CN110917234A - Method for extracting total resin glucoside from Evolvulus alsinoides and application thereof - Google Patents
Method for extracting total resin glucoside from Evolvulus alsinoides and application thereof Download PDFInfo
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Abstract
A method for extracting total resin glucoside from Evolvulus alsinoides and application thereof belong to the technical field of medicines. Extracting the whole Evolvulus alsinoides with a polar solvent, and performing silica gel and gel column chromatography sequentially after extraction to obtain the total resin glucoside extract of Evolvulus alsinoides. The preparation method is simple, the total resin glucoside part in the entire Evolvulus alsinoides herb is extracted, and the total resin glucoside part has obvious antitumor activity and can be used for preparing antitumor drugs.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a total resin glucoside extraction technology in Evolvulus alsinoides and an application technology in preparation of antitumor drugs.
Background
Evolvulus orientalis (Evolvulus alsinoidoids), also known as Potentilla anserine, is a plant of the genus Evolvulus of the family Convolvulaceae. The Tutingui is mainly distributed in Guangxi, Guangdong, Fujian and other places in China. According to records in the Chinese materia medica, the Evolvulus orientalis is used as a whole herb medicine and has the functions of dissipating blood stasis, relieving pain and clearing damp-heat. The plant has medicinal value in India, Central and south peninsula, Malaya and Philippine. Pharmacological research shows that the Evolvulus alsinoides have various biological activities of resisting tumor, oxidation, depression, dementia and the like. At present, the research on the components of the Evolvulus orientalis mainly focuses on chemical components such as alkaloid, flavone, steroid and the like, but the research cannot effectively explain the biological activity of the Evolvulus orientalis.
In earlier researches, the Evolvulus ambrosioides is found to contain a large amount of resin glucoside compounds. Such compounds typically comprise an oligosaccharide chain and a long-chain hydroxy fatty acid, the hydroxy fatty acid acting as an aglycone and forming a lactone with the oligosaccharide chain, with multiple small organic acids present as substituents on the oligosaccharide chain. Due to the variety and connection mode of glycosyl, the change of the position of lactone bond and the position of substituent group, the structure of the resin glucoside compound is rich and diverse. In addition, the resin glucoside compounds have a plurality of significant biological activities, including anti-tumor effect, vasodilation, sedation, anti-depression, antibiosis and the like, and are important active substances.
The resinoid glycosides separated from Evolvulus alsinoides exist in the form of two monomeric resinoid glycosides, and have cytotoxic activity. However, a large amount of resin glycoside compounds still exist in the Evolvulus alsinoides, and the separation difficulty of the monomer resin glycoside compounds is larger.
Although chinese patent document CN106361798A discloses a method for extracting resin glycoside compounds, the method is mainly directed to resin glycoside components with low polarity and capable of being dissolved by dichloromethane, and is not suitable for extraction and separation of resin glycoside components in cinnamomum burmannii.
Researches show that the total resin glucoside part of the Evolvulus alsinoides has higher polarity and obvious antitumor activity.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for extracting total resinoside from Evolvulus alsinoides in order to obtain a corresponding total resinoside extract.
The technical scheme of the invention comprises the following steps:
a) pulverizing Evolvulus alsinoides, extracting with alcohol or alcohol water solution as extraction solvent by cold soaking, infiltration, ultrasonic or hot reflux extraction to obtain extractive solution, and concentrating under reduced pressure to obtain concentrated solution of the extractive solution;
b) dispersing the concentrated solution of the extracting solution in water, sequentially extracting with ethyl acetate and n-butanol to obtain n-butanol extract, and recovering under reduced pressure to obtain extract;
c) performing column chromatography on the extract by using silica gel, eluting by using a mixed solution of chloroform and methanol or a mixed solution of dichloromethane and methanol, collecting eluent, and concentrating to obtain an eluent extract;
d) separating the eluate extract with gel adsorption resin column, eluting with methanol, and collecting the eluted liquid phase to obtain total glycoside extract of Evolvulus alsinoides.
The invention establishes the method for extracting and enriching the total resin glycoside components in the Evolvulus alsinoides, and the method has the advantages of simple operation, mild condition and lower cost. Researches find that the total glycoside part of the Evolvulus alsinoides has obvious antitumor activity and can be used for preparing antitumor drugs.
The total resin glycoside extract of Evolvulus alsinoides is extracted from Chinese medicinal Evolvulus alsinoides, which is the whole herb of Evolvulus alsinoides of Evolvulus of Convolvulaceae.
In the above method, in the step a), the alcohol is ethanol or methanol. The ethanol and the methanol have larger polarity, and can more fully extract the resin glycoside components with large polarity in the Evolvulus alsinoides. In addition, the ethanol and the methanol have low price, and are beneficial to industrial production.
In the step c), the mixing volume ratio of chloroform to methanol in the mixed solution of chloroform and methanol is 2-10: 1. More preferably, the mixing volume ratio of chloroform to methanol is 2: 1. Because the polarity of the resin glucoside components in the Evolvulus alsinoides is higher, the resin glucoside components can be more fully enriched by adopting a high-polarity mixed solvent.
In the step c), the mixing volume ratio of the dichloromethane and the methanol in the mixed solution of the dichloromethane and the methanol is 2-10: 1. More preferably, the mixing volume ratio of dichloromethane to methanol is 2: 1. Because the polarity of the resin glucoside components in the Evolvulus alsinoides is higher, the resin glucoside components can be more fully enriched by adopting a high-polarity mixed solvent.
The invention also provides the application of the total resin glucoside.
Namely, the total resin glucoside prepared by the method is used for preparing the antitumor drugs.
The total glucosides of the Evolvulus alsinoides have obvious antitumor activity, so the invention also provides the application of the total glucosides of the Evolvulus alsinoides extracted from the Evolvulus alsinoides in preparing antitumor drugs.
Drawings
FIG. 1 shows the total resinoside extract obtained by the inventive method1H nuclear magnetic resonance spectrum.
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.
Firstly, preparing the total resin glucoside extract of the Evolvulus alsinoides.
1. Taking 100g of dried Evolvulus alsinoides herb, crushing, cold soaking and infiltrating by adopting 300mL of 95% (v/v) ethanol water solution, extracting for 3 hours by ultrasonic or hot reflux, and filtering to respectively obtain an extracting solution and dregs of a decoction.
Methanol may be used as the above ethanol.
Cold soaking the residue in 300mL 95% (v/v) ethanol water solution, infiltrating, extracting under ultrasonic or hot reflux for 3 hr, and filtering to obtain extractive solution and residue.
Mixing the two extractive solutions, and vacuum concentrating to remove alcohol smell to obtain concentrated solution.
2. Extracting with 500mL ethyl acetate for 3 times, extracting with 500mL n-butanol for 3 times, recovering extractant to obtain n-butanol extractive solution, and recovering under reduced pressure to obtain extract.
3. And (3) performing column chromatography on the extract by using silica gel, and sequentially using a mixed solution of dichloromethane and methanol with the mixing volume of 10:1, 5:1 and 2:1, or a mixed solution of chloroform and methanol as an eluent.
Collecting the eluent obtained by mixing dichloromethane and methanol in a volume ratio of 2:1, and concentrating to obtain an eluent extract for later use.
4. Separating the eluate with gel adsorption resin column, eluting with methanol, mixing the eluates, collecting the eluates, and concentrating under reduced pressure to obtain 0.378g total glycoside resin extract of Evolvulus alsinoides.
5. Extracting cortex Cinnamomi total resin glucoside extract1H nuclear magnetic resonance spectrum analysis:
as shown in figure 1 of the drawings, in which,1in an H NMR spectrogram, signals are mainly concentrated into a sugar residue signal section between 3.4 and 6.5, a fat chain residue signal section between 0.6 and 2.2 and a methylene signal section of a lactone ring between 2.5 and 3.0, and the signals are in accordance with the signal distribution of the resin glycoside components, so that the main component in the extract is the resin glycoside components.
And II, performing qualitative test on the resin glucoside compound.
1. Test subjects:
the product obtained in the above example.
2. The test method comprises the following steps:
the products obtained in the above examples were tested for the detection of glycosides, flavonoids, alkaloids and anthraquinones.
2.1. Glycoside recognition reaction:
to the product obtained in the above example, the Molish reagent was added.
2.2. And (3) flavone identification reaction:
to a solution of the product obtained in the above example in methanol was added a small amount of magnesium powder, and several drops of concentrated hydrochloric acid were added dropwise.
2.3. And (3) alkaloid identification reaction:
to the product obtained in the above example was added a bismuth potassium iodide reagent.
2.4. Anthraquinone identification reaction:
to the product obtained in the above example was added a solution of magnesium acetate in methanol.
3. And (3) test results:
3.1. glycoside recognition reaction:
the Molish reaction, the negative control solution has no purple red ring, the glucose positive control solution has purple red ring, the product obtained in the above example has purple red ring.
The product was confirmed to contain a glycoside compound.
3.2. And (3) flavone identification reaction:
the reaction of the hydrochloric acid and the magnesium powder shows that the reaction of the rutin positive control solution is red, the reaction of the rutin negative control solution is not red, and the reaction of the product solution obtained in the above embodiment also does not generate red.
It was demonstrated that the product obtained in the above example does not contain flavonoids.
3.3. And (3) alkaloid identification reaction:
no precipitate is generated in the reaction of bismuth potassium iodide and the negative control solution, brown yellow precipitate is generated in the reaction of berberine hydrochloride as the positive control solution, and no precipitate is generated in the reaction of the product obtained in the above embodiment.
It was demonstrated that the product obtained in the above example does not contain alkaloid compounds.
3.4. Anthraquinone identification reaction:
the magnesium acetate reacts, the negative control solution has no orange-red color, the emodin positive control solution reacts to present orange-red color, and the product reaction obtained in the above embodiment also has no orange-red color.
It was confirmed that the products obtained in the above examples did not contain anthraquinones.
According to the qualitative analysis, the product purified by the process method provided by the invention contains the resinoid glucoside compounds and does not contain flavonoid, alkaloid and anthraquinone compounds with similar properties, which indicates that the product purified by the process method is the resinoid glucoside compounds and has higher purity.
And thirdly, testing the anti-tumor activity.
The cytotoxic activity of the total cinnamomum subavenium resin glucoside extract on human tumor cells is determined by an MTT method. Taking a proper amount of the total cinnamomum subavenium resin glucoside extract obtained in the example 1, dissolving the extract in DMSO, and respectively measuring the inhibition rates of human non-small cell lung cancer A549 cells, human liver cancer Bel-7402 cells, human cervical cancer Hela cells and human breast cancer MCF-7 cells by using an MTT method.
The experimental method comprises the following steps: a549, Bel-7402, Hela and MCF-7 cells in good state are inoculated in a 96-well plate, 90 mu L of culture medium is added in each well, after 12 hours of culture at 37 ℃, compounds to be tested (1.5625-50 mu g/mL) with different concentrations are added, and 3 parallel wells are arranged in each concentration. After 48h of incubation, adding 20 mu L (5mg/mL) of MTT into each hole, continuing to incubate for 4h, then discarding the culture medium, adding 150 mu L of DMSO into each hole, shaking and shaking the crystals uniformly, measuring the absorbance at 570nm by using an enzyme-labeling instrument, repeating the experiment for three times, taking the average value, and calculating the inhibition rate and the IC50 value under different concentrations. The experiment was repeated three times and the mean value was taken. The results are shown in the following table.
| Cell type | IC50(μg/mL) |
| A549 | 5.40 |
| Bel-7402 | 12.79 |
| Hela | 13.66 |
| MCF-7 | 15.93 |
As can be seen from the above table: the total cinnamomum subavenium resin glycoside extract obtained by the method has a good inhibition effect on various tumor cells, so that the total cinnamomum subavenium resin glycoside prepared by the method can be used for preparing antitumor drugs.
Fourthly, comparison test: the method for extracting the resin glycoside compounds disclosed in Chinese patent CN106361798A is adopted to treat the cinnamomum subavenium medicinal material.
The method comprises the following specific steps:
1. taking 100g of Evolvulus alsinoides, crushing to 20 meshes after crushing to obtain coarse powder, heating and refluxing for extraction by using chloroform-water mixed liquor, wherein the ratio of the amount of the chloroform-water mixed liquor to the material liquid of the coarse powder is 4mL:1g, heating and refluxing for extraction for 2 times, 3 hours each time, filtering, concentrating the extracting solution under reduced pressure, and freeze-drying to obtain a crude product A.
2. And (3) adding methanol into the crude product A to completely dissolve, wherein the liquid-solid ratio of the methanol to the crude product A is 4mL:1g, centrifuging for 15 minutes under the condition of 600 revolutions per minute, taking supernate, concentrating under reduced pressure, cooling and drying to obtain fine powder, adding chloroform into the fine powder for complete dissolution, wherein the liquid-solid ratio of chloroform to crude product A is 4mL:1g, centrifuging at 600 rpm for 10 minutes, and taking supernatant B.
3. And (3) carrying out activated carbon decoloration treatment on the supernatant B for 20 minutes, wherein the granularity of the activated carbon is 20 meshes, filtering, concentrating the filtrate under reduced pressure, and freeze-drying to obtain powder C.
4. And (2) performing silica gel column chromatography on the powder C, mixing the silica gel with the powder C in a weight ratio of 3:1 in the silica gel column chromatography, loading the silica gel into a column with the powder C in a weight ratio of 500:1 and the silica gel with the particle size of 100 meshes, eluting by using a chloroform-methanol mixed solution, collecting eluent, wherein the volume ratio of chloroform to methanol in the chloroform-methanol mixed solution is 5:1, concentrating the eluent under reduced pressure, performing gel chromatography after freeze drying, eluting by using methanol, collecting eluent in the volume of the first column and the second column, combining the eluent of the first column and the eluent of the second column, concentrating under reduced pressure, and freeze drying to obtain an.
5. And carrying out qualitative test on the obtained extract.
The test method comprises the following steps:
and adding a Molish reagent into the extract to perform a detection test of the glucoside compound.
The experimental results are as follows:
the negative control solution has no purple red ring, the glucose positive control solution reacts to generate the purple red ring, and the extract does not generate the purple red ring after Molish reaction.
The extract is proved to contain no glucoside compounds, namely no resin glucoside components.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. A method for extracting total resin glucoside from Evolvulus alsinoides is characterized by comprising the following steps:
a) pulverizing Evolvulus alsinoides, extracting with alcohol or alcohol water solution as extraction solvent by cold soaking, infiltration, ultrasonic or hot reflux extraction to obtain extractive solution, and concentrating under reduced pressure to obtain concentrated solution of the extractive solution;
b) dispersing the concentrated solution of the extracting solution in water, sequentially extracting with ethyl acetate and n-butanol to obtain n-butanol extract, and recovering under reduced pressure to obtain extract;
c) performing column chromatography on the extract by using silica gel, eluting by using a mixed solution of chloroform and methanol or a mixed solution of dichloromethane and methanol, collecting eluent, and concentrating to obtain an eluent extract;
d) separating the eluate extract with gel adsorption resin column, eluting with methanol, and collecting the eluted liquid phase to obtain total glycoside extract of Evolvulus alsinoides.
2. The method of claim l, wherein in step a), the alcohol is ethanol or methanol.
3. The method for extracting total resinoside from Evolvulus alsinoides of claim l, wherein in the step c), the mixing volume ratio of chloroform to methanol in the mixture of chloroform and methanol is 2-10: 1.
4. The method of claim 3, wherein in step c), the volume ratio of chloroform to methanol in the mixture of chloroform and methanol is 2: 1.
5. The method for extracting total resin glycosides from Evolvulus alsinoides of claim 1, wherein in the step c), the mixing volume ratio of dichloromethane to methanol in the mixed solution of dichloromethane and methanol is 2-10: 1.
6. The method of claim 5, wherein in step c), the volume ratio of dichloromethane to methanol in the mixture of dichloromethane and methanol is 2: 1.
7. The total resinoid glycosides prepared by the method of claim 1 are used for preparing antitumor drugs.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018002692A (en) * | 2016-07-08 | 2018-01-11 | ポーラ化成工業株式会社 | Growth hormone receptor expression improver and composition containing the same |
| CN108478619A (en) * | 2018-03-23 | 2018-09-04 | 南通大学 | Total Resin glycoside extract of field bindweed and preparation method thereof and medical usage |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018002692A (en) * | 2016-07-08 | 2018-01-11 | ポーラ化成工業株式会社 | Growth hormone receptor expression improver and composition containing the same |
| CN108478619A (en) * | 2018-03-23 | 2018-09-04 | 南通大学 | Total Resin glycoside extract of field bindweed and preparation method thereof and medical usage |
Non-Patent Citations (2)
| Title |
|---|
| BO-YI FAN等: "Evolvulins I and II, Resin Glycosides with a Trihydroxy Aglycone Unit from Evolvulus alsinoides", 《ORGANIC LETTERS》 * |
| DURAISAMY GOMATHI等: "Secondary metabolite credentials ofEvolvulus alsinoides by high performance thin layer chromatography(HPTLC) ", 《JOURNAL OF BIOMEDICAL RESEARCH》 * |
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