Gynostemma pentaphylla extract as well as preparation method and application thereof

Abstract

The invention relates to a preparation method of a gynostemma pentaphyllum extract, which combines ultrasonic extraction and microwave extraction technologies, and cavitating effect, mechanical effect and heat effect generated by ultrasonic and rapid heating characteristic of microwave can synergistically and effectively destroy plant cell walls of gynostemma pentaphyllum, so that the gynostemma pentaphyllum extract is obtained. The effective components in the cells are easier to dissolve out, so that the extraction time is shortened, the content of the effective components is higher, and the extraction efficiency is improved; according to the preparation method disclosed by the invention, extraction is carried out under the low-temperature condition of 0-4 DEG C, and degradation and oxidation reactions of saponin and flavonoid temperature-sensitive active ingredients caused by high temperature can be greatly reduced, so that the impurity content in the extract is reduced, the purity of the effective ingredients is higher, and the oxidation resistance is stronger; and finally, the preparation method of the gynostemma pentaphylla extract is simple to operate, high in economic benefit and beneficial to popularization.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a gynostemma pentaphyllum extract and a preparation method and application thereof. Background Art

[0002] Gynostemma pentaphyllum is a perennial herbaceous climbing plant belonging to the Cucurbitaceae family. Gynostemma pentaphyllum contains a wide variety of active ingredients, including gypenosides, gypenosides (polysaccharides), water-soluble amino acids, flavonoids, vitamins (such as vitamin C and vitamin K), trace elements, and minerals (such as potassium, magnesium, and calcium) that are beneficial to the human body. Among them, gypenosides are the main active ingredients.

[0003] However, at present, there are many problems in the method of extracting the effective components of Gynostemma pentaphyllum. First, the efficiency of traditional water extraction is low, and it is difficult to fully extract the effective components in Gynostemma pentaphyllum. Secondly, during the high-temperature extraction process, the active components in Gynostemma pentaphyllum, such as total saponins and flavonoids, are easily oxidized, resulting in reduced biological activity. In addition, during the extraction, there are many impurities in the extract, and the purification process is complicated and costly, making it difficult to obtain a high-purity Gynostemma pentaphyllum extract. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a Gynostemma pentaphyllum extract and a preparation method and application thereof.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A preparation method of a gynostemma pentaphyllum extract comprises the following extraction steps: mixing gynostemma pentaphyllum powder with an extraction solvent to obtain a mixed solution, subjecting the mixed solution to ultrasonic treatment, filtration and microwave treatment to obtain a preliminary extract, cooling the preliminary extract, separating it through column chromatography, and vacuum concentrating and drying to obtain the gynostemma pentaphyllum extract.

[0007] Furthermore, the particle size of the Gynostemma pentaphyllum powder is 100-300 meshes.

[0008] Furthermore, the extraction solvent is 50%-70% ethanol solution.

[0009] Furthermore, the ratio of the Gynostemma pentaphyllum powder to the extraction solvent is 1g:(10-20)ml.

[0010] Furthermore, the ultrasonic process parameters are: ultrasonic treatment at a power of 150-250W for 20-40min, and a temperature of 0-4°C during ultrasonic treatment.

[0011] Furthermore, the microwave process parameters are: microwave treatment at a power of 400-500W for 3-5min, and a temperature during microwave treatment of ≤40°C.

[0012] Furthermore, the filtration is vacuum filtration.

[0013] Furthermore, the cooling temperature of the preliminary extract is cooling to room temperature.

[0014] The Jiaogulan extract is prepared according to the above preparation method.

[0015] The invention discloses an application of the gynostemma pentaphyllum extract in preparing anti-inflammatory and antioxidant drugs.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The present invention relates to a method for preparing a gynostemma pentaphyllum extract. The method of the present invention combines ultrasonic extraction and microwave extraction technology. The cavitation effect, mechanical effect and thermal effect generated by ultrasound and the rapid heating characteristics of microwaves can synergistically destroy the cell walls of gynostemma pentaphyllum plants more effectively, making the effective ingredients in the cells easier to dissolve, which not only shortens the extraction time, but also increases the content of the effective ingredient extract and improves the extraction efficiency.

[0018] 2. The present invention relates to a method for preparing a Gynostemma pentaphyllum extract. The method of the present invention extracts under low temperature conditions of 0-4°C, which can effectively reduce the degradation and oxidation reactions of saponins and flavonoid temperature-sensitive active ingredients caused by high temperature, thereby reducing the impurity content in the extract, making the extract more pure and having stronger antioxidant capacity.

[0019] 3. The preparation method of the Gynostemma pentaphyllum extract of the present invention is simple to operate, has high economic benefits, and is conducive to promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation of the present invention, the following will briefly introduce the drawings required for use in the specific implementation. In all the drawings, each element or part is not necessarily drawn according to the actual scale.

[0021] Figure 1 HPLC chromatogram of the Gynostemma pentaphyllum extract extracted in Example 2 of the present invention;

[0022] Figure 2 The HPLC chromatogram of the Gynostemma pentaphyllum extract extracted in Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0023] Source of reagents:

[0024] Gynostemma pentaphyllum raw material: purchased from Bozhou Boyuantong Chinese Medicinal Materials Planting Co., Ltd.

[0025] Example 1 Example 1 is a method for preparing a Gynostemma pentaphyllum extract

[0026] 1) crushing the raw material of Gynostemma pentaphyllum to a particle size of 100 mesh to obtain Gynostemma pentaphyllum powder;

[0027] 2) mixing the Gynostemma pentaphyllum powder with a 50% ethanol solution at a solid-liquid ratio of 1 g:10 ml to obtain a mixed solution;

[0028] 3) Under low temperature conditions of 0°C, using an ultrasonic generator to perform ultrasonic treatment on the mixture A, the ultrasonic power is set to 150 W, and the treatment time is 30 min;

[0029] 4) After the ultrasound is finished, the solid residue is removed by a vacuum filtration system to obtain a preliminary extract;

[0030] 5) placing the preliminary extract obtained in step 4) in a microwave heating device, setting the microwave power to 400 W, and subjecting the extract to microwave treatment for 4 minutes. During heating, the temperature of the extract is monitored in real time by a built-in temperature sensor to ensure that the temperature is at 40° C.;

[0031] 6) After heating, the extract is allowed to cool naturally to 25°C to stabilize the components in the extract;

[0032] 7) The cooled extract was subjected to high performance liquid chromatography, sephadex (cross-linked dextran gel column) was selected as the filling material for column chromatography, the mobile phase was methanol and water, the elution conditions were 10% methanol from 0 min to 10 min, 90% methanol from 10 min to 30 min, the flow rate was 0.7 mL / min, the column temperature was 30° C., the collected components were concentrated under reduced pressure under vacuum conditions (pressure was 80 mbar), the temperature was controlled at 40° C. to avoid high temperature damage to heat-sensitive components, after the concentration was completed, the concentrate was placed in a vacuum drying oven, the temperature was set to 30° C., and the drying time was 8 hours, and finally high-purity total saponins, flavonoids and polysaccharides of Gynostemma pentaphyllum were obtained.

[0033] Example 2 This example is a method for preparing a Gynostemma pentaphyllum extract

[0034] 1) crushing the raw material of Gynostemma pentaphyllum to a particle size of 200 mesh to obtain Gynostemma pentaphyllum powder;

[0035] 2) mixing the Gynostemma pentaphyllum powder with a 60% ethanol solution at a solid-liquid ratio of 1 g:10 ml to obtain a mixed solution;

[0036] 3) Under low temperature conditions of 0°C, using an ultrasonic generator to perform ultrasonic treatment on the mixture A, the ultrasonic power is set to 200 W, and the treatment time is 30 min;

[0037] 4) After the ultrasound is finished, the solid residue is removed by a vacuum filtration system to obtain a preliminary extract;

[0038] 5) placing the preliminary extract obtained in step 4) in a microwave heating device, setting the microwave power to 500 W, and subjecting the extract to microwave treatment for 5 minutes. During heating, the temperature of the extract is monitored in real time by a built-in temperature sensor to ensure that the temperature is at 40° C.;

[0039] 6) After heating, the extract is allowed to cool naturally to 25°C to stabilize the components in the extract;

[0040] 7) The cooled extract was subjected to high performance liquid chromatography, and sephadex LH-20 (cross-linked dextran gel column) was selected as the filling material for column chromatography. The mobile phase was methanol and water, and the elution condition was 10% methanol from 0 min to 10 min. In the initial stage, low-polarity solvents were mainly used to elute impurities with strong polarity; from 10 min to 30 min, the linear gradient increased from 10% methanol to 90% methanol, which was used to gradually elute total saponins, flavonoids, polysaccharides and other target compounds; from 30 min to 40 min, 90% methanol was maintained for isocratic elution to ensure complete separation of highly hydrophobic components; the flow rate was 0.7 mL / min, the column temperature was 40° C., and the collected components were concentrated under vacuum decompression conditions (pressure 80 mbar, temperature 40° C.), and then thoroughly dried in a vacuum drying oven (30° C., drying time 8 hours) to obtain high-purity total saponins, flavonoids and polysaccharides of Gynostemma pentaphyllum.

[0041] The present invention takes Example 2 as an example, and the HPLC chromatogram of the prepared Gynostemma pentaphyllum extract is as follows: Figure 1 As shown in the figure, it can be seen that the target substance mainly has the following peaks:

[0042] Solvent peak: Peak 0.

[0043] The total saponin peaks of Gynostemma pentaphyllum, peak 1: Gynostemma pentaphyllum saponin IV peak; peak 2: Gynostemma pentaphyllum saponin VIII peak; peak 3: Gynostemma pentaphyllum saponin III peak, peak 6: Gynostemma pentaphyllum saponin II peak; the retention time of the total saponin peaks is 15-18 and 20-22 minutes, the peak area is large, and the peak shape is symmetrical, indicating that the total saponins have a higher purity under the elution conditions.

[0044] Flavonoid peak, peak 4, flavonoid quercetin, peak 5: flavonoid rutin; the retention time of the flavonoid peak is 18-20 minutes, and the peak width is narrower, indicating that the elution efficiency of this component is high.

[0045] Polysaccharide peak, peak 7: polysaccharide Gynostemma pentaphyllum acidic polysaccharide (CPS-1); the retention time of the polysaccharide peak is 26-28 minutes, and the peak shape is slightly elongated, which may be related to the wider molecular weight distribution of the polysaccharide.

[0046] Impurity peak analysis: Several smaller peaks can be observed between 0-10 minutes and 28-30 minutes. These peaks correspond to the polar impurities and hydrophobic impurities remaining in the elution process, indicating that the column chromatography has a good separation effect.

[0047] Conclusion: It can be seen intuitively from the chromatogram that the method described in the present invention can effectively separate the main active ingredients (total saponins, flavonoids, polysaccharides) in the Gynostemma pentaphyllum extract, the retention time of each target substance is clear, the peak shape is good, there is little interference from impurities, the purity and separation efficiency of the extract are high, and a reliable material basis is provided for subsequent functional verification.

[0048] Embodiment 3 This embodiment is a method for preparing a Gynostemma pentaphyllum extract

[0049] 1) crushing the raw material of Gynostemma pentaphyllum to a particle size of 300 mesh to obtain Gynostemma pentaphyllum powder;

[0050] 2) mixing the Gynostemma pentaphyllum powder with a 70% ethanol solution at a solid-liquid ratio of 1 g:20 ml to obtain a mixed solution;

[0051] 3) Under low temperature conditions of 4° C., using an ultrasonic generator to perform ultrasonic treatment on the mixture A, the ultrasonic power was set to 250 W, and the treatment time was 35 minutes;

[0052] 4) After the ultrasound is finished, the solid residue is removed by a vacuum filtration system to obtain a preliminary extract;

[0053] 5) placing the preliminary extract obtained in step 4) in a microwave heating device, setting the microwave power to 400 W, and subjecting the extract to microwave treatment for 5 minutes. During heating, the temperature of the extract is monitored in real time by a built-in temperature sensor to ensure that the temperature is at 40° C.;

[0054] 6) After heating, the extract is allowed to cool naturally to 25°C to stabilize the components in the extract;

[0055] 7) The cooled extract was subjected to high performance liquid chromatography, sephadex (cross-linked dextran gel column) was selected as the filling material for column chromatography, the mobile phase was methanol and water, the elution conditions were 10% methanol from 0 min to 10 min, a linear gradient increased from 10% methanol to 90% methanol from 10 min to 30 min, and 90% methanol was maintained for isocratic elution from 30 min to 40 min, the flow rate was 0.7 mL / min, the column temperature was 40° C., and the collected components were concentrated under vacuum decompression conditions (pressure 80 mbar, temperature at 40° C.), and then thoroughly dried in a vacuum drying oven (30° C., drying time for 8 hours) to obtain high-purity total saponins, flavonoids and polysaccharides of Gynostemma pentaphyllum.

[0056] Comparative Example 1

[0057] The preparation method of the Gynostemma pentaphyllum extract provided in this comparative example is basically the same as the preparation method of Example 2, except that the ultrasound is replaced with mechanical stirring at 800 r / min for 30 min, specifically:

[0058] 1) crushing the raw material of Gynostemma pentaphyllum to a particle size of 200 mesh to obtain Gynostemma pentaphyllum powder;

[0059] 2) mixing the Gynostemma pentaphyllum powder with a 60% ethanol solution at a solid-liquid ratio of 1 g:10 ml to obtain a mixed solution;

[0060] 3) Under low temperature conditions of 0°C, the mixture A was mechanically stirred at 800 r / min for 30 min;

[0061] 4) After the mechanical stirring is completed, the solid residue is removed by a vacuum filtration system to obtain a preliminary extract;

[0062] 5) placing the preliminary extract obtained in step 4) in a microwave heating device, setting the microwave power to 500 W, and subjecting the extract to microwave treatment for 5 minutes. During heating, the temperature of the extract is monitored in real time by a built-in temperature sensor to ensure that the temperature is at 40° C.;

[0063] 6) After heating, the extract is allowed to cool naturally to 25°C to stabilize the components in the extract;

[0064] 7) The cooled extract was subjected to high performance liquid chromatography, and sephadex LH-20 (cross-linked dextran gel column) was selected as the filling material for column chromatography. The mobile phase was methanol and water, and the elution condition was 10% methanol from 0 min to 10 min. In the initial stage, low-polarity solvents were mainly used to elute impurities with strong polarity; from 10 min to 30 min, the linear gradient increased from 10% methanol to 90% methanol, which was used to gradually elute total saponins, flavonoids, polysaccharides and other target compounds; from 30 min to 40 min, 90% methanol was maintained for isocratic elution to ensure complete separation of highly hydrophobic components; the flow rate was 0.7 mL / min, the column temperature was 40° C., and the collected components were concentrated under vacuum decompression conditions (pressure 80 mbar, temperature 40° C.), and then thoroughly dried in a vacuum drying oven (30° C., drying time 8 hours) to obtain high-purity total saponins, flavonoids and polysaccharides of Gynostemma pentaphyllum.

[0065] Comparative Example 2

[0066] The preparation method of the Gynostemma pentaphyllum extract provided in this comparative example is basically the same as the preparation method of Example 2, except that microwave extraction is replaced by ordinary heating at a temperature of 40° C., specifically:

[0067] 1) crushing the raw material of Gynostemma pentaphyllum to a particle size of 200 mesh to obtain Gynostemma pentaphyllum powder;

[0068] 2) mixing the Gynostemma pentaphyllum powder with a 60% ethanol solution at a solid-liquid ratio of 1 g:10 ml to obtain a mixed solution;

[0069] 3) Under low temperature conditions of 0° C., using an ultrasonic generator to perform ultrasonic treatment on the mixture A, the ultrasonic power is set to 200 W, and the treatment time is 30 minutes;

[0070] 4) After the ultrasound is finished, the solid residue is removed by a vacuum filtration system to obtain a preliminary extract;

[0071] 5) heating the preliminary extract obtained in step 4) at 40° C. for 5 minutes;

[0072] 6) After heating, the extract is allowed to cool naturally to 25°C to stabilize the components in the extract;

[0073] 7) The cooled extract was subjected to high performance liquid chromatography, and sephadex LH-20 (cross-linked dextran gel column) was selected as the filling material for column chromatography. The mobile phase was methanol and water, and the elution condition was 10% methanol from 0 min to 10 min. In the initial stage, low-polarity solvents were mainly used to elute impurities with strong polarity; from 10 min to 30 min, the linear gradient increased from 10% methanol to 90% methanol, which was used to gradually elute total saponins, flavonoids, polysaccharides and other target compounds; from 30 min to 40 min, 90% methanol was maintained for isocratic elution to ensure complete separation of highly hydrophobic components; the flow rate was 0.7 mL / min, the column temperature was 40° C., and the collected components were concentrated under vacuum decompression conditions (pressure 80 mbar, temperature at 40° C.), and then thoroughly dried in a vacuum drying oven (30° C., drying time for 8 hours) to obtain high-purity total saponins, flavonoids and polysaccharides of Gynostemma pentaphyllum.

[0074] Comparative Example 3

[0075] The preparation method of the Gynostemma pentaphyllum extract provided in this comparative example is basically the same as the preparation method of Example 2, except that the extraction solvent of Gynostemma pentaphyllum is water, and the extraction is carried out by mechanical stirring at high temperature, specifically:

[0076] 1) crushing the raw material of Gynostemma pentaphyllum to a particle size of 200 mesh to obtain Gynostemma pentaphyllum powder;

[0077] 2) mixing the Gynostemma pentaphyllum powder and deionized water at a solid-liquid ratio of 1 g:10 ml to obtain a mixed solution;

[0078] 3) Mixture A was mechanically stirred at 800 r / min for 30 min at 40°C;

[0079] 4) After stirring, the solid residue is removed by a vacuum filtration system to obtain a preliminary extract;

[0080] 5) Cooling the preliminary extract naturally to 25°C to stabilize the components in the extract;

[0081] 7) The cooled extract was subjected to high performance liquid chromatography, and sephadex LH-20 (cross-linked dextran gel column) was selected as the filling material for column chromatography. The mobile phase was methanol and water, and the elution condition was 10% methanol from 0 min to 10 min. In the initial stage, low-polarity solvents were mainly used to elute impurities with strong polarity; from 10 min to 30 min, the linear gradient increased from 10% methanol to 90% methanol, which was used to gradually elute total saponins, flavonoids, polysaccharides and other target compounds; from 30 min to 40 min, 90% methanol was maintained for isocratic elution to ensure complete separation of highly hydrophobic components; the flow rate was 0.7 mL / min, the column temperature was 40° C., and the collected components were concentrated under vacuum decompression conditions (pressure 80 mbar, temperature at 40° C.), and then thoroughly dried in a vacuum drying oven (30° C., drying time for 8 hours) to obtain high-purity total saponins, flavonoids and polysaccharides of Gynostemma pentaphyllum.

[0082] The present invention takes comparative example 3 as an example, and the HPLC chromatogram of the prepared Gynostemma pentaphyllum extract is as follows Figure 2 As shown in the figure, it can be seen that the target substance mainly has the following peaks:

[0083] Solvent peak: Peak 0.

[0084] Total saponin peaks, Peak 1: Gynostemma saponin IV peak; Peak 2: Gynostemma saponin VIII peak; Peak 3: Gynostemma saponin III peak, Peak 6: Gynostemma saponin II peak; Peaks 1 and 2 are obviously low in height, and there is a problem of low extraction yield.

[0085] Flavonoid peak, peak 4, flavonoid quercetin, peak 5: flavonoid rutin; the flavonoid peak has obvious impurities and low extraction content.

[0086] The polysaccharide Gynostemma pentaphyllum was not successfully extracted.

[0087] Test example: Antioxidant capacity test DPPH free radical scavenging experiment

[0088] 1.1 Prepare Gynostemma pentaphyllum extract (mixed extract of total saponins, flavonoids and polysaccharides), DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical solution (0.1 mM, dissolved in anhydrous ethanol); anhydrous ethanol; spectrophotometer; micropipette and EP tube.

[0089] 1.2 Prepare sample solution: accurately weigh the Gynostemma pentaphyllum extract and prepare a series of ethanol solutions with concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL; prepare 0.1 mM DPPH solution and ensure that it is freshly prepared on the day of use.

[0090] 1.3 Mixing reaction: Add 2mL DPPH solution and 2mL sample solution of different concentrations to the EP tube and mix thoroughly; set up a blank control (add 2mL ethanol to replace the sample solution); set up a negative control (add 2mL sample solution to replace the DPPH solution). Place the mixed solution at room temperature in the dark for 30 minutes to ensure that the DPPH free radicals fully react with the sample.

[0091] 1.4 Use a spectrophotometer to measure the absorbance of the sample solution (A1), the absorbance of the blank control (A0), and the absorbance of the negative control (A2) at a wavelength of 517 nm.

[0092] 1.5 Experimental conditions: reaction temperature: 25°C; reaction time: 30 minutes; measurement wavelength: 517 nm.

[0093] 1.6 The free radical scavenging rate was calculated according to the following formula, and the scavenging rates of the Gynostemma pentaphyllum extracts prepared in Examples 1-3 and Comparative Examples 1-3 were calculated to evaluate their antioxidant properties. The data are shown in Table 1.

[0094]

[0095] Test Case

[0096] The present invention also measures the purity of the Gynostemma pentaphyllum extracts prepared in Examples 1-3 and Comparative Examples 1-3, and the test results are shown in Table 1.

[0097] Table 1 Performance test results of Examples 1-3 and Comparative Examples 1-3

[0098]

[0099] From the results in Table 1, it can be seen that the mass of the Gynostemma pentaphyllum extract extracted by the method of the present invention is 8.5-8.8 g, the purity of the Gynostemma pentaphyllum extract is 78.4-81.5%, and the scavenging rate of the prepared Gynostemma pentaphyllum extract on DPPH free radicals is 70.9-73.6%, which is much higher than the effect of Comparative Examples 1-5. It can be seen from the above data that the effective ingredients extracted by the method of the present invention have a synergistic effect.

[0100] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments. On the basis of the present invention, some modifications or improvements can be made thereto, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for preparing a Gynostemma pentaphyllum extract, characterized in that: The extraction steps are as follows: mixing Gynostemma pentaphyllum powder with an extraction solvent to obtain a mixed solution, subjecting the mixed solution to ultrasonic treatment, filtration and microwave treatment to obtain a preliminary extract, cooling the preliminary extract, separating it through column chromatography, and vacuum concentrating and drying to obtain a Gynostemma pentaphyllum extract.

2. The preparation method according to claim 1, characterized in that: The particle size of the Gynostemma pentaphyllum powder is 100-300 meshes.

3. The preparation method according to claim 2, characterized in that: The extraction solvent is 50%-70% ethanol solution.

4. The preparation method according to claim 3, characterized in that: The ratio of the Gynostemma pentaphyllum powder to the extraction solvent is 1g:(10-20)ml.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The ultrasonic process parameters are: ultrasonic treatment at a power of 150-250W for 20-40min, and a temperature of 0-4°C during ultrasonic treatment.

6. The preparation method according to claim 5, characterized in that: The microwave process parameters are: microwave treatment at a power of 400-500W for 3-5min, and a temperature of ≤40°C during microwave treatment.

7. The preparation method according to claim 6, characterized in that: The filtration is vacuum filtration.

8. The preparation method according to claim 7, characterized in that: The cooling temperature of the preliminary extract is cooling to room temperature.

9. The Gynostemma pentaphyllum extract prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the Gynostemma pentaphyllum extract according to claim 9 in the preparation of anti-inflammatory and antioxidant drugs.

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