CN115304651A - Method for extracting verbascoside from paulownia leaves - Google Patents
Method for extracting verbascoside from paulownia leaves Download PDFInfo
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- CN115304651A CN115304651A CN202211114147.4A CN202211114147A CN115304651A CN 115304651 A CN115304651 A CN 115304651A CN 202211114147 A CN202211114147 A CN 202211114147A CN 115304651 A CN115304651 A CN 115304651A
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- verbascoside
- paulownia
- ethanol
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- QFRYQWYZSQDFOS-UHFFFAOYSA-N verbascoside Natural products CC1OC(COC2C(O)C(COC3OC(C(O)C(O)C3O)C(=O)O)OC(Oc4cc(O)cc5OC(=CC(=O)c45)c6ccc(O)c(O)c6)C2O)C(O)C(O)C1O QFRYQWYZSQDFOS-UHFFFAOYSA-N 0.000 title claims abstract description 124
- KDSWDGKIENPKLB-QJDQKFITSA-N verbascoside Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC(=O)CCC=2C=C(O)C(O)=CC=2)[C@@H](CO)O[C@@H](OCCC=2C=C(O)C(O)=CC=2)[C@@H]1O KDSWDGKIENPKLB-QJDQKFITSA-N 0.000 title claims abstract description 92
- 238000000034 method Methods 0.000 title claims abstract description 52
- 240000002834 Paulownia tomentosa Species 0.000 title description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 81
- 244000055346 Paulownia Species 0.000 claims abstract description 74
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- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims abstract description 5
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- FBSKJMQYURKNSU-UKQWSTALSA-N acteoside I Natural products C[C@@H]1O[C@H](O[C@@H]2[C@@H](O)[C@H](OCCc3ccc(O)c(O)c3)O[C@H](CO)[C@H]2OC(=O)C=Cc4ccc(O)c(O)c4)[C@H](O)[C@H](O)[C@H]1O FBSKJMQYURKNSU-UKQWSTALSA-N 0.000 abstract description 31
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/18—Acyclic radicals, substituted by carbocyclic rings
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a method for extracting verbascoside from paulownia leaves, which comprises the following steps: extracting paulownia leaves to obtain a crude extract; separating and purifying the obtained crude extract with macroporous adsorbent resin to obtain acteoside-enriched part; and (3) carrying out liquid chromatography separation on the obtained verbascoside enrichment part to obtain a verbascoside collecting solution, and then concentrating and drying to obtain the verbascoside. 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 20-50 vol% of methanol or 10-40% of acetonitrile solution. The method extracts the verbascoside from the paulownia leaves for the first time, fills up the technical blank in the field, and finally obtains the verbascoside with the purity of more than 98 percent, has simple process and low economic cost, and is easy to realize industrialization and industrialization.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a method for extracting verbascoside from paulownia leaves.
Background
Paulownia (Paulownia) plants are produced in 7 kinds in total, are produced in China, are distributed, cultivated or wild in China except for northern northeast, inner Mongolia, northern Xinjiang, tibet and other areas, and are introduced in some areas. Paulownia as a high-quality fast-growing wood is a common traditional Chinese medicinal material besides being widely applied to agricultural production, and flowers, leaves, barks, roots and fruits of paulownia can be used as the medicine. The compendium of materia medica makes detailed record of pharmacological action of each part of paulownia, and modern medical research also shows that paulownia has the functions of bacteriostasis, inflammation diminishing, tumor resisting and even insect killing. The paulownia trees are luxuriant in branches and leaves, have a plurality of leaves and are rich natural resources. In addition, the paulownia leaves can be used as feed, so that the growth of animals can be promoted, the disease resistance of the animals can be improved, and the paulownia leaves have rich economic value and social value.
Acteoside is called Acteoside, acteoside or Acteoside, is phenylethanoid glycoside compound of 4, 5-dihydroxyphenylethanol (hydroxytyrosol) bonded with rhamnose C1 beta-D-glucopyranose via ester bond and glycosidic bond and C3 glycosidic bond, is named Acteoside, verbasoside or Kusaginin, and has molecular formula of C 29 H 36 O 15 And the molecular weight is 624.59. Phytochemical studies have shown that verbascoside is widely found in a variety of dicotyledonous plants of the families Orobanchaceae, scrophulariaceae, magnoliaceae, and Labiatae. Modern pharmacological researches find that verbascoside has a plurality of pharmacological activities such as remarkable oxidation resistance, anti-inflammation, memory enhancement, nerve protection, tumor resistance and the like, and is widely concerned by researchers at home and abroad due to wide sources, strong biological activity and small adverse reaction.
With the gradual and deep research of pharmaceutical research on phenylpropanoid glycoside compounds such as verbascoside at home and abroad, the medicinal value of the compounds is increasingly emphasized, and the verbascoside and related products thereof are in short supply in the market. Since acteoside is the main active component of cistanche plants, research in recent years is mainly focused on extracting and separating the acteoside from the cistanche plants. It has also been reported that verbascoside can be extracted and separated from other plants such as Osmanthus fragrans, callicarpa nudiflora leaves, campsis, and Buddleja officinalis. However, the extraction and preparation of verbascoside from these plants are difficult to meet the actual demand, and the isolation and purification of verbascoside from these plants usually requires repeated column chromatography to obtain highly pure verbascoside samples, which is a drawback of long time and large solvent consumption. In addition, the biomass of the plants is small, and the extraction preparation needs a large amount of planting, so that the economy is not high.
Disclosure of Invention
Accordingly, the present invention is directed to a method for extracting acteoside from paulownia leaves. The paulownia leaves are rich in content, the verbascoside extracted from the paulownia leaves does not need to be planted in advance in a large amount, the economic value is good, the purity of the extracted verbascoside is high and can reach more than 98%, and the actual requirement 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 adopting macroporous adsorption resin to obtain a verbascoside enrichment part;
(3) Carrying out liquid chromatography separation on the verbascoside enriched part obtained in the step (2) to obtain verbascoside collecting liquid, and then concentrating and drying to obtain verbascoside;
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% of methanol or 10-40 vol% of acetonitrile solution.
Preferably, step (1) is specifically: extracting paulownia leaves by using one or more of water, methanol and ethanol as a solvent to obtain a 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 said extraction in step (1) does not exceed 70 ℃, more preferably is between 40 and 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 step (1) is separated and purified by using macroporous adsorption resin, the elution procedure is as shown in the following table:
elution procedure
| Eluent | Elution volume | Flow rate of eluent |
| Water (W) | 2-4 column volumes | 1-2 column volumes/hour |
| 10vol% ethanol | 2-4 column volumes | 1-2 column volumes/hour |
| 30vol% ethanol | 2-4 column volumes | 1-2 column volumes/hour |
| 50vol% ethanol | 3-5 column volumes | 1-2 column volumes/hour |
Preferably, in the liquid chromatography separation, the mobile phase is 30-35 vol% of methanol or 20-25 vol% of acetonitrile solution.
Preferably, in the liquid chromatographic separation, the flow rate of the mobile phase is 2-15mL/min.
Preferably, in the liquid chromatographic separation, the detection wavelength is 240-370 nm.
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 HPLC spectrogram of paulownia leaves, provides a method for extracting verbascoside from the paulownia leaves on the basis of identifying the components as the verbascoside, fills up the technical blank in the field, widens the plant source of the verbascoside, and has great economic value;
(2) The method for extracting the verbascoside provided by the invention takes paulownia leaves as a raw material, and sequentially carries out crude extraction, macroporous resin adsorption separation and liquid chromatography separation to obtain the verbascoside with the purity of more than 98%.
Drawings
FIG. 1 is a schematic diagram of the structural formula of verbascoside;
FIG. 2 is an HPLC fingerprint of Paulownia leaves;
FIG. 3 is a nuclear magnetic resonance H spectrogram of a peak 11 compound in an HPLC fingerprint of paulownia leaves;
FIG. 4 is a nuclear magnetic resonance C spectrogram of a peak 11 compound in an HPLC fingerprint of paulownia leaves;
FIG. 5 is a diagram of the investigation result of different chromatographic columns of HPLC fingerprints of paulownia leaves;
FIG. 6 is a diagram of the investigation result of different solvent systems of HPLC fingerprints of paulownia leaves;
FIG. 7 is a diagram of the investigation result of different detection wavelengths of HPLC fingerprint spectrum of paulownia leaves;
FIG. 8 is a diagram of the investigation results of different column temperatures of HPLC fingerprints of paulownia leaves;
FIG. 9 is a chromatogram for purity detection of verbascoside obtained in example 4.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The source of all experimental materials is not particularly limited, and the experimental materials can be obtained by purchasing the experimental materials from the market or preparing the experimental materials according to the conventional preparation method well known to those skilled in the art.
The invention is based on the mass study of chromatographic conditions and fingerprint spectrogram of paulownia leaf extract, and the fingerprint spectrogram discovers components with very high separation degree and relative content for the first time and is identified as verbascoside (the structural formula is shown in figure 1) after preparation and separation, and the specific process is as follows:
weighing 0.5g of paulownia fortunei leaf powder, placing the paulownia fortunei leaf powder in a conical flask with a plug, precisely adding 10mL of 70vol% methanol, sealing the conical flask, weighing the conical flask to determine the mass, carrying out ultrasonic treatment (power 500W and frequency 40 kHz) for 30min, cooling the conical flask, complementing the loss reduction mass with 70vol% methanol, taking supernate, and filtering the supernate with a 0.45-micron microporous membrane to obtain a paulownia fortunei leaf sample solution.
Carrying out HPLC detection on the paulownia fortunei leaf sample solution and recording the fingerprint spectrum of the sample solution, wherein the chromatographic conditions are as follows: a chromatographic column: agilent ZORBAX HC-C18 (2) (4.6 mm. Times.250mm, 5 μm); mobile phase: methanol (B) -0.1vol% formic acid buffer solution (a); detection wavelength: 254nm; flow rate: 1mL/min; column temperature: 30 ℃; sample introduction amount: 10 mu L of the solution; gradient elution, the specific elution procedure is shown in the following table:
gradient elution procedure
The HPLC fingerprint spectrum of the paulownia fortunei leaf sample is shown in figure 2, wherein the spectrum presents 23 characteristic peaks, wherein the 11 th peak is a common peak, the intensity is high, the separation degree of the retention time relative to the middle and the rest chromatographic peaks is good, and therefore the 11 th peak is selected as a reference peak. Because the compound represented by the peak is not determined, the compound is rapidly prepared and identified.
Taking a sample solution of paulownia leaves, and obtaining a compound with a peak 11 through semi-preparative liquid chromatography, wherein the chromatographic conditions are as follows: column YMC-Pack Ph (10 mm. Times.250mm, 5 μm); isocratic elution; mobile phase: methanol (B) -water (a) = (33. The detection wavelength is 254nm; the flow rate of the mobile phase is 2.5mL/min; the column temperature is 30 ℃; t is t R =24.9min. The results of the characterization of the compound with peak 11 by nmr spectroscopy are shown in fig. 3-4, where fig. 3 is the nmr H spectrum of the compound with peak 11 and fig. 4 is the nmr C spectrum of the compound with peak 11, and the data are shown below: ESI-MS m/z:647.1940[ 2 ], [ M ] +Na ]] + 。 1 H 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″'). 13 <xnotran> C-NMR (MeOD, 125 MHz) 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 ″'). </xnotran> The above data are in substantial agreement with literature reports (Wang XQ, et al. Phenyl ethyl glycosides from Orobanchpycnostachy Hance and the same chemotaxonomic design [ J]Biochemical systems and ecology), thus final nuclear magnetic characterization of peak 11 compound as verbascoside.
Based on the research, the invention provides a method for extracting verbascoside from paulownia leaves, which comprises the following steps:
(1) Extracting paulownia leaves to obtain a crude extract;
(2) Separating and purifying the crude extract obtained in the step (1) by adopting macroporous adsorption resin to obtain a verbascoside enrichment part;
(3) Performing liquid chromatography separation on the verbascoside enriched part obtained in the step (2) to obtain verbascoside collecting liquid, and then concentrating and drying to obtain verbascoside;
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% of methanol or 10-40 vol% of acetonitrile solution.
In the present invention, one or more of water, methanol or ethanol is preferably used as a solvent, more preferably a 30 to 50vol% ethanol solution, and most preferably a 50vol% ethanol solution is used for extracting paulownia leaves to obtain a crude extract. The extraction method preferably comprises any one or more of immersion, decoction, percolation, reflux extraction, ultrasonic extraction or supercritical extraction, more preferably comprises ultrasonic extraction and/or reflux extraction, and most preferably comprises ultrasonic extraction. In the present invention, when paulownia leaves are extracted, 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. Because acteoside is susceptible to conversion at high temperatures, the extraction temperature is preferably not more than 70 ℃, more preferably 40-70 ℃, and the extraction time is preferably 0.5-2.5 h, more preferably 1-1.5 h, most preferably 1h.
According to the invention, after the crude extract is obtained, macroporous adsorption resin is adopted to separate and purify the crude extract to obtain the verbascoside enrichment part. In the invention, the macroporous adsorption resin preferably 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 AB-8 macroporous adsorption resin or D-101 macroporous adsorption resin, and most preferably D-101 macroporous adsorption resin. The improvement of the sample loading mass concentration is beneficial to improving the service efficiency of the resin. However, when the sample loading mass concentration is too high, the contact time of the sample and the resin is shortened under a certain volume flow, and meanwhile, the sample loading liquid mass concentration is too high, so that the solution is too viscous, viscous adsorption can be formed on the surface of the resin particles, and the adsorption effect is seriously influenced. Therefore, the mass concentration of the sample is preferably 15 to 25mg/mL, more preferably 25mg/mL.
In the present invention, 50vol% ethanol is more preferred as the eluent. However, because impurities exist in the macroporous adsorbent resin column, water, 10vol% ethanol and 30vol% ethanol are preferably used for removing impurities, and then 50vol% ethanol is used for elution. The specific elution procedure is preferably as shown in the following table:
elution procedure
| Eluent solution | Elution volume | Flow rate of eluent |
| Water (I) | 2-4 column volumes | 1-2 column volumes/hour |
| 10vol% ethanol | 2-4 column volumes | 1-2 column volumes/hour |
| 30vol% ethanol | 2-4 column volumes | 1-2 column volumes/hour |
| 50vol% ethanol | 3-5 column volumes | 1-2 column volumes/hour |
According to the invention, the verbascoside enrichment part is subjected to liquid chromatography separation to obtain a verbascoside collecting solution. In the present invention, the liquid chromatography is preferably preparative liquid chromatography. In the liquid chromatography 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 chromatography column is preferably 10 to 50mm, more preferably 10 to 30mm. The mobile phase is preferably 20 to 50vol% methanol solution or 10 to 40vol% acetonitrile solution, more preferably 30 to 40vol% methanol or 20 to 25vol% acetonitrile solution, and most preferably 30 to 35vol% methanol solution. 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, and more preferably 280 to 370nm.
In a particular embodiment, the conditions of the liquid chromatography are preferably:
a chromatographic column: YMC-Pack ODS-A (20 mm. Times.250mm, 5 μm);
mobile phase: methanol (B) -water (a) = (33, volume ratio), isocratic elution;
the detection wavelength is 334nm;
the flow rate of the mobile phase is 10mL/min;
the column temperature is 30 ℃;
tR＝25.0min；
sample introduction amount: 20 μ L.
According to the invention, the verbascoside collected liquid is concentrated and dried to obtain a verbascoside solid. In the present invention, the form of concentration is not particularly limited, and it is preferable to carry out concentration under vacuum at a temperature not exceeding 70 ℃. The concentration is preferably finished when the solid-liquid mass-volume ratio is 1 (4-5) g/mL. The drying form is not particularly limited in the present invention, and preferably any one or more of freeze drying, spray drying or microwave drying is adopted to finally obtain acteoside solid.
The method takes paulownia leaves as raw materials, the mass fraction of verbascoside in a crude extract is not less than 5 percent (the highest can reach 11.1 percent) through conventional primary extraction, the purity of an obtained verbascoside enrichment part is up to more than 27 percent after the verbascoside enrichment part is further separated by macroporous adsorption resin, the purity can reach more than 98 percent after the verbascoside enrichment part is separated by liquid chromatography, and the process is simple. In addition, the method has high acteoside yield (5.5%), low cost, and suitability for industrial application.
It should be noted that although some researchers found low-content verbascoside (von hygienics, lujin, zhangjing, li meng, zhanbei, zheng ke, paulownia flower glycoside components and their antioxidant activities. Chinese patent medicine 2020,42 (02): 369-374) from paulownia alba flowers, this study found for the first time that components very suitable for quantitative analysis were found in HPLC chromatogram of paulownia alba leaves, and identified that verbascoside did not have much reference effect, the reason is two:
firstly, the acteoside is derived from different organs, flowers and leaves of paulownia fortunei are different organs, different organs have different physiological functions and different responses to the environment, and synthesis of secondary metabolites is also emphasized, so that the chemical components of the flowers are different, the lower content of the acteoside found in the flowers does not represent that the leaves contain a large amount of the acteoside, and on the contrary, the content of the acteoside in the leaves is possibly lower or even cannot be detected by a conventional analysis method; secondly, the method for obtaining acteoside is essentially different, the acteoside in the paulownia flower reported in the literature is obtained by a systematic separation and purification method and has larger contingency, but the method is obtained by finding a common chromatographic peak with large content through a fingerprint and carrying out directional separation, preparation and identification on the chromatographic peak, and the common chromatographic peak with large content can not be predicted what chemical components are.
Therefore, the invention discovers for the first time that the paulownia fortunei leaves contain a large amount of verbascoside, widens the plant source of the verbascoside, and the paulownia fortunei belongs to fast-growing plants, can grow three or four meters in one year, has large biomass of the leaves, higher economy and great economic value.
To further illustrate the present invention, the following examples are provided for illustration. The sources of the experimental raw materials used in the following examples of the present invention are not particularly limited, and they may be obtained by commercially available methods or by conventional methods known to those skilled in the art.
The following examples were carried out under the following conditions for HPLC detection:
and (3) chromatographic column: agilent ZORBAX HC-C18 (2) (4.6 mm. Times.250mm, 5 μm);
column temperature: 30 ℃;
sample introduction amount: 10 mu L of the solution;
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
Weighing 0.5g of paulownia fortunei leaf powder, placing the powder in a conical flask with a plug, precisely adding 10mL of 70vol% methanol, sealing the plug, weighing the powder with a fixed mass, carrying out ultrasonic treatment (with the power of 500W and the frequency of 40 kHz) for 30min, cooling, complementing the loss mass with 70vol% methanol, taking supernate, and filtering the supernate with a 0.45-micron microporous filter membrane to prepare a paulownia fortunei leaf sample solution.
Carrying out HPLC detection on the paulownia fortunei leaf sample solution and recording the fingerprint spectrum of the sample solution, wherein the chromatographic conditions are as follows: and (3) chromatographic column: column No. 1: agilent ZORBAX HC-C18 (2) (4.6 mm. Times.250mm, 5 μm); column No. 2: waters Xbridge C18 (4.6 mm. Times.150mm, 3.5 μm); column No. 3: wondaCract C-18WRS (4.6 mm. Times.250mm, 5 μm). (ii) a Mobile phase: methanol (B) -0.1vol% formic acid buffer solution (a); detection wavelength: 254nm; flow rate: 1mL/min; column temperature: 30 ℃; sample injection amount: 10 mu L of the solution; gradient elution, the specific elution procedure is shown in the following table:
gradient elution procedure
| 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 |
As shown in FIG. 5, the results of the separation using column No. 1 showed that the degree of separation of each peak was good, the number of chromatographic peaks was large, and the chromatographic peak profile was relatively good. Therefore, agilent ZORBAX HC-C18 (2) (4.6 mm. Times.250mm, 5 μm) was selected as a column.
Example 2
Referring to the separation method of example 1, the influence of a methanol-water system, a methanol-0.1 vol% formic acid system, a methanol-0.1 vol% acetic acid system, and a methanol-0.1 vol% phosphoric acid system as a flow on the HPLC chromatogram of paulownia leaves was examined.
As shown in FIG. 6, many chromatographic peaks could not be separated and many chromatographic peaks were overlapped by using a methanol-water system, a methanol-0.1 vol% acetic acid system, and a methanol-0.1 vol% phosphoric acid system, but the separation effect was the best in the methanol-0.1 vol% formic acid system, the peak shape was good, and the peak chromatography time was appropriate. Thus a methanol-0.1 vol% formic acid system was chosen as the mobile phase.
Example 3
With reference to the separation method of example 1, HPLC chromatograms of paulownia alba leaves obtained at detection wavelengths of 210nm, 254nm, 270nm, 290nm, 320nm and 365nm were examined.
As shown in FIG. 7, each chromatographic peak at 254nm wavelength has better ultraviolet absorption, the chromatographic information is richer, and the baseline is smoother, so 254nm is selected as the measurement wavelength of the leaves of Paulownia fortunei.
Example 4
The influence of the column temperature (25 ℃,30 ℃ and 35 ℃) on the HPLC chromatogram of Paulownia leaves was examined with reference to the separation method of example 1.
As a result, as shown in fig. 8, when the other chromatographic conditions were the same, the column temperature was determined to be 30 ℃ because the separation effect of each peak was the best when the column temperature was 30 ℃.
In example 1, the HPLC fingerprint of the paulownia tomentosa leaf sample separated by the number 1 chromatographic column is shown in fig. 2, wherein the chromatogram shows 23 characteristic peaks, wherein the number 11 peak is a common peak, the intensity is higher, the separation degree of the retention time relative to the middle and the rest chromatographic peaks is good, and therefore the number 11 peak is selected as a reference peak. Because the compound represented by the peak is not determined, the compound is rapidly prepared and identified. The identification method has been described in detail above, and is not described in detail herein. The peak 11 is identified as verbascoside, and in view of this, the invention provides a method for extracting the verbascoside from paulownia leaves, and a series of optimization is carried out according to corresponding parameters, as described below.
Example 5
This example is intended to investigate the effect of different extraction solvents on the content of verbascoside extracted from paulownia leaves:
taking 0.5g of paulownia leaves, carrying out ultrasonic extraction with 20 times of 70vol% ethanol, 50vol% ethanol, 30vol% ethanol, water, 50vol% methanol and 30vol% methanol at 70 ℃ for 2h respectively, taking filtrate, concentrating and drying to obtain a crude extract.
The results of the measurement show that the content of verbascoside extracted from 70vol% ethanol, 50vol% ethanol, 30vol% ethanol, water, 50vol% methanol and 30vol% methanol is respectively 5.34%, 10.59%, 9.83%, 4.79%, 10.21% and 8.24%, so that the extraction rate of 30-50 vol% ethanol solution is higher, and the extraction rate of 50vol% ethanol is the highest.
Example 6
This example is intended to examine the effect of the extraction mode on the content of verbascoside extracted from paulownia leaves:
reflux extraction: taking 0.5g of paulownia leaves, performing reflux extraction for 2 hours at 70 ℃ by using 50% ethanol in an amount which is 20 times that of the paulownia leaves, concentrating and drying the filtrate to obtain a crude extract;
ultrasonic extraction: taking 0.5g of paulownia leaves, carrying out ultrasonic extraction for 2h at 70 ℃ by using 20 times of 50% ethanol, concentrating and drying the filtrate, and obtaining a crude extract.
The content of verbascoside in the crude extract is determined, and the result shows that the content of verbascoside extracted by ultrasonic extraction and reflux is 11.18% and 8.26% respectively, the extraction amount is higher, but the content of verbascoside extracted by ultrasonic extraction is more under the condition of one-time extraction.
Example 7
This example is intended to examine the effect of the extract liquor on the content of verbascoside extracted from paulownia leaves:
taking 0.5g of paulownia fortunei leaves, adding 50vol% ethanol into the mixture at a ratio of material to liquid (paulownia fortunei leaves: 50vol% ethanol volume, g/mL) of 1.
Measuring the content of acteoside extracted from different extraction liquid ratios. The results showed that the contents of verbascoside extracted at the feed-liquid ratios of 1 (5 g/mL), 1 (10 g/mL), 1 (20 g/mL), 1 (30g/mL) and 1 (50g/mL) were 5.85%, 7.2%, 9.89%, 10.45% and 10.46%, respectively, and thus it was found that the contents of verbascoside extracted at the feed-liquid ratios of 1 (20-50) g/mL were all high. However, the amount of extraction is not increased much after the concentration is higher than 1.
Example 8
This example is intended to examine the effect of extraction time of 50vol% ethanol on the content of verbascoside extracted from paulownia leaves:
extracting Paulownia leaf 0.5g with 20 times of 50vol% ethanol at 70 deg.C under ultrasonic for 30min, 60min, 90min, and 120min, concentrating the filtrate, and drying to obtain crude extract.
The content of acteoside was determined at different extraction times. The results show that the contents of acteoside extracted for 30min, 60min, 90min and 120min are respectively 9.99%, 10.97%, 11.06% and 11.25%, so that the extraction yield is higher in 0.5-2 h, and gradually increases with the extension of the extraction time, but the extraction yield is not greatly increased after 1h, and the most preferable method is 1h for saving the cost.
Example 9
This example is used to examine the effect of water extraction time on the amount of verbascoside extracted from paulownia leaves:
taking 0.5g of paulownia leaves, carrying out ultrasonic extraction for 30min, 60min, 90min, 120min and 150min at 70 ℃ by using 30 times of water, concentrating and drying filtrate, and obtaining crude extract.
The content of verbascoside in different extraction time is measured, and the result shows that the content of verbascoside extracted for 30min, 60min, 90min, 120min and 150min is respectively 7.25%, 7.71%, 8.13%, 8.22% and 8.49%, 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 the most preferable method is 90min for saving the cost.
Example 10
This example is intended to examine the effect of the number of water extractions on the amount of verbascoside extracted from paulownia leaves:
ultrasonic extracting 0.5g of Paulownia leaf with 30 times of water at 70 deg.C for 1, 2, and 3 times, each for 2 hr, concentrating the filtrate, and drying to obtain crude extract.
The content of verbascoside obtained by different extraction times is 7.25%, 10.35%, 10.54% respectively after 1, 2, and 3 times. Thus, the content of verbascoside extracted gradually increases with the increase of the extraction times, but the increase of the extraction content is not significant in the case of 3 extraction times, so that two extractions may be selected to appropriately increase the content of verbascoside extracted when water is used as the extraction solvent.
Example 11
This example investigates the effect of the type of macroporous adsorbent resin on the verbascoside content in the resulting verbascoside-enriched fraction:
100mL of the crude extract solution of the paulownia leaves is respectively added into conical flasks filled with 2.0g of the processed AB-8, DM-301, DA-201, D-101, HPD100 and DM-130 macroporous absorbent resins. The mixture was subjected to shaking adsorption at 25 ℃ for 6 hours, 1mL of the supernatant was collected every 0.5 hour, and the adsorption rate [ adsorption rate = (mass concentration before adsorption-mass concentration after adsorption)/mass concentration before adsorption ] was calculated by HPLC measurement. And (3) washing the adsorbed resin with a proper amount of water, adding 50mL of 50vol% ethanol, carrying out constant-temperature oscillation desorption, taking 1mL of supernatant, and adding 10vol% methanol to reach 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 macroporous adsorbent resins can achieve better separation and purification effects, but compared with the AB-8 type macroporous adsorbent resin, D-101 has better adsorption and desorption effects on acteoside. In addition, compared with AB-8 type macroporous adsorption resin, the D-101 macroporous adsorption resin has the advantage of lower price.
TABLE 2
| Resin type | Adsorption Rate (%) | Desorption ratio (%) |
| 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 this example, the influence of the mass concentration of the sample liquid on the content of verbascoside in the obtained enriched part of verbascoside in the process of separating and purifying crude extract by macroporous adsorption resin is examined:
precisely weighing 3 parts of the treated macroporous adsorption resin (40 g of each part) and respectively filling the obtained product into a column by a wet method. Sample solutions with the mass concentrations of the sample solutions of 15mg/mL, 25mg/mL and 35mg/mL (obtained by dilution of mother liquor) are respectively passed through the macroporous resin column at the same volume flow rate (2 BV/h). After completion of the adsorption, the adsorption rate [ adsorption rate = (mass concentration before adsorption-mass concentration after adsorption)/mass concentration before adsorption ] was calculated by HPLC measurement.
As a result, the adsorption rates of the sample solutions were 72.44%, 73.32%, and 72.56%, respectively, at sample concentration of 15mg/mL, 25mg/mL, and 35 mg/mL. When the mass concentration is 25mg/mL, the adsorption rate reaches the maximum value.
Example 13
In this example, the effect of the eluent on the content of verbascoside in the enriched portion of verbascoside obtained in the process of separating and purifying crude extract by macroporous adsorption resin is examined:
precisely weighing 4 parts of the treated macroporous adsorption resin (40 g of each part), respectively loading the macroporous adsorption resin on a column by a wet method (a loading solution is a crude extract solution with the concentration of 25 mg/mL), eluting by using 3BV of distilled water to remove impurities, respectively eluting by using ethanol solutions with the volume fractions of 10%, 30%, 50% and 70% at the volume flow of 2BV/h, and measuring by using HPLC to calculate the desorption rate [ desorption rate = mass concentration after desorption/(mass concentration before adsorption-mass concentration after adsorption) ].
The results show that when the elution is carried out by ethanol solutions with the volume fractions of 10%, 30%, 50% and 70%, the desorption rates of the elution are respectively 17.25%, 41.63%, 98.16% and 98.58%. The results show that the ethanol with the volume fraction of 10 percent and 30 percent can not be completely eluted, the ethanol with the volume fraction of 50 percent and 70 percent can almost completely elute the verbascoside, but the 70 percent ethanol can elute the verbascoside, and simultaneously, the impurities are increased and the cost is increased. Taken together, 50vol% ethanol is preferred as eluent.
Example 14
In this example, the influence of the amount of eluent on the content of verbascoside in the enriched portion of verbascoside obtained in the process of separating and purifying crude extract by macroporous adsorption resin is examined:
weighing 40g of the treated macroporous adsorption resin, loading the macroporous adsorption resin into a column by a wet method (loading solution is 25mg/mL of crude extract solution), eluting by using 3BV of distilled water to remove impurities, eluting by using 50vol% ethanol, and collecting 1 part of the effluent per 1BV (20 mL). The elution rate (elution rate = mass eluted/mass before elution) was calculated by HPLC measurement.
The elution rates at 1BV, 2BV, 3BV, 4BV, 5BV, 6BV of eluent were 29.49%, 43.49%, 21.36%, 4.45%, 0.42% and 0.11%, respectively. The results show that more than 98% of verbascoside can be eluted when the elution is carried out to 3-4.0 BV, and the elution dosage of 50vol% ethanol is preferably 3-4.0 BV, more preferably 4.0BV, considering the cost comprehensively.
Example 15
In this example, the influence of the volume flow of the eluent on the content of verbascoside in the enriched portion of verbascoside obtained in the process of separating and purifying crude extract by using macroporous adsorption resin is examined:
weighing 40g of treated D-101 type macroporous adsorption resin, loading the treated D-101 type macroporous adsorption resin into a column by a wet method, eluting by using 3BV of distilled water to remove impurities, eluting by using 50vol% ethanol with 3BV at a volume flow rate of 1BV/h, 2BV/h, 3BV/h, 4BV/h, 5BV/h and 6BV/h respectively, and collecting the effluent for 1 time per 1 BV. The elution rate (elution rate = mass eluted/mass before elution) was calculated by HPLC measurement.
The elution rate when the volume of the eluent is 1BV/h, 2BV/h, 3BV/h, 4BV/h, 5BV/h and 6BV/h is respectively 97.78%, 95.32%, 94.69%, 91.53%, 89.77% and 87.41%. The result shows that the elution volume flow is 1BV/h and 2BV/h, and the elution rate is higher. If the elution effect and the time cost are comprehensively considered, the optimal volume flow of the eluent is 2BV/h.
Example 16
In this example, the method for extracting acteoside from paulownia leaves with water comprises the following specific steps:
placing 0.82kg of dried paulownia fortunei leaves in a 30L stainless steel barrel, adding 30L of purified water solution, and performing ultrasonic extraction at 70 ℃ for 2h. And after extraction is finished, filtering an extracting solution, adding 30L of purified water solution again for ultrasonic extraction, combining filtrates, concentrating and drying to obtain 74.3g of the crude extract of the paulownia leaves with the yield of 9.06%. High performance liquid detection shows that in the crude extract of the paulownia leaves extracted by water, the purity of the verbascoside is 10.052 percent.
Example 17
In this example, 50vol% ethanol water solution is used to extract verbascoside in paulownia leaves, and the specific steps are as follows:
placing 0.94kg of dried leaves of Paulownia fortunei in a 30L stainless steel barrel, adding 20L of 50vol% ethanol water solution, and performing ultrasonic extraction for 1h. After extraction is finished, the extracting solution is filtered to obtain 104.7g of the crude extract of the paulownia leaves, and the yield is 11.1%. High performance liquid chromatography detection shows that the purity of verbascoside in the 50vol% ethanol-extracted crude extract of paulownia leaves is 10.514%.
In the crude extracts obtained in comparative examples 1-2, the purity of verbascoside did not differ much. The subsequent extraction 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 with resin to obtain acteoside-enriched fraction, which was prepared by the following steps:
dissolving 55g of the crude extract of paulownia leaves obtained in example 1 in 2.2L of water, applying the solution to D101 macroporous adsorption resin, eluting with an aqueous solution, wherein the elution volume is 3 column volumes, the flow rate is 2 column volumes/hour, eluting with an ethanol aqueous solution with the volume fraction of 10%, the elution volume is 3 column volumes, the flow rate is 2 column volumes/hour, eluting with an ethanol aqueous solution with the volume fraction of 30%, the elution volume is 3 column volumes, the flow rate is 2 column volumes/hour, and finally eluting with an ethanol aqueous solution with the volume fraction of 50%, the elution volume is 4 column volumes, and the flow rate is 2 column volumes/hour. The enriched fraction of acteoside was obtained at a weight of 3.03g, with a yield of 5.51%. The purity of acteoside is 41.6% by high performance liquid detection.
The macroporous adsorption resin in the embodiment can achieve the same effect after being replaced by AB-8, DM-301, DA-201, HPD100, DM-130 and other types of resin.
Example 19
In this example, preparative liquid chromatography (LC-3000A preparative liquid phase in beijing family) was used to further separate and purify the enriched fraction of verbascoside, and the specific steps were as follows:
collecting the enriched part of acteoside obtained in example 18, subjecting to preparative liquid chromatograph to obtain acteoside collecting solution, vacuum concentrating at 70 deg.C until the solid-liquid mass volume ratio is 1 (4-5) g/mL, stopping concentrating, and drying to obtain acteoside solid. The high performance liquid detection and peak area normalization method show that the purity of acteoside is 98.4% as shown in FIG. 9.
Wherein the working conditions of the preparative liquid chromatograph are as follows:
a chromatographic column: YMC-Pack ODS-A (20 mm. Times.250mm, 5 μm);
mobile phase: methanol (B) -water (a) = (33, volume ratio), isocratic elution;
the detection wavelength is 334nm;
the flow rate of the mobile phase is 10mL/min;
the column temperature is 30 ℃;
tR＝25.0min；
sample introduction amount: 20 μ 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 (10)
1. A method for extracting verbascoside from paulownia leaves is characterized by comprising the following steps:
(1) Extracting paulownia leaves to obtain a crude extract;
(2) Separating and purifying the crude extract obtained in the step (1) by adopting macroporous adsorption resin to obtain a verbascoside enrichment part;
(3) Performing liquid chromatography separation on the verbascoside enriched part obtained in the step (2) to obtain verbascoside collecting liquid, and then concentrating and drying to obtain verbascoside;
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% of methanol or 10-40 vol% of acetonitrile solution.
2. The method according to claim 1, wherein step (1) is specifically: extracting paulownia leaves by using one or more of water, methanol and ethanol as a solvent to obtain a crude extract.
3. The method according to claim 2, wherein the solvent is a 30 to 50vol% ethanol solution.
4. The method as claimed in claim 1, wherein 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.
5. The method according to claim 1, wherein the temperature of the extraction in step (1) does not exceed 70 ℃.
6. The method of claim 1, wherein the macroporous adsorbent resin 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.
7. The method of claim 1, wherein when the crude extract obtained in step (1) is separated and purified by using macroporous adsorption resin, the elution procedure is as shown in the following table:
elution procedure
。
8. The method according to claim 1, wherein the mobile phase in the liquid chromatography is 30 to 35vol% methanol or 20 to 25vol% acetonitrile.
9. The method of claim 1, wherein the flow rate of the mobile phase in the liquid chromatography separation is 2-15mL/min.
10. The method according to claim 1, wherein the detection wavelength in the liquid chromatography is 240 to 370nm.
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Non-Patent Citations (4)
| Title |
|---|
| CHUAN-LING SI，等: "Phenylpropanoid Glycosides of Paulownia coreana Uyeki Leaves", 《MOKCHAE KONGHAK》, vol. 34, no. 2, 31 December 2006 (2006-12-31), pages 78 - 82 * |
| JIN-KYU KIM，等: "Phenylpropanoid glycosides from the leaves of Paulownia coreana", 《NATURAL PRODUCT RESEARCH》, vol. 22, no. 3, 15 February 2008 (2008-02-15), pages 241 - 245 * |
| 冯卫生，等: "泡桐花中糖苷类成分及其抗氧化活性", 《中成药》, vol. 42, no. 2, 29 February 2020 (2020-02-29), pages 369 - 374 * |
| 陈娟，等: "高效液相色谱法测定毛泡桐花中麦角甾苷(毛蕊花糖昔)的含量", 《药物分析杂志》, vol. 24, no. 2, 31 December 2004 (2004-12-31), pages 123 - 125 * |
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