Preparation method and application of total diterpene extract of yellow eupatorium flower

Abstract

The application discloses a preparation method and application of total diterpene extract of Daphne giraldii, and belongs to the technical field of traditional Chinese medicine pharmacy. In view of the problems that the content of daphnane diterpenes in Daphne giraldii is low, the structure is complex, and the daphnane diterpenes are difficult to be artificially synthesized, the dried Daphne giraldii is mixed with an extraction solvent, the extraction solvent is recovered under reduced pressure, and then concentrated and dried, and finally, a crude extract of extract is obtained; the crude extract of extract is added into a certain amount of water to prepare a sample solution which is added into a D101 macroporous resin column which has been treated to perform dynamic adsorption; in desorption, an organic solvent and water are used to perform first elution for not less than 10 column volumes (BV), impurities with relatively large polarity in the resin column are removed, then the organic solvent and water are used to perform second elution and collection, the solvent is recovered under reduced pressure, and then dried to obtain the crude extract; the crude extract is added into silica gel for sample mixing, and then uniformly added into a silica gel column which has been treated by a wet method to perform elution, the eluate is collected, the solvent is recovered under reduced pressure, and then dried to obtain the total diterpene extract.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and particularly relates to a method for preparing total diterpene extract from traditional Chinese medicine Wikstroemia chamaedaphne Meissn. and application of the total diterpene extract in treating AIDS. BACKGROUND

[0002] Wikstroemia chamaedaphne Meissn. is a perennial erect shrub plant (Wikstroemia) of Thymelaeaceae, which is originally from North China and West China, including Shanxi, Hebei, Gansu and Sichuan provinces, and the dried flower buds thereof are a traditional Chinese herbal medicine, which is called Wikstroemia chamaedaphne Meissn. Wikstroemia chamaedaphne Meissn. is a traditional Chinese herbal medicine in folk, which has the effects of relieving cough and asthma and purging water. In addition, modern pharmacological research shows that Wikstroemia chamaedaphne Meissn. has the effects of resisting early pregnancy, schizophrenia, anti-arrhythmia, anti-hepatitis virus and activating latent HIV. At present, the compounds separated from Wikstroemia chamaedaphne Meissn. mainly include diterpenes, flavonoids, phenolic acids, lignins and sesquiterpenes. The diterpenes are the most representative compounds in Wikstroemia chamaedaphne Meissn., and the diterpene components identified therefrom show good potential in activating latent HIV in HeLa-NH2 cell models. Wikstroelide E, pimelea factor P8, pimelea factor P0, simplexin and pimelea factor S7 show significant activation activity in activating latent HIV, and the activation effects of wikstroelide E, pimelea factor P8 and pimelea factor P2 reach the nanomolar level, and the activation potential of wikstroelide E is 500 times that of prostratin. However, the content of the diterpenes in Wikstroemia chamaedaphne Meissn. is low, and the structure thereof is complex and difficult to be artificially synthesized. Therefore, it is of great practical significance to establish a simple and rapid diterpene component enrichment method from Wikstroemia chamaedaphne Meissn. for developing total diterpene extract in treating AIDS. SUMMARY

[0003] In view of the problems of low content, complex structure and difficulty in artificial synthesis of Wikstroemia chamaedaphne Meissn., on the basis of the previous research on chemical components and activation of latent HIV in Wikstroemia chamaedaphne Meissn., the application provides a method for rapidly preparing total diterpene extract from traditional Chinese medicine Wikstroemia chamaedaphne Meissn. by macroporous resin combined with silica gel column chromatography enrichment and application of the total diterpene extract in preparing latent activator in a “shock and kill” strategy.

[0004] In order to achieve the above object, the following technical scheme is adopted in the application.

[0005] A preparation method of total diterpene extract of yellow flower, comprising the following steps:

[0006] Step 1, taking dry yellow flower medicinal materials, crushing, and then mixing with extraction solvent, extracting twice by percolation or hot reflux, combining the extraction liquid, recovering the solvent under reduced pressure and concentrating and drying, and finally obtaining the crude extract of extract;

[0007] Step 2, adding a certain amount of water to the crude extract of extract obtained in step 1 to prepare a sample loading solution and adding it to the treated D101 macroporous resin column for dynamic adsorption; next, in the desorption process, first eluting not less than 10 column volumes (BV) of organic solvent and water to remove water-soluble components in the resin column, and then eluting and collecting the second elution of organic solvent and water, recovering the solvent under reduced pressure, and drying to obtain the crude extract;

[0008] Step 3, adding the crude extract obtained in step 2 to silica gel and uniformly adding it to the silica gel column chromatography treated by wet method for elution; the elution program is: first gradient elution with petroleum ether: acetone, elution volume not less than 40 BV; second gradient elution with petroleum ether: acetone, elution volume not less than 50 BV, collecting the eluate, recovering the solvent under reduced pressure, and drying to obtain the total diterpene extract.

[0009] Further, the total diterpene extract includes four diterpene components pimelotide C, pimelotide A, simplexin, and 6α, 7α-epoxy-5β-hydroxy-12-deoxyphorbol-13-decanoate, and the total content of the four components is not less than 80%.

[0010] Further, the ratio of the use amount of the yellow flower to the extraction solvent is 1:5-1:10.

[0011] Further, the extraction solvent is ethanol or methanol; and the organic solvent is ethanol or methanol.

[0012] Further, the volume ratio of the organic solvent to water in the first elution in step 2 is 1:1.

[0013] Further, the volume ratio of the organic solvent to water in the second elution in step 2 is 15-17:3-5.

[0014] Further, the volume ratio of petroleum ether to acetone in the first gradient in step 3 is 12:1-10:1.

[0015] Further, the volume ratio of petroleum ether to acetone in the second gradient in step 3 is 6:1-4:1.

[0016] Use of a total diterpene extract of pycnalonga in the treatment of HIV latent activation agent drugs in the AIDS "shock and kill" strategy.

[0017] Compared with the prior art, the present application has the following advantages:

[0018] The present application establishes a method for rapidly enriching and preparing a total diterpene extract from pycnalonga, which is simple in operation, time-saving and labor-saving, efficient and fast, cost-saving, suitable for industrialized preparation and production, and has better activation of latent HIV activity, and can be used for preparing an HIV latent activation agent. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Structure diagrams of pimelotide C (1), pimelotide A (2), simplexin (3), and 6α, 7α-epoxy-5β-hydroxy-12-deoxyphorbol-13-decanoate (4).

[0020] Figure 2 It is a flow chart of the enrichment method of Example 3.

[0021] Figure 3 It is a high performance liquid chromatogram of the mixed reference substance and the total diterpene extract. DETAILED DESCRIPTION

[0022] Example 1: Enrichment and preparation method of total diterpene extract of pycnalonga

[0023] Take 500g of the medicinal material of yellow flower, dry and crush, put in a 10L round bottom flask, add 5L of 75% ethanol for heating reflux extraction, continuous extraction for 2 times, 1h each time. The obtained extract was concentrated under reduced pressure until no alcohol taste, add 7L of distilled water, stir and mix, stand for 12h, discard the upper solution, dry the precipitate to obtain the crude extract 26.4g. Add appropriate amount of ethanol to the crude extract to assist dissolution, add a certain amount of distilled water to prepare a sample solution with a mass concentration of 16.67mg / mL. Then add the prepared sample solution to the treated D101 macroporous resin column (diameter-height ratio = 1:5) at a loading adsorption speed of 1.8BV / h, the amount of sample solution is 5BV. In the desorption process, the elution speed is 6BV / h, first elute with 50% ethanol for 10BV to remove the larger polarity impurities in the resin column, then elute with 75% ethanol for 40BV, collect and recover the solvent, dry to obtain 75% ethanol fraction 12.5g. Add appropriate amount of methanol to fully dissolve the 80% ethanol fraction, take 12.5g of silica gel for sample mixing, dry loading, and uniformly add to the treated silica gel column chromatography (wet loading), the elution program is: first gradient elution with petroleum ether: acetone (10:1) for 40BV to remove smaller polarity impurities; second gradient elution with petroleum ether: acetone (6:1) for 50BV and collection, reduce pressure to recover the solvent, dry to obtain diterpene extract 2.8g; by HLPC content determination, the total content of 4 diterpenes pimelotide C (1), pimelotide A (2), simplexin (3), 6α,7α-epoxy-5β-hydroxy-12-deoxyphorbol-13-decanoate (4) in the extract is 80.6%.

[0024] Example 2: Enrichment preparation method of total diterpene extract of yellow flower

[0025] Take 1 kg of the medicinal material of yellow flower, dry and crush, put in 10 L round bottom flask for two times, add 5 L of 75% ethanol for heating reflux extraction, continuous extraction for 2 times, 1 h each time. The obtained extract was concentrated under reduced pressure to stop when there was no alcohol taste, add 15 L of distilled water, stir and mix, stand for 12 h, discard the upper solution, dry the precipitate to obtain the extract 62.0 g. Add appropriate amount of ethanol to the extract to assist dissolution, add a certain amount of distilled water to prepare a sample solution with a mass concentration of 16.67 mg / mL. Then add the prepared sample solution to the treated D101 macroporous resin column (diameter-height ratio = 1:5) at a loading adsorption speed of 1.8 BV / h. The sample solution is 5 BV. In the desorption process, the elution flow rate is 6 BV / h. First, 50% ethanol is used to elute 10 BV to remove the impurities with larger polarity in the resin column, and then 80% ethanol is used to elute 40 BV. The solvent is collected and recovered, and then dried to obtain 80% ethanol fraction 27.6 g. Add appropriate amount of ethanol to fully dissolve the 80% ethanol fraction, take 27.6 g of silica gel for sample mixing, dry loading, and uniformly add to the treated silica gel column chromatography (wet loading). The elution program is as follows: first gradient, petroleum ether: acetone (12:1) elution 42 BV to remove small polarity impurities; second gradient, petroleum ether: acetone (4:1) elution 53 BV and collection, reduced pressure recovery of solvent, and drying to obtain diterpene extract 6.3 g; by HLPC content determination, the total content of 4 diterpenes pimelotide C (1), pimelotide A (2), simplexin (3), 6α,7α-epoxy-5β-hydroxy-12-deoxyphorbol-13-decanoate (4) in the extract is 81.3%.

[0026] Example 3: Enrichment preparation method of total diterpene extract of yellow flower

[0027] Take 1 kg of the medicinal material of yellow flower, dry and crush, put in 10 L round bottom flask for two times, add 5 L of 75% ethanol for heating reflux extraction, continuous extraction for 2 times, 1 h each time. The obtained extract was concentrated under reduced pressure to stop when there was no alcohol taste, add 15 L of distilled water, stir and mix, stand for 12 h, discard the upper solution, dry the precipitate to obtain the extract 61.0 g. Add appropriate amount of ethanol to the extract to assist dissolution, add a certain amount of distilled water to prepare a sample solution with a mass concentration of 16.67 mg / mL. Then add the prepared sample solution to the treated D101 macroporous resin column (diameter-height ratio = 1:5) at a loading adsorption speed of 1.8 BV / h. The sample solution is 5 BV. In the desorption process, the elution flow rate is 6 BV / h. First, 50% ethanol is used to elute 10 BV to remove the impurities with larger polarity in the resin column, and then 85% ethanol is used to elute 40 BV. The solvent is collected and recovered, and then dried to obtain 80% ethanol fraction 27.0 g. Add appropriate amount of ethanol to fully dissolve the 80% ethanol fraction, take 27.0 g of silica gel for sample mixing, dry loading, and uniformly add to the treated silica gel column chromatography (wet loading). The elution program is as follows: first gradient, petroleum ether: acetone (12:1) elution 40 BV to remove small polarity impurities; second gradient, petroleum ether: acetone (4:1) elution 50 BV and collection, reduce pressure to recover the solvent, and dry to obtain diterpene extract 6.0 g. The total content of 4 diterpenes pimelotide C (1), pimelotide A (2), simplexin (3), 6α,7α-epoxy-5β-hydroxy-12-deoxyphorbol-13-decanoate (4) in the extract is 80.5% by HLPC content determination.

[0028] Example 4: Enrichment preparation method of total diterpene extract of yellow flower

[0029] Take 20 kg of the medicinal material of yellow flower, dry, crush, and put into a percolation barrel. Pour in appropriate amount of 90-95% ethanol solution, mix well, and let stand for 12 hours. Then start percolation, and percolate every 24 hours. Continue percolation for 24 days. Concentrate the percolation liquid under reduced pressure, and recover ethanol. Pour the recovered ethanol into the percolation barrel again to percolate again. Continue such operation until 2.4 kg of crude extract is obtained. Add appropriate amount of ethanol to the crude extract to assist dissolution, and add a certain amount of distilled water to prepare a sample solution with a mass concentration of 16.67 mg / mL. Then add the prepared sample solution to a D101 macroporous resin column (diameter-height ratio = 1:5) at a flow rate of 1.8 BV / h to perform dynamic adsorption. The amount of sample solution is 5 BV. During desorption, the flow rate is 6 BV / h. First, elute with 50% ethanol for 10 BV to remove impurities with large polarity. Then elute with 85% ethanol for 40 BV. Collect and recover the solvent, and dry to obtain 80% ethanol fraction 486.0 g. Add appropriate amount of dichloromethane to dissolve the 80% ethanol fraction. Take 486 g of silica gel to mix with the 80% ethanol fraction, dry, and add to a silica gel column (wet column) to perform chromatography. The elution program is as follows: first gradient, elute with petroleum ether: acetone (12:1) for 45 BV to remove impurities with small polarity; second gradient, elute with petroleum ether: acetone (5:1) for 55 BV and collect. Recover the solvent under reduced pressure, and dry to obtain diterpene extract 124.0 g. The total content of four diterpenes pimelotide C (1), pimelotide A (2), simplexin (3), and 6α,7α-epoxy-5β-hydroxy-12-deoxyphorbol-13-decanoate (4) in the extract is 80.2% as determined by HPLC.

[0030] Example 5: Method for enriching and preparing total diterpene extract of yellow flower

[0031] Take 20 kg of yellow-flowered herb medicine, dry and crush, put into percolation barrel, pour in appropriate amount of 90-95% ethanol solution, mix well, stand for 12 h, then start percolation, percolate every 24 h, continuously percolate for 24 d. The obtained percolation liquid is concentrated under reduced pressure and ethanol is recovered, the recovered ethanol is poured into the percolation barrel again for a new round of percolation, and the operation is continued, finally 2.4 kg of extract crude extract is obtained. Add appropriate amount of ethanol to assist dissolution in the extract crude extract, add a certain amount of distilled water to prepare a sample solution with a mass concentration of 16.67 mg / mL. Then add the prepared sample solution to the treated D101 macroporous resin column (diameter-height ratio = 1:5) at a loading adsorption flow rate of 1.8 BV / h to perform dynamic adsorption, and the amount of sample solution is 5 BV. During the desorption process, the elution flow rate is 6 BV / h, first elute 10 BV of 50% methanol to remove impurities with larger polarity in the resin column, then elute 40 BV of 85% methanol, collect and recover the solvent, and dry to obtain 80% ethanol fraction 485.0 g. Add appropriate amount of dichloromethane to fully dissolve the 80% ethanol fraction, take 485 g of silica gel for sample mixing, dry loading, and uniformly add to the treated silica gel column chromatography (wet loading), the elution program is: first gradient elution with petroleum ether: acetone (12:1) for 45 BV to remove impurities with smaller polarity; second gradient elution with petroleum ether: acetone (5:1) for 55 BV and collection, reduce pressure to recover the solvent, and dry to obtain diterpene extract 122.0 g; the total content of 4 diterpenes pimelotide C (1), pimelotide A (2), simplexin (3), 6α, 7α-epoxy-5β-hydroxy-12-deoxyphorbol-13-decanoate (4) in the extract is 81.2% by HLPC content determination.

[0032] Example 6: Activation of latent HIV activity test of each sample obtained by enrichment method

[0033] NH2 cells are cultured at 37℃, 5% CO2, and when the cells are stable in DMEM medium and in logarithmic growth phase, the cells are prepared into cell suspension and inoculated into 96-well plates at 100 μL per well, with three groups in parallel, and the blank group is the same as the above except that no drug is added; after the cells are attached to the wall for 6 h, different drug treatments are performed, and after the drug administration, the cells are cultured for another 12 h. Then, under the condition of avoiding light, the activation of latent HIV is determined. First, the DMEM medium is aspirated, and the drug administration group and the blank group are washed twice with PBS buffer. After the PBS buffer is aspirated, 100 μL of cell lysate is added to each well, and after being frozen at -80℃ for 1 h, the cells are thawed in a 37℃ incubator. After the DMEM medium is melted, the cells are lysed at 4℃ for 10 min. During this period, a white 96-well cell culture plate is prepared, 50 μL of the lysed cell solution is added to the white culture plate, 50 μL of luciferase detection reagent is added, and the fluorescence value is determined on an enzyme marker. The change multiple of the luminescence signal of the drug administration group and the blank group is used to predict the activation level of the drug on the HIV provirus.

[0034] Table 1: Activation of latent HIV activity of each sample obtained by the enrichment method

[0035]

[0036]

[0037] The contents not described in detail in the specification of the present application belong to the prior art known to those skilled in the art. Although the above describes the specific embodiments of the present application in a descriptive manner, so as to facilitate the understanding of the present application by those skilled in the art, it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are included in the protection.

Claims

1. A preparation method of total diterpene extract of Eupatorium fortunei, characterized in that, The method comprises the following steps: Step 1, taking dry yellow flower medicine, crushing, and then mixing with extraction solvent, extracting twice by percolation or hot reflux, combining the extract, recovering the solvent under reduced pressure and concentrating and drying, and finally obtaining the crude extract of the extract; Step 2, adding a certain amount of water to the crude extract obtained in step 1 to prepare a sample solution, and adding the sample solution to a D101 macroporous resin column treated in advance for dynamic adsorption; in the desorption process, first, elute not less than 10 column volumes (BV) of the mixed solvent of organic solvent and water to remove water-soluble substances in the resin column, and then elute and collect the mixed solvent of organic solvent and water, recover the solvent under reduced pressure, and dry to obtain the crude extract; Step 3, adding the crude extract obtained in step 2 to silica gel, and uniformly adding the silica gel to a silica gel column treated in advance for elution; the elution program is as follows: first gradient elution with petroleum ether: acetone, elution volume not less than 40 BV; second gradient elution with petroleum ether: acetone, elution volume not less than 50 BV, collect the eluate, recover the solvent under reduced pressure, and dry to obtain the total diterpene extract; The total diterpene extract comprises four diterpene components, and the total content of pimelotide C, pimelotide A, simplexin and 6α,7α-epoxy-5β-hydroxy-12-deoxyphorbol-13-decanoate is not less than 80%; The extraction solvent is ethanol or methanol; the organic solvent is ethanol or methanol; The volume ratio of the organic solvent to water in the first elution in step 2 is 1:1; The volume ratio of the organic solvent to water in the second elution in step 2 is 15-17:3-5; The volume ratio of petroleum ether to acetone in the first gradient in step 3 is 12:1-10:1; The volume ratio of petroleum ether to acetone in the second gradient in step 3 is 6:1-4:

1.

2. The preparation method of the total diterpenes extract of Isodon japonicus according to claim 1, characterized in that: The ratio of the amount of yellow flower to the extraction solvent is 1:5-1:

10.

3. A total diterpene extract of yellow flower prepared by the preparation method according to any one of claims 1-2.

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