A novel triterpenoid compound and its epimers from Acanthopanax senticosus, along with their extraction, separation methods, and applications.

Abstract

This invention belongs to the field of traditional Chinese medicine chemistry and natural product separation technology, specifically relating to a novel triterpenoid compound and its epimerical form from *Eleutherococcus senticosus*, along with its extraction and separation methods and applications. The new compound is named 22α-Hydroxy-19-epi-chiisanogenin, and its epimerical form is named 22α-Hydroxychiisanogenin. The extraction and separation method provided by this invention employs acid hydrolysis / ethanol reflux extraction, macroporous resin column adsorption, silica gel column chromatography, ODS column chromatography, and high-performance liquid chromatography for separation, purification, and preparation. The compound was successfully extracted and separated. This method involves only five steps, is simple and rapid, and mainly uses conventional reagents such as ethanol, dichloromethane, and methanol. The process is simple and environmentally friendly, and the purity of the compound obtained by this method is high, exceeding 90%. Furthermore, studies have shown that this compound has anti-inflammatory effects. Therefore, the compound of this invention can be used as a lead compound for the synthesis of other compounds, as well as a raw material for new drug development and pharmacological activity research, and can also be used to prepare anti-inflammatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine chemistry and natural product separation technology, specifically involving a new triterpenoid compound and its epimers from Acanthopanax senticosus, as well as their extraction and separation methods and applications. Background Technology

[0002] Short-stemmed Acanthopanax senticosus Acanthopanax sessiliflorus *Acanthopanax senticosus* (Rupr. et Maxim.) Seem is a plant belonging to the genus *Acanthopanax* of the family Araliaceae. Also known as stalkless eleutherococcus or crow's seed, it is mainly distributed in Northeast my country. It has medicinal properties, including dispelling wind and dampness, tonifying the liver and kidneys, strengthening muscles and bones, and promoting blood circulation. Its roots, stems, leaves, and fruits can all be used medicinally. Besides its medicinal uses, its tender stems have long been a popular and prized wild vegetable. In 2008, *Acanthopanax senticosus* was approved as a new resource food, and its stems, leaves, and fruits have become a potential raw material for "preventive medicine," showing promising development prospects.

[0003] Currently, the chemical components isolated and identified from Acanthopanax senticosus mainly include triterpenoids, phenylpropanoids, phenolic acids, volatile oils, flavonoids, and steroidal compounds. Studies have reported that it has a variety of pharmacological activities, such as sedation and hypnosis, anti-oxidation, anti-tumor, immunomodulation, anti-fatigue and anti-hypoxia, anti-inflammatory and analgesic, antithrombotic and antiplatelet aggregation, antihypertensive, anti-obesity, and anti-osteoporosis. It has the potential to be developed into a drug and health product.

[0004] Most of the chemical components isolated from Acanthopanax senticosus are known and have low structural novelty. Therefore, it is urgent to develop novel compounds with better activity from Acanthopanax senticosus and its processed products. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a novel triterpenoid compound from Acanthopanax senticosus and its epimeric form, along with its extraction and separation methods and applications. This invention extracts a novel compound, 22α-Hydroxy-19-epi-chiisanogenin, and its epimeric form, 22α-Hydroxychiisanogenin, from Acanthopanax senticosus, Acanthopanax senticosus, and their processed stem and leaf products prepared by steaming and drying. Studies have shown that the compound of this invention possesses anti-inflammatory properties. Furthermore, this invention provides a simple, rapid, environmentally friendly, and highly pure extraction and separation method for the compound.

[0006] To achieve the above-mentioned objectives of this invention, the present invention provides the following technical solutions.

[0007] This invention provides a novel triterpenoid compound and its epimer, with the molecular formula C0. 30 H 44O6, the compound is named 22α-Hydroxy-19-epi-chiisanogenin, and its epimeric form is named 22α-Hydroxychiisanogenin. Its chemical structure is as follows: .

[0008] This invention also provides a novel method for extracting and separating the triterpenoid compound 22α-Hydroxy-19-epi-chiisanogenin and its epimeric form 22α-Hydroxychiisanogenin, comprising the following steps: Step 1: Weigh the raw material powder of Acanthopanax gracilistylus and perform acid hydrolysis to obtain hydrolysate. After the hydrolysate is cooled, add sodium carbonate to adjust the pH to 7. After centrifugation, add alcohol to the residue for ultrasonic extraction. After evaporation under reduced pressure, dissolve in boiling water and transfer to a beaker to obtain concentrated solution for later use. Alternatively, take the raw material powder of Acanthopanax genus plants, extract it multiple times by reflux with alcohol, concentrate the extract under reduced pressure, and then use the concentrate. Step 2: The concentrate obtained in Step 1 is adsorbed and enriched by macroporous resin column. First, it is eluted with water to remove impurities, and then isocratic eluted with 95% ethanol. The eluent is collected, concentrated and dried to obtain the concentrated and dried product. Step 3: Dissolve the concentrated dried product obtained in Step 2 in methanol, mix it with silica gel, and then perform silica gel column chromatography. Elute with a dichloromethane-methanol gradient and collect the target eluent. Step 4: The target eluent obtained in Step 3 is subjected to ODS column chromatography and eluted with a methanol-water gradient. The target eluent is collected and concentrated to dryness under reduced pressure to obtain a concentrate for later use. Step 5: The concentrate obtained in Step 4 is separated and purified by high performance liquid chromatography to obtain the compound and its epimers.

[0009] Furthermore, in step 1, the Acanthopanax species are Acanthopanax breviscapus, Acanthopanax senticosus, or their stem and leaf processing products prepared by steaming and drying.

[0010] Furthermore, in step 1, the acid used for acid hydrolysis is hydrochloric acid aqueous solution or sulfuric acid aqueous solution with a concentration of 0.05-0.2 mol / L, the hydrolysis temperature is 50-90℃, and the hydrolysis time is 2-8h; the ratio of raw material powder to acid is 1:2 (g / mL).

[0011] Further, in step 1, the alcohol is a methanol aqueous solution or ethanol aqueous solution with a mass fraction of 50-95%, and the ratio of raw material powder to alcohol is 1:2 to 1:20 (g / mL).

[0012] Furthermore, in step 1, the ultrasonic extraction time is 30 min, and the vacuum evaporation temperature is 45℃.

[0013] Furthermore, in step 1, the alcohol reflux extraction is performed for 2 hours each time.

[0014] Furthermore, in step 2, the amount of 95% ethanol used is 5 times the volume of the macroporous adsorption resin; the amount of water used is 2 times the volume of the macroporous adsorption resin.

[0015] Further, in step 3, the dichloromethane-methanol gradient elution includes volume ratios of 100:0, 50:1, 20:1, 10:1, and 5:1, with the target elution fraction obtained from the 50:1 and 20:1 elution stages.

[0016] Furthermore, in step 4, the methanol-water gradient elution includes volume ratios of 30:70, 70:30, and 100:0, with the target elution fraction obtained from the 70:30 elution stage.

[0017] Furthermore, in step 5, the high-performance liquid chromatography separation uses a methanol-water (60%-75% by volume) or acetonitrile-water (38%-45% by volume) system as the mobile phase for isocratic elution; the retention times of the two compounds are between 30.42 and 50.36 min.

[0018] The above-mentioned compounds and their epimers are used in the preparation of products for the prevention or treatment of inflammatory diseases; the efficacy of the products is demonstrated by their inhibition of LPS-induced release of inflammatory factors such as TNF-α and IL-6 from RAW264.7 macrophages.

[0019] The present invention also discloses a pharmaceutical composition comprising the above-described 22α-Hydroxy-19-epi-chiisanogenin and its epimeric form 22α-Hydroxychiisanogenin or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier or excipient.

[0020] Furthermore, the use of the pharmaceutical composition described above in the preparation of a drug for treating inflammation.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0022] The isolation and pharmacological activity studies of the novel compound 22α-Hydroxy-19-epi-chiisanogenin and its epimeric form 22α-Hydroxychiisanogenin described in this invention have not been reported in the prior art. This invention provides an extraction and separation method for this novel compound, employing acid hydrolysis / ethanol reflux extraction, macroporous resin column adsorption, silica gel column chromatography, ODS column chromatography, and high-performance liquid chromatography for separation, purification, and preparation. The compound was successfully extracted and separated. This method involves only five steps and is simple and rapid. The extraction and separation process mainly uses conventional reagents such as ethanol, dichloromethane, and methanol. The process is simple and environmentally friendly, and the compounds obtained by this method have high purity, all greater than 90%. In addition, studies have shown that this compound has anti-inflammatory effects. Therefore, the compound 22α-Hydroxy-19-epi-chiisanogenin and its epimeric form 22α-Hydroxychiisanogenin can be used as lead compounds for the synthesis of other compounds, as well as raw materials for new drug development and pharmacological activity research. They can also be used to prepare anti-inflammatory drugs. Attached Figure Description

[0023] Figure 1 This is a high-resolution mass spectrum of the compound 22α-Hydroxy-19-epi-chiisanogenin of the present invention.

[0024] Figure 2 The compound 22α-Hydroxy-19-epi-chiisanogenin of this invention 13 C-NMR spectrum.

[0025] Figure 3 The compound 22α-Hydroxy-19-epi-chiisanogenin of this invention 1 H-NMR spectrum.

[0026] Figure 4 The image shows the HSQC spectrum of the compound 22α-Hydroxy-19-epi-chiisanogenin of this invention.

[0027] Figure 5 The HMBC spectrum of the compound 22α-Hydroxy-19-epi-chiisanogenin of this invention is shown.

[0028] Figure 6 The NOESY diagram is for the compound 22α-Hydroxy-19-epi-chiisanogenin of this invention.

[0029] Figure 7This is a NOESY diagram of the compound 22α-Hydroxychiisanogenin of this invention. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0031] Example 1.

[0032] This invention provides a novel triterpenoid compound, 22α-Hydroxy-19-epi-chiisanogenin, and its epimeric form, 22α-Hydroxychiisanogenin, with the molecular formula C. 30 H 44 O6, the chemical structural formula of which is shown below: .

[0033] The above compounds were named 22α-Hydroxy-19-epi-chiisanogenin and 22α-Hydroxychiisanogenin based on their structures. Table 1 shows the NMR data of these compounds. 1 H-NMR (600MHz) and 13 C-NMR (150MHz) in C5D5N.

[0034] Table 1. NMR data of compounds 22α-Hydroxy-19-epi-chiisanogenin and 22α-Hydroxychiisanogenin of the present invention.

[0035] The structure of the novel compound 22α-Hydroxy-19-epi-chiisanogenin of this invention was identified and deduced.

[0036] The compound is a white powder, readily soluble in pyridine, but sparingly soluble in water. HR-ESI-MS yielded a quasi-molecular ion peak at m / z 501.3214 [M+H]. + The calculated value was 501.3216, and a quasi-molecular ion peak of 483.3109 [M+H-H2O] was observed. + 1001.6353 [2M+H] + 1023.6172 [2M+Na] + 1039.5924 [2M+K] + The final molecular formula of the new compound was determined to be C64.30 H 44 O6. Compound 13 The C-NMR spectrum (C5D5N, 150MHz) yielded 30 carbon signals: δ179.00 and 173.13 were carbonyl carbon signals, and four alkene carbon signals were also observed, δ147.84, 151.53, 113.86, and 110.75. Four alkoxy carbon signals were also observed, δ70.59, 75.68, and 75.81. In addition, there was a carbon signal at δ62.77, which was the 17th carbon signal (connected to a carboxyl group). 1 1H-NMR spectrum (C5D5N, 600MHz) shows that the compound contains four double-bonded hydrogen signals at 5.15 (1H, s), 5.04 (1H, s), 5.08 (1H, d, J=1.98 Hz), 4.73 (1H, dq, J=1.20, 2.50 Hz), three oxymethimide hydrogens at 3.75 (1H, d, J=8.04 Hz), 4.69 (1H, ddd, J=9.03 Hz), 4.83 (1H, d, J=5.28 Hz), and five typical methyl hydrogens at 1.89 (3H, s), 1.03 (3H, s), 1.10 (3H, s), 1.23 (3H, s), 2.00 (3H, s). Comprehensive analysis of the hydrocarbon data revealed that the compound is highly similar to the known compound 22α-Hydroxychiisanogenin (Shirasuna K, Miyakoshi M, Mimoto S, et al. Lupane triterpenoid glycosylesters from leaves of Acanthopanax divaricatus[J]. Phytochemistry,1997,45(3):579-584), differing only in the chemical shifts of carbons 11, 16, and 20, which differ by 0.2-0.3. Furthermore, no NOE effect was observed at δ 3.72 (H-19) and 3.21 (H-13) in the ROESY spectrum, indicating that H-19 and H13 are on opposite sides. In conclusion, the compound is a diastereomer of similar compounds, and the structure of the compound is determined to be 22α-Hydroxy-19-epi-chiisanogenin.

[0037] Structural identification of the compound 22α-Hydroxychiisanogenin of this invention.

[0038] This compound is a white powder, readily soluble in pyridine, but sparingly soluble in water. 13The C-NMR spectrum (C5D5N, 150MHz) yielded 30 carbon signals: carbonyl carbon signals at δ179.00 and 173.10, four alkene carbon signals at δ147.80, 151.18, 113.90, and 110.95, four alkoxy carbon signals at δ70.57, 75.47, and 75.56, and a carbon signal at δ62.61, which is the 17th carbon signal (connected to a carboxyl group). 1 1H-NMR spectrum (C5D5N, 600MHz) showed that the compound contained four double-bonded hydrogen signals at 5.15 (1H, s), 5.04 (1H, s), 5.08 (1H, d, J=2.04 Hz), and 4.72 (1H, dq, J=1.15, 2.3 Hz), three oxymethyl hydrogens at 3.75 (1H, d, J=8.10 Hz), 4.67 (1H, ddd, J=8.84 Hz), and 4.86 (1H, d, J=5.22 Hz), and five typical methyl hydrogens at 1.89 (3H, s), 1.03 (3H, s), 1.09 (3H, s), 1.21 (3H, s), and 1.99 (3H, s). Comprehensive analysis of the hydrocarbon data revealed that the compound is basically consistent with the known compound 22α-Hydroxychiisanogenin (Shirasuna K, Miyakoshi M, Mimoto S, et al. Lupane triterpenoid glycosyl esters from leaves of Acanthopanax divaricatus[J]. Phytochemistry,1997,45(3):579-584), and is also highly similar to the new compound 22α-Hydroxy-19-epi-chiisanogenin. However, its proton NMR data differs significantly from those of 22α-Hydroxy-19-epi-chiisanogenin, especially at δ 3.18 (1H, t, J=14.58 Hz, H-13) and 3.67 (1H, dt, J=4.82, 11.04 Hz, H-19). The NOE effect observed at 3.67 (H-19) and 3.18 (H-13) on the ROESY spectrum indicates that H-19 and H-13 are on the same side. Therefore, the structure of the compound is determined to be 22α-Hydroxychiisanogenin.

[0039] This invention also provides a method for extracting and separating the above-mentioned compounds, the specific steps of which are as follows: Step 1: Prepare concentrated extract using Acanthopanax gracilistylus stems and leaves: Weigh 500g of dried Acanthopanax gracilistylus powder, add 1000mL of 80% ethanol, reflux extract twice, 2h each time, concentrate the extract under reduced pressure, and set aside the concentrate for use.

[0040] Step 2: Inject the concentrated solution from Step 1 into a 1L AB-8 macroporous resin column for adsorption, and elute sequentially with water (2L) and 95% ethanol (5L). Concentrate and dry the 95% ethanol eluent to obtain the concentrated and dried product.

[0041] Step 3: Dissolve the concentrated dried product obtained in Step 2 in methanol, mix with 100-200 mesh silica gel, and then separate using a 200-300 mesh silica gel column. Elute using a dichloromethane-methanol gradient (100:0, 50:1, 20:1, 10:1, 5:1, v / v). Develop the eluted portions of dichloromethane-methanol (50:1) and dichloromethane-methanol (20:1) with vanillin-concentrated sulfuric acid colorimetric reagent. Combine the same components, concentrate under reduced pressure to dryness, and set aside for later use.

[0042] Step 4: The product obtained in Step 3 is further separated by pretreated ODS column (Octadecylsilyl, octadecylsilane bonded silica gel packing material) chromatography, eluted with methanol-water gradient (10:90, 30:70, 70:30, 100:0, v / v) to obtain 4 elution fractions. The 70:30 methanol-water elution fraction is concentrated to dryness under reduced pressure to obtain the concentrate for later use.

[0043] Step 5: The concentrate obtained in Step 4 was separated by HPLC (high performance liquid chromatography). Isocratic elution was performed using 70% methanol-water as the mobile phase at a flow rate of 2 mL / min and a detection wavelength of 210 or 203 nm. Finally, the above-mentioned new compound 22α-Hydroxy-19-epi-chiisanogenin and its epimeric form 2α-Hydroxychiisanogenin were obtained. The purity of both was determined to be above 98% after normalization.

[0044] Example 2: Anti-inflammatory effect of the compound of the present invention.

[0045] 1. Main materials.

[0046] 1.1 Cell lines Mouse mononuclear macrophage leukemia cells RAW264.7 (TCM13) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0047] 1.2 Reagents Indomethacin (Sigma); MTT (Amreso); DMSO (Sigma); DMEM medium (HyClone); trypsin (HyClone); fetal bovine serum (Gibco); penicillin antibody (Hyclone); PBS phosphate buffer (Solarbio); lipopolysaccharide (LPS) (Sigma); IL-6 ELISA kit (Beijing Chenglin Biotechnology Co., Ltd.); TNF-α ELISA kit (Beijing Chenglin Biotechnology Co., Ltd.).

[0048] 1.3 Preparation of main reagents DMEM culture medium: Take an appropriate amount of DMEM culture medium, add 10% fetal bovine serum inactivated at 56 ℃ and 1% penicillin antibiotics, filter through a 0.22μm filter membrane, and store at 4 ℃ for later use.

[0049] MTT solution: Dissolve 50 mg MTT in 10 mL of PBS buffer (pH 7.4, 0.01 mol / L), filter through a 0.22 μm filter membrane, aliquot, and store at -20 ℃ protected from light for later use.

[0050] Preparation of the stock solution of the test compound: Accurately weigh a certain amount of the test compound and dissolve it in DMSO. Prepare a high-concentration stock solution according to the table below. Store at -20℃ in the dark. When conducting the experiment, dilute with culture medium to the required concentration and use after sterilization by 0.22 μm filtration.

[0051] Table 2. Preparation of the mother liquor for the test sample.

[0052] 2 Experimental Methods 2.1 Cell Culture Place the cells in a 37°C incubator containing 5% CO2. The cells will be round and adhere to the culture vessel. Change the medium daily. When 70-80% of the cells have adhered, discard the old culture medium, add 5 mL of fresh culture medium, gently pipette the cells, and then passage them into new culture flasks. The passage ratio is 1:3, and the cells are passaged every two days.

[0053] 2.2 Effect of the compound on the proliferation of RAW264.7 cells was detected by MTT assay.

[0054] RAW264.7 cells in logarithmic growth phase were selected and the cell density was adjusted to 2 × 10⁻⁶. 5Cells were seeded at a concentration of 100 μL / mL in each well of a 96-well plate. When the cells reached 70%-80% confluence, 22α-Hydroxy-19-epi-chiisanogenin and 22α-Hydroxyepi-chiisanogenin were added to each well at final concentrations of 200 μmol / L, 100 μmol / L, 50 μmol / L, 20 μmol / L, 10 μmol / L, and 1 μmol / L, respectively. A negative control group (cells only) and a blank control group (no cells, only culture medium) were also included. After 24 h, 10 μL of 5 mg / mL MTT was added to each well, and the plates were incubated for another 4 h. The MTT-containing culture medium was then aspirated, and 100 μL of DMSO was added to each well. The plates were shaken for 10 min, and the absorbance was measured at 492 nm (reference wavelength 630 nm) using a microplate reader. The cell proliferation inhibition rate was calculated. Proliferation inhibition rate (%) = ((1-(A)) 实验组 -A 空白对照组 ) / (A 阴性对照组 -A 空白对照组 ))×100%.

[0055] 2.3 ELISA method for detecting the release of IL-6 and TNF-α 2.3.1 Collection of cell supernatant RAW264.7 cells in the logarithmic growth phase were harvested and the cell density was adjusted to 1×10⁻⁶. 5 Cells were seeded at a rate of 1 mL / well in a 24-well plate. When cells reached 70%-80% confluence, the following groups were established: a negative control group (cells only), a model group (cells seeded with LPS only), a positive control group (cells seeded with indomethacin and LPS), and an experimental group (cells seeded with the analyte and LPS). The positive control group received indomethacin at a final concentration of 200 μmol / L. The experimental groups received 2 hours of pretreatment with 22α-Hydroxy-19-epi-chiisanogenin and 22α-Hydroxyepi-chiisanogenin at final concentrations of 10 μmol / L, 30 μmol / L, and 60 μmol / L, respectively. After this pretreatment, except for the blank control group, each well received LPS at a final concentration of 10 mg / L. After 24 hours of incubation, the cell supernatant from each well was collected, centrifuged at 3000 rpm for 20 minutes, and the supernatant was carefully collected for ELISA kit analysis.

[0056] 2.3.2 Detection methods for ELISA kits Add 40 μL of sample diluent and 10 μL of cell supernatant to an enzyme-labeled plate, cover with a sealing film, and incubate at 37 ℃ for 30 min. Remove the sealing film, add 250 μL of washing buffer to each well, wash 5 times, and blot dry. Add 50 μL of enzyme-labeled reagent to each well, cover with a sealing film, incubate at 37 ℃ for 30 min, remove the sealing film, add 250 μL of washing buffer to each well, wash 5 times, and blot dry. Add 50 μL each of chromogenic reagent A and B to each well, vortex to mix, and incubate at 37 ℃ in the dark for 15 min. Stop the reaction by adding 50 μL of stop solution to each well. Measure the absorbance of each group at 450 nm. Calculate the inhibitory effect on COX-2 enzyme activity based on the PGE2 release of each monomer compound using the following formula: Inhibition rate % = (A LPS组 –A 实验组 ) / (A LPS组 -A 阴性对照组 ) × 100%.

[0057] 2.4 Data Processing All experimental data were processed using Excel and expressed as mean ± standard deviation. (±s) represents the proliferation inhibition rate, cytokine release, and COX-2 enzyme activity inhibition rate. Intergroup comparisons were performed using one-way ANOVA in SPSS 20.0, with LSD analysis for statistical analysis.

[0058] 3. Experimental Results 3.1 Effects of the compound on the proliferation of RAW264.7 cells Following the experimental procedure described above, the inhibition rate of the compound on the proliferation of RAW264.7 cells is shown in the table.

[0059] Table 3. Inhibition rate of different concentrations of compounds on cell proliferation (%) ±s, n=3) Note: ** P<0.01, compared with the negative control group.

[0060] Table 4. Effects of compounds on LPS-induced release of IL-6 and TNF-α from RAW264.7 cells ( ±s, n=3) Note: ## P<0.01, compared with the negative control group; ** P<0.01, compared with the LPS model group.

[0061] Experimental results show that the 22α-Hydroxy-19-epi-chiisanogenin and 22α-Hydroxyepi-chiisanogenin at various concentrations of the present invention have a significant inhibitory effect on the release of IL-6 and TNF-α induced by LPS in RAW264.7 cells, and the effect is significantly enhanced with increasing concentration, showing strong anti-inflammatory activity.

[0062] In summary, this invention provides a method for the extraction and separation of novel compounds and their epimers. The method employs ethanol reflux extraction, macroporous resin column adsorption, silica gel column chromatography, ODS column chromatography, and high-performance liquid chromatography for separation. A pair of compounds with anti-inflammatory activity were successfully separated. This method is simple, rapid, and environmentally friendly, and the compounds obtained by this method have high purity. Due to the unique chemical structure of the obtained novel compounds, which are reported for the first time and extracted from the hydrolysis products of Acanthopanax senticosus and related traditional Chinese medicines, and possessing anti-inflammatory effects, the compounds and their compositions of this invention have broad prospects for developing new traditional Chinese medicines as natural products.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A novel triterpenoid compound and its epimer from Acanthopanax senticosus, characterized in that, The molecular formula is C 30 H 44 O6, the compound is named 22α-Hydroxy-19-epi-chiisanogenin, and its epimeric form is named 22α-Hydroxychiisanogenin. Its chemical structure is as follows: 。 2. The method for extracting and separating a novel triterpenoid compound and its epimers from Acanthopanax senticosus according to claim 1, characterized in that, Includes the following steps: Step 1: Weigh the raw material powder of Acanthopanax gracilistylus and perform acid hydrolysis to obtain hydrolysate. After the hydrolysate is cooled, add sodium carbonate to adjust the pH to 7. After centrifugation, add alcohol to the residue for ultrasonic extraction. After evaporation under reduced pressure, dissolve in boiling water and transfer to a beaker to obtain concentrated solution for later use. Alternatively, take the raw material powder of Acanthopanax genus plants, extract it multiple times by reflux with alcohol, concentrate the extract under reduced pressure, and then use the concentrate. Step 2: The concentrate obtained in Step 1 is adsorbed and enriched by macroporous resin column. First, it is eluted with water to remove impurities, and then isocratic eluted with 95% ethanol. The eluent is collected, concentrated and dried to obtain the concentrated and dried product. Step 3: Dissolve the concentrated dried product obtained in Step 2 in methanol, mix it with silica gel, and then perform silica gel column chromatography. Elute with a dichloromethane-methanol gradient and collect the target eluent. Step 4: The target eluent obtained in Step 3 is subjected to ODS column chromatography and eluted with a methanol-water gradient. The target eluent is collected and concentrated to dryness under reduced pressure to obtain a concentrate for later use. Step 5: The concentrate obtained in Step 4 is separated and purified by high performance liquid chromatography to obtain the compound and its epimers.

3. The extraction and separation method according to claim 2, characterized in that, In step 1, the acid used for acid hydrolysis is hydrochloric acid aqueous solution or sulfuric acid aqueous solution with a concentration of 0.05-0.2 mol / L, the hydrolysis temperature is 50-90℃, and the hydrolysis time is 2-8h; the ratio of raw material powder to acid is 1:2 g / mL.

4. The extraction and separation method according to claim 2, characterized in that, In step 1, the alcohol is a methanol aqueous solution or ethanol aqueous solution with a mass fraction of 50-95%, and the ratio of raw material powder to alcohol is 1:2 to 1:20 g / mL.

5. The extraction and separation method according to claim 2, characterized in that, In step 1, the ultrasonic extraction time is 30 min, the vacuum evaporation temperature is 45℃, and the alcohol reflux extraction is performed for 2 h each time.

6. The extraction and separation method according to claim 2, characterized in that, In step 3, the dichloromethane-methanol gradient elution includes volume ratios of 100:0, 50:1, 20:1, 10:1, and 5:1, with the target elution fraction obtained from the 50:1 and 20:1 elution stages.

7. The extraction and separation method according to claim 2, characterized in that, In step 4, the methanol-water gradient elution includes volume ratios of 30:70, 70:30, and 100:0, and the target elution fraction is obtained from the 70:30 elution stage.

8. The extraction and separation method according to claim 2, characterized in that, In step 5, high-performance liquid chromatography separation uses a methanol-water or acetonitrile-water system as the mobile phase for isocratic elution; the retention times of the two compounds are between 30.42 and 50.36 min.

9. The use of a triterpenoid compound as described in claim 1 and its epimers in the preparation of anti-inflammatory drugs.

10. A pharmaceutical composition, characterized in that, It includes the aforementioned triterpenoid compound and its epimers or pharmaceutically acceptable salts or esters thereof, as well as a pharmaceutically acceptable carrier or excipient.

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