Euphorbia kanane type triterpenoids as well as preparation method and application thereof

Abstract

The invention discloses a euphorbia kanane type triterpenoid compound as well as a preparation method and application thereof, and relates to the technical field of natural product chemistry, the compound has at least one structural formula as shown in the specification: # imgabs0 #; the preparation method comprises the following steps: by taking Xiangluxiang as a raw material, extracting by using a mixed solution containing methanol and ethyl acetate, removing insoluble impurities and lower brown oily substances, and concentrating filtrate under reduced pressure to obtain an extract; and separating the extract through normal-phase silica gel column chromatography and / or MCI column chromatography, reverse-phase silica gel column chromatography and semi-preparative high performance liquid chromatography to obtain the compound. The invention also provides application of the compound in preparation of anti-inflammatory drugs. The preparation method has the beneficial effects that four euphorbia kanane type triterpenoids which are rich in content and separated from the champignon have no obvious cytotoxicity on RAW264.7 cells, can inhibit the generation of NO in the RAW264.7 cells to different degrees, and can be used for preparing anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product chemistry, specifically to the field of extraction, separation and preparation technology of effective ingredients of natural products, and in particular to the extraction and separation of four gansuline-type triterpenoid compounds with abundant content and pharmacological activity from the medicinal material Xunluxiang, as well as a preparation method and application thereof. Background Art

[0002] Mastic is a species of Pistacia chinensis in the Anacardiaceae family. Pistacia lentiscus L.) Plant viscose: The colorless, translucent natural resin that flows out of the trunk of the frankincense tree after being cut, also known as foreign frankincense.

[0003] Scholars at home and abroad have conducted systematic research on Xunluxiang from the aspects of phytochemistry and pharmacology, and discovered more than 160 chemical components, mainly including natural polymers, monoterpenes, sesquiterpenes, triterpenes and polyphenol compounds. It was also shown that the crude extract of Xunluxiang and some monomer compounds have antibacterial, anti-inflammatory, anti-ulcer, anti-cancer and liver protection activities.

[0004] Xunluxiang has high safety in clinical use and few side effects. Adults took Xunluxiang (2g / d) for 3 months continuously without any adverse reactions. It is effective in treating diarrhea and ulcerative colitis when used alone or in combination.

[0005] At present, the existing technology has little research on the chemical components contained in Xunluxiang, and how to separate effective chemical components from Xunluxiang is of great significance. Summary of the invention

[0006] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a gansuline-type triterpenoid compound and a preparation method and application thereof.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a gansuline-type triterpenoid compound having at least one structural formula as shown below: .

[0008] In a second aspect, the present invention provides a method for preparing the gansuline-type triterpenoid compound according to claim 1, comprising the following steps: Using the radix oxycarpa as raw material, extracting with a mixture of methanol and ethyl acetate, removing insoluble impurities and the lower brown oily substance, and concentrating the filtrate under reduced pressure to obtain an extract; The extract is separated by normal phase silica gel column chromatography and / or MCI column chromatography, reverse phase silica gel column chromatography and semi-preparative high performance liquid chromatography to obtain gansuline-type triterpenoid compounds as shown in formula I, formula II, formula III and formula IV.

[0009] Optionally, before separating the extract by normal phase silica gel column chromatography and / or MCI column chromatography, reverse phase silica gel column chromatography and high performance liquid chromatography, the preparation method further comprises the following steps: The extract is dispersed with a 4-6% mass concentration Na2CO3 aqueous solution, extracted with ethyl acetate 4-6 times, and the ethyl acetate phases are combined; the extract is back-extracted with a 4-6% mass concentration Na2CO3 aqueous solution 1-3 times, and the ethyl acetate phase is concentrated under reduced pressure to obtain a neutral partial extract; the aqueous phases are combined, acidified with dilute hydrochloric acid to pH = 2-3, and further extracted with ethyl acetate 4-6 times, and the ethyl acetate phase is concentrated under reduced pressure to obtain an acidic partial extract.

[0010] Optionally, before separating the extract by normal phase silica gel column chromatography and / or MCI column chromatography, reverse phase silica gel column chromatography and high performance liquid chromatography, the preparation method further comprises the following steps: The extract is dispersed in water, extracted with petroleum ether for 4 to 6 times, the petroleum ether phases are combined and concentrated under reduced pressure to obtain a petroleum ether extracted extract and an aqueous phase; the aqueous phase is extracted with ethyl acetate for 4 to 6 times, the ethyl acetate phases are combined and concentrated under reduced pressure to obtain an ethyl acetate extracted extract.

[0011] Optionally, the normal phase silica gel column chromatography is a pressurized column chromatography, the filler is 300-400 mesh normal phase silica gel, the eluent is a petroleum ether-ethyl acetate system, and the volume ratio is 14-16:1 isocratic elution; The MCI column chromatography method is a pressurized column chromatography, the filler is an MCI small-pore resin, and the eluent is a gradient elution of a methanol aqueous solution with a volume concentration of 80% to 100%; The reverse phase column chromatography is a pressurized column chromatography, the filler is reverse phase C18 silica gel, and the eluent is a gradient elution of methanol aqueous solution with a volume concentration of 80% to 100%.

[0012] Optionally, the amount of the mixed solution is 45 to 55 times the volume of the Xunluxiang, the volume ratio of the methanol to the ethyl acetate is 2 to 4:1, and the extraction time is 1 to 3 days.

[0013] Optionally, the preparation method further comprises the following steps: The separated products were followed and detected by thin layer chromatography and high performance liquid chromatography.

[0014] Optionally, the developing solvent in the thin layer chromatography is petroleum ether: ethyl acetate = volume ratio of 3 to 5: 1, and the relative migration value of the gansuane-type triterpenoid compound is R fBetween 0.15 - 0.25, there is no absorption at 254 nm and 365 nm in the UV dark box, and rose-red spots appear after color development with 10% sulfuric acid ethanol.

[0015] Optionally, the HPLC detection uses an Agilent SB-C18 column with a particle size of 5 μ m, size 4.6×250mm, mobile phase is acetonitrile water system, flow rate 0.75 mL / min, method: 0-15 min, 90%-100% acetonitrile; 15-35 min, 100%-100% acetonitrile; 35-36 min, 100%-90% acetonitrile; 36-42 min, 90%-90% acetonitrile, retention time of gansuline-type triterpenoid compounds shown in formula I, formula II, formula III and formula IV t R 31.5 min, 34.2 min, 20.6 min and 21.6 min respectively. The maximum ultraviolet absorption λ of the gansuline-type triterpenoid compounds shown in Formula I, Formula II, Formula III and Formula IV is max They are 213.7 nm, 209.8 nm, 212.2 nm and 207.5 nm respectively.

[0016] Optionally, the high performance liquid chromatography detection uses a Hitachi primaide high performance liquid chromatograph equipped with a 1210 quaternary solvent management system, a 1220 automatic injection system, a 1230 column temperature management unit and a 1430 diode array detection system.

[0017] In a third aspect, the present invention provides the use of the above-mentioned gansuican-type triterpenoid compound or the gansuican-type triterpenoid compound obtained by the above-mentioned preparation method in the preparation of anti-inflammatory drugs.

[0018] Optionally, the present invention provides the use of the above-mentioned gansuican-type triterpenoid compounds in the preparation of drugs for treating gastrointestinal inflammatory diseases.

[0019] Optionally, the present invention provides the use of a gansuican-type triterpenoid compound as shown in formula III in the preparation of a drug for treating inflammatory bowel disease.

[0020] The present invention has at least one of the following beneficial effects: 1. The present invention uses Xanthan gum as raw material, and separates 4 gansuline-type triterpenoid compounds with relatively rich contents by three or four methods including solvent extraction, acid-base treatment, extraction with solvents of different polarities, normal phase and reverse phase silica gel column chromatography, MCI column chromatography and semi-preparative high performance liquid chromatography. Compound I is (3 S )-3-acetoxy-7,24( Z)-dien-26-oic acid, compound II is (3 S )-3-acetoxy-8,24( Z )-dien-26-oic acid, compound III is mastadienonic acid, and compound IV is anwuweizonic acid.

[0021] 2. Test results of the cytotoxicity of the four gansuline-type triterpenoids isolated by the present invention on RAW264.7 cells and the inhibitory activity of NO production in RAW264.7 cells in vitro. The results show that the four gansuline-type triterpenoids isolated from Fuluxiang have no obvious cytotoxicity to RAW264.7 cells and inhibit the production of NO in RAW264.7 cells to varying degrees. Therefore, they can be used to prepare anti-inflammatory drugs.

[0022] 3. The compound III isolated by the present invention inhibits systemic inflammation by inhibiting NF-κB and MAPK signaling pathways and reducing pro-inflammatory cytokines, and can also exhibit barrier protection properties by activating the Nrf2 pathway and upregulating tight junction proteins. Therefore, the compound III isolated from the flavonoids can be used as a promising therapeutic drug for controlling inflammatory bowel disease or other diseases involving inflammation and barrier dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a liquid chromatogram extracted at 210 nm when analyzing compounds I-IV in Examples 1-4 of the present invention; Figure 2 This is a liquid chromatogram extracted at 210 nm during the preparation of compounds I and II in Examples 1-4 of the present invention; Figure 3 This is a liquid chromatogram extracted at 210 nm during the preparation of compounds III and IV in Examples 1-4 of the present invention; Figure 4 This is a high-resolution mass spectrum of compound I in the embodiment of the present invention; Figure 5 This is a high-resolution mass spectrum of compound II in the embodiment of the present invention; Figure 6 This is a high-resolution mass spectrum of compound III in the examples of the present invention; Figure 7 This is a high-resolution mass spectrum of compound IV in the examples of the present invention; Figure 8 is the compound I in the embodiment of the present invention 1 H NMR (400 MHz, deuterated chloroform); Fig. 9 is the compound I in the embodiment of the present invention 13C NMR (100 MHz, deuterated chloroform); Fig.10 is the compound II in the embodiment of the present invention 1 H NMR (400 MHz, deuterated chloroform); Fig.11 is the compound II in the embodiment of the present invention 13 C NMR (100 MHz, deuterated chloroform); Fig.12 is the compound III in the embodiment of the present invention 1 H NMR (400 MHz, deuterated methanol); Fig.13 is the compound III in the embodiment of the present invention 13 C NMR (100 MHz, deuterated methanol); Fig.14 is the compound IV in the embodiment of the present invention 1 H NMR (400 MHz, deuterated methanol); Fig.15 is the compound IV in the embodiment of the present invention 13 C NMR (100 MHz, deuterated methanol). DETAILED DESCRIPTION

[0024] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] The present invention is further described in detail below with specific examples, but the present invention is not limited to the following specific examples.

[0026] Example 1 a. Take 60 g of Xunluxiang, extract it with 50 times the volume of methanol: ethyl acetate = 3:1 at room temperature for 2 days, remove insoluble impurities and the lower brown oil, and concentrate the filtrate under reduced pressure to obtain 50.4 g of extract.

[0027] b. The extract obtained in step a was dispersed with 500 mL of 5% Na2CO3 aqueous solution, and then extracted with an equal volume of ethyl acetate for 5 times. The ethyl acetate phase was combined, concentrated, and then back-extracted twice with 500 mL of 5% Na2CO3 aqueous solution. The ethyl acetate phase was concentrated under reduced pressure to obtain 29.6 g of the neutral part. The aqueous phases in the above steps were combined, acidified to pH = 2-3 with dilute hydrochloric acid, and then extracted with an equal volume of ethyl acetate for 5 times. The ethyl acetate phase was concentrated under reduced pressure to obtain 18.8 g of the acidic part.

[0028] c. The acidic portion obtained in step b was separated by normal phase silica gel column chromatography, eluted with petroleum ether-ethyl acetate system, 300-400 mesh silica gel wet column, 200-300 mesh silica gel dry loading, column inner diameter 46 mm, column height 40.4 cm, sample height 3.6 cm, PE:EA = 15:1 isocratic elution, thin layer chromatography tracking detection, to obtain fraction 1-A (1.2 g) containing compound I-IV; the fraction was then separated by ODS column chromatography, column inner diameter 26 mm, dry loading, column height 40.2 cm, sample height 2.6 cm, with a gradient elution of methanol-water at volume concentrations of 80%, 85%, 90%, 95%, and 100%, and high performance liquid chromatography tracking detection. In the high performance liquid chromatography detection, a Hitachi primaide high performance liquid chromatograph was used, equipped with a 1210 quaternary solvent management system, a 1220 automatic injection system, a 1230 column temperature management unit, and a 1430 diode array detection system. An Agilent SB-C18 column with a particle size of 5 μ m, size 4.6×250mm, mobile phase is acetonitrile water system, flow rate 0.75 mL / min, method: 0-15 min, 90%-100% acetonitrile; 15-35 min, 100%-100% acetonitrile; 35-36 min, 100%-90% acetonitrile; 36-42 min, 90%-90% acetonitrile, to obtain two fractions 1-B (658.2 mg) 1-C (821.3 mg); fraction 1-C was separated by semi-preparative HPLC (90% isocratic acetonitrile-water, flow rate 3 mL / min) to obtain compound I (167.3 mg, t R = 28.1min) and compound II (149.8 mg, t R = 30.6min), fraction 1-B was separated by semi-preparative HPLC (90% acetonitrile-water, 3 mL / min) to obtain compound III (443.7 mg, t R = 13.9 min) and compound IV (179 mg, t R = 14.8 min), wherein a phenomenex kinetex-C18 column with a particle size of 5 μ m, size 10.0×250 mm. The maximum ultraviolet absorption λ of the gansuline-type triterpenoid compounds represented by formula I, formula II, formula III and formula IV max They are 213.7 nm, 209.8 nm, 212.2 nm and 207.5 nm respectively.

[0029] Example 2 a. Take 60 g of Xunluxiang, extract it with 50 times the volume of methanol: ethyl acetate = 3:1 at room temperature for 2 days, remove insoluble impurities and the lower brown oil, and concentrate the filtrate under reduced pressure to obtain 51.4 g of extract.

[0030] b. Disperse the extract obtained in step a in 200 mL of distilled water, extract with an equal volume of petroleum ether 5 times, combine the petroleum ether phases and concentrate under reduced pressure to obtain 856.2 mg of petroleum ether extract; extract the remaining aqueous phase with an equal volume of ethyl acetate 5 times, combine the ethyl acetate phases and concentrate under reduced pressure to obtain 49.6 g of ethyl acetate extract; extract the remaining aqueous phase with an equal volume of n-butanol twice, combine the n-butanol phases and concentrate under reduced pressure to obtain 423.8 mg of n-butanol extract.

[0031] c. Separate the ethyl acetate extract obtained in step b by normal phase silica gel column chromatography, use petroleum ether-ethyl acetate system for elution, 300-400 mesh silica gel wet column, 200-300 mesh silica gel dry loading, column inner diameter 46 mm, column height 38.6 cm, sample height 4.5 cm, PE:EA = 15:1 isocratic elution, thin layer chromatography tracking detection, obtain fraction 2-A (2.1 g) containing compound I-IV; then separate the fraction by ODS column chromatography, column inner diameter 46 mm, dry loading, column height 28 cm, sample height 2.0 cm, with a volume concentration of 80%, 85%, 90%, 95%, 100% methanol water gradient elution, and high performance liquid chromatography tracking detection, wherein, in the high performance liquid chromatography detection, a Hitachi primaide high performance liquid chromatograph, an Agilent SB-C18 chromatographic column, a particle size of 5 μ m, size 4.6×250 mm, flow rate 0.75 mL / min, method: 0-15 min, 90%-100% acetonitrile; 15-35 min, 100%-100% acetonitrile; 35-36 min, 100%-90% acetonitrile; 36-42 min, 90%-90% acetonitrile, to obtain two fractions 2-B (1.0 g) and 2-C (763.4 mg); fraction 2-C was separated by semi-preparative HPLC (90% acetonitrile-water, 3 mL / min) to obtain compound I (456.2 mg, t R = 28.1 min) and compound II (382.5 mg, t R = 30.6 min), fraction 2-B was separated by semi-preparative HPLC (90% acetonitrile-water, 3 mL / min) to obtain compound III (465.1 mg, t R = 13.9min) and compound IV (218.0 mg, t R= 14.8 min), wherein a phenomenex kinetex-C18 column with a particle size of 5 μ m, specification: 10.0×250mm.

[0032] Example 3 a. Take 60 g of Xunluxiang, extract it with 50 times the volume of methanol: ethyl acetate = 3:1 at room temperature for 2 days, remove insoluble impurities and the lower brown oil, and concentrate the filtrate under reduced pressure to obtain 49.6 g of extract.

[0033] b. The extract obtained in step a was separated by normal phase silica gel column chromatography, eluted with petroleum ether-ethyl acetate system, 300-400 mesh silica gel wet column, 200-300 mesh silica gel dry loading, column inner diameter 46 mm, column height 39.5 cm, sample height 8.5 cm, PE:EA = 15:1 isocratic elution, thin layer chromatography tracking detection, to obtain fraction 3-A (2.0 g) containing compound I-IV; the fraction was then separated by ODS column chromatography, column inner diameter 46 mm, dry loading, column height 26.9 cm, sample height 2.4 cm, gradient elution with methanol water at volume concentrations of 80%, 85%, 90%, 95%, 100%, and high performance liquid chromatography tracking detection, wherein, in the high performance liquid chromatography detection, a Hitachi primaide high performance liquid chromatograph, an Agilent SB-C18 chromatographic column, a particle size of 5 μ m, size 4.6×250 mm, flow rate 0.75 mL / min, method: 0-15 min, 90%-100% acetonitrile; 15-35 min, 100%-100% acetonitrile; 35-36 min, 100%-90% acetonitrile; 36-42 min, 90%-90% acetonitrile, to obtain two fractions 3-B (1.6 g) and 3-C (1.1 g); fraction 3-C was separated by semi-preparative HPLC (90% acetonitrile-water, 3 mL / min) to obtain compound I (612.6 mg, t R = 28.1 min) and compound II (432.8 mg, t R = 30.6 min), fraction 3-B was separated by semi-preparative HPLC (90% acetonitrile-water, 3 mL / min) to obtain compound III (525.5 mg, t R = 13.9 min) and compound IV (274.8 mg, t R = 14.8min), wherein a phenomenex kinetex-C18 column with a particle size of 5 μ m, specification: 10.0×250mm.

[0034] Example 4 a. Take 60 g of Xunluxiang, extract it with 50 times the volume of methanol: ethyl acetate = 3:1 at room temperature for 2 days, remove insoluble impurities and the lower brown oil, and concentrate the filtrate under reduced pressure to obtain 48.9 g of extract.

[0035] b. The extract obtained in step a was separated by MCI column chromatography, with a column inner diameter of 46 mm, a column height of 39.7 cm, a sample height of 9.2 cm, and 80-100% methanol water isocratic elution, and high performance liquid chromatography tracking detection was performed to obtain fraction 3-A (8.1 g) containing compound I-IV; fraction 3-A was separated by ODS column chromatography, with a column inner diameter of 46 mm, dry loading, a column height of 42.8 cm, and a sample height of 4.4 cm, and gradient elution with methanol water at volume concentrations of 80%, 85%, 90%, 95%, and 100%, and high performance liquid chromatography tracking detection was performed, wherein the high performance liquid chromatography detection used a Hitachi primaide high performance liquid chromatograph, an Agilent SB-C18 chromatographic column, and a particle size of 5 μ m, size 4.6×250 mm, mobile phase is acetonitrile-water system, flow rate 0.75 mL / min, method: 0-15 min, 90%-100% acetonitrile; 15-35 min, 100%-100% acetonitrile; 35-36 min, 100%-90% acetonitrile; 36-42 min, 90%-90% acetonitrile, to obtain two fractions 3-B (1.9 g) and 3-C (2.1 g); fraction 3-C was separated by semi-preparative HPLC (90% acetonitrile-water, 3 mL / min) to obtain compound I (650.6 mg, t R = 28.1 min) and compound II (442.8 mg, t R = 30.6 min), fraction 3-B was separated by semi-preparative HPLC (90% acetonitrile-water, 3 mL / min) to obtain compound III (483.3 mg, t R =13.9 min) and compound IV (256.2 mg, t R = 14.8 min), wherein a phenomenex kinetex-C18 column with a particle size of 5 μ m, specification: 10.0×250mm.

[0036] Example 5 like Figures 4 to 15 As shown, the structures of compounds I-IV were determined by combining high-resolution mass spectrometry data with NMR data from relevant literature.

[0037] Compound I was identified as (3 S )-3-acetoxy-7,24( Z)-dien-26-oic acid, white amorphous powder, HR-ESI-MS m / z 497.3632[MH] - (Calculated value C 32 H 49 O4, 497.3631) to determine its molecular formula is C 32 H 50 O4, unsaturation is 8; 1 H NMR and 13 C NMR data are shown in Table 1.

[0038] Compound II was identified as (3 S )-3-acetoxy-8,24( Z )-dien-26-oic acid, white amorphous powder, HR-ESI-MS m / z 497.3634[MH] - (Calculated value C 32 H 49 O4, 497.3631) to determine its molecular formula is C 32 H 50 O4, unsaturation is 8; 1 H NMR and 13 C NMR data are shown in Table 1.

[0039] Compound III was identified as mastadienonic acid, white needle crystals (methanol), HR-ESI-MS m / z 499.3427 [M+COOH] - (Calculated value C 31 H 47 O5, 499.3423) to determine its molecular formula is C 30 H 46 O3, unsaturation is 8; 1 HNMR and 13 C NMR data are shown in Table 1.

[0040] Compound IV was identified as anwuweizonic acid, white amorphous powder, HR-ESI-MS m / z 499.3426 [M+COOH] - (Calculated value C 32 H 49 O4, 497.3631) to determine its molecular formula is C 32 H 50 O4, unsaturation is 8. 1 HNMR and 13 C NMR data are shown in Table 1.

[0041] Table 1 Key to compounds I-IV 1H NMR, 13 C NMR data Note: 1) Solvent: deuterated chloroform; 2) Solvent: deuterated methanol Example 6 The compounds I-IV obtained in Examples 1-4 were subjected to biological activity testing: in combination with their application in gastrointestinal inflammatory diseases, a method combining inflammatory activity guidance with modern chromatographic technology was used to identify compounds with anti-inflammatory activity in Rhizoma Xanthophyllae, and their anti-inflammatory mechanism was studied.

[0042] The present invention conducts RAW264.7 cytotoxicity test and in vitro activity determination of inhibiting pro-inflammatory factor NO on the obtained compounds I-IV.

[0043] First, the CCK-8 method was used to evaluate the effects of compounds I-IV on the activity of mouse macrophage RAW264.7. RAW264.7 cells with good growth conditions were taken and approximately 1.5 × 10 4 The cells were inoculated into 96-well plates at a density of 100 cells / well and incubated at a constant temperature for 24 h. The supernatant was removed by aspiration and 80% lysate was added to RAW264.7 cells. μ mol / L complete medium of the sample to be tested was incubated for 24 h, and 6 replicate wells were set for each sample. After the incubation was completed, the supernatant was aspirated, washed once with PBS, and 100 μL of complete medium containing 10% CCK-8 was added to each well and incubated for 2 h. The absorbance value was measured at 450 nm by an ELISA reader ( A ). The results show that 80 μ mol / L, the survival rates of compounds I-IV on RAW264.7 cells were 60.3%, 65.6%, 73.4% and 85.7%, respectively, showing no obvious cytotoxicity.

[0044] Secondly, the potential anti-inflammatory activity of compounds I-IV was evaluated by measuring the effects of compounds I-IV on the secretion level of pro-inflammatory factor NO in RAW264.7 cells induced by LPS. Cytotoxicity experiments showed that compounds I-IV μ mol / L had no significant growth inhibitory effect on RAW264.7 cells, which could exclude false positive experimental results. 4The cells were inoculated at a density of 100 / well in a 96-well plate and cultured in a cell culture incubator at 37 °C and 5% CO for 24 h. The supernatant was removed and 50 μL of the sample to be tested at the corresponding concentration (0, 10, 20, 40, 80 μmol / L) was added. Three replicates were made for each concentration. The dexamethasone group was used as the positive control, and the group without sample was used as the experimental control group. After incubation in the cell culture incubator for 1 h, LPS with a mass concentration of 1.0 μg / mL was added to stimulate the cells. After incubation for 24 h, 50 μL of the supernatant was aspirated into a new 96-well plate, and the absorbance value was measured at 540 nm using the Griess kit operation method ( A ), detect the content of NO in the cell culture medium. 50 The values ​​were calculated by GraphpadPrism (version 8.0). The results showed that compared with the positive control Dexamethasone (IC 50 = 9.93 ± 1.17 μmol / L), the compound showed a certain inhibitory effect on the production of NO in RAW264.7 cells, IC 50 18.91 ± 2.57 μ mol / L, 27.07 ± 2.73 μ mol / L, 16.43 ± 3.30 μ mol / L, 19.13 ± 0.52 μ mol / L. Example 7 The compound III obtained in Examples 1-4 was used in mouse experiments to explore the potential role and mechanism of the compound in the pathogenesis of inflammatory bowel disease (IBD).

[0045] To evaluate the potential of compound III to alleviate colitis, in this example, an acute colitis model was established in C57BL / 6J mice by continuous administration of 3% dextran sulfate sodium (DSS). Mice were pretreated with different concentrations of compound III for 7 days before DSS induction. From day 3, the body weight of mice treated with DSS decreased significantly compared with the control group. Both low doses (10 mg / kg / day) and high doses (100 mg / kg / day) significantly reduced the severity of colitis, with increased body weight and colon length compared with mice given only 3% DSS. From day 4, clinical symptoms of rectal bleeding and diarrhea were observed in all mice treated with DSS. However, these symptoms were not found in mice treated with high concentrations of compound III. The spleen is the largest organ in lymphoid tissue, and splenomegaly is a potential symptom associated with the severity of inflammatory diseases, including IBD. In this study, the spleen weight of mice treated with compound III was significantly lower than that of mice treated with DSS, suggesting that compound III may play a role in protecting the colon from inflammatory damage. In summary, MDA (compound III) can alleviate the occurrence of experimental colitis in mice.

[0046] Excessive secretion of proinflammatory factors is a hallmark of IBD. In this example, serum inflammatory markers, including IL-1 β , IL-6 and TNF- α The severity of colitis was assessed by the level of DSS. Mice exposed to DSS showed more severe inflammatory responses than mice in the control group, as evidenced by significantly increased levels of proinflammatory cytokines. Compared with the DSS group, both low-dose (10 mg / kg / day) and high-dose (100 mg / kg / day) compound III significantly reduced proinflammatory cytokines in serum.

[0047] MAPK and NF- κ Activation of the B signaling pathway is usually associated with the inflammatory state of DSS-induced colitis. In this example, the levels of phosphorylated p38, ERK, JNK, IκBα, and p65 in the colon tissue of DSS-treated mice were significantly increased compared with those in the control group. Both low-dose (10 mg / kg / day) and high-dose (100 mg / kg / day) compound III inhibited the expression of ERK, IκBα, and p65. κ B α The results showed that compound III may inhibit the phosphorylation of MAPKs and NF- κ B signaling pathway to partially improve the DSS-induced inflammatory response.

[0048] The integrity and function of the intestinal barrier are maintained by tight junction proteins such as ZO-1 and occludin. Under IBD conditions, the expression levels of these proteins are significantly downregulated, leading to barrier dysfunction and systemic inflammation. In the DSS-induced colitis model, the expression levels of ZO-1 and occludin were significantly reduced. Compared with the DSS-treated group, oral administration of compound III significantly upregulated the expression of ZO-1 and occludin in colonic tissues, suggesting that compound III may have a beneficial effect on preventing colitis by effectively restoring intestinal barrier function. To explore the exact mechanism by which compound III induces the upregulation of occludin and ZO-1, the expression of Nrf2 and its target gene HO-1 in colonic tissues of different groups was detected. Both low-dose (10 mg / kg / day) and high-dose (100 mg / kg / day) compound III treatments significantly increased the protein levels of Nrf2 and HO-1, suggesting that the Nrf2 signaling pathway may be required for compound III-mediated upregulation of occludin and ZO-1.

[0049] This example shows that compound III inhibits systemic inflammation by inhibiting NF-κB and MAPK signaling pathways and reducing proinflammatory cytokines. In addition, compound III exhibits barrier protection properties by activating the Nrf2 pathway and upregulating tight junction proteins. Therefore, MDA (compound III) isolated from Fructus oxyphyllae can be used as a promising therapeutic agent for the control of IBD or other diseases involving inflammation and barrier dysfunction.

[0050] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A gansuline-type triterpenoid compound, characterized in that: Has at least one structural formula as shown below: 。 2. The method for preparing the gansuline-type triterpenoid compound according to claim 1, characterized in that: The following steps are involved: Using the radix oxycarpa as raw material, extracting with a mixture of methanol and ethyl acetate, removing insoluble impurities and the lower brown oily substance, and concentrating the filtrate under reduced pressure to obtain an extract; The extract is separated by normal phase silica gel column chromatography and / or MCI column chromatography, reverse phase silica gel column chromatography and semi-preparative high performance liquid chromatography to obtain gansuline-type triterpenoid compounds as shown in formula I, formula II, formula III and formula IV.

3. The preparation method according to claim 2, characterized in that: Before separating the extract by normal phase silica gel column chromatography and / or MCI column chromatography, reverse phase silica gel column chromatography and high performance liquid chromatography, the preparation method further comprises the following steps: The extract is dispersed with a 4-6% mass concentration Na2CO3 aqueous solution, extracted with ethyl acetate 4-6 times, and the ethyl acetate phases are combined; the Na2CO3 aqueous solution with a mass concentration of 4-6% is used for back extraction 1-3 times, and the ethyl acetate phase is concentrated under reduced pressure to obtain a neutral partial extract; the aqueous phases are combined, acidified with dilute hydrochloric acid to pH = 2-3, and further extracted with ethyl acetate 4-6 times, and the ethyl acetate phase is concentrated under reduced pressure to obtain an acidic partial extract.

4. The preparation method according to claim 2, characterized in that: Before separating the extract by normal phase silica gel column chromatography and / or MCI column chromatography, reverse phase silica gel column chromatography and high performance liquid chromatography, the preparation method further comprises the following steps: The extract is dispersed in water, extracted with petroleum ether for 4 to 6 times, the petroleum ether phases are combined and concentrated under reduced pressure to obtain a petroleum ether extracted extract and an aqueous phase; the aqueous phase is extracted with ethyl acetate for 4 to 6 times, the ethyl acetate phases are combined and concentrated under reduced pressure to obtain an ethyl acetate extracted extract.

5. The preparation method according to claim 2, characterized in that: The normal phase silica gel column chromatography is a pressurized column chromatography, the filler is 300-400 mesh normal phase silica gel, the eluent is a petroleum ether-ethyl acetate system, and the volume ratio is 14-16:1 isocratic elution; The MCI column chromatography method is a pressurized column chromatography, the filler is an MCI small-pore resin, and the eluent is a gradient elution of a methanol aqueous solution with a volume concentration of 80% to 100%; The reverse phase column chromatography is a pressurized column chromatography, the filler is reverse phase C18 silica gel, and the eluent is a gradient elution of methanol aqueous solution with a volume concentration of 80% to 100%.

6. The preparation method according to claim 2, characterized in that: The dosage of the mixed solution is 45 to 55 times the volume of the Xunluxiang, the volume ratio of the methanol to the ethyl acetate is 2 to 4:1, and the extraction time is 1 to 3 days.

7. The preparation method according to claim 2, characterized in that: The preparation method further comprises the following steps: The separated products were followed and detected by thin layer chromatography and high performance liquid chromatography.

8. The preparation method according to claim 7, characterized in that: The developing solvent in the thin layer chromatography method is petroleum ether: ethyl acetate = volume ratio of 3 to 5:1, and the transfer value of the gansuline-type triterpenoid compound is R f Between 0.15 - 0.25, there is no absorption at 254 nm and 365 nm in the UV dark box, and rose-red spots appear after color development with 10% sulfuric acid ethanol.

9. The preparation method according to claim 7, characterized in that: The HPLC method uses an Agilent SB-C18 column with a particle size of 5 μ m, size 4.6×250mm, mobile phase is acetonitrile water system, flow rate 0.75 mL / min, method: 0-15 min, 90%-100% acetonitrile; 15-35 min, 100%-100% acetonitrile; 35-36 min, 100%-90% acetonitrile; 36-42 min, 90%-90% acetonitrile, retention time of gansuline-type triterpenoid compounds shown in formula I, formula II, formula III and formula IV t R 31.5 min, 34.2 min, 20.6 min and 21.6 min respectively. The maximum ultraviolet absorption λ of the gansuline-type triterpenoid compounds shown in Formula I, Formula II, Formula III and Formula IV is max They are 213.7 nm, 209.8 nm, 212.2 nm and 207.5 nm respectively.

10. Use of the gansuline-type triterpenoid compound as claimed in claim 1 or the gansuline-type triterpenoid compound obtained by the preparation method as claimed in any one of claims 2 to 9 in the preparation of anti-inflammatory drugs.

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