A terpenoid compound isolated from purslane and its preparation method and application

Abstract

The present invention belongs to the field of medical technology, and specifically relates to a terpene compound isolated from purslane, a preparation method, and an application thereof. The chemical structural formula of the terpene compound is shown in the following formula 1). The present invention first extracts a total extract of purslane, and then extracts the total extract of purslane with organic solvents of different polarities to obtain a petroleum ether layer, an ethyl acetate layer, and an n-butanol layer. The ethyl acetate layer is repeatedly eluted with a silica gel column, an ODS reverse phase column, and a gel column. The collected identical fractions are then isocratically eluted with an HPLC column to separate the terpene compound. This terpene compound is obtained for the first time and exhibits significant anti-inflammatory activity in vitro. The present invention provides a new option for the development of natural anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a terpenoid compound separated from purslane, a preparation method and an application thereof. Background Art

[0002] Portulaca oleracea L. is a widely distributed plant with both medicinal and edible uses. It belongs to the genus Portulaca in the family Portulacaceae. The Compendium of Materia Medica states that purslane can "dissipate blood and reduce swelling, promote bowel movements and abortion, and detoxify and relieve stranguria." The Newly Revised Compendium of Materia Medica states that it "treats various swellings, fistulas, warts, stranguria, and bleeding from wounds." It is used to treat a variety of inflammatory conditions, including enteritis, dysentery, appendicitis, and urinary tract infections, and is known as a "natural antibiotic."

[0003] Modern research shows that purslane is rich in chemical components, such as alkaloids, flavonoids, terpenoids, coumarins, and organic acids, and has multiple pharmacological effects, including anti-inflammatory, antibacterial, anti-tumor, regulation of blood sugar and lipid levels, and immune enhancement. However, the anti-inflammatory effects of purslane have not yet been fully elucidated, and the anti-inflammatory active components of purslane are still unclear. Summary of the Invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a terpenoid compound separated from Portulaca oleracea and a preparation method and application thereof.

[0005] To achieve the above objectives, the technical solutions of the present invention are as follows.

[0006] First, the present invention provides a terpene compound isolated from Portulaca oleracea, the chemical structure of which is shown in the following formula 1):

[0007]

[0008] Secondly, the present invention provides a method for preparing the terpenoid compound, comprising the following steps:

[0009] S1. Using purslane as raw material, extracting with ethanol solution by heating, combining the extracts, filtering, and evaporating the ethanol to obtain purslane total extract;

[0010] S2. suspending the purslane total extract in water to obtain a suspension;

[0011] S3, extracting the suspension with petroleum ether, ethyl acetate, and n-butanol as extraction solvents in sequence, concentrating and drying the extracts under reduced pressure to obtain a petroleum ether extract, an ethyl acetate extract, and an n-butanol extract;

[0012] S4. Load the ethyl acetate extract onto a silica gel column, and after elution, color development, and merging, obtain nine eluted products, Fr.A to Fr.I. Gradient elution is performed using a dichloromethane-methanol elution system. The volume ratios of dichloromethane and methanol during elution are 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, and 0:1, respectively.

[0013] S5. The eluted products Fr.B, Fr.E, Fr.F, and Fr.G were combined and loaded onto an ODS column. After elution, color development, and combination, 19 eluted products Fr.1 to Fr.19 were obtained. A methanol-water elution system was used for gradient elution. The volume ratios of methanol and water during elution were 0:1, 1:9, 1:4, 2:3, 3:2, 4:1, and 1:0, respectively.

[0014] S6. Load the eluted product Fr.5 onto a silica gel column, and after elution and color development, combine to obtain 10 eluted products Fr.5-1 to Fr.5-10; use a dichloromethane-methanol elution system for gradient elution, with the volume ratio of dichloromethane to methanol being 100:1 and 80:1, respectively;

[0015] S7, loading the eluted product Fr.5-5 onto a gel column, performing isocratic elution with methanol, and combining the eluted products to obtain five eluted products Fr.5-5-1 to Fr.5-5-5;

[0016] S8. Separate and purify the eluted product Fr.5-5-2 by semi-preparative HPLC to obtain the terpenoid compound, and the separating liquid is methanol / ammonia solution.

[0017] Furthermore, in S1, the volume fraction of the ethanol solution is 85%, the ratio of purslane to ethanol is 1 g: 5-15 mL, the heating extraction times are 1-3 times, each time for 60-90 min, and the temperature is 65-70°C.

[0018] Furthermore, in S2, 38-40°C water is added for suspension.

[0019] Furthermore, in S3, the extraction condition is to extract 5 to 6 times according to a volume ratio of the suspension to the extraction solvent of 2:1.

[0020] Furthermore, in S8, the volume fraction of aqueous ammonia is 0.2%, and the volume ratio of methanol to aqueous ammonia is 40:60.

[0021] Furthermore, in S8, the flow rate was 2.0 mL / min, and the effluent with a retention time of 18.1 min was collected, concentrated, and dried to obtain the terpenoid compound.

[0022] Furthermore, the present invention provides the use of the terpenoid compound or a pharmaceutically acceptable salt thereof in the preparation of an anti-inflammatory drug.

[0023] Furthermore, the drug also contains pharmaceutically acceptable excipients.

[0024] The present invention also provides an anti-inflammatory drug, which contains the terpenoid compound as an effective ingredient and also contains pharmaceutically acceptable excipients.

[0025] The present invention has the following beneficial effects:

[0026] 1. The present invention is the first to isolate a novel terpenoid compound (1S,3R,5R,6S)-1-carboxy-3-hydroxy-5,6-epoxy-6-(3-methyl-2-(Z)-4-(E)-dien-valeric acid)-ionone from Portulaca oleracea. This terpenoid compound has a clear structure and strong anti-inflammatory activity, and has the potential to be made into anti-inflammatory drugs.

[0027] 2. The present invention first extracts the total extract of purslane, and extracts the total extract of purslane with organic solvents of different polarities to obtain a petroleum ether layer, an ethyl acetate layer, and an n-butanol layer. The ethyl acetate layer is repeatedly eluted with a silica gel column, an ODS reverse phase column, and a gel column. The collected identical fractions are then isocratically eluted with an HPLC column. The present invention uses a dichloromethane-methanol system as a mobile phase during silica gel column separation. The mobile phase system is low in cost and easy to obtain. The method of the present invention is simple to operate, has low raw material cost, and has high purity of the separated compound, which is beneficial for large-scale promotion and use after drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure is a high-resolution mass spectrum of the compound separated by the present invention.

[0029] Figure 2 The figure is the UV spectrum of the compound separated by the present invention.

[0030] Figure 3 The infrared spectrum of the compound separated by the present invention is shown in FIG.

[0031] Figure 4 The 1H-NMR spectrum (600 MHz) of the compound separated in the present invention.

[0032] Figure 5 The 13C-NMR spectrum (150 MHz) of the compound separated in the present invention.

[0033] Figure 6 The DEPT spectrum of the compound separated by the present invention is shown in FIG.

[0034] Figure 7 The HSQC spectrum of the compound separated in the present invention is shown in FIG.

[0035] Figure 8 The figure is the HMBC spectrum of the compound separated by the present invention.

[0036] Figure 9 1H-1H COSY spectrum of the compound separated by the present invention.

[0037] Figure 10 The NOESY spectrum of the compound separated by the present invention is shown in FIG.

[0038] Figure 11 is a coupling correlation diagram of the compounds of the present invention.

[0039] Figure 12 The measured ECD and calculated ECD spectra of the compounds of the present invention are shown in FIG.

[0040] Figure 13 Schematic diagram of the molecular structure of the compound of the present invention.

[0041] Figure 14 This is a flow chart for the preparation of the compound of Example 2 of the present invention.

[0042] Figure 15 This figure shows the effect of the compounds of the present invention on the survival rate of RAW264.7 cells.

[0043] Figure 16 IC of the compounds of the present invention for inhibiting NO 50 picture.

[0044] Figure 17 This is a graph showing the effect of the compounds of the present invention on TNF-α concentration.

[0045] Figure 18 This is a graph showing the effect of the compounds of the present invention on IL-6 concentration. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0047] Example 1

[0048] A method for preparing terpenoid compounds extracted and separated from purslane comprises the following steps:

[0049] S1. Grind 9.5 kg of dried Portulaca oleracea medicinal material, add 85% (v / v) ethanol solution at a material-liquid ratio of 1 g:5 mL, heat and extract three times, each extraction for 60 min, combine the three extracts, and recover ethanol from the filtrate until there is no alcohol taste to obtain Portulaca oleracea total extract (1.18 kg).

[0050] S2. Add 13 L of warm water (40° C.) to the total extract of Portulaca oleracea prepared in S1 and suspend the mixture to obtain a suspension.

[0051] S3. The suspension obtained in S2 was extracted with extraction solvents of petroleum ether, ethyl acetate, and n-butanol in sequence for a total of 5 times. The volume ratio of the suspension to each extraction solvent was 2:1. The extracts were combined, concentrated under reduced pressure, and dried to obtain 253.5 g of petroleum ether layer, 29.4 g of ethyl acetate layer, and 39.7 g of n-butanol layer.

[0052] S4. Dissolve the ethyl acetate layer from S3 in 60 mL of methanol and dry-blend with silica gel at a mass ratio of 1:1.2. Load the sample onto a silica gel column with a diameter-to-height ratio of 1:8. Gradient elution is performed using, in order, 11 L of dichloromethane-methanol (volume ratio) of 100:1, 9 L of dichloromethane-methanol (volume ratio) of 80:1, 6 L of dichloromethane-methanol (volume ratio) of 60:1, 10 L of dichloromethane-methanol (volume ratio) of 40:1, 12 L of dichloromethane-methanol (volume ratio) of 20:1, 8 L of dichloromethane-methanol (volume ratio) of 10:1, 11 L of dichloromethane-methanol (volume ratio) of 5:1, and 1 L of pure methanol. The effluent is analyzed by thin-layer chromatography. Fractions with the same dominant spot are combined and the solvent is evaporated to dryness to obtain nine eluted products, Fr.A-I.

[0053] S5. Combine the eluted products Fr.B, Fr.E, Fr.F, and Fr.G in S4 (26.2 g) and dissolve them in 55 mL of methanol. Dry mix them with silica gel at a mass ratio of 1:1.2, load them onto an ODS column with a diameter-to-height ratio of 1:7, and gradient elute with 2.5 L of methanol-water at a volume ratio of 0:1, 4 L of methanol-water at 1:9, 4 L of methanol-water at 1:4, 6 L of methanol-water at 2:3, 5 L of methanol-water at 3:2, 5 L of methanol-water at 4:1, and 6 L of 100% methanol. Detect the effluent by thin layer chromatography. After combining the fractions with the same main spot, evaporate the solvent to obtain 19 eluted products Fr.1 to Fr.19.

[0054] S6. Dissolve the Fr.5 portion in S5 with methanol, dry-mix it with silica gel at a mass ratio of 1:1.2, load it onto a silica gel column with a diameter-to-height ratio of 1:12, and gradient elute it with 2 L of dichloromethane-methanol at a volume ratio of 100:1 and 0.8 L of dichloromethane-methanol at a volume ratio of 80:1. Detect the effluent by thin layer chromatography, combine the fractions with the same main spot, and evaporate the solvent to obtain 10 eluted products Fr.5-1 to Fr.5-10.

[0055] S7. Dissolve the Fr.5-5 portion in S6 with methanol, wet-load it onto a Sephadex LH-20 gel column with a diameter-to-height ratio of 1:20, and perform isocratic elution with 100% methanol. Detect the effluent by thin layer chromatography, combine the fractions, and evaporate the solvent to obtain five eluted products Fr.5-5-1 to Fr.5-5-5.

[0056] S8. The eluted product Fr.5-5-2 from S7 was partially dissolved in chromatographic methanol, passed through a 0.45 μm microporous filter membrane, and further purified by semi-preparative HPLC at a flow rate of 2.0 mL / min. Isocratic elution was performed using a chromatographic methanol-ammonia (0.2% by volume) elution system with a volume ratio of 40:60. The effluent at a retention time of 18.1 min was collected, and the solvent was evaporated to dryness to finally obtain 1.3 mg of the compound. The liquid chromatography column for high performance liquid chromatography separation was a C18 HPLC column, and the UV detector was a PDA diode array detector.

[0057] Example 2

[0058] A method for preparing terpenoid compounds extracted and separated from Portulaca oleracea, see Figure 14 The flowchart includes the following steps:

[0059] S1. Grind 9.5 kg of dried Portulaca oleracea medicinal material, add 85% (v / v) ethanol solution at a material-liquid ratio of 1 g:10 mL, heat and extract three times, each extraction for 75 min, combine the three extracts, and recover ethanol from the filtrate until there is no alcohol taste to obtain Portulaca oleracea total extract (mass 1.54 kg).

[0060] S2. Add 15 L of warm water (40° C.) to the total extract of Portulaca oleracea prepared in S1 and suspend the mixture to obtain a suspension.

[0061] S3. The suspension obtained in S2 was extracted with extraction solvents of petroleum ether, ethyl acetate, and n-butanol in sequence for a total of 5 times, with the volume ratio of the suspension to each extraction solvent being 2:1. Each extract was concentrated under reduced pressure and dried to obtain 280.2 g of petroleum ether layer, 37.5 g of ethyl acetate layer, and 50.1 g of n-butanol layer.

[0062] S4. Dissolve the ethyl acetate layer from S3 in 85 mL of methanol and dry-blend with silica gel at a mass ratio of 1:1.2. Load the sample onto a silica gel column with a diameter-to-height ratio of 1:8. Gradient elution is performed using, in order, 11 L of dichloromethane-methanol (volume ratio) of 100:1, 9 L of dichloromethane-methanol (volume ratio) of 80:1, 6 L of dichloromethane-methanol (volume ratio) of 60:1, 10 L of dichloromethane-methanol (volume ratio) of 40:1, 12 L of dichloromethane-methanol (volume ratio) of 20:1, 8 L of dichloromethane-methanol (volume ratio) of 10:1, 11 L of dichloromethane-methanol (volume ratio) of 5:1, and finally 1 L of pure methanol. The effluent is analyzed by thin-layer chromatography. After combining fractions with the same dominant spot, the solvent is evaporated to dryness to obtain nine eluted products, Fr.A-I.

[0063] S5. Combine the eluted products Fr.B, Fr.E, Fr.F, and Fr.G in S4 (27.5 g) and dissolve them in 65 mL of methanol. Dry mix them with silica gel at a mass ratio of 1:1.2, load them onto an ODS column with a diameter-to-height ratio of 1:7, and gradient elute with 2.5 L of methanol-water at a volume ratio of 0:1, 4 L of methanol-water at 1:9, 4 L of methanol-water at 1:4, 6 L of methanol-water at 2:3, 5 L of methanol-water at 3:2, 5 L of methanol-water at 4:1, and 6 L of 100% methanol. Detect the effluent by thin layer chromatography. After combining the fractions with the same main spot, evaporate the solvent to obtain 19 eluted products Fr.1 to Fr.19.

[0064] S6. Dissolve the Fr.5 portion in S5 with methanol, dry-mix it with silica gel at a mass ratio of 1:1.2, load it onto a silica gel column with a diameter-to-height ratio of 1:12, and gradient elute it with 2 L of dichloromethane-methanol at a volume ratio of 100:1 and 0.8 L of dichloromethane-methanol at a volume ratio of 80:1. Detect the effluent by thin layer chromatography, combine the fractions with the same main spot, and evaporate the solvent to obtain 10 eluted products Fr.5-1 to Fr.5-10.

[0065] S7. Dissolve the Fr.5-5 portion in S6 with methanol, wet-load it onto a Sephadex LH-20 gel column with a diameter-to-height ratio of 1:20, and perform isocratic elution with 100% methanol. Detect the effluent by thin layer chromatography, combine the fractions, and evaporate the solvent to obtain five eluted products Fr.5-5-1 to Fr.5-5-5.

[0066] S8. The eluted product Fr.5-5-2 from S7 was partially dissolved in chromatographic methanol, passed through a 0.45 μm microporous filter membrane, and further purified by semi-preparative HPLC at a flow rate of 2.0 mL / min. Isocratic elution was performed using a chromatographic methanol-ammonia (volume fraction 0.2%) elution system with a volume ratio of 40:60. The effluent at a retention time of 18.1 min was collected, and the solvent was evaporated to dryness to finally obtain 1.8 mg of the compound. The liquid chromatography column for high performance liquid chromatography separation was a C18 HPLC column, and the UV detector was a PDA diode array detector.

[0067] Example 3

[0068] A method for preparing terpenoid compounds extracted and separated from purslane comprises the following steps:

[0069] S1. Grind 9.5 kg of dried Portulaca oleracea medicinal material, add 85% (v / v) ethanol solution at a material-liquid ratio of 1 g:15 mL, heat and extract three times, each extraction for 90 min, combine the three extracts, and recover ethanol from the filtrate until there is no alcohol taste to obtain Portulaca oleracea total extract (mass 1.69 kg).

[0070] S2. Add 18 L of warm water (40° C.) to the total extract of Portulaca oleracea prepared in S1 and suspend the mixture to obtain a suspension.

[0071] S3. The suspension obtained in S2 was extracted with extraction solvents of petroleum ether, ethyl acetate, and n-butanol in sequence for a total of 5 times, with the volume ratio of the suspension to each extraction solvent being 2:1. Each extract was concentrated and dried under reduced pressure to obtain 295.5 g of the petroleum ether layer, 46.8 g of the ethyl acetate fraction, and 61.3 g of the n-butanol fraction.

[0072] S4. Dissolve the ethyl acetate layer from S3 in 90 mL of methanol and dry-blend with silica gel at a mass ratio of 1:1.2. Load the sample onto a silica gel column with a diameter-to-height ratio of 1:8. Gradient elution is performed using, in order, 11 L of dichloromethane-methanol (volume ratio) of 100:1, 9 L of dichloromethane-methanol (volume ratio) of 80:1, 6 L of dichloromethane-methanol (volume ratio) of 60:1, 10 L of dichloromethane-methanol (volume ratio) of 40:1, 12 L of dichloromethane-methanol (volume ratio) of 20:1, 8 L of dichloromethane-methanol (volume ratio) of 10:1, 11 L of dichloromethane-methanol (volume ratio) of 5:1, and 1 L of pure methanol. The effluent is analyzed by thin-layer chromatography. After combining fractions with the same dominant spot, the solvent is evaporated to dryness to obtain nine eluted products, Fr.A-I.

[0073] S5. Combine the eluted products Fr.B, Fr.E, Fr.F, and Fr.G in S4 (28.9 g) and dissolve them in 70 mL of methanol. Dry mix them with silica gel at a mass ratio of 1:1.2 and load them onto an ODS column with a diameter-to-height ratio of 1:7. Gradient elution is performed with 2.5 L of methanol-water at a volume ratio of 0:1, 4 L of methanol-water at a volume ratio of 1:9, 4 L of methanol-water at a volume ratio of 1:4, 6 L of methanol-water at a volume ratio of 2:3, 5 L of methanol-water at a volume ratio of 3:2, 5 L of methanol-water at a volume ratio of 4:1, and 6 L of 100% methanol. The effluent is detected by thin layer chromatography. After combining the fractions with the same main spot, the solvent is evaporated to obtain 19 eluted products Fr.1 to Fr.19.

[0074] S6. Dissolve the Fr.5 portion in S5 with methanol, dry-mix it with silica gel at a mass ratio of 1:1.2, and load it onto a silica gel column with a diameter-to-height ratio of 1:12. Detect the effluent by thin-layer chromatography using 2 L of dichloromethane-methanol at a volume ratio of 100:1 and 0.8 L of dichloromethane-methanol at a volume ratio of 80:1, respectively. After combining the fractions with the same main spot, evaporate the solvent to obtain 10 eluted products Fr.5-1 to Fr.5-10.

[0075] S7. Dissolve the Fr.5-5 portion in S6 with methanol, wet-load it onto a Sephadex LH-20 gel column with a diameter-to-height ratio of 1:20, and perform isocratic elution with 100% methanol. Detect the effluent by thin layer chromatography, combine the fractions, and evaporate the solvent to obtain five eluted products Fr.5-5-1 to Fr.5-5-5.

[0076] S8. The eluted product Fr.5-5-2 from S7 was partially dissolved in chromatographic methanol, passed through a 0.45 μm microporous filter membrane, and further purified by semi-preparative HPLC at a flow rate of 2.0 mL / min. Isocratic elution was performed using a chromatographic methanol-ammonia (0.2% by volume) elution system with a volume ratio of 40:60. The effluent at a retention time of 18.1 min was collected, and the solvent was evaporated to dryness to finally obtain 2.5 mg of the compound. The liquid chromatography column for the HPLC separation was a C18 HPLC column, and the UV detector was a PDA diode array detector.

[0077] The terpenoid compounds of Examples 1-3 were identified as the same substance and named (1S,3R,5R,6S)-1-carboxy-3-hydroxy-5,6-epoxy-6-(3-methyl-2-(Z)-4-(E)-dien-valeric acid)-ionone. The specific determination and experimental results are as follows:

[0078] (1S,3R,5R,6S)-1-carboxy-3-hydroxy-5,6-epoxy-6-(3-methyl-2-(Z)-4-(E)-dien-valeric acid)-ionone, white powder, easily soluble in methanol.

[0079] Figure 1 is the high resolution mass spectrum (HRESIMS) of the compound. Figure 1 High resolution mass spectrometry (HRESIMS) gave the quasi-molecular ion peak of the compound as m / z 295.11877[MH]+(calculated value C 15 H 19 O6,295.11871), combined Figure 2 and Figure 5 of 1 H-NMR, 13 C-NMR suggests that its molecular formula is C 15 H 20 O6.

[0080] Figure 2 is the ultraviolet (UV) spectrum of the compound, Figure 3 is the infrared (IR) spectrum of the compound. Figure 2 and Figure 3 It can be seen that the UV spectrum has maximum absorption at 202nm and 256nm, and the IR spectrum has maximum absorption at 1760cm -1 There is an absorption peak at 3339cm -1 There is an absorption peak at , indicating the presence of hydroxyl groups in the compound.

[0081] Figure 4 For this compound 1 H-NMR spectrum (600 MHz); Figure 5 For this compound 13 C-NMR spectrum (150 MHz); Figure 6 is the DEPT spectrum of the compound.

[0082] Figure 4 of 1 The H-NMR (CD3OD, 600 MHz) spectrum gave three double bond hydrogen signals (δ H 7.92,d,J=15.8Hz,H-4';δ H 6.34,d,J=15.8Hz,H-5';δ H 5.82,s,H-2'); hydrogen signals of 3 methyl groups δ H 2.04 (3H, s, H-6'), δ H1.34 (3H, s, H-9), δ H 1.07 (3H, s, H-8); hydrogen signals of two methylene groups δ H 2.25(1H,dd,J=14.8,7.5Hz,H-4), δ H 1.91(1H,dd,J=15.4,8.1Hz,H-2), δ H 1.85(1H,dd,J=14.8,10.8Hz,H-4), δ H 1.74 (1H, dd, J = 15.4, 10.3 Hz, H-2); 1 hydrogen signal of methine δ H 3.84 (1H, m, H-3).

[0083] Figure 5 of 13 C-NMR and Figure 6 The DEPT spectrum (CD3OD, 150MHz) showed 15 carbon atoms, including the carbon signal of the carboxyl group δ C 181.06 (C-7); carbon signal of 4 double bonds δ C 146.78 (C-3'), δ C 133.70(C-4'), δ C 130.44 (C-5') and δ C 123.80 (C-2'); 3 quaternary carbon signals δ C 89.92(C-5), δ C 82.81(C-6), δ C 53.46 (C-1); δ of the carbon signal of the 3 methyl groups C 20.80(C-6'), δ C 18.49(C-9), δ C 14.55 (C-8); carbon signal of two methylene groups δ C 42.29(C-4), δ C 40.97 (C-2); 1 methine carbon signal δ C 65.27(C-3).

[0084] The compound 1 H-NMR and 13 The relevant data of C-NMR are shown in Table 1.

[0085] Table 1 Compounds 1 H-NMR and 13 C-NMR data

[0086]

[0087] Figure 7 is the HSQC spectrum of the compound; Figure 8 is the HMBC spectrum of the compound; Figure 9 For this compound 1 H- 1 HCOSY spectrum; Figure 10 is the NOESY spectrum of the compound.

[0088] Figure 9 of 1 H- 1 δ in the H COSY spectrum H 7.92(H-4') and δ H 6.34 (H-5') related, indicating that C-4' is connected to C-5'; δ H 3.84(H-3) and δ H 1.91(H-2) and 1.74(H-2) are correlated, indicating that C-2 is connected to C-3; δ H 2.25(H-4) and δ H 1.85(H-4) and δ H 3.84 (H-3) correlation, indicating that C-3 is connected to C-4.

[0089] Figure 8 In the HMBC spectrum of H 3.84(H-3) and δ C 42.29 (C-4) related; δ H 2.25(H-4) and δ C 82.81(C-6), δ C 40.97(C-2), δ C 18.49 (C-9) related; δ H 1.74(H-2) and δ C 82.81(C-6), δ C 42.29(C-4), δ C 181.14(C-7), δ C 14.55 (C-8) is related, indicating that it may contain a six-membered ring; δ H 5.84(H-2') and δ C 133.70(C-4'), δ C 20.80 (C-6') related; δ H 7.92(H-4') and δ C 123.80(C-2'), δ C 146.78 (C-3'), δ C 130.44(C-5'), δC 82.81(C-6) related; δ H 6.34(H-5') and δ C 146.78 (C-3'), δ C 53.46(C-1), δ C 89.92 (C-5), indicating that a methyl-substituted pentadienoic acid fragment is connected to the six-membered ring.

[0090] Figure 10 In the NOESY spectrum of δ H 5.84(H-2) and δ H 2.04 (H-6), it is inferred that the double bond at position 2 is of Z type.

[0091] In summary, the structure of the compound was determined to be 1-carboxy-3-hydroxy-5,6-epoxy-6-(3-methyl-2-(Z)-4-(E)-dien-valeric acid)-ionone. It is similar in structure to the known compound (2Z,4E)-5-[(1S,4R,6R)-4-hydroxy-2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl]-3-methyl-2,4-pentadienoic acid, except that the C-2' position is substituted by a carboxyl group. Therefore, the planar structure of the compound was determined. The above inference was further confirmed by high-resolution mass spectrometry. Therefore, the planar structure of the compound was determined, and the molecular structure of the compound is shown in FIG. Figure 13 .

[0092] Figure 11 is the coupling correlation diagram of the compound. Figure 11 It can be seen that the relative configuration of the compound can be determined by hydrogen spectrum and NOESY spectrum. H 6.34(H-5') and δ H 1.34(H-9), δ H 1.07 (H-8) correlation, δ H 2.25(H-4) and δ H 1.34(H-9), δ H 3.84 (H-3) correlation, that is, H-5', H-3, H-8 and H-9 are in the same plane, so the relative configuration of the compound is determined. By comparing the calculated ECD with the experimental ECD curve, it is found that the negative Cotton effect produced by the experimental ECD and the calculated ECD (1S, 3R, 5R, 6S) at 250nm is the same, as shown in Figure 2. Figure 12Therefore, the absolute configuration of the compound is 1S, 3R, 5R, 6S. A systematic literature search revealed that the compound is an unreported terpenoid.

[0093] Example 4

[0094] Anti-inflammatory function test

[0095] The novel terpenoid compound isolated and extracted from Portulaca oleracea in Examples 1 to 3 of the present invention exhibited significant anti-inflammatory activity. The specific determination and results of the anti-inflammatory activity are as follows:

[0096] 1. Prepare sample solution

[0097] The compound was prepared into a 100 mmol / L solution using DMSO as solvent, and diluted 1000 times by adding DMEM high glucose medium to prepare a solution with a concentration of 100 μmol / L, which was used as the initial screening concentration. The solution was then prepared into solutions with concentrations of 100, 50, 25, 12.5, and 6.25 μmol / L, which were used as the IC 50 The drug concentration value.

[0098] 2 cell lines

[0099] Macrophage RAW264.7 cells.

[0100] 3 Experimental instruments

[0101] Clean bench (HS, HJCLEAN TECH), CO2 incubator (D180-P, RWD), inverted microscope (CX43, OLYMPUS), microplate reader (infinite F50, TECAN), analytical balance (AS 220.X2, RADWAG WagiElektroniczne).

[0102] 4 Cell culture

[0103] Macrophage RAW264.7 cells were cultured in DMEM complete medium (containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin double antibody solution) and placed in a cell culture incubator at 37° C. and 5% CO 2 .

[0104] 5 Anti-inflammatory tests

[0105] When the cells reached the logarithmic growth phase, they were cultured at 5×10 5Cells were inoculated at a concentration of 1 μg / mL in a 96-well plate at 100 μL / well and placed in a cell culture incubator containing 5% CO2 and a constant temperature of 37°C for 24 hours. A blank control group (added with 200 μL of culture medium), a model group (added with 100 μL of LPS with a final concentration of 1 μg / mL and 100 μL of culture medium), a positive control group (added with 100 μL of 100 μmol / L dexamethasone and 100 μL of LPS with a final concentration of 1 μg / mL), and a drug group (added with 100 μL of the test sample and 100 μL of LPS with a final concentration of 1 μg / mL) were set up, with 5 replicates per group. After the 96-well plate was placed in a 37°C, 5% CO2 incubator for another 24 hours, the cell supernatant was aspirated and the concentration of each inflammatory factor was determined according to the instructions of the NO, TNF-α, and IL-6 detection kits.

[0106] 6. Cell activity test

[0107] Add 20 μL of MTT solution (MTT solvent is added to MTT powder to prepare a 5 mg / mL solution) to the 96-well plate described above, then place the 96-well plate in a cell culture incubator. After incubation at 37°C and 5% CO₂ for 4 hours, aspirate all the liquid from the wells, add 150 μL / well of DMSO, measure the absorbance of each well at 490 nm, and calculate cell viability.

[0108] 7 Experimental results

[0109] The effects of the compounds on the survival rate of macrophage RAW264.7 were detected by MTT assay. Figure 15 As shown, the test compounds of Examples 1 to 3 had no effect on the survival rate of macrophages RAW264.7. The results of the initial screening of NO inhibition rate showed that the test compounds of Examples 1 to 3 had an inhibition rate of 70.67% on the production of NO by macrophages RAW264.7 stimulated by LPS.

[0110] By measuring the IC of the test compounds of Examples 1 to 3 50 The anti-inflammatory activity of Figure 16 、 Figure 17 As shown, the test compounds of Examples 1 to 3 exhibited strong anti-inflammatory activity and could significantly reduce the NO content produced by LPS-stimulated macrophage RAW264.7 cells. 50 The value was 42.15 μmol / L. Taking dexamethasone as the positive drug, when the compound concentration was 12.5, 25, and 50 μmol / L, compared with 610.32 ng / mL of TNF-α and 154.25 ng / mL of IL-6 in the LPS model group, it could significantly reduce the concentrations of TNF-α and IL-6, and showed a dose-dependent effect.

[0111] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0112] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0113] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A terpenoid compound isolated from Portulaca oleracea, characterized in that: Its chemical structure is shown in the following formula 1): Formula 1).

2. The method for preparing the terpenoid compound according to claim 1, characterized in that: The following steps are involved: S1. Using purslane as raw material, extracting with ethanol solution by heating, combining the extracts, and evaporating the filtrate to dry the ethanol to obtain purslane total extract; S2. suspending the purslane total extract in water to obtain a suspension; S3, extracting the suspension with petroleum ether, ethyl acetate, and n-butanol as extraction solvents in sequence, concentrating and drying the extracts under reduced pressure to obtain a petroleum ether extract, an ethyl acetate extract, and an n-butanol extract; S4. Load the ethyl acetate extract onto a silica gel column, and after elution, color development, and merging, obtain nine eluted products, Fr.A to Fr.I. Gradient elution is performed using a dichloromethane-methanol elution system. The volume ratios of dichloromethane and methanol during elution are 100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, and 0:1, respectively. S5. The eluted products Fr.B, Fr.E, Fr.F, and Fr.G were combined and loaded onto an ODS column. After elution, color development, and combination, 19 eluted products Fr. 1 to Fr. 19 were obtained. Gradient elution was performed using a methanol-water elution system. The volume ratios of methanol and water during elution were 0:1, 1:9, 1:4, 2:3, 3:2, 4:1, and 1:0, respectively. S6. Load the eluted product Fr. 5 onto a silica gel column. After elution and color development, 10 eluted products Fr.5-1 to Fr.5-10 are obtained. Gradient elution is performed using a dichloromethane-methanol elution system. The volume ratios of dichloromethane and methanol during elution are 100:1 and 80:1, respectively. S7, load the eluted product Fr.5-5 onto a gel column, perform isocratic elution with methanol, and after color development and merging, obtain five eluted products Fr.5-5-1 to Fr.5-5-5; S8. Separate and purify the eluted product Fr. 5-5-2 by semi-preparative HPLC to obtain the terpenoid compound, and the separating liquid is methanol / ammonia solution.

3. The method for preparing terpenoid compounds according to claim 2, characterized in that: In S1, the volume fraction of the ethanol solution was 85%, the ratio of purslane to ethanol was 1 g: 5-15 mL, the heating extraction times were 1-3 times, each time for 60-90 min, and the temperature was 65-70°C.

4. The method for preparing terpenoid compounds according to claim 3, characterized in that: Add 38~40℃ water to S2 and suspend.

5. The method for preparing terpenoid compounds according to claim 4, characterized in that: In S3, the extraction condition is to extract 5 to 6 times according to the volume ratio of suspension to extraction solvent of 2:

1.

6. The method for preparing terpenoid compounds according to claim 5, characterized in that: In S8, the volume fraction of aqueous ammonia is 0.2%, and the volume ratio of methanol to aqueous ammonia is 40:

60.

7. The method for preparing terpenoid compounds according to claim 6, characterized in that: In S8, the flow rate was 2.0 mL / min, and the effluent with a retention time of 18.1 min was collected, concentrated, and dried to obtain the terpenoid compound.

8. Use of the terpenoid compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of anti-inflammatory drugs.

9. Use of the terpenoid compound or a pharmaceutically acceptable salt thereof in the preparation of an anti-inflammatory drug according to claim 8, characterized in that: The drug further comprises pharmaceutically acceptable excipients.

10. An anti-inflammatory drug, characterized in that The drug contains the terpenoid compound according to claim 1 as an active ingredient and further contains pharmaceutically acceptable excipients.

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