CN115304651B

CN115304651B - Method for extracting acteoside from paulownia leaves

Info

Publication number: CN115304651B
Application number: CN202211114147.4A
Authority: CN
Inventors: 魏华; 郭洪伟; 周水生; 向帮清; 李国栋
Original assignee: Xiangxi Natural Biotechnology Co ltd
Current assignee: Xiangxi Natural Biotechnology Co ltd
Filing date: 2022-09-14
Publication date: 2024-06-28
Anticipated expiration: 2042-09-14

Abstract

The invention provides a method for extracting acteoside from paulownia leaves, which comprises the following steps: extracting paulownia leaves to obtain a crude extract; separating and purifying the obtained crude extract by macroporous adsorption resin to obtain acteoside enriched part; and (3) carrying out liquid chromatography separation on the obtained acteoside enrichment part to obtain acteoside enrichment liquid, and concentrating and drying to obtain acteoside. In the liquid chromatographic separation, the chromatographic column filler is any one of octadecyl bonded silica gel, octaalkyl bonded silica gel, tetraalkyl bonded silica gel or phenyl bonded silica gel, and the mobile phase is methanol with the concentration of 20-50 vol% or acetonitrile solution with the concentration of 10-40%. The method extracts the acteoside from the paulownia leaves for the first time, fills the technical blank in the field, and finally obtains the acteoside with the purity of more than 98 percent, and has the advantages of simple process, low economic cost and easy realization of industrialization.

Description

Method for extracting acteoside from paulownia leaves

Technical Field

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a method for extracting acteoside from paulownia leaves.

Background

The Paulownia (Paulownia) plants are 7 species and are produced in China, and the Paulownia plants are distributed, cultivated or wild throughout the country except for northeast, inner Mongolia, northern Xinjiang, tibet and other areas, and some areas are also being introduced. Paulownia as one kind of high quality fast growing wood material is used widely in agricultural production and is one kind of common Chinese medicine material, including flower, leaf, bark, root and fruit of Paulownia. The outline of the materia medica makes detailed records on the pharmacological actions of all parts of paulownia, and modern medical researches also show that the paulownia has the functions of bacteriostasis, anti-inflammation, anti-tumor and even disinsection. The paulownia tree has luxuriant branches and leaves and a plurality of leaves, and is a rich natural resource. In addition, the paulownia leaves can be used as feed, so that not only can the growth of animals be promoted, but also the disease resistance of the animals can be improved, and the paulownia leaves have rich economic and social values.

Acteoside, acteoside or acteoside is a phenethyl alcohol glycoside compound of 4, 5-dihydroxyphenethyl alcohol (hydroxytyrosol) combined with rhamnose C1 beta-D-glucopyranose by C3 glycosidic bond through ester bond and glycosidic bond, its English name is Acteoside, verbascoside or Kusaginin, its molecular formula is C ₂₉H₃₆O₁₅, and molecular weight is 624.59. Phytochemical studies have shown that acteoside is widely found in a variety of dicotyledonous plants of the family Orobanchaceae, scrophulariaceae, magnoliaceae, and Labiatae. Modern pharmacological researches show that the acteoside has remarkable antioxidant, anti-inflammatory, memory-enhancing, nerve-protecting and antitumor activities, and is widely focused by researchers at home and abroad due to wide sources, strong biological activity and small adverse reaction.

With the gradual deep research of the pharmaceutical of the phenylpropanoid glycoside compounds such as the acteoside and the like at home and abroad, the medical value of the acteoside is increasingly valued, and the acteoside and related products thereof are not supplied to the market. Since acteoside is the main active ingredient of cistanche, research in recent years has focused on extraction and separation of acteoside from cistanche. It has also been reported that acteoside can be extracted and separated from other plants such as osmanthus fragrans, callicarpa nudiflora, campsis plant, buddleia, etc. However, the actual needs are hardly met by extracting and preparing acteoside from the plants, and the method for separating and purifying acteoside from the plants often needs to adopt a repeated column chromatography method to obtain high-purity acteoside samples, and has the defects of long time consumption and high solvent consumption. In addition, the biomass of the plants is small, a large amount of plants are needed for extraction and preparation, and the economy is not high.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for extracting acteoside from paulownia leaves. The paulownia leaves are rich in content, the acteoside extracted from the paulownia leaves does not need to be planted in a large amount in advance, the method has good economic value, and the purity of the extracted acteoside is high and can reach more than 98%, so that the actual requirements can be met.

In a first aspect, the present invention provides a method for extracting acteoside from paulownia leaves, comprising the steps of:

(1) Extracting paulownia leaves to obtain a crude extract;

(2) Separating and purifying the crude extract obtained in the step (1) by using macroporous adsorption resin to obtain a acteoside enriched part;

(3) Performing liquid chromatography separation on the acteoside enriched part obtained in the step (2) to obtain acteoside collected liquid, and concentrating and drying to obtain acteoside;

In the liquid chromatographic separation, the chromatographic column filler is any one of octadecyl bonded silica gel, octaalkyl bonded silica gel, tetraalkyl bonded silica gel or phenyl bonded silica gel;

in the liquid chromatographic separation, the mobile phase is 20-50 vol% methanol or 10-40 vol% acetonitrile solution.

Preferably, the step (1) specifically comprises: extracting paulownia leaves with one or more solvents selected from water, methanol or ethanol to obtain crude extract.

Preferably, the solvent is 30 to 50vol% ethanol solution.

Preferably, in the extraction of the paulownia leaves, the ratio of the mass of the paulownia leaves to the volume of the solvent is 1 (20-50) g/mL.

Preferably, the temperature of the extraction in step (1) does not exceed 70 ℃, more preferably 40-70 ℃.

Preferably, the macroporous adsorption resin comprises any one of AB-8 macroporous adsorption resin, DM-301 macroporous adsorption resin, DA-201 macroporous adsorption resin, D-101 macroporous adsorption resin, HPD100 macroporous adsorption resin or DM-130 macroporous adsorption resin, more preferably D-101 macroporous adsorption resin or AB-8 macroporous adsorption resin, and most preferably D-101 macroporous adsorption resin.

Preferably, when the crude extract obtained in the step (1) is separated and purified by using macroporous adsorption resin, the elution procedure is as follows:

Elution procedure

Eluent (eluent)	Elution volume	Eluent flow rate
			Water and its preparation method	2 To 4 column volumes	1-2 Column volumes/hr
10Vol% ethanol	2 To 4 column volumes	1-2 Column volumes/hr
			30Vol% ethanol	2 To 4 column volumes	1-2 Column volumes/hr
50Vol% ethanol	3 To 5 column volumes	1-2 Column volumes/hr

Preferably, in the liquid chromatographic separation, the mobile phase is 30 to 35vol% methanol or 20 to 25vol% acetonitrile solution.

Preferably, in the liquid chromatographic separation, the flow rate of the mobile phase is 2-15mL/min.

Preferably, in the liquid chromatography separation, the detection wavelength is 240 to 370nm.

Compared with the prior art, the invention has the beneficial effects that:

(1) The invention discovers components with very high separation degree and relative content in an HPLC spectrogram of paulownia fortunei leaves, provides a method for extracting the acteoside from the paulownia fortunei leaves on the basis of identifying the acteoside, fills up the technical blank in the field, widens the plant source of the acteoside, and has great economic value;

(2) The method for extracting acteoside provided by the invention takes the paulownia leaves as raw materials, and sequentially carries out crude extraction, macroporous resin adsorption separation and liquid chromatography separation, so that the purity of the obtained acteoside is more than 98 percent.

Drawings

FIG. 1 is a schematic structural diagram of acteoside;

FIG. 2 is an HPLC fingerprint of paulownia tomentosa leaves;

FIG. 3 is a nuclear magnetic resonance H-spectrum of the No. 11 peak compound in the HPLC fingerprint of paulownia fortunei leaves;

FIG. 4 is a nuclear magnetic resonance C-spectrum of the No. 11 peak compound in the HPLC fingerprint of paulownia fortunei leaves;

Fig. 5 is a diagram of the results of different chromatographic column inspections of HPLC finger print of paulownia fortunei leaves;

fig. 6 is a diagram of the results of different solvent system investigation of HPLC fingerprint of paulownia fortunei leaves;

fig. 7 is a diagram of the results of different detection wavelengths of HPLC fingerprint of paulownia fortunei leaves;

fig. 8 is a diagram of different column temperature investigation results of HPLC finger print of paulownia fortunei leaves;

FIG. 9 is a purity detection chromatogram of the acteoside obtained in example 4.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The sources of all experimental raw materials are not particularly limited, and can be purchased from the market or prepared according to conventional preparation methods well known to those skilled in the art.

The invention is based on the mass researches of chromatographic conditions and fingerprint spectrograms of paulownia leaf extract, and components with very high separation degree and relative content are discovered in the fingerprint spectrograms for the first time, and are identified as acteoside (the structural formula is shown in figure 1) after preparation and separation, and the specific process is as follows:

0.5g of paulownia fortunei leaf powder is weighed, placed in a conical flask with a plug, 10mL of 70vol% methanol is precisely added, the mixture is sealed, the mass is weighed, ultrasonic treatment (power 500W, frequency 40 kHz) is carried out for 30min, cooling is carried out, the mass which is reduced is complemented by 70vol% methanol, supernatant fluid is taken and filtered by a microporous filter membrane with the thickness of 0.45 mu m, and the paulownia fortunei leaf sample solution is prepared.

HPLC detection is carried out on the paulownia fortunei leaf sample solution, and the fingerprint spectrum is recorded, and the chromatographic conditions are as follows: chromatographic column: agilent ZORBAX HC-C18 (2) (4.6 mm. Times.250 mm,5 μm); mobile phase: methanol (B) -0.1vol% formic acid buffer (a); detection wavelength: 254nm; flow rate: 1mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; gradient elution, specific elution procedure is shown in the following table:

Gradient elution procedure

The HPLC fingerprint of the paulownia tomentosa leaf sample is shown in figure 2, wherein the spectrum shows 23 characteristic peaks, wherein the No. 11 peak is a common peak, the intensity is higher, the retention time is relatively centered and the separation degree from the rest chromatographic peaks is good, so the No. 11 peak is selected as a reference peak. The compound represented by this peak was rapidly prepared and identified because it was not determined.

Taking a paulownia fortunei leaf sample solution, and carrying out semi-preparative liquid chromatography to obtain a No. 11 peak compound, wherein the chromatographic conditions are as follows: column YMC-Pack Ph (10 mm. Times.250 mm,5 μm); isocratic elution; mobile phase: methanol (B) -water (a) = (33:67, volume ratio). A detection wavelength of 254nm; the flow rate of the mobile phase is 2.5mL/min; column temperature is 30 ℃; t _R = 24.9min. The nuclear magnetic resonance spectrometer is adopted to carry out qualitative analysis on the No. 11 peak compound, the results are shown in fig. 3-4, wherein fig. 3 is a nuclear magnetic resonance H spectrum of the No. 11 peak compound, fig. 4 is a nuclear magnetic resonance C spectrum of the No. 11 peak compound, the data are shown as follows, and the data above ：ESI-MS m/z:647.1940[M+Na]⁺.¹H NMR(600MHz,MeOD)δ:7.59(1H,d,J＝16.2Hz,H-7'),7.05(1H,d,J＝1.8Hz,H-2'),6.95(1H,dd,J＝7.8,1.8Hz,H-6'),6.77(1H,d,J＝7.8Hz,H-5'),6.69(1H,d,J＝2.4Hz,H-2),6.67(1H,d,J＝7.8Hz,H-5),6.56(1H,dd,J＝8.0,1.8Hz,H-6),6.27(1H,d,J＝15.6Hz,H-8'),5.18(1H,d,J＝1.8Hz,H-1″'),4.37(1H,d,J＝7.8Hz,H-1″),4.05(1H,m,H-8β),3.71(1H,m,H-8α),2.79(2H,m,H-7),1.09(3H,d,J＝6.0Hz,H-6″').¹³C-NMR(MeOD,125MHz)δ:166.9(C-9'),148.5(C-4'),146.7(C-7'),145.5(C-3'),144.8(C-3),143.3(C-4),130.0(C-1),126.2(C-1'),121.9(C-6'),119.8(C-6),115.7(C-2),115.1(C-5'),114.9(C-5),113.7(C-8'),113.2(C-2'),102.8(C-1″),101.7(C-1″'),80.3(C-3″),74.8(C-2″),74.6(C-5″),72.4(C-4″'),71.0(C-2″'),70.9(C-3″'),70.6(C-5″'),69.1(C-4″),69.0(C-5″'),60.9(C-6″),35.2(C-7),17.0(C-6″'). are basically consistent with the literature report (Wang XQ,et al.Phenylethaniod glycosides from Orobanchepycnostachya Hance and their chemotaxonomic significance[J].Biochemical Systematics andEcology.),, so that the final nuclear magnetic qualitative identification of the No. 11 peak compound is acteoside.

Based on the above study, the present invention provides a method for extracting acteoside from paulownia leaves, comprising the following steps:

(1) Extracting paulownia leaves to obtain a crude extract;

In the present invention, the paulownia leaves are preferably extracted with one or more of water, methanol or ethanol as a solvent, more preferably with 30 to 50vol% ethanol solution, most preferably with 50vol% ethanol solution, to obtain a crude extract. The means of extraction preferably comprises any one or more of maceration, decoction, percolation, reflux extraction, ultrasonic extraction or supercritical extraction, more preferably comprises ultrasonic extraction and/or reflux extraction, most preferably ultrasonic extraction. In the present invention, the ratio of the mass of paulownia leaves to the volume of the solvent is preferably 1 (20 to 50) g/mL, more preferably 1 (20 to 30) g/mL, and most preferably 1:30g/mL. Since acteoside is easily converted at high temperature, the extraction temperature is preferably not more than 70 ℃, more preferably 40-70 ℃, and the extraction time is preferably 0.5-2.5 hours, more preferably 1-1.5 hours, and most preferably 1 hour.

According to the invention, after the crude extract is obtained, macroporous adsorption resin is adopted to separate and purify the crude extract, thus obtaining the acteoside enriched part. In the present invention, the macroporous adsorbent resin preferably comprises any one of AB-8 macroporous adsorbent resin, DM-301 macroporous adsorbent resin, DA-201 macroporous adsorbent resin, D-101 macroporous adsorbent resin, HPD100 macroporous adsorbent resin or DM-130 macroporous adsorbent resin, more preferably AB-8 macroporous adsorbent resin or D-101 macroporous adsorbent resin, and most preferably D-101 macroporous adsorbent resin. The improvement of the loading mass concentration is beneficial to improving the use efficiency of the resin. However, when the mass concentration of the sample is too high, the contact time between the sample and the resin is shortened under a certain volume flow, and meanwhile, when the mass concentration of the sample solution is too high, the solution is too viscous, viscous adsorption can be formed on the surface of the resin particles, and the adsorption effect is seriously affected. Therefore, the mass concentration of the sample solution is preferably 15 to 25mg/mL, more preferably 25mg/mL.

In the present invention, 50vol% ethanol is more preferable as the eluent. However, since impurities are present in the macroporous adsorbent resin column, it is preferable to remove impurities by using water, 10vol% ethanol, 30vol% ethanol, and then to elute with 50vol% ethanol. The specific elution procedure is preferably as shown in the following table:

Elution procedure

According to the invention, the acteoside enrichment part is subjected to liquid chromatography separation to obtain acteoside enrichment liquid. In the present invention, the liquid chromatography is preferably a preparative liquid chromatography. In the liquid chromatographic separation, the chromatographic column packing is preferably any one of octadecyl bonded silica gel, octaalkyl bonded silica gel, tetraalkyl bonded silica gel or phenyl bonded silica gel. The inner diameter of the column is preferably 10 to 50mm, more preferably 10 to 30mm. The mobile phase is preferably a methanol solution of 20 to 50vol% or an acetonitrile solution of 10 to 40vol%, more preferably a methanol solution of 30 to 40vol% or an acetonitrile solution of 20 to 25vol%, and most preferably a methanol solution of 30 to 35 vol%. The flow rate of the mobile phase is preferably 2 to 15mL/min, more preferably 5 to 15mL/min. In the liquid chromatography, the detection wavelength is preferably 240 to 370nm, more preferably 280 to 370nm.

In a specific embodiment, the conditions of the liquid chromatography are preferably:

chromatographic column: YMC-Pack ODS-A (20 mm. Times.250 mm,5 μm);

mobile phase: methanol (B) -water (a) = (33:67, volume ratio), isocratic elution;

The detection wavelength is 334nm;

the flow rate of the mobile phase is 10mL/min;

column temperature is 30 ℃;

tR＝25.0min；

Sample injection amount: 20. Mu.L.

According to the present invention, after obtaining the collection liquid of acteoside, concentration and drying are carried out to obtain the acteoside solid. In the present invention, the form of concentration is not particularly limited, and vacuum concentration is preferably performed at a temperature of not more than 70 ℃. The concentration is preferably finished when the solid-liquid mass volume ratio is 1 (4-5) g/mL. The present invention is not particularly limited in terms of the dried form, and preferably any one or more of freeze-drying, spray-drying or microwave-drying is used to finally obtain the acteoside solid.

The invention takes paulownia leaves as raw materials, the mass fraction of acteoside in the crude extract is not less than 5% (up to 11.1%) through conventional primary extraction, the purity of the acteoside enriched part obtained after further separation by using macroporous adsorption resin is up to more than 27%, the purity can be up to more than 98% after separation by liquid chromatography, and the process is simple. In addition, the method has high yield (5.5%) of the acteoside product and low cost, and is suitable for industrial application.

Although scholars found very low amounts of acteoside from paulownia fortunei (Feng Weisheng, lv Jinjin, zhang Jingke, li Meng, zhang Beibei, zheng Xiaoke. Glycoside components of paulownia fortunei and their antioxidant activity. Chinese patent No. 2020,42 (02): 369-374), this study was a first study to find out components in the HPLC profile of paulownia fortunei that are very suitable for quantitative analysis and identified that acteoside does not have much reference effect for two reasons:

firstly, the acteoside is derived from different organs, the flowers and leaves of the paulownia fortunei are different organs, the physiological functions of the different organs are different, the responses to the environment are different, the synthesis of secondary metabolites is also emphasized, so that the composition difference of chemical components is caused, the acteoside with lower content is found in the flowers, and does not represent the leaves, but contains a large amount of acteoside, and the content in the opposite leaves is possibly lower or even not detected at all by a conventional analysis method; the obtaining method of the acteoside is essentially different, and acteoside in the white paulownia flowers reported in the literature is obtained by a systematic separation and purification method, so that the acteoside has high contingency, and the acteoside is obtained by finding a common chromatographic peak with high content through fingerprint and carrying out directional separation, preparation and identification on the chromatographic peak, wherein the chemical component of the common chromatographic peak with high content is unpredictable.

Therefore, the invention discovers that the white paulownia leaves contain a large amount of acteoside for the first time, widens the plant sources of the acteoside, and the white paulownia belongs to fast-growing plants, can be even three and four meters high in one year, has large biomass of the leaves, higher economy and great economic value.

In order to further illustrate the present invention, the following examples are provided. The sources of the experimental raw materials used in the following examples of the present invention are not particularly limited, and may be purchased from the market or prepared according to conventional preparation methods well known to those skilled in the art.

The conditions for high performance liquid phase detection in the following examples are as follows:

Chromatographic column: agilent ZORBAX HC-C18 (2) (4.6 mm. Times.250 mm,5 μm);

Column temperature: 30 ℃;

Sample injection amount: 10. Mu.L;

Flow rate: 1mL/min;

detection wavelength: 254nm;

mobile phase: methanol (B) -0.1vol% formic acid buffer (a), gradient elution, see table 1 below:

TABLE 1

Time (min)	A(vol％)	B(vol％)
			0	90	10
8	68	32
			44	50	50
52	0	100
			65	0	100
66	90	10
			76	90	10

Example 1

HPLC detection is carried out on the paulownia fortunei leaf sample solution, and the fingerprint spectrum is recorded, and the chromatographic conditions are as follows: chromatographic column: column No. 1: agilent ZORBAX HC-C18 (2) (4.6 mm. Times.250 mm,5 μm); column No. 2: waters XBiridge C18 (4.6mm. Times.150 mm,3.5 μm); column No. 3: wondaCract C-18WRS (4.6 mm. Times.250 mm,5 μm). ; mobile phase: methanol (B) -0.1vol% formic acid buffer (a); detection wavelength: 254nm; flow rate: 1mL/min; column temperature: 30 ℃; sample injection amount: 10. Mu.L; gradient elution, specific elution procedure is shown in the following table:

Gradient elution procedure

As a result, as shown in FIG. 5, the separation was carried out by using a No. 1 column, and the separation degree of each peak was good, the number of the chromatographic peaks was large and the shape of the chromatographic peak was relatively good. Thus Agilent ZORBAX HC-C18 (2) (4.6mm. Times.250 mm,5 μm) was chosen as the column.

Example 2

With reference to the separation method of example 1, the effect of methanol-water system, methanol-0.1 vol% formic acid system, methanol-0.1 vol% acetic acid system, methanol-0.1 vol% phosphoric acid system as a flow against the HPLC chromatogram of paulownia fortunei leaf was examined.

As a result, as shown in FIG. 6, the methanol-water system, the methanol-0.1 vol% acetic acid system and the methanol-0.1 vol% phosphoric acid system were used, many chromatographic peaks could not be separated and the number of overlapping chromatographic peaks was large, but the separation effect of the methanol-0.1 vol% formic acid system was the best, the chromatographic peak shape was good, and the time for the chromatographic peak was appropriate. Thus methanol-0.1 vol% formic acid system was chosen as mobile phase.

Example 3

HPLC chromatograms of the leaves of paulownia fortunei were obtained by examining the separation method of example 1 at detection wavelengths of 210nm, 254nm, 270nm, 290nm, 320nm and 365 nm.

As shown in FIG. 7, each chromatographic peak at 254nm has better ultraviolet light absorption, and the chromatographic information is rich and the base line is stable, so 254nm is selected as the measurement wavelength of the paulownia fortunei leaves.

Example 4

The effect of column temperature (25 ℃,30 ℃ and 35 ℃) on the HPLC chromatogram of the paulownia tomentosa leaf was examined with reference to the separation method of example 1.

As a result, as shown in fig. 8, when the column temperature was 30 ℃ in the case where other chromatographic conditions were uniform, the separation effect of each peak was optimal, and therefore, the column temperature was determined to be 30 ℃.

In example 1, the HPLC fingerprint of the paulownia tomentosa leaf sample separated by the chromatographic column No.1 is shown in fig. 2, wherein the spectrum shows 23 characteristic peaks, wherein the peak No. 11 is a common peak, the intensity is higher, the retention time is relatively good in the separation degree from the rest of chromatographic peaks, and therefore the peak No. 11 is selected as a reference peak. The compound represented by this peak was rapidly prepared and identified because it was not determined. The authentication method is described in detail above and will not be described in detail here. The peak 11 is identified as acteoside, and in view of this, the present invention provides a method for extracting acteoside from paulownia leaves, and a series of optimization is performed for the corresponding parameters, as follows.

Example 5

This example was used to examine the effect of different extraction solvents on the content of acteoside extracted from paulownia leaves:

Taking 0.5g of paulownia tomentosa leaves, respectively carrying out ultrasonic extraction for 2 hours at 70 ℃ by using 20 times of 70vol% ethanol, 50vol% ethanol, 30vol% ethanol, water, 50vol% methanol and 30vol% methanol, taking filtrate, concentrating and drying to obtain crude extract.

As a result of measurement, it was found that the extraction rates of the ethanol solutions of 30 to 50vol% were high and the extraction rates of the ethanol solutions of 50vol% were highest, respectively, since the content of the acteoside extracted from the ethanol solutions of 70vol% ethanol, 50vol% ethanol, 30vol% ethanol, water, 50vol% methanol, and 30vol% methanol was 5.34%, 10.59%, 9.83%, 4.79%, 10.21%, and 8.24%, respectively.

Example 6

This example was used to examine the effect of the extraction mode on the content of acteoside extracted from paulownia leaves:

Reflux extraction: reflux-extracting 0.5g of paulownia fortunei leaves with 20 times of 50% ethanol at 70 ℃ for 2 hours, concentrating and drying the filtrate to obtain a crude extract;

ultrasonic extraction: taking 0.5g of paulownia fortunei leaves, carrying out ultrasonic extraction for 2 hours at 70 ℃ by using 20 times of 50% ethanol, concentrating and drying filtrate to obtain crude extract.

The content of acteoside in the crude extract is measured, and the results show that the content of acteoside extracted by ultrasonic extraction and reflux extraction is respectively 11.18% and 8.26%, the extraction amount is higher, but the content of acteoside extracted by ultrasonic extraction is more in the case of once extraction.

Example 7

The present example was used to examine the effect of the extract liquid comparison on the content of acteoside extracted from paulownia leaves:

taking 0.5g of paulownia fortunei leaves, adding 50vol% ethanol into the solution for reflux extraction for 2 hours at 70 ℃ according to the feed liquid ratio of (the paulownia fortunei leaves: 50vol% ethanol volume, g/mL) of 1:5, 1:10, 1:20, 1:30 and 1:50, and concentrating and drying the filtrate to obtain a crude extract.

And measuring the content of the acteoside extracted under different extraction liquid ratios. The results show that the content of the extracted acteoside is 5.85%, 7.2%, 9.89%, 10.45% and 10.46% respectively in the feed liquid ratio of 1:5g/mL, 1:10g/mL, 1:20g/mL, 1:30g/mL and 1:50g/mL, and therefore, the content of the extracted acteoside is higher when the feed liquid ratio is 1 (20-50) g/mL. However, the extraction amount increases little after more than 1:30g/mL, so that a feed-to-liquid ratio of 1:30g/mL is more preferable from the viewpoint of saving the solvent.

Example 8

This example was used to examine the effect of 50vol% ethanol extraction time on the amount of acteoside extracted from paulownia leaves:

taking 0.5g of paulownia tomentosa leaves, carrying out ultrasonic extraction on the paulownia tomentosa leaves by 20 times of 50vol% ethanol at 70 ℃ for 30min, 60min, 90min and 120min, concentrating and drying filtrate to obtain crude extract.

And measuring the content of the acteoside at different extraction times. The results show that the content of the acteoside is 9.99%, 10.97%, 11.06% and 11.25% respectively in 30min, 60min, 90min and 120min, so that the extraction yield is higher in 0.5-2 h, and the extraction yield gradually increases along with the extension of the extraction time, but the extraction yield does not increase greatly after 1h, and most preferably 1h can be adopted for saving the cost.

Example 9

This example was used to examine the effect of water extraction time on the content of acteoside extracted from paulownia leaves:

Taking 0.5g of paulownia tomentosa leaves, ultrasonically extracting with 30 times of water at 70 ℃ for 30min, 60min, 90min, 120min and 150min, concentrating and drying the filtrate to obtain a crude extract.

The content of the acteoside in different extraction times is measured, and the results show that the content of the acteoside in extraction is 7.25%, 7.71%, 8.13%, 8.22% and 8.49% in 30min, 60min, 90min, 120min and 150min respectively, so that the extraction yield is higher in 30-150 min, the extraction yield is gradually increased along with the extension of the extraction time, but the extraction yield is not greatly increased after 90min, and 90min is most preferable for saving the cost.

Example 10

This example was used to examine the effect of water extraction times on the content of acteoside extracted from paulownia leaves:

taking 0.5g of paulownia fortunei leaves, carrying out ultrasonic extraction for 1 time, 2 times and 3 times with 30 times of water at 70 ℃, extracting for 2 hours each time, concentrating and drying filtrate to obtain a crude extract.

The content of the acteoside in different times of extraction is measured, and the results show that the content of the acteoside in the extraction for 1 time, 2 times and 3 times is 7.25%, 10.35% and 10.54% respectively. Thus, the content of acteoside extracted gradually increases with the increase of the number of extractions, but in the case of 3 extractions, the increase of the extraction content is not significant, so that the extraction can be selected twice to appropriately increase the content of acteoside extracted when water is used as the extraction solvent.

Example 11

The influence of the type of macroporous adsorption resin on the acteoside content in the obtained acteoside enriched fraction was examined in this example:

100mL of crude extract solution of paulownia tomentosa leaves was added to a conical flask containing 2.0g of treated macroporous adsorption resins AB-8, DM-301, DA-201, D-101, HPD100, DM-130, respectively. The adsorption was performed at 25℃for 6 hours with shaking, 1mL of the supernatant was taken every 0.5 hour, and the adsorption rate was calculated by HPLC measurement [ adsorption rate= (mass concentration before adsorption-mass concentration after adsorption)/mass concentration before adsorption ]. And (3) taking the adsorbed resin, washing with a proper amount of water, adding 50mL of 50vol% ethanol, performing constant-temperature oscillation desorption, taking 1mL of supernatant, and adding 10vol% methanol to a constant volume of 10mL. The desorption rate [ desorption rate=mass concentration after desorption/(mass concentration before adsorption-mass concentration after adsorption) ] was calculated by HPLC measurement.

As shown in Table 2, the above macroporous adsorbent resins can achieve better separation and purification effects, but compared with the AB-8 type macroporous adsorbent resins, D-101 has better adsorption and desorption effects on acteoside. In addition, the D-101 macroporous adsorption resin has the advantage of lower price compared with AB-8 macroporous adsorption resin.

TABLE 2

Resin model	Adsorption Rate (%)	Desorption rate (%)
			D-101	73.21	64.54
AB-8	72.72	63.89
			DM-301	62.97	51.35
DA-201	61.56	51.21
			HPD100	60.88	51.02
DM-130	61.34	51.22

Example 12

In the process of separating and purifying the crude extract by using the macroporous adsorption resin, the influence of the mass concentration of the loading liquid on the content of acteoside in the obtained acteoside enrichment part is examined in the embodiment:

Precisely weighing 3 parts of treated macroporous adsorption resin (40 g each part), and respectively filling into columns by wet method. Sample solutions (obtained by mother liquor dilution) with mass concentrations of 15mg/mL, 25mg/mL and 35mg/mL respectively are passed through a macroporous resin column at the same volume flow rate (2 BV/h). After the adsorption was completed, the adsorption rate [ adsorption rate= (mass concentration before adsorption-mass concentration after adsorption)/mass concentration before adsorption ] was calculated by HPLC measurement.

The results show that the adsorption rates of the sample solutions are 72.44%, 73.32% and 72.56% respectively under the conditions that the mass concentration of the sample solution is 15mg/mL, 25mg/mL and 35 mg/mL. The adsorption rate reached a maximum value at a mass concentration of 25 mg/mL.

Example 13

In the process of separating and purifying the crude extract by using the macroporous adsorption resin, the influence of the eluent on the content of acteoside in the obtained acteoside enriched part is examined in the embodiment:

Precisely weighing 4 parts of treated macroporous adsorption resin (40 g each), respectively loading on a wet method column (the loading liquid is a crude extract solution of 25 mg/mL), eluting with 3BV distilled water to remove impurities, eluting with 3BV ethanol solution with volume fractions of 10%, 30%, 50% and 70% at volume flow rate of 2BV/h, and calculating desorption rate [ desorption rate=mass concentration after desorption/(mass concentration before adsorption-mass concentration after adsorption) ] by adopting HPLC.

The results showed that when elution was performed with 10%, 30%, 50%, 70% ethanol solutions, the desorption rates of elution were 17.25%, 41.63%, 98.16%, 98.58%, respectively. The results show that the ethanol with the volume fraction of 10 percent and 30 percent is not completely eluted, the acteoside can be almost completely eluted by the ethanol with the volume fraction of 50 percent and 70 percent, but the acteoside is eluted by the ethanol with the volume fraction of 70 percent, and the cost is increased due to the increase of impurities. In combination, 50vol% ethanol is more preferred as the eluent.

Example 14

In the process of separating and purifying the crude extract by using the macroporous adsorption resin, the influence of the using amount of the eluent on the content of acteoside in the obtained acteoside enrichment part is examined in the embodiment:

40g of the treated macroporous adsorption resin is weighed and loaded on a wet method column (the loading liquid is a crude extract solution of 25 mg/mL), 3BV distilled water is used for eluting impurities, 50vol% ethanol is used for eluting, and 1 part of effluent is collected every 1BV (20 mL). The elution rate (elution rate=eluted mass/pre-elution mass) was calculated using HPLC measurement.

The elution rates for the eluent volumes of 1BV, 2BV, 3BV, 4BV, 5BV, 6BV were 29.49%, 43.49%, 21.36%, 4.45%, 0.42% and 0.11%, respectively. As a result, it was found that 98% or more of the acteoside could be eluted to 3 to 4.0BV, and the eluting amount of 50vol% ethanol was preferably 3 to 4.0BV, more preferably 4.0BV, in consideration of the cost.

Example 15

In the process of separating and purifying the crude extract by using the macroporous adsorption resin, the influence of the volume flow of the eluent on the content of acteoside in the obtained acteoside enrichment part is examined in the embodiment:

40g of the treated D-101 macroporous adsorption resin is weighed and loaded on a wet column (the loading liquid is a crude extract solution of 25 mg/mL), 3BV distilled water is used for eluting to remove impurities, 3BV 50vol% ethanol is used for eluting at volume flows of 1BV/h, 2BV/h, 3BV/h, 4BV/h, 5BV/h and 6BV/h, and the effluent liquid is collected for 1 time every 1 BV. The elution rate (elution rate=eluted mass/pre-elution mass) was calculated using HPLC measurement.

The elution rates at the eluent volumes of 1BV/h, 2BV/h, 3BV/h, 4BV/h, 5BV/h and 6BV/h are 97.78%, 95.32%, 94.69%, 91.53%, 89.77% and 87.41% respectively. The results show that the eluting volume flow rate is 1BV/h and 2BV/h, and the eluting rate is higher. If the eluting effect and the time cost are comprehensively considered, the optimal volume flow of the eluent is 2BV/h.

Example 16

The embodiment adopts water to extract the acteoside in the paulownia leaves, and comprises the following specific steps:

0.82kg of dried paulownia tomentosa leaves are placed in a 30L stainless steel barrel, 30L purified water solution is added, and ultrasonic extraction is carried out at 70 ℃ for 2 hours. After the extraction is finished, filtering out the extracting solution, adding 30L of purified water solution again for ultrasonic extraction, combining the filtrates, concentrating and drying to obtain 74.3g of the crude extract of the paulownia fortunei leaves, wherein the yield is 9.06%. Through high performance liquid phase detection, the purity of the acteoside in the crude extract of the paulownia tomentosa leaves extracted by water is 10.052 percent.

Example 17

In this example, 50vol% ethanol aqueous solution was used to extract acteoside from paulownia leaves, and the specific steps were as follows:

0.94kg of dried leaves of paulownia fortunei are placed in a 30L stainless steel barrel, 20L of 50vol% ethanol aqueous solution is added for ultrasonic extraction for 1h. After the extraction is finished, the extracting solution is filtered out to obtain 104.7g of crude extract of the paulownia fortunei leaves, and the yield is 11.1%. Through high performance liquid phase detection, the purity of the acteoside in the crude extract of the paulownia fortunei leaves extracted by 50vol% ethanol is 10.514%.

The crude extracts obtained in comparative examples 1 to 2 were not very different in the purity of the acteoside. The subsequent extraction and purification was carried out with the crude extract obtained in example 1.

Example 18

In this example, the crude extract obtained in example 16 was separated and purified by using a resin to obtain a acteoside enriched fraction, which comprises the following steps:

55g of the crude extract of paulownia tomentosa leaves obtained in example 1 was taken and dissolved in 2.2L of water, and the solution was applied to D101 macroporous adsorption resin, and the solution was first eluted with an aqueous solution having an elution volume of 3 column volumes and a flow rate of 2 column volumes/hour, then eluted with an aqueous solution of 10% by volume, the solution was eluted with an elution volume of 3 column volumes and a flow rate of 2 column volumes/hour, then eluted with an aqueous solution of 30% by volume, the solution was eluted with an elution volume of 3 column volumes and a flow rate of 2 column volumes/hour, and finally eluted with an aqueous solution of 50% by volume, the elution volume was 4 column volumes and the flow rate was 2 column volumes/hour. The acteoside enriched part is obtained, the weight is 3.03g, and the yield is 5.51%. Through high performance liquid phase detection, the purity of the acteoside reaches 41.6%.

The same effect can be achieved by replacing the macroporous adsorption resin with AB-8, DM-301, DA-201, HPD100, DM-130, etc.

Example 19

The preparation liquid chromatography (preparation liquid phase of LC-3000A type of Beijing) of the embodiment further separates and purifies the obtained acteoside enrichment part, and the specific steps are as follows:

Taking the acteoside enriched part obtained in the example 18, obtaining acteoside collected liquid after entering a preparation liquid chromatograph, carrying out vacuum concentration at 70 ℃ until the solid-liquid mass volume ratio is 1 (4-5) g/mL, stopping concentration, and then drying to obtain acteoside solid. Through high performance liquid phase detection, the purity of the acteoside reaches 98.4% as calculated by a peak area normalization method and shown in a figure 9.

Wherein, the working conditions of the preparation liquid chromatograph are as follows:

chromatographic column: YMC-Pack ODS-A (20 mm. Times.250 mm,5 μm);

The detection wavelength is 334nm;

the flow rate of the mobile phase is 10mL/min;

column temperature is 30 ℃;

tR＝25.0min；

Sample injection amount: 20. Mu.L.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting acteoside from paulownia tomentosa leaves, which is characterized by comprising the following steps:

(1) Extracting paulownia fortunei leaves by using 30-50 vol% ethanol solution as a solvent to obtain a crude extract;

the macroporous adsorption resin comprises AB-8 macroporous adsorption resin or D-101 macroporous adsorption resin;

2. The method according to claim 1, wherein in the extraction of the paulownia fortunei leaves, the ratio of the mass of the paulownia fortunei leaves to the volume of the solvent is 1 (20-50) g/mL.

3. The method according to claim 1, wherein the temperature of the extraction in step (1) does not exceed 70 ℃.

4. The method according to claim 1, wherein the crude extract obtained in step (1) is separated and purified by using macroporous adsorbent resin, and the elution procedure is as follows:

Elution procedure

。

5. The method according to claim 1, wherein in the liquid chromatography separation, the mobile phase is 30 to 35vol% methanol or 20 to 25vol% acetonitrile solution.

6. The method according to claim 1, wherein the flow rate of the mobile phase in the liquid chromatography separation is 2-15mL/min.

7. The method according to claim 1, wherein the detection wavelength in the liquid chromatography separation is 240 to 370nm.