Method for preparing kocodiol and arjunolic acid

Abstract

The invention discloses a preparation method for preparing kok glycol and arjunolic acid. According to the invention, the traditional Chinese medicinal material euonymus alatus is used as a raw material for the first time to prepare the compounds, i.e., the kok-diol and the arjunolic acid. In the prior art, it is not reported that the ramulus euonymi contains a compound kukodiol or arjiurolic acid, and it is also not reported that other plants in the euonymus family where the ramulus euonymi contains a compound kukodiol or arjiurolic acid. The method provided by the invention widens the raw material sources of the kok glycol and the arjunolic acid.

Description

Technical Field

[0001] The invention belongs to the field of natural drug chemistry and relates to a separation and preparation method of a known compound, and specifically relates to a method for preparing compound erythrodiol and compound arjuna acid from a traditional Chinese medicine Euphorbia obesa. Background Art

[0002] The compounds elaboradiol and arjunolic acid are natural products with the following chemical structures:

[0003]

[0004] Euonymus alatus (Thunb.) Siebold, E.alatus belongs to the winged branches or winged appendages of Euonymus of the genus Euonymus of the family Celastraceae. It was first recorded in Shennong's Herbal Classic as a medium-quality product. It is bitter and cold in nature and enters the liver and spleen meridians. It has the effects of promoting blood circulation, relieving blood stasis and relieving pain, and killing insects. The plant has a wide distribution range and is most common in East my country, Central China, North China and Southwest China. It is rich in resources and can be harvested all year round.

[0005] Studies have found that elaborandiol has multiple pharmacological activities such as anti-tumor, anti-inflammatory and antioxidant; arjunolic acid has multiple pharmacological activities such as anti-inflammatory, anti-cancer and hypoglycemic.

[0006] Although erythrodiol and arjunol acid have good activity, the raw material sources are few. In order to expand the raw material sources of erythrodiol and arjunol acid, the present invention is specially proposed. Summary of the invention

[0007] The present invention aims to overcome the deficiencies of the prior art and provide a method for preparing erythrodiol and arjuna acid.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] A method for preparing erythrodiol and / or arjuna acid, the chemical structures of erythrodiol and arjuna acid are as follows, and the Chinese medicinal material Euphorbia obesa is used as the separation raw material:

[0010]

[0011] Preferably, the method for preparing tricadoline comprises the following steps:

[0012] Step S1, crushing the Chinese medicinal material Psoralea corylifolia, extracting it with 95% ethanol at room temperature, and concentrating the extract to obtain a crude extract of Psoralea corylifolia;

[0013] Step S2, suspending the crude extract of Psoralea corylifolia with an appropriate amount of water, and then extracting with petroleum ether and dichloromethane in sequence, and concentrating the dichloromethane extract under reduced pressure to obtain a dichloromethane extract extract;

[0014] Step S3, loading the dichloromethane extract onto a normal phase silica gel column chromatography, gradient eluting with a dichloromethane-methanol mixed solvent, and merging by TLC detection to obtain 7 fractions Fr.1-7, and the Fr.2 fraction is used for subsequent separation; wherein, the normal phase silica gel column chromatography gradient elution ratio, volume and sequence are: 100:0 elution 1 column volume → 100:1 elution 2 column volumes → 50:1 elution 3 column volumes → 30:1 elution 3 column volumes → 20:1 elution 3 column volumes → 10:1 elution 2 column volumes → 5:1 elution 2 column volumes;

[0015] Step S4, loading the Fr.2 fraction onto a normal phase silica gel column chromatography, eluting with a petroleum ether-ethyl acetate mixed solvent in a gradient manner, and combining the fractions by TLC detection to obtain 11 fractions Fr.2.1-2.11, and the Fr.2.9 fraction is used for subsequent separation; wherein, the normal phase silica gel column chromatography gradient elution ratio, volume, and sequence are: 100:0 elution for 1 column volume → 20:1 elution for 2 column volumes → 15:1 elution for 6 column volumes → 10:1 elution for 4 column volumes → 6:1 elution for 4 column volumes → 4:1 elution for 3 column volumes → 2:1 elution for 2 column volumes → 1:1 elution for 1 column volume;

[0016] Step S5, loading the Fr.2.9 fraction onto an ODS column chromatography, gradient eluting with a methanol-water mixed solvent, and merging the fractions by TLC detection to obtain 6 fractions Fr.2.9.1 to 2.9.6, wherein the Fr.2.9.6 fraction is used for subsequent separation; wherein the gradient elution ratio, volume, and sequence of the ODS column chromatography are: 30:70 elution for 1 column volume → 45:55 elution for 1 column volume → 60:40 elution for 1 column volume → 75:25 elution for 4 column volumes → 90:10 elution for 8 column volumes → 100:0 elution for 3 column volumes;

[0017] Step S6, subjecting the Fr.2.9.6 fraction to preparative HPLC chromatography with isocratic elution using methanol-0.1% acetic acid water in a volume ratio of 96:4, collecting the fractions at the elution peak of trichomediol, concentrating and drying to obtain trichomediol.

[0018] Preferably, the method for preparing tricadoline comprises the following steps:

[0019] Step S1, crushing the Chinese medicinal material Psoralea corylifolia, extracting it with 95% ethanol at room temperature, and concentrating the extract to obtain a crude extract of Psoralea corylifolia;

[0020] Step S2, suspending the crude extract of Psoralea corylifolia with an appropriate amount of water and then extracting it with petroleum ether, dichloromethane and ethyl acetate, and concentrating the ethyl acetate extract under reduced pressure to obtain the ethyl acetate extract;

[0021] Step S3, loading the ethyl acetate extract onto a normal phase silica gel column chromatography, gradient eluting with a dichloromethane-methanol mixed solvent, and merging the fractions after TLC detection to obtain 10 fractions Fr.1-10, with the Fr.7 fraction used for subsequent separation; wherein the gradient elution ratio, volume, and sequence of the normal phase silica gel column chromatography are: 100:0 elution for 1 column volume → 200:1 elution for 2 column volumes → 100:1 elution for 2 column volumes → 50:1 elution for 2 column volumes → 30:1 elution for 3 column volumes → 20:1 elution for 3 column volumes → 10:1 elution for 3 column volumes → 5:1 elution for 2 column volumes;

[0022] Step S4, loading the Fr.7 fraction onto a normal phase silica gel column chromatography, eluting with a dichloromethane-methanol mixed solvent in a gradient manner, and combining the fractions detected by TLC to obtain 7 fractions Fr.7.1 to 7.7, wherein the Fr.7.7 fraction is used for subsequent separation; wherein the normal phase silica gel column chromatography gradient elution ratio, volume, and sequence are: 100:0 elution for 1 column volume → 60:1 elution for 2 column volumes → 30:1 elution for 4 column volumes → 15:1 elution for 4 column volumes → 10:1 elution for 4 column volumes → 5:1 elution for 2 column volumes;

[0023] Step S5, loading the Fr.7.7 fraction onto a Sephadex LH-20 column, eluting with a dichloromethane-methanol mixed solvent with a volume ratio of 1:1, and combining the fractions after TLC detection to obtain five fractions Fr.7.7.1 to 7.7.5, wherein the Fr.7.7.5 fraction is used for subsequent separation;

[0024] Step S6, subjecting the Fr.7.7.5 fraction to preparative HPLC chromatography with isocratic elution using methanol-0.1% acetic acid water in a volume ratio of 70:30, collecting the fractions at the elution peak of arjuna acid, concentrating and drying to obtain arjuna acid.

[0025] More preferably, step S1 is performed by leaching three times, filtering the extracts separately and then combining them.

[0026] Beneficial effects:

[0027] The present invention discloses a method for preparing erythrodiol and arjunalic acid. The present invention uses the Chinese medicinal material Euphorbia obesa as a raw material to prepare compounds erythrodiol and arjunalic acid for the first time. The prior art has not reported that Euphorbia obesa contains compounds erythrodiol or arjunalic acid, nor has it been reported that other plants in the Celastraceae family, where Euphorbia obesa is located, contain compounds erythrodiol or arjunalic acid. The method provided by the present invention broadens the raw material sources of erythrodiol and arjunalic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the mass spectrum detection diagram of compound tricadiol;

[0029] Figure 2is the H-NMR spectrum of compound tricadiol;

[0030] Figure 3 This is the C NMR spectrum of compound tricadiol;

[0031] Figure 4 This is the mass spectrum detection diagram of the compound arjunolic acid;

[0032] Figure 5 is the H NMR spectrum of the compound arjunolic acid;

[0033] Figure 6 This is the NMR carbon spectrum of the compound arjunolic acid. DETAILED DESCRIPTION

[0034] The essential contents of the present invention are described in detail below with reference to the accompanying drawings and embodiments, but the protection scope of the present invention is not limited thereto.

[0035] General steps: 10.0 kg of Chinese medicinal material Herba Lycopodii was crushed and then added with 95% ethanol (5 L) and extracted at room temperature for 3 times, for 7 days, 5 days and 3 days respectively. The extracts were filtered and combined, and then concentrated under reduced pressure to obtain 243.8 g of Herba Lycopodii crude extract. The Herba Lycopodii crude extract was suspended with an appropriate amount of water and extracted with petroleum ether, dichloromethane, ethyl acetate and water-saturated n-butanol in turn.

[0036] Embodiment 1:

[0037] The method for preparing the compound erythrodiol from the Chinese medicinal material Euphorbia obesa comprises the following steps:

[0038] According to the general steps, the dichloromethane extract was collected and concentrated under reduced pressure to an extract. The dichloromethane extract of Psoralea corylifolia (133.4 g) was loaded onto a normal phase silica gel column chromatography, and gradient eluted with a dichloromethane-methanol mixed solvent (100:0×1→100:1×2→50:1×3→30:1×3→20:1×3→10:1×2→5:1×2), and detected by TLC. According to the TLC detection results, the same parts were combined to obtain 7 fractions Fr.1-7. After HPLC analysis of the 7 fractions Fr.1-7, the Fr.2 fraction was an enrichment containing cycodiol;

[0039] The fraction Fr.2 (6.73 g) was loaded onto a normal phase silica gel column chromatography, and gradient eluted with a petroleum ether-ethyl acetate mixed solvent (100:0×1→20:1×2→15:1×6→10:1×4→6:1×4→4:1×3→2:1×2→1:1×1), and detected by TLC. According to the TLC detection results, the same parts were combined to obtain 11 fractions Fr.2.1 to 2.11, of which the fraction Fr.2.9 was an enrichment containing tricadiol;

[0040] The fraction Fr.2.9 (1 g) was loaded onto an ODS column chromatography, and gradient eluted with a methanol-water mixed solvent (30:70×1→45:55×1→60:40×1→75:25×4→90:10×8→100:0×3), and detected by TLC. Based on the TLC detection results, the same parts were combined to obtain 6 fractions Fr.2.9.1 to 2.9.6, of which the fraction Fr.2.9.6 contained scopolamine-enriched substances;

[0041] The fraction Fr.2.9.6 (930.2 mg) was separated by preparative HPLC chromatography (methanol-0.1% acetic acid water, volume ratio 96:4), and the fraction with a peak at 26.2 min was concentrated under reduced pressure and dried to obtain compound scopolamine (14.3 mg) with a purity of 98%.

[0042] Structure confirmation: white powder. ESI-MS m / z 443.35[M+H] + . 1 H-NMR (500MHz,CDCl3)δ H ; 13 C NMR (125MHz,CDCl3)δ C :38.7(C-1),27.3(C-2),79.1(C-3),38.9(C-4),55.3(C-5),18.5(C-6),32.7(C-7),39.9(C-8), 47.7(C-9),37.1(C-10),23.7(C-11),122.5(C-12),144.3(C-13),41.8(C-14),25.7(C-15),22. 1(C-16),37.0(C-17),42.5(C-18),46.6(C-19),31.2(C-20),34.2(C-21),31.1(C-22),28.2(C-23),15.7(C-24),15.6(C-25),16.9(C-26),26.1(C-27),69.8(C-28),33.3(C-29),23.7(C-30). The above data are basically consistent with the literature report (Xu Shaoying et al., A new cinnamate derivative from maple resin), Chinese Journal of Traditional Chinese Medicine. Therefore, the compound was identified as erythrodiol, and the structure is as follows:

[0043]

[0044] Figure 1 is the mass spectrum of compound erythrodiol; Figure 2 is the H-NMR spectrum of compound tricadiol; Figure 3 This is the carbon NMR spectrum of the compound erythrodiol.

[0045] Embodiment 2:

[0046] The method for preparing the compound arjuna acid from the Chinese medicinal material Euphorbia obesa comprises the following steps:

[0047] According to the general steps, the ethyl acetate extract was collected and concentrated under reduced pressure to an extract. The ethyl acetate extract of Psoralea corylifolia (37.0 g) was loaded onto a normal phase silica gel column chromatography, and gradient eluted with a dichloromethane-methanol mixed solvent (100:0×1→200:1×2→100:1×2→50:1×2→30:1×3→20:1×3→10:1×3→5:1×2), and detected by TLC. According to the TLC detection results, the same parts were combined to obtain 10 fractions Fr.1 to 10. After HPLC analysis of the 10 fractions Fr.1 to 10, the fraction Fr.7 was an enrichment containing arjunic acid;

[0048] The fraction Fr.7 (668.3 mg) was loaded onto a normal phase silica gel column chromatography, and gradient eluted with a dichloromethane-methanol mixed solvent (100:0×1→60:1×2→30:1×4→15:1×4→10:1×4→5:1×2), and detected by TLC. According to the TLC detection results, the same parts were combined to obtain 7 fractions Fr.7.1 to 7.7, among which the fraction Fr.7.7 was an enrichment containing arjunic acid;

[0049] The fraction Fr.7.7 (108.7 mg) was loaded onto a Sephadex LH-20 column chromatography column, eluted with a dichloromethane-methanol mixed solvent at a volume ratio of 1:1, and detected by TLC. Five fractions Fr.7.7.1 to 7.7.5 were obtained based on the TLC detection results, among which the fraction Fr.7.7.5 was an enrichment containing arjunic acid;

[0050] The fraction Fr.7.7.5 (76 mg) was separated by preparative HPLC chromatography (methanol-0.1% acetic acid water, volume ratio of 70:30), and the fraction with a peak at 34.0 min was concentrated under reduced pressure and dried to obtain the compound arjuna acid (3 mg) with a purity of 99%.

[0051] Structure confirmation: white powder. ESI-MS m / z 487.42[MH] - . 1 H-NMR (500MHz,CDCl3) 1H-NMR (500MHz, CD3OD)δ H :5.25(1H,t,J=3.7Hz,H-12),3.69(1H,ddd,J=11.2,9.5,4.6Hz,H-2-α-OH),3.35(1H,d,J=9.6Hz,H-3-β-OH),3.50(1H,d,H-23a),δ H 3.27(1H,d,H-23b),1.18,1.03,0.95,0.91,0.83,0.70(each,3H,s,6×CH3,Me-24-27,29,30; 13 C-NMR (125MHz, CD3OD)δ C :47.9(C-1),69.7(C-2),78.2(C-3),43.0(C-4),48.2(C-5),19.1(C-6),33.9(C-7),40.6(C- 8),48.8(C-9),39.0(C-10),24.1(C-11),123.3(C-12),145.6(C-13),42.8(C-14),28.8(C-1 5), 24.6 (C-16), 47.7 (C-17), 44.1 (C-18), 47.3 (C-19), 31.6 (C-20), 35.0 (C-21), 33.6 (C-22), 66.3 (C-23), 13.9 (C-24), 17.8 (C-25), 17.5 (C-26), 26.5 (C-27), 33.4 (C-29), 24.0 (C-30). The above data are basically consistent with those reported in the literature (Yang Yao et al., Study on the Chemical Components of Triterpenes in Syzygium wilfordii, Journal of Yunnan Normal University (Natural Science Edition, 2021). Therefore, the compound was identified as arjunolic acid, and its structure is as follows:

[0052]

[0053] Figure 4 is the mass spectrum of the compound arjunolic acid; Figure 5 is the H NMR spectrum of the compound arjunolic acid; Figure 6 This is the NMR carbon spectrum of the compound arjunolic acid.

[0054] In summary, the present invention is the first method to prepare the compound erythrodiol and the compound arjunal acid using the Chinese medicinal material Euphorbia obesa as raw material. The prior art has not reported that Euphorbia obesa contains the compound erythrodiol and the compound arjunal acid, nor has it been reported that other plants in the Celastraceae family, where Euphorbia obesa is located, contain the compound erythrodiol and the compound arjunal acid. The method of the present invention broadens the raw material sources of the compound erythrodiol and the compound arjunal acid.

[0055] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. A method for preparing erythrodiol and / or arjunal acid, wherein the chemical structures of erythrodiol and arjunal acid are as follows, characterized in that: Using the Chinese medicinal material Herba Astragali as the raw material for separation:

2. The method according to claim 1, characterized in that The method for preparing tricadoline comprises the following steps: Step S1, crushing the Chinese medicinal material Psoralea corylifolia, extracting it with 95% ethanol at room temperature, and concentrating the extract to obtain a crude extract of Psoralea corylifolia; Step S2, suspending the crude extract of Psoralea corylifolia with an appropriate amount of water, and then extracting with petroleum ether and dichloromethane in sequence, and concentrating the dichloromethane extract under reduced pressure to obtain a dichloromethane extract extract; Step S3, loading the dichloromethane extract onto a normal phase silica gel column chromatography, gradient eluting with a dichloromethane-methanol mixed solvent, and merging by TLC detection to obtain 7 fractions Fr.1-7, and the Fr.2 fraction is used for subsequent separation; wherein, the normal phase silica gel column chromatography gradient elution ratio, volume and sequence are: 100:0 elution 1 column volume → 100:1 elution 2 column volumes → 50:1 elution 3 column volumes → 30:1 elution 3 column volumes → 20:1 elution 3 column volumes → 10:1 elution 2 column volumes → 5:1 elution 2 column volumes; Step S4, loading the Fr.2 fraction onto a normal phase silica gel column chromatography, eluting with a petroleum ether-ethyl acetate mixed solvent in a gradient manner, and combining the fractions by TLC detection to obtain 11 fractions Fr.2.1-2.11, and the Fr.2.9 fraction is used for subsequent separation; wherein, the normal phase silica gel column chromatography gradient elution ratio, volume, and sequence are: 100:0 elution for 1 column volume → 20:1 elution for 2 column volumes → 15:1 elution for 6 column volumes → 10:1 elution for 4 column volumes → 6:1 elution for 4 column volumes → 4:1 elution for 3 column volumes → 2:1 elution for 2 column volumes → 1:1 elution for 1 column volume; Step S5, loading the Fr.2.9 fraction onto an ODS column chromatography, gradient eluting with a methanol-water mixed solvent, and merging the fractions by TLC detection to obtain 6 fractions Fr.2.9.1 to 2.9.6, wherein the Fr.2.9.6 fraction is used for subsequent separation; wherein the gradient elution ratio, volume, and sequence of the ODS column chromatography are: 30:70 elution for 1 column volume → 45:55 elution for 1 column volume → 60:40 elution for 1 column volume → 75:25 elution for 4 column volumes → 90:10 elution for 8 column volumes → 100:0 elution for 3 column volumes; Step S6, subjecting the Fr.2.9.6 fraction to preparative HPLC chromatography with isocratic elution using methanol-0.1% acetic acid water in a volume ratio of 96:4, collecting the fractions at the elution peak of trichomediol, concentrating and drying to obtain trichomediol.

3. The method according to claim 1, characterized in that The method for preparing tricadoline comprises the following steps: Step S1, crushing the Chinese medicinal material Psoralea corylifolia, extracting it with 95% ethanol at room temperature, and concentrating the extract to obtain a crude extract of Psoralea corylifolia; Step S2, suspending the crude extract of Psoralea corylifolia with an appropriate amount of water and then extracting it with petroleum ether, dichloromethane and ethyl acetate, and concentrating the ethyl acetate extract under reduced pressure to obtain the ethyl acetate extract; Step S3, loading the ethyl acetate extract onto a normal phase silica gel column chromatography, gradient eluting with a dichloromethane-methanol mixed solvent, and merging the fractions after TLC detection to obtain 10 fractions Fr.1-10, with the Fr.7 fraction used for subsequent separation; wherein the gradient elution ratio, volume, and sequence of the normal phase silica gel column chromatography are: 100:0 elution for 1 column volume → 200:1 elution for 2 column volumes → 100:1 elution for 2 column volumes → 50:1 elution for 2 column volumes → 30:1 elution for 3 column volumes → 20:1 elution for 3 column volumes → 10:1 elution for 3 column volumes → 5:1 elution for 2 column volumes; Step S4, loading the Fr.7 fraction onto a normal phase silica gel column chromatography, eluting with a dichloromethane-methanol mixed solvent in a gradient manner, and combining the fractions detected by TLC to obtain 7 fractions Fr.7.1 to 7.7, wherein the Fr.7.7 fraction is used for subsequent separation; wherein the normal phase silica gel column chromatography gradient elution ratio, volume, and sequence are: 100:0 elution for 1 column volume → 60:1 elution for 2 column volumes → 30:1 elution for 4 column volumes → 15:1 elution for 4 column volumes → 10:1 elution for 4 column volumes → 5:1 elution for 2 column volumes; Step S5, loading the Fr.7.7 fraction onto a Sephadex LH-20 column, eluting with a dichloromethane-methanol mixed solvent with a volume ratio of 1:1, and combining the fractions after TLC detection to obtain five fractions Fr.7.7.1 to 7.7.5, wherein the Fr.7.7.5 fraction is used for subsequent separation; Step S6, subjecting the Fr.7.7.5 fraction to preparative HPLC chromatography with isocratic elution using methanol-0.1% acetic acid water in a volume ratio of 70:30, collecting the fractions at the elution peak of arjuna acid, concentrating and drying to obtain arjuna acid.

4. The method according to claim 2 or 3, characterized in that: Step S1 is to extract three times, filter them separately and then combine them.

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