Proterpane type triterpenoids in rhizoma alismatis as well as extraction method and application of prototerpane type triterpenoids

Abstract

The invention discloses a prototerpane type triterpenoid compound in rhizoma alismatis as well as an extraction method and application thereof, belongs to the field of traditional Chinese medicine extraction, and particularly relates to a prototerpane type triterpenoid compound which is separated from the rhizoma alismatis and is shown in general formulas (I)-(VI), an extraction method of the prototerpane type triterpenoid compound and application of the prototerpane type triterpenoid compound in preparation of anti-inflammatory drugs. The structural diversity of active substances in the rhizoma alismatis is further enriched, on the basis, a foundation is laid for carrying out related biological activity tests on subsequently obtained monomeric compounds, an active lead compound is provided for new drug development, and meanwhile, a theoretical basis is provided for deep research and development of dry rhizoma alismatis tuberous root medicinal materials.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine extraction, and particularly relates to a proto-terpane triterpenoid compound separated from Alisma orientale (Sam.) Juzep and an extraction method thereof and application of the compound in preparation of anti-inflammatory drugs. BACKGROUND

[0002] The chemical components in Alisma orientale mainly include triterpenes, sesquiterpenes, aliphatic hydrocarbons and derivatives thereof, saccharides and alkaloids, etc. (Liu S S. Chemical constituents from Alismatis Rhizoma and their anti-inflammatory activities [D]. China Academy of Chinese Medical Sciences, 2019.), and experiments prove that Alisma orientale has anti-inflammatory activity (Liu S S, Sheng W L, Li Y, et al. Chemical constituents from Alismatis Rhizoma and their anti-inflammatory activities in vitro and in vivo [J]. Bioorganic Chemistry, 2019, 92: 103226.). SUMMARY

[0003] The primary object of the present application is to provide a proto-terpane triterpenoid compound in Alisma orientale or an isomer of the compound or a pharmaceutically acceptable salt of the compound.

[0004] The second object of the present application is to provide an extraction method of the proto-terpane triterpenoid compound in Alisma orientale.

[0005] The third object of the present application is to provide a pharmaceutical composition containing the proto-terpane triterpenoid compound in Alisma orientale.

[0006] The fourth object of the present application is to provide application of the proto-terpane triterpenoid compound in Alisma orientale or an isomer of the compound or a pharmaceutically acceptable salt of the compound or a pharmaceutical composition containing the compound in preparation of anti-inflammatory drugs.

[0007] The technical scheme of the present application comprises the following: The proto-terpane triterpenoid compound as shown in general formula (I)-(VI) or an isomer of the compound or a pharmaceutically acceptable salt of the compound.

[0008] The general formula (I), (II), (III), (IV), (V) and (VI) are as follows:

[0009] R1 is H or α -hydroxyl; R2 is H or hydroxyl;

[0010] R1 is α -hydroxyl or β -acetoxy; R2 is terminal double bond or hydroxyl; R3, R4 are each independently H or form double bond;

[0011] R1, R2 are each independently H or β -hydroxyl; R3 is carbonyl or H; R4 is carbonyl or α -hydroxyl; R5 and R6 form double bond or R5 is α -hydroxyl; R6 is ethoxyl;

[0012] R1 is H or acetoxy;

[0013] R1, R2 are each independently H or form double bond; R3 is carbonyl or α -acetoxy; R4 is H or hydroxyl;

[0014] R1, R2 are each independently H or form double bond; R3 is hydroxyl or carbonyl.

[0015] Further, the proto-terpanoid triterpenoid compound is any one of the compounds shown in the following structural formulae 1-10 or an isomer of the compound or any one of the pharmaceutically acceptable salts of the compound.

[0016] The pharmaceutically acceptable salt includes sodium salt, potassium salt, ammonia salt, hydrochloride and sulfate formed by the proto-terpanoid triterpenoid compound and pharmaceutically acceptable inorganic acid or organic acid or inorganic base or organic base.

[0017] The isomer includes optical isomer, cis-trans isomer, racemate and mixture thereof.

[0018] The application further provides an extraction method of the proto-terpanoid triterpenoid compound, which comprises the following steps: (1) Using the dried tuberous roots of Alisma plantago-aquatica as raw material, add 5 to 15 times the volume of methanol of the raw material, reflux extract 2 to 4 times, extract for 2 to 4 hours each time, combine the extracts, recover the solvent under reduced pressure, concentrate to obtain total extract; (2) Disperse the total extract into 5 to 10 times its mass volume of water, extract with ethyl acetate, concentrate the extract to recover the solvent, and obtain ethyl acetate extract concentrate and aqueous phase respectively; (3) The ethyl acetate extract concentrate was separated by silica gel column chromatography, using petroleum ether-ethyl acetate with a volume ratio of 100:1 to 0:1 as the eluent gradient, and the fraction E2 with a volume ratio of 50:1 was collected. (4) The concentrated fraction E2 was separated by silica gel column chromatography and eluted with petroleum ether-acetone in a volume ratio of 30:1 to 0:1. Further purification yielded proterpane triterpenoid compounds 1 to 10.

[0019] In the above extraction method, the term "mass-to-volume ratio" refers to the volume-to-mass ratio of solvent to material, expressed in L / kg. For example, "5~15 mass-to-volume ratio" means that the volume-to-mass ratio of added methanol to raw material is (5~15):1, expressed in L / kg; similarly, "5~10 mass-to-volume ratio" means that the volume-to-mass ratio of added water to total extract is (5~10):1, expressed in L / kg. Any similar expressions of "mass-to-volume ratio" appearing elsewhere in this invention have the same meaning as defined above, i.e., corresponding to the volume-to-mass ratio of solvent to material, expressed in L / kg.

[0020] In the above extraction method, the specific separation and purification process of fraction E2 in step (4) is as follows: After concentration, fraction E2 was separated by silica gel column chromatography, using a gradient elution of petroleum ether-acetone at a volume ratio of 30:1 to 0:1. The fraction with a petroleum ether-acetone volume ratio of 20:1 was collected and designated as E22, the fraction with a volume ratio of 10:1 was designated as E23, and the fraction with a volume ratio of 3:1 was designated as E25.

[0021] After concentration, fraction E22 was separated by ODS column chromatography, using a gradient elution of methanol-water with a volume ratio of 50:50 to 100:0. The fraction with a volume ratio of 60:40 was collected and designated as E222.

[0022] After concentration, fraction E222 was purified by preparative HPLC with methanol-water (80:20 v / v) as the mobile phase to obtain compounds 1, 2, 5, 7, 8 and 9.

[0023] After concentration, fraction E23 was separated by ODS column chromatography, using a gradient elution of methanol-water with a volume ratio of 50:50 to 100:0. The fraction with a volume ratio of 80:20 was collected and designated as E233; the fraction with a volume ratio of 90:10 was collected and designated as E234.

[0024] After concentration, fraction E233 was separated by ODS column chromatography, using a gradient elution of methanol-water with a volume ratio of 50:50 to 100:0. The fraction with a volume ratio of 50:50 was collected and designated as E2331, and the fraction with a volume ratio of 80:20 was designated as E2334.

[0025] Fraction E2331 was concentrated and purified by preparative HPLC with acetonitrile-water (50:50 v / v) as the mobile phase to obtain compound 10.

[0026] After concentration, fraction E2334 was purified by preparative HPLC using a methanol-water mixture with a volume ratio of 80:20 as the mobile phase to obtain compound 4.

[0027] Fraction E234 was separated by silica gel column chromatography, using a gradient elution of petroleum ether-ethyl acetate with a volume ratio of 10:1 to 0:100. The fraction with a volume ratio of petroleum ether-ethyl acetate of 3:1 was collected and designated as E2345.

[0028] Fraction E2345 was concentrated and purified by preparative HPLC with acetonitrile-water as the mobile phase at a volume ratio of 70:30 to obtain compound 3.

[0029] After concentration, fraction E25 was separated by ODS column chromatography, using a gradient elution of methanol-water with a volume ratio of 30:70 to 100:0. The fraction with a volume ratio of 80:20 was collected and designated as E255.

[0030] After concentration, fraction E255 was separated by ODS column chromatography, using a gradient elution of methanol-water with a volume ratio of 50:50 to 70:30. The fraction with a volume ratio of 65:35 was collected and designated as E2553.

[0031] Fraction E2553 was separated by silica gel column chromatography, using a gradient elution of petroleum ether-ethyl acetate with a volume ratio of 20:1 to 10:1. The fraction with a petroleum ether-ethyl acetate volume ratio of 10:1 was collected and designated as E25539.

[0032] Fraction E25539 was concentrated and purified by preparative HPLC with acetonitrile-water as the mobile phase at a volume ratio of 75:25 to obtain compound 6.

[0033] The proterane-type triterpenoid compound is an extract from the dried tuberous root of Alisma plantago-aquatica.

[0034] The application of the dried tuberous root extract of Alisma plantago-aquatica in the preparation of anti-inflammatory drugs.

[0035] A pharmaceutical composition comprising one or more of the aforementioned proterane-type triterpenoid compound, or an isomer of the compound, or a pharmaceutically acceptable salt of the compound; further comprising one or a combination of a pharmaceutically acceptable carrier, excipient, and diluent. The pharmaceutical composition is administered orally or by injection, and in dosage forms including tablets, capsules, powders, syrups, and injections.

[0036] The present invention also provides the use of the proterane-type triterpenoid compound, or an isomer of the compound, or a pharmaceutically acceptable salt of the compound, or the pharmaceutical composition thereof, in the preparation of anti-inflammatory drugs.

[0037] The beneficial effects of this invention are: The present invention relates to the inhibitory effect of proterane-type triterpenoid compounds or their isomers or pharmaceutically acceptable salts, or pharmaceutical compositions containing such compounds, on LPS-induced NO production in RAW264.7 cells, and applies this to the preparation of anti-inflammatory drugs. The method of this invention further enriches the structural diversity of active substances in Alisma plantago-aquatica, laying the foundation for subsequent bioactivity testing of the obtained monomeric compounds, providing active lead compounds for new drug development, and also providing a theoretical basis for in-depth research and development of dried tuberous roots of Alisma plantago-aquatica. Attached Figure Description

[0038] Figure 1 The inhibitory activity of compounds 1-10 of this invention on LPS-induced NO production in RAW264.7. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the raw materials and reagents used in the present invention can be obtained through commercial channels.

[0040] Example 1

[0041] The extraction method for proterane-type triterpenoids from Alisma plantago-aquatica includes the following steps: (1) Using 100.0 kg of dried tuberous roots of Alisma plantago-aquatica as raw material, add 8 times the mass volume of methanol (800 L) of the raw material, reflux extract 3 times, extract for 3 hours each time, combine the extracts, recover the solvent under reduced pressure, concentrate and obtain total extract (20.0 kg).

[0042] (2) Disperse the total extract into 5 times the volume of water (100 L), extract with an equal volume of ethyl acetate, concentrate the extract to recover the solvent, and obtain ethyl acetate extract concentrate (2.2 kg) and aqueous phase respectively.

[0043] (3) The ethyl acetate extract concentrate was separated by silica gel column chromatography, and the petroleum ether-ethyl acetate was used as the eluent in a gradient elution with a volume ratio of 100:1 to 0:1. The fraction E2 with a volume ratio of 50:1 was collected.

[0044] (4) The concentrated fraction E2 was separated by silica gel column chromatography and eluted with a gradient of petroleum ether-acetone at a volume ratio of 30:1 to 0:1. Further purification yielded 34.8 mg compound 1, 8.8 mg compound 2, 36.2 mg compound 3, 35.5 mg compound 4, 6.7 mg compound 5, 63.1 mg compound 6, 20.7 mg compound 7, 35.3 mg compound 8, 6.7 mg compound 9, and 21.8 mg compound 10.

[0045] The specific separation and purification process of fraction E2 in step (4) is as follows: After concentration, fraction E2 was separated by silica gel column chromatography, using a gradient elution of petroleum ether-acetone at a volume ratio of 30:1 to 0:1. The fraction with a petroleum ether-acetone volume ratio of 20:1 was collected and designated as E22, the fraction with a volume ratio of 10:1 was designated as E23, and the fraction with a volume ratio of 3:1 was designated as E25.

[0046] After concentration, fraction E22 was separated by ODS column chromatography, using methanol-water gradient elution with volume ratios of 50:50, 60:40, 70:30, 80:20, and 100:0. The fraction with a volume ratio of 60:40 was collected and designated as E222.

[0047] After concentration, fraction E222 was purified by preparative HPLC with methanol-water (80:20 v / v) as the mobile phase to obtain compounds 1, 2, 5, 7, 8 and 9.

[0048] After concentration, fraction E23 was separated by ODS column chromatography, using a gradient elution with methanol-water eluent at volume ratios of 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0. The fraction with a volume ratio of 80:20 was collected and designated as E233; the fraction with a volume ratio of 90:10 was collected and designated as E234.

[0049] After concentration, fraction E233 was separated by ODS column chromatography, using methanol-water gradient elution with volume ratios of 50:50, 60:40, 70:30, 80:20, and 100:0. The fraction with a volume ratio of 50:50 was collected and designated as E2331, and the fraction with a volume ratio of 80:20 was designated as E2334.

[0050] Fraction E2331 was concentrated and purified by preparative HPLC with acetonitrile-water (50:50 v / v) as the mobile phase to obtain compound 10.

[0051] After concentration, fraction E2334 was purified by preparative HPLC using a methanol-water mixture with a volume ratio of 80:20 as the mobile phase to obtain compound 4.

[0052] Fraction E234 was separated by silica gel column chromatography, eluted sequentially with petroleum ether-ethyl acetate eluent at volume ratios of 10:1, 8:1, 6:1, 3:1, and 0:100. The fraction with a petroleum ether-ethyl acetate volume ratio of 3:1 was collected and designated as E2345.

[0053] Fraction E2345 was concentrated and purified by preparative HPLC with acetonitrile-water as the mobile phase at a volume ratio of 70:30 to obtain compound 3.

[0054] After concentration, fraction E25 was separated by ODS column chromatography, using methanol-water gradient elution with volume ratios of 30:70, 50:50, 60:40, 70:30, 80:20, and 100:0. The fraction with a volume ratio of 80:20 was collected and designated as E255.

[0055] After concentration, fraction E255 was separated by ODS column chromatography, using methanol-water gradient elution with volume ratios of 50:50, 60:40, 65:35, and 70:30. The fraction with a volume ratio of 65:35 was collected and designated as E2553.

[0056] Fraction E2553 was separated by silica gel column chromatography, eluted sequentially with petroleum ether-ethyl acetate eluent at volume ratios of 20:1, 15:1, and 10:1. The fraction with a petroleum ether-ethyl acetate volume ratio of 10:1 was collected and designated as E25539.

[0057] Fraction E25539 was concentrated and purified by preparative HPLC with acetonitrile-water as the mobile phase at a volume ratio of 75:25 to obtain compound 6.

[0058] The structures of the extracted proterane-type triterpenoid compounds 1-10 were identified, and the specific physicochemical data are as follows: Compound 1: Colorless, transparent crystals (methanol). HR-ESI-MSm / z 495.3445 [M + Na] + (calcd. forC 30 H 48 O4Na + , 495.3445), molecular formula C 30 H 48 O4. + 43.422 ( c = 0.10, CH3OH). 1 H-NMR (600MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 1.

[0059] Compound 2: Colorless, transparent crystals (methanol). HR-ESI-MS m / z 455.3521 [M + H] + (calcd. forC 30 H 47 O3 + (455.3525), molecular formula C 30 H 46 O3. :- 55.770 ( c = 0.10, CH3OH). 1 H-NMR (600MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 2.

[0060] Compound 3: Colorless oil. HR-ESI-MS m / z 513.3579 [M + H] + (calcd. for C 32 H 49 O5 + (,513.3580), molecular formula C 32 H 48 O5. :- 32.838 ( c = 0.10, CH3OH). 1 H-NMR (600 MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 3.

[0061] Compound 4: Colorless oil. HR-ESI-MS m / z 453.3366 [M + H] +(calcd. for C 30 H 45 O3 + ,453.3368), molecular formula C 30 H 44 O3. :+ 64.734 ( c = 0.10, CH3OH). 1 H-NMR (600 MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 4.

[0062] Compound 5: Colorless oil. HR-ESI-MS m / z 547.4021 [M + HCOOH-H] - (calcd. forC 32 H 55 O6 - , 547.3999), molecular formula C 32 H 54 O4. :+ 79.672 ( c = 0.10, CH3OH). 1 H-NMR (600MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 5.

[0063] Compound 6: White amorphous powder (methanol). HR-ESI-MS m / z [M + Na] + 557.3818 (calcd.for C 32 H 54 O6Na + (, 557.3817), molecular formula is C 32 H 54 O6. :+ 40.800 ( c = 0.10, CH3OH). 1 H-NMR (600 MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 6.

[0064] Compound 7: White amorphous powder (methanol). HR-ESI-MS m / z 517.3290 [M + Na] + (calcd.for C 32 H46 O4Na + , 517.3294), molecular formula C 32 H 46 O4. :+ 23.902 ( c = 0.10, CH3OH). 1 H-NMR (600 MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 7.

[0065] Compound 8: Colorless oil. HR-ESI-MS m / z 513.3572 [M + H] + (calcd. for C 32 H 49 O5 + (,513.3580), molecular formula is C 32 H 48 O5. :- 3.568 ( c = 0.10, CH3OH). 1 H-NMR (600 MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 8.

[0066] Compound 9: White amorphous powder (methanol). HR-ESI-MS m / z 471.3469 [M + H] + (calcd. forC 30 H 47 O4 + , 471.3474), molecular formula C 30 H 46 O4. :+ 4.709 ( c = 0.10, CH3OH). 1 H-NMR (600 MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 9.

[0067] Compound 10: Colorless oil. HR-ESI-MS m / z 327.2316 [M + H] + (calcd. for C 22 H 31 O2 + (327.2324), molecular formula C22 H 30 O2. :+ 91.224 ( c = 0.10, CH3OH). 1 H-NMR (600 MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3) data are shown in Table 10.

[0068] Table 1. Carbon and proton spectrum data of compound 1

[0069] Table 2. Carbon and proton spectrum data of compound 2

[0070] Table 3. Carbon and hydrogen spectral data of compound 3

[0071] Table 4. Carbon and proton spectrum data of compound 4

[0072] Table 5. Carbon and hydrogen spectral data of compound 5

[0073] Table 6. Carbon and proton spectrum data of compound 6

[0074] Table 7. Carbon and hydrogen spectral data of compound 7

[0075] Table 8. Carbon and proton spectrum data of compound 8

[0076] Table 9. Carbon and hydrogen spectral data of compound 9

[0077] Table 10 Carbon and proton spectrum data of compound 10

[0078] Using physicochemical data and modern spectroscopic techniques (HRESIMS and NMR), combined with relevant data from published literature, the structures of the above compounds were identified, confirming that compounds 1-10 are all novel compounds not previously reported in the literature, as shown below:

[0079] Example 2

[0080] The extraction method for proterane-type triterpenoids from Alisma plantago-aquatica includes the following steps: (1) Using 50.0 kg of dried tuberous roots of Alisma plantago-aquatica as raw material, methanol (250 L) with a mass of 5 times the volume of raw material was added, and the mixture was refluxed and extracted 3 times, each time for 3 hours. The extracts were combined, the solvent was recovered under reduced pressure, and the total extract (11.0 kg) was obtained after concentration. (2) The total extract obtained was dispersed in 5 times the mass volume of water (55 L), and extracted with an equal mass volume of ethyl acetate. The extract was concentrated to recover the solvent, and ethyl acetate extract concentrate (1 kg) and aqueous phase were obtained respectively. (3) The ethyl acetate extract concentrate was separated by silica gel column chromatography, using petroleum ether-ethyl acetate with a volume ratio of 100:1 to 0:1 as the eluent gradient, and the fraction E2 with a volume ratio of 50:1 was collected. (4) After concentrating fraction E2, 390.0 g of concentrate was obtained. The concentrate was further purified by silica gel column chromatography to obtain 16.8 mg of compound 1, 4.1 mg of compound 2, 18.5 mg of compound 3, 17.3 mg of compound 4, 3.2 mg of compound 5, 33.1 mg of compound 6, 9.8 mg of compound 7, 17.6 mg of compound 8, 3.5 mg of compound 9, and 10.2 mg of compound 10. The specific separation and purification process is the same as in Example 1.

[0081] Example 3

[0082] The extraction method for proterane-type triterpenoids from Alisma plantago-aquatica includes the following steps: (1) Using 20.0 kg of dried tuberous roots of Alisma plantago-aquatica as raw material, add 10 times the volume of methanol (200 L) of the raw material, reflux extract 3 times, extract for 3 hours each time, combine the extracts, recover the solvent under reduced pressure, concentrate and obtain total extract (4.2 kg). (2) The total extract obtained was dispersed in 5 times the mass volume of water (21 L), and extracted with an equal mass volume of ethyl acetate. The extract was concentrated to recover the solvent, and ethyl acetate extract concentrate (0.45 kg) and aqueous phase were obtained respectively. (3) The ethyl acetate extract concentrate was separated by silica gel column chromatography, using petroleum ether-ethyl acetate with a volume ratio of 100:1 to 0:1 as the eluent gradient, and the fraction E2 with a volume ratio of 50:1 was collected. (4) After concentrating fraction E2, 390.0 g of concentrate was obtained. The concentrate was further purified by silica gel column chromatography to obtain 6.8 mg of compound 1, 1.7 mg of compound 2, 7.3 mg of compound 3, 6.9 mg of compound 4, 1.3 mg of compound 5, 12.6 mg of compound 6, 4.0 mg of compound 7, 7.1 mg of compound 8, 1.3 mg of compound 9, and 4.5 mg of compound 10. The specific separation and purification process is the same as in Example 1.

[0083] Example 4

[0084] The proterane-type triterpenoid compounds 1-10 obtained above were tested for their inhibitory effect on LPS-induced NO production in RAW264.7 cells, as follows: (1) Cell culture; Mouse mononuclear macrophages RAW264.7 were cultured in DMEM medium containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 U / mL penicillin sodium, and incubated at 37°C in a constant temperature incubator with 5% CO2.

[0085] (2) The effect of compounds 1-10 on inhibiting the release of nitric oxide (NO) from mouse RAW264.7 macrophages induced by lipopolysaccharide was detected by the Griess method.

[0086] 1. Principle: Excessive LPS induces the activation and expression of nitric oxide synthase (NOS) in macrophages, leading to the production of NO. NO then reacts with oxygen free radicals to rapidly form NO2. - NO2 formed - It can be quantitatively detected by Griess Reagent under acidic conditions. First, NO2 - It undergoes a diazotization reaction with sulfanilamide, followed by reaction with hydrochloric acid. N -1-Naphthyl-ethylenediamine ( N The azo compound (-1-naphthylethylene-diaminedihydrochloride) undergoes a coupling reaction to form a purple-red azo compound. Finally, the absorbance value of the compound is measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Combined with the standard curve, the NO content in the sample can be determined.

[0087] 2. Methods: RAW264.7 mouse mononuclear macrophages in the logarithmic growth phase were collected and their concentration adjusted to 3.5 × 10⁻⁶ cells. 4Cells / wells were seeded in 96-well plates, with 100 μL of cell suspension added to each well. The experiment included a control group (RAW264.7 cells, DMSO), a model group (RAW264.7 cells, DMSO, 0.5 μg / mL LPS), a positive control group (RAW264.7 cells, dexamethasone (20 μM), 0.5 μg / mL LPS), and a test drug group (RAW264.7 cells, each test compound (20 μM), 0.5 μg / mL LPS). Cells were incubated in a 5% CO2, 37℃ incubator for 24 h. Then, 40 μL of cell supernatant was transferred to an ELISA plate, and an equal volume of Griess reagent was added. The accumulation of nitrite in the culture medium was measured at 540 nm using an ELISA reader. Results are as follows: Figure 1 As shown in Table 11, compounds 3, 4, 5, 8 and 9 exhibit NO release inhibition activity comparable to that of the positive control.

[0088] Table 11 Effects of compounds 1-10 on LPS-induced RAW264.7 cell viability

[0089] The above description of the embodiments is only for the purpose of helping to understand the method and central idea of ​​the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the present invention.

Claims

1. Proteraneous triterpenoids as shown in formulas (I) to (VI), or their pharmaceutically acceptable salts: in: R1 is H or α -Hydroxy group; R2 is H or hydroxyl group; Where: R1 is α -hydroxyl or β - Acetoxy group; R2 is a terminal double bond or hydroxyl group; R3 and R4 are each independently H or form a double bond; Where: R1 and R2 are each independently H or β -hydroxyl group; R3 is carbonyl or H; R4 is carbonyl or α -hydroxyl group; R5 forms a double bond with R6 or R5 is a hydroxyl group; α -hydroxyl group; R6 is ethoxy group; Wherein: R1 is H or acetoxy; Wherein: R1 and R2 are each independently H or form a double bond; R3 is a carbonyl group or α -Acetoxy; R4 is H or hydroxyl; Wherein: R1 and R2 are each independently H or form a double bond; R3 is a hydroxyl or carbonyl group.

2. The proterane-type triterpenoid compound or its pharmaceutically acceptable salt according to claim 1, characterized in that, The compound is any one of the compounds shown in structural formulas 1 to 10 below or a pharmaceutically acceptable salt thereof; 。 3. A method for extracting the proterane-type triterpenoid compound of claim 2 or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: (1) Using the dried tuberous roots of Alisma plantago-aquatica as raw material, add 5 to 15 times the volume of methanol of the raw material, reflux extract 2 to 4 times, extract for 2 to 4 hours each time, combine the extracts, recover the solvent under reduced pressure, concentrate to obtain total extract; (2) Disperse the total extract into 5 to 10 times its mass volume of water, extract with ethyl acetate, concentrate the extract to recover the solvent, and obtain ethyl acetate extract concentrate and aqueous phase respectively; (3) The ethyl acetate extract concentrate was separated by silica gel column chromatography, using petroleum ether-ethyl acetate with a volume ratio of 100:1 to 0:1 as the eluent gradient, and the fraction E2 with a volume ratio of 50:1 was collected. (4) The concentrated fraction E2 was separated by silica gel column chromatography and eluted with petroleum ether-acetone in a volume ratio of 30:1 to 0:1 to obtain proterpane-type triterpenoids 1 to 10.

4. The extraction method according to claim 3, characterized in that, The specific separation and purification process of fraction E2 in step (4) is as follows: After concentration, fraction E2 was separated by silica gel column chromatography, using a gradient elution of petroleum ether-acetone at a volume ratio of 30:1 to 0:

1. The fraction with a petroleum ether-acetone volume ratio of 20:1 was collected and designated as E22, the fraction with a volume ratio of 10:1 was designated as E23, and the fraction with a volume ratio of 3:1 was designated as E25. After concentration, fraction E22 was separated by ODS column chromatography with a gradient elution of methanol-water at a volume ratio of 50:50 to 100:

0. The fraction with a volume ratio of 60:40 was collected and designated as E222. After concentration, fraction E222 was purified by preparative HPLC with methanol-water (80:20 v / v) as the mobile phase to obtain compounds 1, 2, 5, 7, 8 and 9. After concentration, fraction E23 was separated by ODS column chromatography with a gradient elution of methanol-water at a volume ratio of 50:50 to 100:

0. The fraction with a volume ratio of 80:20 was collected and designated as E233; the fraction with a volume ratio of 90:10 was collected and designated as E234. After concentration, fraction E233 was separated by ODS column chromatography with a gradient elution of methanol-water at a volume ratio of 50:50 to 100:

0. The fraction with a volume ratio of 50:50 was collected and designated as E2331, and the fraction with a volume ratio of 80:20 was designated as E2334. After concentration, fraction E2331 was purified by preparative HPLC with acetonitrile-water as the mobile phase at a volume ratio of 50:50 to obtain compound 10. Fraction E2334 was concentrated and purified by preparative HPLC with methanol-water (80:20 v / v) as the mobile phase to obtain compound 4. Fraction E234 was separated by silica gel column chromatography, using a gradient elution of petroleum ether-ethyl acetate with a volume ratio of 10:1 to 0:

100. The fraction with a volume ratio of petroleum ether-ethyl acetate of 3:1 was collected and designated as E2345. Fraction E2345 was concentrated and purified by preparative HPLC with acetonitrile-water as the mobile phase at a volume ratio of 70:30 to obtain compound 3. After concentration, fraction E25 was separated by ODS column chromatography with a gradient elution of methanol-water at a volume ratio of 30:70 to 100:

0. The fraction with a volume ratio of 80:20 was collected and designated as E255. After concentration, fraction E255 was separated by ODS column chromatography with a gradient elution of methanol-water at a volume ratio of 50:50 to 70:

30. The fraction with a volume ratio of 65:35 was collected and designated as E2553. Fraction E2553 was separated by silica gel column chromatography, using a gradient elution of petroleum ether-ethyl acetate with a volume ratio of 20:1 to 10:

1. The fraction with a volume ratio of petroleum ether-ethyl acetate of 10:1 was collected and designated as E25539. Fraction E25539 was concentrated and purified by preparative HPLC with acetonitrile-water as the mobile phase at a volume ratio of 75:25 to obtain compound 6.

5. A pharmaceutical composition, characterized in that, It includes one or more of the proterane-type triterpenoids as described in claim 1 or 2 or their pharmaceutically acceptable salts; it also includes one or a combination of pharmaceutically acceptable carriers, excipients, diluents.

6. The pharmaceutical composition according to claim 5, characterized in that, The drug composition is administered orally or by injection, and the dosage forms include tablets, capsules, powders, syrups, and injections.

7. The use of the proterane-type triterpenoid compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, in the preparation of an anti-inflammatory drug.

8. Use of the pharmaceutical composition according to claim 5 or 6 in the preparation of an anti-inflammatory drug.

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