CN111269277B

CN111269277B - Method for extracting and separating isocorilagin from Geranium strictipes

Info

Publication number: CN111269277B
Application number: CN202010266533.XA
Authority: CN
Inventors: 项勋; 常华; 代飞燕; 段纲; 江华超; 杨林富; 段博芳; 曾邦全
Original assignee: Yunnan Agricultural University
Current assignee: Yunnan Agricultural University
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2023-02-28
Anticipated expiration: 2040-04-07
Also published as: CN111269277A

Abstract

The invention relates to a method for extracting and separating isocorilagin from garden burnet, which belongs to the technical field of medicines.

Description

Method for extracting and separating isocorilagin from Geranium strictipes

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a method for extracting and separating isocorilagin from Geranium strictipes.

Background

Geranium strictipes R.Knuth is a specific plant in Geraniaceae, and is distributed in Yunnan, sichuan and other places. Geranium strictipes grows in areas with an altitude of 2,700-3,000 meters, often grows in hilly grassland, under forest and in shrubs, is mainly distributed in places such as Zhongdian, lijiang, dali, kunming and the like in Yunnan, is a common herbal medicine used by white and Yi nationalities to separate mountains and disappear, is bitter, slightly astringent and sour in taste and slightly cold in nature, and has the main functions of clearing heat and promoting diuresis, and activating blood and stopping bleeding (Yunnan herbal medicine). The white nationality and the Yi nationality have multiple purposes for treating anorexia, dysentery, chronic gastritis, abdominal pain, irregular menstruation and epistaxis; it can also be used externally to treat traumatic injury. However, the Geranium strictipes extract is difficult to separate due to its complex composition. For example, in patent CN201210179223, it is studied to extract an effective component of geranium strictipes, which is effective in inhibiting staphylococcus and escherichia coli, with ethanol, and to use the effective component in the treatment of multiple drug-resistant bacteria infection diseases. Although the active ingredients of sanguisorba officinalis have good medicinal value, the active ingredients in the sanguisorba officinalis extract are complex, and the value of the active ingredients in the medical treatment as the active ingredients is not known. At present, researches on Geranium strictipes are mostly limited to the extraction of active ingredients and the medicinal effect thereof, and no relevant reports are found on which active ingredient actually acts and how to separate the active ingredients of the extract. If the effective components of Geranium strictipes can be successfully extracted and separated, the method is undoubtedly a great technical contribution to explaining the pharmaceutical principle of Geranium strictipes.

Isocoryzanol, white powder, chemical formula C27H20O18, molecular weight 632, structural formula is iso-1-O-galloyl-3,6- (R) -O-hexahydroxydiphenoyl-alpha-D-glucose (iso-1-O-galloyl-3, 6- (R) -O-hexahydroxydiphenoyl-alpha-D-glucose), which is a polyphenol tannic acid compound. The anti-inflammatory, anti-diabetic, anti-proliferative, antioxidant and anti-tumor activities are reported in the literature.

Disclosure of Invention

In order to overcome the problems in the background art, the invention provides a method for extracting and separating isocorilagin from sanguisorba officinalis, which comprises the steps of leaching with ethanol, primarily extracting with petroleum ether to remove impurities, extracting with ethyl acetate to obtain a primary isolate containing a high amount of isocorilagin, extracting effective components from sanguisorba officinalis through HPLC-UV operation control, and separating the isocorilagin from sanguisorba officinalis for the first time, thereby providing a basis for explaining the pharmaceutical principle of the sanguisorba officinalis.

In order to realize the purpose, the invention is realized by the following technical scheme:

the method for extracting and separating isocorilagin from Geranium strictipes comprises the following steps:

(1) Extracting Geranium strictipes dry root powder with ethanol to obtain ethanol extract;

(2) Extracting with petroleum ether, and separating ethanol extract to obtain water phase;

(3) Extracting the water phase after petroleum ether extraction by ethyl acetate for coarse separation to obtain an ethyl acetate extract;

(4) Separating the ethyl acetate extract by HPLC-UV, and preparing to obtain isocorilagin.

Further, the HPLC separation conditions in the step (4) are as follows: phenomenexC ₁₈ Chromatography column (250 mm. Times.20mm, 5 μm); the mobile phase is 0.5% of AAqueous acid solution (A) -methanol (B), gradient elution (0-10min, A90% → 82%, 10-40min, A82% → 75%, 40-90min, A75% → 50%, 90-100min, A50% → 0%); the volume flow is 8mL/min; the column temperature is 30 ℃; the detection wavelength is 254nm; the sample injection amount is 500 mu L; elution time 120min.

Further, the preparation method of the ethanol extract in the step (1) comprises the following steps: pulverizing dried root of Geranium strictipes, extracting with 55% -95% ethanol at 25-45 deg.C for 2-4 times at a material-liquid ratio of 1-18-1; the step (2) of extracting the ethanol extract by using petroleum ether refers to the following steps: adding deionized water into the ethanol extract according to the mass ratio of 1.

Further, the preparation method of the ethanol extract in the step (1) comprises the following steps: pulverizing dried root of Geranium strictipes, extracting with 95% ethanol at 45 deg.C for 4 times at a ratio of 1.

Further, in the step (4), before HPLC-UV separation and preparation, drying the ethyl acetate extract, adding chromatographic methanol into the dried powder, dissolving for 30min by ultrasonic oscillation, and then centrifuging and precipitating at 4000r/min to remove insoluble impurities; filtering each supernatant with 0.45 μm organic solvent microporous membrane, and loading into liquid-phase sample bottle for sample introduction and separation.

The invention has the beneficial effects that:

the method obtains the Geranium strictipes extract by optimizing the extraction process, and separates the isocorilagin from the Geranium strictipes extract for the first time by removing impurities and using an HPLC-UV separation technology; the invention extracts the effective components in the Geranium strictipes to the maximum extent by optimizing extraction data, which has great significance for pharmacological research of the Geranium strictipes.

Drawings

FIG. 1 is a chromatogram of an ethanol extract of the present invention;

FIG. 2 is a chromatogram peak of the ethyl acetate extract of example 1 of the present invention;

FIG. 3 is a chromatogram peak of the ethyl acetate extract of example 1 of the present invention;

FIG. 4 is a hydrogen spectrum (1H-NMR) chart of isocorylazin isolated in example 1 of the present invention;

FIG. 5 is a graph of carbon spectrum (13C-NMR) of isocorylazin isolated in example 1 of the present invention;

FIG. 6 is an ethyl acetate extract chromatogram of example 2 of the present invention;

fig. 7 is a comparison of fig. 2 and 6.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding of the skilled person.

(1) Extracting Geranium strictipes with ethanol to obtain ethanol extract; the main function of the step is to extract the effective components from the garden burnet root. The extraction process comprises the following steps: pulverizing dried root of Geranium strictipes, extracting with ethanol, filtering to remove insoluble impurities, vacuum distilling the clear liquid, concentrating to obtain ethanol extract, and recovering ethanol from the extract during vacuum distillation and concentration.

(2) Adding deionized water into the ethanol extract according to the mass ratio of 1. Extracting the ethanol extract with petroleum ether, and extracting the interfering impurities to obtain petroleum ether water phase.

(3) Extracting the water phase after petroleum ether extraction with ethyl acetate to obtain an ethyl acetate extract; according to the principle of 'similar solubility' of two substances, namely, different solutes of chemical components in the garden burnet are replaced from one solvent to the other solvent according to the polarity condition by utilizing the difference of the solubility or the partition coefficient of the solutes in two solvents which are not soluble (or slightly soluble) mutually; because our target substance belongs to a substance with moderate polarity, most of small polar impurities can be removed after petroleum ether extraction in the early stage, so that the viscosity of a subsequent ethyl acetate extract is reduced, the subsequent ethyl acetate extract is more easily dissolved in methanol, and more target substances are promoted to be enriched in ethyl acetate with moderate polarity because of the reduction of the impurities.

(4) Separating the ethyl acetate extract by using HPLC to obtain isocorylixin, wherein the HPLC separation conditions are as follows: phenomenexC ₁₈ Chromatography column (250 mm. Times.10mm, 5 μm); the mobile phase was methanol (a) -0.5% formic acid aqueous solution (B), gradient elution (0-10min, 10% → 18% a, 10-40min, 18% → 25% a, 40-90min, 25% → 50% a, 50% → 100% a; the volume flow is 8.0mL/min; the column temperature is 30 ℃; the detection wavelength is 254nm; the sample injection amount is 500 mu L; the elution time was 120min.

The preparation method of the ethanol extract in the step (1) comprises the following steps: pulverizing dried root of Geranium strictipes into 40 mesh, extracting with 55% -95% ethanol at 25-45 deg.C for 2-4 times at a material-liquid ratio of 1.

In the step (4), the ethyl acetate extract is dried before HPLC separation, and if the ethyl acetate extract is not dried, substances remained in the extract during separation can influence the separation effect, even the isocorylixin cannot be separated, and the inventor proves that the influence can be eliminated by drying through a large number of experiments. Adding chromatographic methanol into the dry powder, dissolving for 30min by ultrasonic oscillation, and centrifuging at 4000r/min for precipitation to remove insoluble impurities; filtering each supernatant with 0.22 μm microporous membrane for sterilization, and loading into liquid-phase sample bottle for sample introduction and separation.

Example 1

Geranium strictipes medicinal material is dug in Shangrila, di Qing, yunnan province, and the dug Geranium strictipes root is cleaned, aired, dried to constant weight, crushed to 40 meshes, and then subpackaged in plastic self-sealing bags for later use.

Taking Geranium strictipes dry powder, extracting with 95% ethanol at a material-liquid ratio of 1.

Accurately weighing 100g of geranium strictipes ethanol extract by using a high-precision electronic counting balance, adding deionized water according to a material-liquid ratio of 1 (g: mL) to dilute the ethanol extract, and pouring the diluted ethanol extract into a 5000mL separating funnel; then adding petroleum ether into the mixture according to the liquid-liquid ratio of 1:2 (mL: mL) for extraction for 4-5 times, and drying to obtain 2g of petroleum ether phase extract. And (3) extracting the residual water phase by using petroleum ether, adding ethyl acetate into the mixture according to the liquid-liquid ratio of 1 (mL: mL) to extract for 4-5 times, combining the extracts obtained after 4 times of extraction, distilling and concentrating the mixture at 60 ℃ under reduced pressure to obtain a paste, and drying the paste to obtain 45g of ethyl acetate phase extract. The chromatographic peak patterns of the ethyl acetate phase extract are shown in figure 2 and figure 3. FIG. 2 is a chromatogram of a single extract, and FIG. 3 is a chromatogram of a mixture of multiple extracts. Fig. 3 is a more disordered view than fig. 2, mainly along with the increase of the amount of the ethyl acetate phase extract, although the peak shape of the target substance is more prominent, the impurities are also enriched to a certain extent, but far less than the enrichment degree of the target substance. It can be seen from figure 3 that there are mainly 1, 2, 3, 4, 5, five major fractions.

2g of ethyl acetate phase extract is taken and filled in a 50mL centrifuge tube, then 20mL of chromatographic grade methanol is added, and the mixture is subjected to ultrasonic treatment for 30min and then centrifuged at 4000r/min for 10min; 2mL of each supernatant is taken, filtered by a 0.45 mu m organic solvent microporous filter membrane and then filled in a liquid phase sample injection bottle for sample injection analysis; the chromatographic analysis conditions were: phenomenexC ₁₈ A chromatography column, (150 mm. Times.4.6 mm,5 μm); the mobile phase is 0.5% formic acid water solution (A) -methanol (B), and gradient elution is carried out (0-10min, A90% → 82%, 10-40min, A82% → 75%, 40-90min, A75% → 50%, 90-100min, A50% → 0%); the volume flow is 0.75mL/min; the column temperature is 30 ℃; the detection wavelength is 254nm; the sample injection amount is 10 mu L; the elution time was 120min.

The main fraction 5 is separated from Geranium strictipes Maxim by the following scheme 3, wherein the 3 is obtained by first separating Geranium strictipes Maxim, the hydrogen spectrum thereof is shown in the following diagram 4, the carbon spectrum thereof is shown in the following diagram 5, and the structural formula of the 3 substance is shown by detection:

substance No. 5 was detected as gallic acid.

Separation and preparation of main monomer components in geranium strictipes ethyl acetate extract by high performance liquid chromatography-ultraviolet spectroscopy detection method (HPLC-UV)

Separating the components in the extract by HPLC, wherein the separation method comprises the following steps:

the preparation chromatographic conditions are as follows: phenomenexC18 column (250 mm. Times.10mm, 5 μm); the mobile phase was methanol (a) -0.5% formic acid aqueous solution (B), gradient elution (0-10min, 10% → 18% a, 10-40min, 18% → 25% a, 40-90min, 25% → 50% a, 50% → 100% a; the volume flow is 3.0mL/min; the column temperature is 30 ℃; the detection wavelength is 254nm. The sample injection amount is 500 mu L; elution time 120min.

Regression analysis of chromatographic conditions: phenomenexC ₁₈ Chromatography column (250 mm. Times.4.6 mm,5 μm); the mobile phase is methanol (A) -0.5% formic acid aqueous solution (B), and the gradient elution is carried out (0-20min, A8% → 40%, 20-30min, A40% → 100%); the volume flow is 0.75ml/min; the column temperature is 30 ℃; the detection wavelength is 254nm; the sample injection amount is 10 mu L; the elution time was 50min.

Selecting 5 peaks according to the size, the uniformity and the appearance time of the peaks for sample inoculation, respectively combining and concentrating each inoculated sample, and drying to constant weight.

And (3) detecting the separated materials: 2.2g of gallic acid and 1.7g of isocorilagin can be separated from 100g of Geranium strictipes, and finally the purity of the monomer is calculated by an area normalization method through integral processing of a chromatographic peak diagram.

Actual purity of monomer component = measured monomer component content actual purity of reference standard drug of monomer component/measured drug content of reference standard of monomer component.

The purity of the separated isocorylixin is analyzed as follows: = 74.32%/98%/79.86% =91.20%.

Example 2

Accurately weighing 100g of geranium strictipes ethanol extract by using a high-precision electronic counting balance, adding deionized water according to a material-liquid ratio of 1 (g: mL) to dilute the ethanol extract, and pouring the diluted ethanol extract into a separating funnel with the volume of 5000 mL; extracting with ethyl acetate; obtaining ethyl acetate extract, distilling the ethyl acetate extract under reduced pressure, concentrating to paste, drying to constant weight to obtain 47g of ethyl acetate extract, wherein the viscosity of the obtained ethyl acetate extract is obviously greater than that of example 1, and the chromatogram thereof is shown in FIG. 6. From the comparison of the chromatographic peak diagrams, compared with the chromatographic peak diagram shown in FIG. 6, the peaks in the base diagram shown in FIG. 2 are smaller, fewer and more stable, and the target peaks are larger, higher and more broad, which shows that the petroleum ether extraction can eliminate a large amount of impurities.

Sample introduction detection: 1mL of chromatographic grade methanol ethyl acetate extract liquid medicine is filled in a 1.5mL liquid phase sample injection bottle and is placed in a liquid chromatograph for sample injection detection; then comparing and analyzing chromatographic peak diagrams of chemical components in the total extract and the ethyl acetate extract, and estimating the content; selecting the single and complete chromatographic peak with peak time over 1min in the most extracted ethyl acetate as the main monomer component to be prepared.

the preparation chromatographic conditions are as follows: phenomenexC18 column (250 mm. Times.10mm, 5 μm); the mobile phase was methanol (a) -0.5% formic acid aqueous solution (B), gradient elution (0-10min, 10% → 18% a, 10-40min, 18% → 25% a, 40-90min, 25% → 50% a, 50% → 100% a; the volume flow is 3.0mL/min; the column temperature is 30 ℃; the detection wavelength is 254nm. The sample injection amount is 500 mu L; the elution time was 120min.

Regression analysis chromatographic conditions: phenomenexC ₁₈ Chromatography column (250 mm. Times.4.6 mm,5 μm); the mobile phase is methanol (A) -0.5% formic acid aqueous solution (B), and the gradient elution is carried out (0-20min, A8% → 40%, 20-30min, A40% → 100%); the volume flow is 0.75mL/min; the column temperature is 30 ℃; the detection wavelength is 254nm; the sample injection amount is 10 mu L; the elution time was 50min.

The purity of the isolated iso-corilagin is analyzed as follows: = 74.32%/98%/79.86% =91.20%.

This example is mainly used to demonstrate that without petroleum ether extraction for decontamination, the present scheme is more difficult to prepare and decontaminate the target and more time consuming for the separation process. Firstly, the increase in viscosity affects the service life and stability of the instrument; secondly, the increase of impurities can affect the preparation efficiency of equipment; finally, the amount of methanol which can be dissolved by 1mL originally needs a volume which is more than 1mL due to the increase of impurities and viscosity, but the sample volume of the device is fixed, and only the sample introduction times can be increased along with the increase of the volume of the liquid medicine, thereby being more time-consuming. The whole process has little influence on the preparation rate and the purity of the target product.

Because the impurity (and viscosity) is increased, the solubility (distribution coefficient) is reduced, and more methanol is needed for dissolving the same amount of ethyl acetate extract, so that the sample injection volume is increased, but because the sample injection amount of the device is a fixed value, and the time of each elution is 120min, the sample injection times can be increased when the target object with the same sample amount is prepared, so that the preparation time is greatly increased, and the experimental process is more time-consuming.

The calculations illustrate with example 1 and example 2: the extraction rate of the ethyl acetate extract in example 1 is 45%, in this experiment, 100mg/mL of drug concentration, 8 mL/time of sample injection amount and 120min of elution time are used for sample injection separation to prepare a target object, namely 45g of ethyl acetate extract can be obtained from 100g of geranium strictipes crude drug powder, 450mL of methanol is needed to be added to dissolve the ethyl acetate extract to prepare 100mg/mL of liquid medicine, 56.25 times of sample injection amount is needed for 8 mL/time of sample injection amount, and 6750min is needed for 120 min/time of elution; the extraction rate of the ethyl acetate extract of example 2 was 47%, and it took 6900min and more 150min, similarly calculated.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. The method for extracting and separating isocorilagin from Geranium strictipes is characterized by comprising the following steps: the method comprises the following steps:

(1) Pulverizing dried root of Geranium strictipes, extracting with 95% ethanol at a ratio of material to liquid of 1;

(2) Diluting the ethanol extract with deionized water, and extracting with petroleum ether;

(4) Separating and preparing the ethyl acetate extract by using HPLC-UV to obtain isocorilagin; the HPLC-UV separation conditions are as follows: "PhenomenexC ₁₈ Chromatographic column, specification: 250mm is multiplied by 20mm,5 mu m; the mobile phase is 0.5% formic acid water solution (A) -methanol (B), "gradient elution is 0-10 min, A90% → 82%; 10-40 min, A82% → 75%; 40-90 min, A75% → 50%; 90-100 min, A50% → 0% "; the volume flow is 8mL/min; the column temperature is 30 ℃; the detection wavelength is 254nm; the sample injection amount is 500 mu L; elution time 120min.

2. The method for extracting and separating isocorilagin from Geranium strictipes as claimed in claim 1, wherein the extraction is carried out after the dried root of Geranium strictipes is crushed to 40 mesh in step (1).