CN110563781A

CN110563781A - Preparation method of albizzia bark neolignan monomer compound

Info

Publication number: CN110563781A
Application number: CN201910944153.4A
Authority: CN
Inventors: 邱丽颖; 史学林; 艾敏; 蔡维维; 李忠杰; 刘艺筱
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-09-30
Filing date: 2019-09-30
Publication date: 2019-12-13
Anticipated expiration: 2039-09-30
Also published as: CN110563781B

Abstract

The invention discloses a preparation method of a albizzia bark neolignan monomer compound, belonging to the technical field of biochemistry. The compound prepared by the invention can effectively treat FFAs-induced lipid metabolism disorder and steatosis, and the administration concentration is 20 mu M, and the lipid drop area can be reduced by about 3 times after administration for 24 hours; and can obviously enhance the cell activity of HUVEC and obviously promote the self-replication of cells, thereby promoting the proliferation of endothelial cells HUVEC. At 40 μ M, 24h after administration, cell viability reached 242.233% of control. The compound prepared by the invention can obviously inhibit the generation of Reactive Oxygen Species (ROS) induced by HG (mercury-induced killer) of HUVEC (human embryonic kidney) cells, and can reduce the average fluorescence intensity of HG groups from 818.37% to 176.36%.

Description

Preparation method of albizzia bark neolignan monomer compound

Technical Field

The invention relates to a preparation method of a monomer compound of cortex albiziae neolignanoid, belonging to the technical field of biochemistry.

background

Cortex Albizziae (Albiziae Cortex) is the bark of Albizzia julibrissin Durazz (Albzia julibrissin Durazz) belonging to Leguminosae, is a common traditional Chinese medicine, has sweet and mild properties and taste, and has the effects of resolving stagnation, regulating blood, calming heart and relieving swelling. In recent years, with the intensive research on albizzia julibrissin durazzini by scholars, chemical components and pharmacological and medicinal effects of albizzia julibrissin durazzini are continuously discovered.

At present, the active compounds in the cortex albiziae are mainly researched by chemical extraction and separation, for example, extraction with organic solvents such as ethanol and the like, and elution with macroporous resin, silica gel column chromatography, reverse C18 chromatographic separation and other technologies. Hitherto, compounds such as triterpenes, flavones and lignans have been isolated from cortex albiziae, and the pharmacological and pharmacological activities of the compounds are not clear, and the molecular weights and functional group structures of the similar compounds are similar, so that it is difficult to specifically isolate and extract a specific compound.

Disclosure of Invention

The first object of the present invention is to provide a method for isolating a neolignan compound of albizzia julibrissin durazzi, comprising the steps of: (1) crushing cortex albiziae, mixing with 70-80% ethanol according to a material-liquid ratio of 1: 4-10, and performing reflux extraction at 70-90 ℃ for 1-3 times, 1-3 hours each time; (2) filtering to remove cortex Albizziae residues, mixing the extractive solutions obtained in step (1), freeze drying, collecting crude extract, suspending, and sequentially extracting with ethyl acetate and n-butanol; (3) recovering n-butanol extractive solution, and drying; (4) d101 macroporous adsorption resin is adopted, ethanol with different volume fractions is used for elution and separation, and elution sections are respectively enriched; (5) performing silica gel column chromatography on each elution section, performing gradient elution by using dichloromethane-methanol mixed solution with different volume fractions, combining the same components through TLC detection, and collecting each elution section.

In one embodiment, the albizzia bark neolignan compound comprises

In one embodiment, the step (4) is performed by eluting and separating with 30%, 50%, 70% and 95% ethanol by volume fraction, and the 30% ethanol elution section, the 50% ethanol elution section, the 70% ethanol elution section and the 95% ethanol elution section are respectively enriched.

In one embodiment, the step (5) is to perform silica gel column chromatography on the elution segments, perform gradient elution by using dichloromethane-methanol mixed solution of 40:1,20:1,16:1,10:1,8:1 and 6:1, collect each elution segment and separate the albizia julibrissin lipid compounds.

In one embodiment, step (5) collects the 10:1 eluate fraction, further purifies the 10:1 eluate fraction by reverse phase silica gel column, and finally adopts X-bridge-18 (5 μm, 10X 250mm) chromatographic column with flow rate of 4mL/min and CH₃OH is phase A, water is phase B, and elution is carried out according to the following semi-preparative liquid phase separation conditions:

time of day	CH₃Volume percent OH	H₂Volume percent of O
			0	10％	90％
4	10％	90％
			5	26％	74％
45	28％	72％
			60	32％	68％
70	50％	50％
			80	100％	0％

Collecting the compound with retention time of 26.50min

in one embodiment, step (5) collects the 8:1 eluate fraction, and the 8:1 eluate fraction is further purified by reverse phase silica gel column chromatography using an X-bridge-18 (5 μm, 10X 250mm) chromatography column at a flow rate of 4mL/min in CH₃OH is phase A, water is phase B, and semi-preparative elution separation is carried out according to the following elution conditions:

Collecting the part with retention time of 31.75minTo obtain a compound of

in one embodiment, step (5) collects the 8:1 eluate fraction on a C18 column with CH₃OH is A phase, water is B phase, elution is carried out according to the following elution conditions:

Time of day	CH₃Volume percent OH	H₂Volume percent of O
			0	10％	90％
4	10％	90％
			5	23％	77％
50	23％	77％
			55	100％	0％
60	100％	0％

Collecting the compound with retention time of 35.20min to obtain

In one embodiment, step (5) collects the 8:1 eluate fraction, further purifies with a reverse phase silica gel column, and finally adopts an X-bridge-18 (5 μm, 10X 250mm) chromatographic column with a flow rate of 4mL/min and CH₃OH is phase A, water is phase B, and semi-preparative elution separation is carried out according to the following elution conditions:

Collecting the compound with retention time of 41.0min to obtain

Time of day	CH₃Volume percent OH	H₂Volume percent of O
			0	10％	90％
4	10％	90％
			5	27％	73％
50	27％	73％
			55	100％	0％
60	100％	0％

Collecting the compound with retention time of 37.25min as shown in FIG. 5

Has the advantages that: the invention provides a preparation method of a albizzia julibrissin durazzini derived compound, which can be separated in a targeted manner the purity of the five compounds can reach over 96 percent.

Drawings

FIG. 1 is a 10:1 elution fraction analytical high performance liquid chromatogram.

FIG. 2 shows a 10:1 component Davisil C18 reverse column 42% CH₃And (3) performing a semi-preparative high performance liquid chromatogram of the OH elution section.

FIG. 3 is a drawing of compound Aj4¹H-NMR chromatogram.

FIG. 4 is a drawing of compound Aj4¹³C-NMR chromatogram.

FIG. 5 is an HR-ESI-MS chromatogram of albicide A.

FIG. 6 is an 8:1 elution fraction analytical high performance liquid chromatogram.

FIG. 7 is a Davisil C18 reverse column 32% CH₃And (3) performing chromatogram of the component Aj5 of the OH elution section under semi-preparative high performance liquid chromatography detection.

FIG. 8 is an ESI-MS chromatogram of compound (+) -Lyoniresinol-9' -O-glucoside (Aj 5).

FIG. 9 shows (+) -Lyoniresinol-9' -O-glucoside (Aj5)¹³C-NMR chromatogram.

FIG. 10 is a HR-ESI-MS chromatogram of (+) -Lyoniresinol-9' -O-glucoside (Aj 5);

FIG. 11 is a Davisil C18 reverse column 32% CH₃And (3) performing chromatogram of the component Aj6 of the OH elution section under semi-preparative high performance liquid chromatography detection.

FIG. 12 is an ESI-MS chromatogram of (-) -Lyoniresinol-9' -O-glucoside (Aj 6).

FIG. 13 is a schematic representation of (-) -Lyoniresinol-9' -O-glucoside (Aj6)¹HNMR spectrogram.

FIG. 14 shows a sequence of (-) -Lyoniresinol-9' -O-glucoside (Aj6)¹³CNMR spectrogram.

FIG. 15 is a Davisil C18 reverse column 32% CH₃And (3) performing chromatogram of the component Aj7 of the OH elution section under semi-preparative high performance liquid chromatography detection.

FIG. 16 is an ESI-MS chromatogram of compound Picraquassioside C (Aj 7).

FIG. 17 is a drawing of compound Picraquassioside C (Aj7)¹HNMR spectrogram.

FIG. 18 is a drawing of compound Picraquassioside C (Aj7)¹³CNMR spectrogram.

FIG. 19 is Davisil C18 reverse column 36% CH₃And (3) performing chromatogram of the component Aj8 of the OH elution section under semi-preparative high performance liquid chromatography detection.

FIG. 20 is compound Icariside E₅ESI-MS chromatogram of (Aj 8).

FIG. 21 shows Compound Icariside E₅(Aj8)¹HNMR spectrogram.

FIG. 22 shows compound Icariside E₅(Aj8)¹³CNMR spectrogram.

Detailed Description

EXAMPLE 1 preparation of the Compound Aj4

(1) Extraction: pulverizing 20kg of dried cortex Albizziae, extracting with 5 times of 75% ethanol (water) 100L each time under reflux at 80 deg.C for 2 times, each time for 2 hr. Filtering to remove cortex Albizziae residue, mixing 75% ethanol extractive solutions of cortex Albizziae, and freeze drying to obtain 1.6kg ethanol crude extract of cortex Albizziae. The crude extract was triturated and suspended in 2L of deionized water to dissolve it as far as possible. Suspending, sequentially extracting with ethyl acetate and saturated n-butanol, and mixing ethyl acetate phase and saturated n-butanol phase extractive solutions to obtain ethyl acetate extract and n-butanol extract.

(2) Separation: taking 254g of n-butanol, dissolving and suspending with deionized water, separating and purifying by using D101 macroporous adsorption resin, and performing gradient elution by using an ethanol-water mixed solution with 2-3 times of column volume as a mobile phase to enrich a 30% ethanol elution section, a 50% ethanol elution section, a 70% ethanol elution section and a 95% ethanol elution section. Subjecting 30% ethanol eluate to silica gel column chromatography, taking dichloromethane-methanol mixed solution as mobile phase, and gradient solvent dichloromethane (CH)₂Cl₂): methanol (CH)₃And (4) performing gradient elution (the volume of a mobile phase is 3 times of the volume of a column, and whether a target product is obtained or not is detected by TLC in real time) on OH) which is (40:1,20:1,16:1,10:1,8:1 and 6:1), performing elution by the mobile phase with the volume of about 4 times of the volume of the column, collecting eluates by 250ml conical flasks respectively, and merging the same components through TLC detection to obtain elution sections.

Carrying out analytical High Performance Liquid Chromatography (HPLC) detection on the silica gel 10:1 elution section (the chromatographic column is X-Bridge C18,5 mu m, 4.6X 250mm, the flow rate is 1ml/min, the column temperature is 30 ℃), the ultraviolet detection wavelength is 254nm, and the elution conditions are as follows:

Time CH₃OH H₂O

0min 5％ 95％

60min 100％ 0％

The results are shown in FIG. 1. According to the figure1 liquid phase detection, determining the retention time of the compound on C18 reverse column (Davisil C18,50 μm) corresponding to the elution conditions of silica gel 10:1, and the methanol concentration corresponding to each retention time is 41% CH, and the retention time is 23min, 30min, 36min, and 60min₃OH，52％CH₃OH，62％CH₃OH，100％CH₃OH。

The 10:1 fraction was passed through a reverse phase silica gel column (Davisil C18,50 μm) and eluted with methanol and water as the eluent phase. Since FIG. 1 is an analytical liquid chromatogram with a column X-Bridge C18(5 μm, 4.6X 250mm) and 50 μm packing for a Davisil C18 reverse phase column, the mobile phase methanol concentrations were all 10% less. Thus, it was determined from FIG. 1 that the elution phase of X-Bridge C18 column chromatography was 31% CH₃OH,42％CH₃OH,52％CH₃OH,100％CH₃And (5) OH. HPLC detection was carried out on the eluted fractions, respectively, and the results showed 42% CH₃The ultraviolet absorption peak intensity of OH elution section is larger and easier to separate, and 42 percent CH is groped according to an analytical liquid phase₃The separation conditions of the OH elution section, the optimum separation conditions were determined as shown in Table 1. Last 42% CH₃the OH fraction was subjected to semipreparative liquid phase separation (column X-Bridge C18,5 μm, 10X 250mm, flow rate 4ml/min, column temperature 30 ℃ C.), and the result was shown in FIG. 2, and the compound having a retention time t 26.50min was named as albicide A (Aj 4).

TABLE 1 different separation conditions for semi-preparative liquid chromatography

Among the separation conditions shown in table 1, condition one, condition two, and condition three failed to effectively separate Aj4, and there was a peak interference around the retention time, and condition four was able to more effectively separate compound Aj4, and the separation conditions adopted in this experiment were further optimized based on condition four, and the separation effect is shown in fig. 2. Collecting the retention time t_RThe compound at 26.50min was rotary evaporated at 75 ℃ under reduced pressure, the solvent was recovered, and dried in a vacuum oven to obtain Aj4 monomeric compound.

(3) Structural identification(shown in FIGS. 3 to 5): aj4 is light yellow powder, detected by UPLC-ESI-MS, ESI-MS M/z 633.1932[ M + Cl^-]The molecular formula of the compound is determined to be C by Monosotopic Mass, Even Electron Ions₂₈H₃₈O₁₄。

Dissolving the sample Aj4 in a nuclear magnetic tube by using deuterated methanol, and determining by using a full-digital nuclear magnetic resonance spectrometer¹HNMR、¹³CNMR, the results are as follows:

¹H NMR(400MHz,CD₃OD)δ6.76(2H,s,H-2’,6’),6.74(2H,s,H-2,6),6.56(1H,d,J＝16.0Hz,H-7’),6.34(1H,dt,J＝15.8,5.5Hz,H-8’),4.81(1H,d,J＝7.2Hz,H-7),4.65(4H,s),4.29(1H,dd,J＝9.0,4.8Hz,H-1”),4.24(2H,d,J＝5.4Hz,H-9’),3.96–3.88(2H,m),3.85(6H,s,3,5-OMe),3.83(6H,s,3’,5’-OMe),3.77(1H,s),3.73–3.63(4H,m),3.60(1H,dd,J＝8.4,3.9Hz),3.56–3.39(4H,m),3.22(s,1H).¹³C NMR(101MHz,CD₃OD)δ153.08(C-3’,5’),152.39(C-3,5),138.12(C-4’),134.99(C-4),134.13(C-1’),133.30(C-1),129.95(C-7’),128.51(C-8’),104.62(C-2,6),104.26(C-1”),103.51(C-2’,6’),85.66(C-8),76.94(C-3”),76.37(C-5”),74.32(C-2”),72.64(C-7),69.92(C-4”),62.16(C-9),61.17(C-9’),60.23(C-6”),55.61(3’,5’-OMe),55.28(3,5-OMe)。

Based on mass spectrum and¹HNMR、¹³CNMR finally determines its structure as follows,Chemical name 2- [4- (3-Hydroxy-1-propenyl) -2,6-dimethoxy-phenoxy]-3-hydroxy-3- (4-hydroxy-3, 5-methoxy-phenyl) propyl-beta-D-glucopyranoside, which is named albizim new lignan glycoside A (albicide A, Aj 4).

EXAMPLE 2 preparation of the Compound Aj5

Procedures for extraction and separation referring to example 1, the retention time of each component in the analytical high performance liquid chromatogram of FIG. 6 was used to determine CH₂Cl₂:CH₃the OH 8:1 elution fraction was subjected to a reverse phase silica gel column (Davisil C18,50 μm) under the conditions of t_RCH corresponding to 22min, 24min, 26min, 60min₃OH concentrations 39.8%, 42.9%, 46.2%, 100%, CH since the C18 filler used was 50 μm in diameter₃The OH concentrations are reduced by 10%, i.e. 29.8%, 32.9%, 36.2%, 100%. Eluting with methanol and water as eluting phase. The elution phase composition was determined to be 29% CH according to FIG. 6₃OH(H₂O),32％CH₃OH(H₂O),36％CH₃OH(H₂O),100％CH₃oh. analytical HPLC assay was performed on each eluted fraction separately, and the separation conditions were groped to determine the optimum separation method (as shown in table 2). For 32% CH₃The OH elution fraction was subjected to semi-preparative liquid phase separation, as shown in FIG. 7 (UV detection wavelength 290nm), retention time t_RThe compound at 31.75min was (+) -Lyoniresinol-9' -O-glucoside (Aj 5).

TABLE 2

Although the target monomeric compound can be separated under the conditions of one to four in the liquid phase separation method described in the present study, the purity of the obtained product Aj5 is poor due to the interference of impurity peaks, and the optimal separation conditions optimized on the basis can eliminate the interference of the impurity peaks on the target product, so that the high-purity monomeric compound is separated, and the semi-preparative separation chromatogram is shown in fig. 7.

(3) Structural identification

Aj5 is white powder, and its mass spectrum is shown in FIG. 8 by UPLC-ESI-MS detection. ESI-MS M/z 617[ M + Cl ]^-]The molecular formula of the compound is determined to be C by Monosotopic Mass, Even Electron Ions₂₈H₃₈O₁₃。

Dissolving the sample Aj5 in a nuclear magnetic tube by using deuterated methanol, and determining by using a full-digital nuclear magnetic resonance spectrometer¹HNMR、¹³The results of CNMR are as follows (FIGS. 9 to 10):

¹HNMR(400MHz,MeOD)δ6.60(1H,s,H-8),6.45(2H,s,H-2’,6’),4.44(1H,d,J＝6.2Hz,H-4),4.30(1H,d,J＝7.7Hz,anomeric-H),3.95–3.81(6H,m,5,7-OMe),3.76(6H,s,3’,5’-OMe),3.67(2H,dd,J＝11.8,4.9Hz,H-3a),3.56(1H,dd,J＝10.9,6.6Hz),3.47(1H,dd,J＝9.8,3.9Hz),3.39(1H,t,J＝7.7Hz),3.36(3H,s),3.28–3.23(2H,m,H-2a),2.77–2.59(2H,m,H-1),2.09(1H,d,J＝5.7Hz,H-3),1.73(1H,s,H-2).¹³CNMR(101MHz,MeOD)δ147.59(C-3’,5’),147.24(C-5),146.19(C-7),137.95(C-1’),137.51(C-6),133.10(C-4’),128.81(C-9),125.03(C-10),106.47(C-8),105.55(C-2’,6’),103.43(C-1”),76.85(C-5”),76.54(C-3”),73.79(C-2”),70.27(C-4”),70.11(C-3α),64.84(C-2α),61.44(C-6”),58.80(5-OMe),55.48(3’,5’-OMe),55.23(7-OMe),45.30(C-3),41.39(C-4),39.22(C-2),32.43(C-1).

Based on mass spectrum and¹HNMR、¹³CNMR finally determines its structure asThe chemical name (+) -Lyoniresinol 9' -O-glucoside.

EXAMPLE 3 preparation of the Compound Aj6

The extraction and separation conditions were the same as in example 2, and the semi-preparative separation chromatogram is shown in FIG. 11. For 32% CH₃The OH eluate fraction was subjected to semi-preparative liquid phase separation (column chromatography X-Bridge C18,5 μm, 10X 250mm, flow rate of 4ml/min, column temperature 30 deg.C), as shown in FIG. 15 (UV detection wavelength 290nm), and the retention time t was collected_RThe compound at 35.20min was rotary evaporated under reduced pressure at 75 ℃ to recover the solvent, and dried in a vacuum oven to obtain Aj6 monomeric compound.

and (3) structural identification: aj6 is light yellow powder, and its mass spectrum is shown in FIG. 12 by UPLC-ESI-MS detection. ESI-MS M/z 617[ M + Cl ]^-]The molecular formula of the compound is determined to be C by Monosotopic Mass, Even Electron Ions₂₈H₃₈O₁₃。

Dissolving the sample Aj5 in a nuclear magnetic tube by using deuterated methanol, and determining by using a full-digital nuclear magnetic resonance spectrometer¹HNMR、¹³The results of CNMR are as follows (FIGS. 13 to 14):

¹H NMR(400MHz,CD₃OD)δ6.59(1H,s,H-8),6.43(2H,s,H-2’,6’),4.24(1H,d,J＝5.3Hz,H-4),4.15(1H,d,J＝7.7Hz,H-1”),3.87(3H,s,-OCH₃),3.84(3H,d,J＝3.6Hz,-OCH₃),3.77(6H,s,3’,5’-OCH₃),3.72(1H,d,J＝5.2Hz),3.69(1H,d,J＝5.1Hz),3.62(5H,dt,J＝22.8,6.5Hz),3.49(1H,dd,J＝12.5,7.2Hz),3.44–3.35(2H,m),3.30–3.13(3H,m),2.70(2H,t,J＝8.0Hz,H),2.13(1H,dd,J＝13.1,6.9Hz,H-3),1.70(1H,d,J＝6.3Hz,H-1).¹³C NMR(101MHz,CD₃OD)δ147.61(C-3’,5’),147.29(C-5),146.15(C-7),138.06(C-1’),137.49(C-6),133.21(C-4’),128.83(C-9),124.84(C-10),106.41(C-8),105.72(C-2’,6’),102.85(C-1”),76.79(C-5”),76.58(C-3”),73.67(C-2”),70.61(C-3α),70.16(C-4”),64.83(C-2α),61.31(C-6”),58.72(5-OMe),55.53(7-OMe),55.23(3’,5’-OMe),45.19(C-3),41.83(C-4),39.85(C-2),32.43(C-1)。

Based on mass spectrum and¹HNMR、¹³CNMR finally determines its structure asThe chemical name is (-) -Lyoniresinol-9' -O-glucoside.

EXAMPLE 4 preparation of the Compound Aj7

Extraction and isolation procedure referring to example 1, isolation procedure was the same as example 2. For 32% CH₃The OH eluate fraction was subjected to semi-preparative liquid phase separation (column chromatography X-Bridge C18,5 μm, 10X 250mm, flow rate of 4ml/min, column temperature 30 deg.C), as shown in FIG. 15 (UV detection wavelength 290nm), and retention time t_RThe compound at 41.0min was dendrobium candidum C (picraquassiside C) (Aj7),

Aj7 is white powder, and its mass spectrum is shown in FIG. 16 by UPLC-ESI-MS detection. HR-ESI-MS M/z633.1688[ M + Cl ]]^-The molecular formula of the compound is determined to be C by Monosotopic Mass, Even Electron Ions₂₈H₃₈O₁₄。

dissolving the sample Aj7 in a nuclear magnetic tube by using deuterated methanol, and determining by using a full-digital nuclear magnetic resonance spectrometer¹H-NMR、¹³C-NMR results were as follows (FIGS. 17 to 18):

¹H-NMR(400MHz,CD₃OD)δ6.76(2H,s,H-2,6),6.74(2H,s,H-2’,6’),6.57(1H,d,J＝15.8Hz,H-7’),6.33(1H,dt,J＝15.8,5.5Hz,H-8’),4.94(1H,d,J＝5.6Hz,H-7),4.81(1H,d,J＝7.3Hz,H-1”),4.29(1H,dd,J＝9.0,4.8Hz,H-8),4.24(1H,d,J＝5.4Hz,H-9’),3.98–3.89(2H,m),3.87(1H,s),3.85(6H,s,3,5-OMe),3.83(6H,s,3’,5’-OMe),3.77(1H,s),3.73–3.58(4H,m),3.52–3.41(4H,m),3.37(1H,s),3.22(s,2H).¹³C-NMR(101MHz,CD₃OD)δ153.09(C-3’,5’),152.40(C-3,5),138.12(C-1),135.02(C-4’),134.17(C-4),133.31(C-1’),129.96(C-8’),128.53(C-7’),104.66(C-2,6),104.29(C-1”),103.55(C-2’,6’),85.68(C-8),76.94(C-5”),76.39(C-3”),74.34(C-2”),72.67(C-7),69.95(C-4”),62.17(C-9’),61.20(C-6”),60.25(C-9),55.63(3,5-OMe),55.31(3’,5’-OMe)。

Based on mass spectrum and¹HNMR、¹³CNMR finally determines the structure as follows, the Chinese name is Dendrobii herba C, the English name is Picraquassiside C, and albizim new lignanoid A (albizide A) are isomers.

EXAMPLE 5 preparation of the Compound Aj8

Extraction and isolation procedure referring to example 1, isolation procedure was the same as example 2. For 36% CH₃The OH eluate fraction was subjected to semi-preparative liquid phase separation (column chromatography X-Bridge C18,5 μm, 10X 250mm, flow rate of 4ml/min, column temperature 30 deg.C), as shown in FIG. 19 (UV detection wavelength 254nm), and retention time t_RThe compound at 37.25min is icariside E₅(Aj8)。

TABLE 3 conditions of liquid chromatography separation

In the separation conditions shown in table 3, condition one, condition two and condition three can not effectively separate Aj8, and the retention time is near to have strong interference of miscellaneous peak₅(Aj8), and its semi-preparative chromatogram is shown in FIG. 19. Collecting the retention time t_R37.25min under reduced pressure at 75 deg.CAnd (4) performing rotary evaporation, recovering the solvent, and drying in a vacuum drying oven to obtain the Aj8 monomer compound.

(3) Structural identification

Aj8 is white powder, and its mass spectrum is shown in FIG. 20 by UPLC-ESI-MS detection. ESI-MS M/z557[ M + Cl ]^-]is combined with¹H-NMR and¹³C-NMR (FIGS. 21 to 22) confirmed that the molecular formula is C₂₆H₃₄O₁₁。

Dissolving the sample Aj8 in a nuclear magnetic tube by using deuterated methanol, and determining by using a full-digital nuclear magnetic resonance spectrometer¹HNMR、¹³CNMR, the results are as follows:¹H NMR(400MHz,CD₃OD)δ6.94(2H,d,J＝4.8Hz,H-2,6),6.59(3H,t,J＝7.0Hz,H-2’,5’,6’),6.50(1H,d,J＝8.0Hz,H-7),6.32(1H,dt,J＝15.6,5.6Hz,H-8),4.69(2H,d,J＝7.2Hz),4.24(2H,d,J＝5.5Hz,H-9),4.03–3.94(1H,m),3.92–3.85(1H,m),3.84(3H,s,3-OMe),3.82–3.74(3H,m),3.72(1H,s),3.71(3H,s,3’-OMe),3.67(1H,d,J＝7.2Hz),3.62–3.35(4H,m),3.19–3.11(1H,m),2.99(1H,dd,J＝13.8,5.5Hz),2.74(1H,dd,J＝13.7,9.4Hz).¹³CNMR(101MHz,CD₃OD)δ152.05(C-3),147.02(C-3’),143.94(C-4’),143.61(C-4),137.53(C-5),134.01(C-1),131.82(C-1’),130.10(C-8),128.29(C-7),121.22(C-6’),117.76(C-6),114.29(C-5’),112.41(C-2’),107.75(C-2),103.97(C-1”),76.67(C-5”),76.46(C-3”),74.54(C-2”),69.85(C-4”),65.45(C-9’),62.27(C-9),61.06(C-6”),55.02(3’-OCH₃),54.89(5’-OCH₃) 41.39(C-8 '), 37.76 (C-7'). According to¹HNMR、¹³CNMR and mass spectrum finally determined that Aj8 is Icariside E5(Icariside E)₅). The results are as follows:

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The method for separating the albizzia julibrissin neolignan compound is characterized by comprising the following steps of: (1) crushing cortex albiziae, mixing with 70-80% ethanol according to a material-liquid ratio of 1: 4-10, and performing reflux extraction at 70-90 ℃ for 1-3 times, 1-3 hours each time; (2) filtering to remove cortex Albizziae residues, mixing the extractive solutions obtained in step (1), freeze drying, collecting crude extract, suspending, and sequentially extracting with ethyl acetate and n-butanol; (3) recovering n-butanol extractive solution, and drying; (4) d101 macroporous adsorption resin is adopted, ethanol with different volume fractions is used for elution and separation, and elution sections are respectively enriched; (5) performing silica gel column chromatography on each elution section, performing gradient elution by using dichloromethane-methanol mixed solution with different volume fractions, combining the same components through TLC detection, and collecting each elution section.

2. The method of claim 1, wherein the albizzia bark neolignan compound comprises

3. The method according to claim 1 or 2, wherein the step (4) is performed by eluting with ethanol with a volume fraction of 30%, 50%, 70% and 95%.

4. the method as claimed in claim 3, wherein the 30% ethanol elution fraction is enriched, silica gel column chromatography is performed, gradient elution is performed with dichloromethane-methanol mixed solution of 40:1,20:1,16:1,10:1,8:1 and 6:1, and the elution fractions are collected and the albizia bark lignan compound is separated.

5. The method of claim 4, wherein 10:1 elution of dichloromethane-methanol mixture is collected, 10:1 elution is passed through a reverse silica gel column, and CH is added₃OH is an A phase, water is a B phase, and liquid phase detection is carried out according to the following elution conditions: c18 chromatographic column with flow rate of 1-1.2mL/min, the column temperature is 29.5-30.5, and the elution phase is subjected to gradient elution according to the following procedure:

time A phase B phase

0min 5％ 95％

60min 100％ 0％。

6. the method of claim 5, wherein the ratio of A to A is 42% CH₃Elution separation was performed according to the following semi-preparative separation conditions for the elution peak at OH:

Time/min CH₃Volume percent OH H₂Volume percent of O 0 10％ 90％ 4 10％ 90％ 5 26％ 74％ 45 28％ 72％ 60 32％ 68％ 70 50％ 50％ 80 100％ 0％

Collecting the compound with retention time of 26.50min

7. The method of claim 4, wherein the dichloromethane-methanol mixture is collected at 8:1 elution, the 8:1 elution is passed through a reverse silica gel column, and CH is added₃OH is an A phase, water is a B phase, and liquid phase detection is carried out according to the following elution conditions: c18 chromatographic column, the flow rate is 1-1.2 mL/min, the column temperature is 29.5-30.5, and the elution phase is eluted according to the following program gradient:

Time A phase B phase

0min 5％ 95％

60min 100% 0%; for 32% CH₃The elution peak for OH was separated by semipreparative elution according to the following elution conditions:

Time/min CH₃Volume percent OH H₂Volume percent of O 0 10％ 90％ 4 10％ 90％ 5 23％ 77％ 50 23％ 77％ 55 100％ 0％ 60 100％ 0％

Collecting the target compound.

8. the method of claim 7, wherein the compound is collected at a retention time of 31.75minor collecting the compound with retention time of 35.20minOr collecting the compound with retention time of 41.0min

9. the method of claim 4, wherein step (5) collects the 8:1 fractions, which are eluted with a reverse phase silica gel column and finally with CH₃OH is an A phase, water is a B phase, and liquid phase detection is carried out according to the following elution conditions: c18 chromatographic column, the flow rate is 1-1.2 mL/min, the column temperature is 29.5-30.5, and the elution phase is eluted according to the following program gradient:

Time A phase B phase

0min 50％ 95％

60min 100% 0%; for 36% CH₃The elution peak for OH was separated by semipreparative elution according to the following elution conditions:

Time/min CH₃Volume percent OH H₂Volume percent of O 0 10％ 90％ 4 10％ 90％ 5 27％ 73％ 50 27％ 73％ 55 100％ 0％ 60 100％ 0％

Collecting the compound with retention time of 37.25min to obtain

10. Use of the method according to any one of claims 1 to 9 for the preparation of a cortex albiziae derived compound and derivatives thereof.