (40 μM, 50 μM, 60 μM, 70 μM, and 80 μM) on NO production was then investigated, and the results are as follows: Figure 10 As shown in B.

[0097] Depend on Figure 10 It can be seen that Pogostesquines E can effectively reduce the NO content in the supernatant of BV2 cells, and its inhibitory effect is concentration-dependent.

[0098] 4. CCK-8 assay for cytotoxicity

[0099] Observe the growth density of BV2 cells. When the density reaches approximately 80%, digest the cells with trypsin, then centrifuge, resuspend, and count them. Then, seed the BV2 cells into 96-well plates at a density of 5 × 10⁶ cells / well. 3 Cells were cultured per well in DMEM medium containing 10% FBS and 1g penicillin-streptomycin at 37°C and 5% CO2 for 24 hours. After incubation for another 24 hours, 10μL of CCK-8 reagent was added to each well, and the cells were incubated for another 2 hours. OD values ​​were measured at 450nm using a microplate reader. Cytotoxicity assays included three groups: a blank group (medium medium + CCK-8 solution), a control group (medium medium + CCK-8 solution + cells), and an experimental group (medium medium + CCK-8 solution + cells + test sample). Cell viability was calculated as [(experimental group - medium) / (control group - medium)] × 100%, as shown in the figure. Figure 11 As shown.

[0100] Depend on Figure 11 It was found that Pogostesquines E showed no cytotoxicity below 80 μM. Further testing was conducted on the IC50 of Pogostesquines E.50 Pogostesquines E showed better anti-inflammatory activity in the LPS-induced BV2 cell inflammation model, with an IC 50 value of 84.17 ± 5.97 μM.

[0101] 5. Western blot was used to detect the expression of inflammatory factors and related proteins in the inflammation model

[0102] BV2 cells were seeded in 6-well plates (1 x 10 6 cells / well) and cultured for 24 h. The old culture medium was then removed and replaced with fresh culture medium containing different concentrations of Pogostesquines E (20 and 40 μM). After 2 h, 2 μL of LPS was added to each well, and the cells were incubated for 1 h. The 6-well plates were removed, the culture medium was removed, and the cells were washed three times with 3 mL of cold PBS, transferred to EP tubes, and lysed with 300 μL of RIPA buffer. The cells were incubated in an ice bath for 30 min and centrifuged at 4°C and 13,000 rpm for 15 min. The protein concentration was determined using a BCA protein assay kit, and the proteins were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with 5% skim milk for 2 h, and the milk was removed. The membrane was washed three times with TBST for 5 min each time. Then, the diluted primary antibody was added to the PVDF membrane, and the membrane was incubated at 4°C overnight. The primary antibody was recovered, and the membrane was washed three times with TBST for 5 min each time. After washing, the secondary antibody was added, and the membrane was incubated at room temperature for 90 min. The secondary antibody was recovered, and the membrane was washed three times with TBST for 5 min each time. The working concentration of the developing solution was prepared according to the instructions in the ECL reagent. The band gray value was analyzed using Image J software, and the results are shown in Figure 12 and Figure 13 .

[0103] Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are typical pro-inflammatory factors involved in various inflammatory responses.

[0104] As can be seen from Figure 12 , compared with the control group, the model group significantly increased the secretion of inflammatory factors IL-6 and TNF-α in BV2 cells after LPS induction (P < 0.001). After administration, Pogostesquines E significantly reduced the secretion of inflammatory factors IL-6 and TNF-α in LPS-induced BV2 cells compared with the model group.

[0105] Prostaglandins (PGs) are important lipid mediators of inflammation, formed from arachidonic acid catalyzed by cyclooxygenase (COX). Cyclooxygenase is divided into COX-1 and COX-2. COX-2 has very low activity in normal tissue cells, and when stimulated by external stimuli such as LPS, the expression level of COX-2 in the body will rise exponentially, leading to the release of a large amount of prostaglandin, inducing inflammatory response and tissue damage. Under inflammatory conditions, COX-2 and iNOS are often expressed simultaneously.

[0106] As can be seen from Figure 13 Pogostesquines E can significantly reduce the expression of inflammation-related proteins COX-2 and iNOS in LPS-induced BV2 cells, and the inhibitory effect on COX-2 is more obvious.

[0107] 6. Western blot detection of the activation of NF-κB and MAPK signaling pathways in the inflammation model

[0108] The nuclear factor-κB (NF-κB) transcription factor family is an important regulator of inflammatory response and a key player in innate and adaptive immune responses, which can be activated by pro-inflammatory stimuli. Free NF-κB is produced in an unbound form, then transported to the nucleus, binds to DNA binding sites, leading to the production of pro-inflammatory mediators and cytokines, and participates in the regulation of immune response. NF-κB / p65 is a key functional isoform, and its nuclear translocation level is proportional to the degree of NF-κB activation. Therefore, this embodiment uses Western blot to detect the activation of the NF-κB signaling pathway, and the detection method is as described in the method for detecting inflammatory factors in the foregoing, and the results are shown in Figure 14 .

[0109] As can be seen from Figure 14 Compared with the LPS group, Pogostesquines E inhibits the activation of the NF-κB signaling pathway, and is concentration-dependent (40 μM and 80 μM).

[0110] The mitogen-activated protein kinase (MAPK) signaling pathway is involved in the initiation, progression and regulation of inflammatory response by regulating the activation of various signal transduction, and plays an important role in inflammatory response. This signaling pathway is composed of several key components, including p38 and JNK. Studies have shown that p38 and JNK are activated in glial cells, affecting the secretion of inflammatory factors. These signaling pathways play a crucial role in regulating the survival, death, proliferation and differentiation of neurons. Therefore, this embodiment uses Western blot to detect the activation of the MAPK signaling pathway, and the detection method is as described in the method for detecting inflammatory factors in the foregoing, and the results are shown in Figure 15 .

[0111] As can be seen fromFigure 15 It can be known that Pogostesquines E can significantly inhibit the activation of MAPK signal pathway, and the phosphorylation levels of p38 and JNK are reduced.

[0112] The above describes the embodiments of the present application in detail in combination with the drawings, but the present application is not limited to the above-described embodiments, and various changes can be made within the knowledge range of the ordinary skill in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A sesquiterpene compound, characterized by, The sesquiterpenes have a structure as shown in Formula I:

2. The sesquiterpene compound according to claim 1, characterized in that, The sesquiterpenes are separated from Pogostemon esquirolii of Lamiaceae.

3. The method of preparing the sesquiterpene compound as claimed in claim 1 or 2, characterized in that, The preparation method comprises the following steps: S1: taking dry Pogostemon esquirolii whole grass to extract with 85%-100% ethanol, and concentrating the combined extract to obtain extract; dissolving the extract in water and sequentially extracting with petroleum ether, ethyl acetate and n-butanol to collect the petroleum ether layer; S2: taking the petroleum ether layer to perform column chromatography separation with n-hexane-ethyl acetate as eluent for gradient elution, and collecting 11 components PA1-PA11; S3: taking component PA6 obtained in step S2 to perform column chromatography separation with methanol aqueous solution as eluent for gradient elution, and collecting 3 components PA6B1-PA6B3; S4: taking component PA6B2 obtained in step S3 to perform column chromatography separation with n-hexane-ethyl acetate as eluent for gradient elution, and collecting 2 components PA6B2C1-PA6B2C2; S5: taking component PA6B2C2 obtained in step S4 to perform column chromatography separation with methanol aqueous solution as eluent for gradient elution, and collecting 6 components PA6B2C2D1-PA6B2C2D6; S6: taking component PA6B2C2D3 obtained in step S5 to perform liquid chromatography separation with 20%-30% acetonitrile aqueous solution for isocratic elution, and collecting 3 components PA6B2C2D3E1-PA6B2C2D3E3; S7: taking component PA6B2C2D3E1 obtained in step S6 to perform liquid chromatography separation with 40%-60% acetonitrile aqueous solution for isocratic elution to obtain the sesquiterpenes Pogostesquines E.

4. The production method according to claim 3, characterized by, The column chromatography separation in step S2 adopts normal phase silica gel column chromatography; Preferably, the column chromatography separation in step S2 adopts dry loading; Preferably, the volume ratio of n-hexane-ethyl acetate in step S2 is pure n-hexane, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, pure ethyl acetate in turn.

5. The preparation method according to claim 3, characterized in that, The column chromatography separation in step S3 adopts small-pore resin chromatography; Preferably, the column chromatography separation in step S3 adopts dry loading; Preferably, the concentration of methanol aqueous solution in step S3 is 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol, 100% methanol in turn.

6. The preparation method according to claim 3, characterized in that, The column chromatography separation in step S4 adopts normal phase silica gel column chromatography; Preferably, the column chromatography separation in step S4 adopts dry loading; Preferably, the volume ratio of n-hexane-ethyl acetate in step S4 is 20:1, 15:1, 10:1, 8:1, 5:1, 2:1 in turn.

7. The preparation method according to claim 3, characterized in that, The column chromatography separation in step S5 adopts C18 reverse phase silica gel column chromatography; Preferably, the column chromatography separation in step S5 adopts wet loading; Preferably, the concentration of methanol aqueous solution in step S5 is 50% methanol, 60% methanol, 70% methanol in turn.

8. The preparation method according to claim 3, characterized in that, The liquid chromatography in step S6 comprises semi-preparative liquid chromatography; Preferably, the liquid chromatography in step S7 comprises semi-preparative liquid chromatography.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the sesquiterpene compound according to claim 1 or 2 and pharmaceutically acceptable excipients.

10. Use of the sesquiterpene compound according to claim 1 or 2 or the sesquiterpene compound prepared by the method according to any one of claims 3 to 8 in the preparation of an anti-inflammatory drug.

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