Terpenoid separated from purslane and preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to terpenoids separated from purslane as well as a preparation method and application of the terpenoids. The chemical structural formula of the terpenoid is as shown in formula 1), a total extract of portulaca oleracea is firstly extracted, organic solvents with different polarities are used for extracting the total extract of the portulaca oleracea to obtain a petroleum ether layer, an ethyl acetate layer and an n-butyl alcohol layer, the ethyl acetate layer is repeatedly eluted through a silica gel column, an ODS reversed-phase column and a gel column, and the terpenoid is obtained. According to the present invention, the terpenoid is obtained through the isocratic elution of the collected same fraction through the HPLC chromatographic column and separation, and the terpenoid is obtained for the first time and shows the significant anti-inflammatory activity in vitro, such that the new choice is provided for the development of the natural anti-inflammatory drug. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to a terpene compound separated from purslane and a preparation method and application thereof. Background Art

[0002] Portulaca oleracea L. is a widely distributed plant with both medicinal and edible value, belonging to the genus Portulaca of the family Portulacaceae. The Compendium of Materia Medica records that Portulaca oleracea can "dissipate blood and reduce swelling, facilitate bowel movements, detoxify and relieve stranguria", and the New Compendium of Materia Medica says that it "treats all kinds of swelling, fistulas, warts, stranguria, and bleeding from wounds". It can be used to treat a variety of inflammatory diseases such as enteritis, dysentery, appendicitis, urinary tract infection, etc., and is also known as a "natural antibiotic".

[0003] Modern research shows that Purslane contains rich chemical components, such as alkaloids, flavonoids, terpenoids, coumarins and organic acids, and has a variety of pharmacological effects, such as anti-inflammatory, antibacterial, anti-tumor, regulating blood sugar and blood lipid levels, and enhancing immune function. However, the anti-inflammatory effect of Purslane has not been fully elucidated, and the anti-inflammatory active components of Purslane are not yet clear. Summary of the invention

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

[0005] To achieve the above objectives, the technical solution of the present invention is 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 by heating with ethanol solution, combining the extracts, filtering, and evaporating the ethanol to obtain purslane total extract;

[0010] S2. suspending the total extract of Portulaca oleracea with water to obtain a suspension;

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

[0012] S4, the ethyl acetate layer extract was loaded onto a silica gel column, and after elution, color development and merging, 9 elution products Fr.A~I were obtained; a dichloromethane-methanol elution system was used for gradient elution, and the volume ratios of dichloromethane and methanol during elution were 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 the 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, loading the eluted product Fr.5 onto a silica gel column, and after elution, color development and merging, 10 eluted products Fr.5-1 to Fr.5-10 were obtained; a dichloromethane-methanol elution system was used for gradient elution, and the volume ratio of dichloromethane to methanol during elution was 100:1 and 80:1 respectively;

[0015] S7, loading the eluted product Fr.5-5 onto a gel column, isocratically eluted with methanol, and after color development and merging, five eluted products Fr.5-5-1 to Fr.5-5-5 are obtained;

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

[0017] Furthermore, in S1, the volume fraction of the ethanol solution is 85%, the usage 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 is 2.0 mL / min, and the effluent with a retention time of 18.1 min is collected, concentrated, and dried to obtain the terpene compound.

[0022] Again, 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 medicine also contains pharmaceutically acceptable excipients.

[0024] The present invention also provides an anti-inflammatory drug, which uses 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 separates a novel terpene 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 for the first time. The terpene 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, and the ethyl acetate layer is repeatedly eluted with a silica gel column, an ODS reverse phase column, and a gel column, and the collected same 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, and the mobile phase system is low in cost and easy to obtain. The method of the present invention is simple to operate, low in raw material cost, and the purity of the separated compound is high, which is conducive to 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.

[0034] Figure 7 The HSQC spectrum of the compound separated by 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] Fig. 9 The 1H-1H COSY spectrum of the compound separated by the present invention.

[0037] Fig.10 The NOESY spectrum of the compound separated by the present invention.

[0038] Fig.11 It is the coupling correlation diagram of the compound of the present invention.

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

[0040] Fig.13 The molecular structure diagram of the compound of the present invention.

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

[0042] Fig.15 This is a graph showing the effect of the compounds of the present invention on the survival rate of RAW264.7 cells.

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

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

[0045] Fig.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 is described in detail below in conjunction with the accompanying drawings and specific examples, but 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 purslane medicinal material, add 85% (v / v) ethanol solution at a solid-liquid ratio of 1 g:5 mL, heat and extract 3 times, each extraction for 60 min, combine the 3 extracts, recover ethanol from the filtrate until there is no alcohol taste, and obtain purslane total extract (1.18 kg).

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

[0051] S3. The suspension obtained in S2 was extracted with extraction solvents such as petroleum ether, ethyl acetate and n-butanol in turn for a total of 5 times. The volume ratio of the suspension to each extraction solvent was 2:1. The extracts were combined, concentrated and dried under reduced pressure 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 in S3 with 60 mL of methanol, dry-mix 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, and gradiently elute with 11 L of dichloromethane-methanol with a volume ratio of 100:1, 9 L of dichloromethane-methanol with a volume ratio of 80:1, 6 L of dichloromethane-methanol with a volume ratio of 60:1, 10 L of dichloromethane-methanol with a volume ratio of 40:1, 12 L of dichloromethane-methanol with a volume ratio of 20:1, 8 L of dichloromethane-methanol with a volume ratio of 10:1, 11 L of dichloromethane-methanol with a volume ratio of 5:1, and 1 L of pure methanol. Detect the effluent by thin layer chromatography, combine the fractions with the same main spot, evaporate the solvent, and obtain 9 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), dissolve them in 55 mL of methanol, dry-mix them with silica gel at a mass ratio of 1:1.2, load them on an ODS column with a diameter-to-height ratio of 1:7, and gradient elute with 2.5 L of methanol-water with a volume ratio of 0:1, 4 L of methanol-water with a volume ratio of 1:9, 4 L of methanol-water with a volume ratio of 1:4, 6 L of methanol-water with a volume ratio of 2:3, 5 L of methanol-water with a volume ratio of 3:2, 5 L of methanol-water with a volume ratio of 4:1, and 6 L of 100% methanol. Detect the effluent by thin layer chromatography, combine 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, respectively. Detect the effluent by thin layer chromatography, combine the fractions with the same main spot, 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 5 eluted products Fr.5-5-1 to Fr.5-5-5.

[0056] S8, the eluted product Fr.5-5-2 in S7 was partially dissolved with 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, and isocratic elution was performed using a chromatographic methanol-ammonia water (volume fraction 0.2%) elution system with a volume ratio of 40:60, and the effluent at a retention time of 18.1 min was collected, and the solvent was evaporated to finally obtain 1.3 mg of the compound. Among them, the liquid chromatography column during high performance liquid chromatography separation was a C18 HPLC column, and the ultraviolet detector was a PDA diode array detector.

[0057] Example 2

[0058] A method for preparing terpenoid compounds extracted and separated from purslane, see Fig.14 The flowchart includes the following steps:

[0059] S1. Grind 9.5 kg of dried purslane medicinal material, add 85% (v / v) ethanol solution at a solid-liquid ratio of 1 g:10 mL, heat and extract 3 times, each extraction for 75 min, combine the 3 extracts, recover ethanol from the filtrate until there is no alcohol taste, and obtain purslane total extract (mass 1.54 kg).

[0060] S2. Add 15 L of warm water (40° C.) to the total extract of Portulaca oleracea 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 turn for a total of 5 times. The volume ratio of the suspension to each extraction solvent was 2:1. Each extract was concentrated and dried under reduced pressure 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 in S3 with 85 mL of methanol, dry-mix 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, and gradiently elute with 11 L of dichloromethane-methanol with a volume ratio of 100:1, 9 L of dichloromethane-methanol with a volume ratio of 80:1, 6 L of dichloromethane-methanol with a volume ratio of 60:1, 10 L of dichloromethane-methanol with a volume ratio of 40:1, 12 L of dichloromethane-methanol with a volume ratio of 20:1, 8 L of dichloromethane-methanol with a volume ratio of 10:1, 11 L of dichloromethane-methanol with a volume ratio of 5:1, and 1 L of pure methanol. Detect the effluent by thin layer chromatography, combine the fractions with the same main spot, evaporate the solvent, and obtain 9 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), dissolve them in 65 mL of methanol, dry-mix them with silica gel at a mass ratio of 1:1.2, load them on an ODS column with a diameter-to-height ratio of 1:7, and gradient elute with 2.5 L of methanol-water with a volume ratio of 0:1, 4 L of methanol-water with a volume ratio of 1:9, 4 L of methanol-water with a volume ratio of 1:4, 6 L of methanol-water with a volume ratio of 2:3, 5 L of methanol-water with a volume ratio of 3:2, 5 L of methanol-water with a volume ratio of 4:1, and 6 L of 100% methanol. Detect the effluent by thin layer chromatography, combine 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, respectively. Detect the effluent by thin layer chromatography, combine the fractions with the same main spot, 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 5 eluted products Fr.5-5-1 to Fr.5-5-5.

[0066] S8, the eluted product Fr.5-5-2 in S7 was partially dissolved with 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, and isocratic elution was performed using a chromatographic methanol-ammonia water (volume fraction 0.2%) elution system with a volume ratio of 40:60, and the effluent at a retention time of 18.1 min was collected, and the solvent was evaporated to finally obtain 1.8 mg of the compound. Among them, the liquid chromatography column during high performance liquid chromatography separation was a C18 HPLC column, and the ultraviolet 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 purslane medicinal material, add 85% (v / v) ethanol solution at a solid-liquid ratio of 1 g:15 mL, heat and extract 3 times, each extraction for 90 min, combine the 3 extracts, recover ethanol from the filtrate until there is no alcohol taste, and obtain purslane total extract (mass 1.69 kg).

[0070] S2. Add 18 L of warm water (40° C.) to the total extract of Portulaca oleracea 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 turn for a total of 5 times. The volume ratio of the suspension to each extraction solvent was 2:1. Each extract was concentrated and dried under reduced pressure to obtain 295.5 g of petroleum ether layer, 46.8 g of ethyl acetate fraction and 61.3 g of n-butanol fraction.

[0072] S4, dissolve the ethyl acetate layer in S3 with 90 mL of methanol, dry-mix 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, and gradiently elute with 11 L of dichloromethane-methanol with a volume ratio of 100:1, 9 L of dichloromethane-methanol with a volume ratio of 80:1, 6 L of dichloromethane-methanol with a volume ratio of 60:1, 10 L of dichloromethane-methanol with a volume ratio of 40:1, 12 L of dichloromethane-methanol with a volume ratio of 20:1, 8 L of dichloromethane-methanol with a volume ratio of 10:1, 11 L of dichloromethane-methanol with a volume ratio of 5:1, and 1 L of pure methanol. Detect the effluent by thin layer chromatography, combine the fractions with the same main spot, evaporate the solvent, and obtain 9 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), dissolve them in 70 mL of methanol, dry-mix them with silica gel at a mass ratio of 1:1.2, load them on an ODS column with a diameter-to-height ratio of 1:7, and gradient elute with 2.5 L of methanol-water with a volume ratio of 0:1, 4 L of methanol-water with a volume ratio of 1:9, 4 L of methanol-water with a volume ratio of 1:4, 6 L of methanol-water with a volume ratio of 2:3, 5 L of methanol-water with a volume ratio of 3:2, 5 L of methanol-water with a volume ratio of 4:1, and 6 L of 100% methanol. Detect the effluent by thin layer chromatography, combine the fractions with the same main spot, evaporate the solvent 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, load it onto a silica gel column with a diameter-to-height ratio of 1:12, and perform thin layer chromatography on the effluent using 2 L of dichloromethane-methanol with a volume ratio of 100:1 and 0.8 L of dichloromethane-methanol with 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 5 eluted products Fr.5-5-1 to Fr.5-5-5.

[0076] S8, the eluted product Fr.5-5-2 in S7 was partially dissolved with 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, and isocratic elution was performed using a chromatographic methanol-ammonia water (volume fraction 0.2%) elution system with a volume ratio of 40:60, and the effluent at a retention time of 18.1 min was collected, and the solvent was evaporated to finally obtain 2.5 mg of the compound. Among them, the liquid chromatography column during high performance liquid chromatography separation was a C18 HPLC column, and the ultraviolet detector was a PDA diode array detector.

[0077] The terpenoid compounds of Examples 1-3 were identified as the same substance, 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 The high resolution mass spectrometry (HRESIMS) of the compound gave the quasi-molecular ion peak 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 (600MHz); 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'); 3 methyl hydrogen signals 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) shows 15 carbons, 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); 3 methyl carbon signals δ C 20.80(C-6'), δ C 18.49(C-9),δ C 14.55 (C-8); 2 methylene carbon signals δ C 42.29(C-4),δ C 40.97 (C-2); 1 carbon signal of methine 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; Fig. 9 For this compound 1 H- 1 HCOSY spectrum; Fig.10 is the NOESY spectrum of the compound.

[0088] Fig. 9 of 1 H- 1 δ in H COSY spectrum H 7.92(H-4') and δ H 6.34 (H-5'), 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 related, 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), 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 attached to the six-membered ring.

[0090] Fig.10 In the NOESY spectrum of 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 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 C-2' is replaced 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.13 .

[0092] Fig.11 is the coupling correlation diagram of the compound. Fig.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) related, δ H 2.25(H-4) and δ H 1.34(H-9),δ H 3.84 (H-3), 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, such as Fig.12As shown. Therefore, the absolute configuration of the compound is 1S, 3R, 5R, 6S. After systematic literature search, the compound is a terpene compound that has not been reported.

[0093] Example 4

[0094] Anti-inflammatory function test

[0095] A new terpenoid compound extracted and separated from Portulaca oleracea in Examples 1 to 3 of the present invention exhibits 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 then 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 value of drug concentration.

[0098] 2 Cell lines

[0099] Macrophages RAW264.7.

[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] The 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 5The cells were inoculated in a 96-well plate at a concentration of 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 (200 μL of culture medium) was added, a model group (100 μL of LPS with a final concentration of 1 μg / mL and 100 μL of culture medium was added), a positive control group (100 μL of dexamethasone with a concentration of 100 μmol / L and 100 μL of LPS with a final concentration of 1 μg / mL were added), and a drug group (100 μL of the sample to be tested and 100 μL of LPS with a final concentration of 1 μg / mL were added), and 5 replicate wells were set for each group. After the 96-well plate was placed in a 37°C, 5% CO2 incubator for 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] In the above 96-well plate, add 20 μL of MTT solution (add MTT solvent to MTT powder to prepare a 5 mg / mL solution), and then put the 96-well plate into a cell culture incubator. After continuing to culture for 4 hours at 37°C and 5% CO2, all the liquid in the wells is aspirated, 150 μL / well DMSO is added, and the absorbance of each well is measured at a wavelength of 490 nm to calculate the cell survival rate.

[0108] 7 Experimental results

[0109] The MTT method was used to detect the effects of the compounds on the survival rate of macrophage RAW264.7 cells. Fig.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 LPS-stimulated macrophages RAW264.7.

[0110] By measuring the IC of the test compounds of Examples 1 to 3 50 The anti-inflammatory activity of Fig.16 , Fig.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. 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 concentration 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 a preferred embodiment.

[0112] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other 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 can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

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

2. The method for preparing terpenoid compounds according to claim 1, characterized in that: The following steps are involved: S1. Using purslane as raw material, extracting by heating with ethanol solution, combining the extracts, filtering, and evaporating the ethanol to obtain purslane total extract; S2. suspending the total extract of Portulaca oleracea with water to obtain a suspension; S3, extracting the suspension with extraction solvents of petroleum ether, ethyl acetate, and n-butanol in sequence, concentrating and drying each extract under reduced pressure to obtain a petroleum ether layer extract, an ethyl acetate layer extract, and an n-butanol layer extract; S4, the ethyl acetate layer extract was loaded onto a silica gel column, and after elution, color development and merging, 9 elution products Fr.A~I were obtained; a dichloromethane-methanol elution system was used for gradient elution, and the volume ratios of dichloromethane and methanol during elution were 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 the 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. S6, loading the eluted product Fr.5 onto a silica gel column, and after elution, color development and merging, 10 eluted products Fr.5-1 to Fr.5-10 were obtained; a dichloromethane-methanol elution system was used for gradient elution, and the volume ratio of dichloromethane to methanol during elution was 100:1 and 80:1 respectively; S7, loading the eluted product Fr.5-5 onto a gel column, isocratically eluted with methanol, and after color development and merging, five eluted products Fr.5-5-1 to Fr.5-5-5 are obtained; S8. Separate and purify the eluted product Fr.5-5-2 by semi-preparative HPLC to obtain the terpene compound, and the separation 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 is 85%, the usage 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.

4. The method for preparing terpenoid compounds according to claim 3, characterized in that: In S2, add 38-40℃ water to 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 is 2.0 mL / min, and the effluent with a retention time of 18.1 min is 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 an anti-inflammatory drug.

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 described in claim 1 as an active ingredient and further contains pharmaceutically acceptable excipients.

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