CN106176873B - Capsella bursa-pastoris total alkaloid extract and preparation method and application thereof - Google Patents

Capsella bursa-pastoris total alkaloid extract and preparation method and application thereof Download PDF

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CN106176873B
CN106176873B CN201610650471.6A CN201610650471A CN106176873B CN 106176873 B CN106176873 B CN 106176873B CN 201610650471 A CN201610650471 A CN 201610650471A CN 106176873 B CN106176873 B CN 106176873B
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shepherd
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马勤阁
魏荣锐
高丽
桑志培
柳文敏
宋忠雯
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Nanyang Normal University
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2236/333Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50Methods involving additional extraction steps
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50Methods involving additional extraction steps
    • A61K2236/55Liquid-liquid separation; Phase separation

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Abstract

The invention provides a preparation method of a shepherd's purse total alkaloid extract, which comprises the following steps: (1) pulverizing aerial parts of dried herba Capsellae, extracting with ethanol under reflux, collecting extractive solution, and concentrating to obtain extract; (2) adding HCl into the extract, stirring to obtain uniform dispersion liquid, and filtering to obtain filtrate; (3) adjusting the pH of the filtrate to be 8-10, extracting with chloroform, mixing the extracts, and concentrating to obtain chloroform extract; (4) subjecting the chloroform extract to silica gel column chromatography, and gradient eluting with petroleum ether-ethyl acetate mixture at volume ratio of 10:1 → 6:1 → 4: 1; (5) concentrating petroleum ether-ethyl acetate elution fraction at a volume ratio of 6:1, loading on Sephadex LH-20 gel adsorption resin column chromatography, eluting with methanol, and collecting elution fraction to obtain herba Capsellae total alkaloid extract. The extract has good effects of protecting liver and reducing blood fat, and the preparation method is simple.

Description

Capsella bursa-pastoris total alkaloid extract and preparation method and application thereof
Technical Field
The invention belongs to the technical field of traditional Chinese medicine extracts, and particularly relates to a shepherd's purse total alkaloid extract and a preparation method and application thereof.
Background
Hepatitis b is a well-known infectious disease that presents a significant health hazard to humans. Hepatitis b is an infectious disease caused by Hepatitis B Virus (HBV), mainly as a hepatitis virus and eventually leading to multiple organ damage, and even some patients may turn to cirrhosis or liver cancer. At present, China is a high-development country of hepatitis B virus carriers, and according to statistics, the number of asymptomatic hepatitis B virus carriers in China is 1.3 hundred million, and the number of hepatitis B patients is 3000 ten thousand. Approximately 23.7 million people die each year from hepatitis b-related diseases, of which 15.6 die from liver cancer. Therefore, the preparation of a novel hepatitis B treatment medicament with remarkable curative effect and small toxic and side effects as soon as possible is an important task to be solved urgently by the medical scientists in China at present.
Meanwhile, with the improvement of living standard of people, the eating quality and habits of many people are changed, the number of people suffering from hyperlipidemia is increased year by year, hyperlipidemia becomes a global disease, and the disease trend is more and more younger, so that the prospect is worried. In addition, hyperlipidemia can cause complications such as coronary heart disease and atherosclerosis, which have serious threats to the physical and mental health of patients. Therefore, the research and development of the novel hypolipidemic drug have great social and economic benefits.
At present, the drugs related to the treatment of hepatitis B and hyperlipidemia in the market are mainly chemical synthetic drugs. It is known that the long-term administration of these chemicals will cause great harm to human body. It is appreciated that natural Chinese herbs have unique advantages in this respect. The shepherd's purse, a wild vegetable in folk, is very rich in nutrient substances and is deeply favored by people. According to the principle of homology of medicine and food, the capsella bursa-pastoris has the advantages of greenness, naturalness, small toxic and side effects, safety, reliability and the like in the aspect of medicine development. Modern pharmacological research shows that the shepherd's purse has the effects of resisting aging, inflammation and cancer, reducing blood pressure and the like. Researches also find that the shepherd's purse mainly contains chemical components such as flavonoids, organic acids, alkaloids and the like. At present, people do not deeply research the chemical components of the shepherd's purse, and particularly, other aspects of liver protection and blood fat reduction are not reported. Therefore, the shepherd's purse has a great research space in the aspect of drug development, the effective components of the shepherd's purse need to be further excavated, the relation between the pharmacological activity and the effective components is determined, and an important theoretical basis is laid for the human beings to find new drugs with liver protection activity and blood fat reduction from natural plants.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a shepherd's purse total alkaloid extract which has good liver protection and blood fat reduction effects.
The invention provides a preparation method of a shepherd's purse total alkaloid extract, which comprises the following steps:
(1) pulverizing aerial parts of dried herba Capsellae, extracting with ethanol under reflux, collecting extractive solution, and concentrating under reduced pressure at low temperature to obtain extract;
(2) adding HCl into the extract obtained in the step (1), stirring to obtain uniform dispersion liquid, standing, and filtering to obtain filtrate;
(3) adjusting the pH of the filtrate obtained in the step (2) to be 8-10, extracting with chloroform until no alkaloid reaction exists, combining the extract solutions, and concentrating to obtain a chloroform extract;
(4) performing silica gel column chromatography on the chloroform extract obtained in the step (3), and performing gradient elution by using petroleum ether-ethyl acetate mixed liquor with the volume ratio of 10:1 → 6:1 → 4:1 in sequence to obtain elution parts;
(5) concentrating the fraction eluted with petroleum ether-ethyl acetate with the volume ratio of 6:1 obtained in the step (4), loading on Sephadex LH-20 gel adsorption resin column chromatography, eluting with methanol with the mass fraction of 85-95%, and collecting the eluted fraction to obtain the capsella bursa-pastoris total alkaloid extract.
Preferably, the ethanol reflux extraction in the step (1) is heating reflux extraction for 2 to 3 times with ethanol with the mass percent of 80 to 95 percent, and each time lasts for 1.5 to 2.5 hours.
Preferably, the volume ratio of the extract to HCl in the step (2) is 1: 1; the mass percent of HCl is 1.0-2.0%.
Preferably, the standing time in the step (2) is 24 to 30 hours.
Preferably, the step (5) further comprises concentrating and drying under reduced pressure after collecting the eluted fraction.
The invention also provides the shepherd's purse total alkaloid extract prepared by the method.
The invention also provides application of the shepherd's purse total alkaloid extract in preparing liver-protecting medicines or foods.
The preparation method comprises the following steps.
In the preparation of food, the shepherd's purse total alkaloid extract is added into food raw materials and prepared according to a conventional method.
The invention also provides application of the shepherd's purse total alkaloid extract in preparation of blood fat reducing medicines or foods.
The invention also provides a medicament, the effective component of which is the shepherd's purse total alkaloid extract of claim 6.
Preferably, the composition also comprises medically acceptable auxiliary materials.
The invention has the advantages that the shepherd's purse with dual purposes of medicine and food' is selected as a raw material source, the preparation method is simple and convenient, the raw materials are saved, the preparation process is easy to control, the yield is high, the production process is green and environment-friendly, can be circularly carried out, is suitable for batch production, and has good effects of protecting liver and reducing blood fat.
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The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a flow chart of the preparation method of the shepherd's purse total alkaloid extract of the present invention.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. 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.
The preparation method of the shepherd's purse total alkaloid extract comprises the following steps:
(1) taking dried herba Capsellae aerial part as raw material, pulverizing to 0.5-1.0 cm, adding 80-95% ethanol by mass, and extracting under heating and refluxing for 2-3 times, each for 1.5-2.5 hr. Filtering, collecting filtrate, and concentrating under reduced pressure at low temperature (40-55 deg.C) to obtain extract;
(2) adding 1.0-2.0% of HCl (extract: HCl =1:1, V/V) in percentage by mass into the extract obtained in the step (1), stirring to obtain uniform dispersion, standing for 24-30 hours, and filtering to obtain filtrate for later use; namely, an acid-soluble alkali-precipitation method is adopted for extraction; the hydrochloric acid can completely dissolve the capsella bursa-pastoris total alkali;
(3) adjusting the pH of the filtrate obtained in the step (2) to 8-10 by using ammonia water, extracting by using chloroform until no alkaloid reaction exists, combining the extract liquor and concentrating to obtain a chloroform extract;
(4) performing silica gel (100-200 mesh) column chromatography on the chloroform extract obtained in the step (3), sequentially performing gradient elution by using petroleum ether-ethyl acetate mixed liquor with the volume ratio of 10:1 → 6:1 → 4:1 to obtain elution parts, and concentrating the elution parts into thick paste for later use;
(5) and (3) loading the petroleum ether-ethyl acetate elution fraction obtained in the step (4) in a volume ratio of 6:1 onto SephadexLH-20 gel adsorption resin column chromatography, eluting with 85-95% by mass of methanol, combining the obtained elution fractions, and concentrating and drying under reduced pressure to obtain the capsella bursa-pastoris total alkaloid extract with the extraction rate of 0.5-1%.
Tests prove that the shepherd's purse total alkaloid extract prepared by the method has the effects of protecting the liver and reducing the lipid activity.
Example 1
Pulverizing the above-ground part of dried herba Capsellae to 0.6 cm, extracting with 85% ethanol under reflux for 2 times (each for 1.5 hr), filtering, collecting filtrate, and concentrating under reduced pressure at 50 deg.C to obtain soft extract. Then adding 1.2% of HCl (extract: HCl =1:1, V/V) in percentage by mass, stirring to obtain a uniform dispersion, standing for 26 hours, and filtering to obtain a filtrate; adjusting pH of the filtrate to =8 with ammonia water, extracting with chloroform until no alkaloid reaction occurs to obtain extract, mixing the extracts, concentrating, and drying to obtain extract. Subjecting the extract to silica gel (100-; separating petroleum ether-ethyl acetate elution fraction with volume ratio of 6:1 on Sephadex LH-20 gel adsorption resin column chromatography, eluting with 85% methanol by mass, mixing the obtained elution fractions, and finally concentrating and drying under reduced pressure to obtain the shepherd's purse total alkaloid extract with extraction rate of 0.9%.
Example 2
Pulverizing the above-ground part of dried herba Capsellae to 0.8 cm, extracting with 90% ethanol under reflux for 2 times (2.0 hr each time), filtering, collecting filtrate, and concentrating under reduced pressure at 40 deg.C to obtain soft extract. Then adding 1.6% of HCl (extract: HCl =1:1, V/V) in percentage by mass, stirring to obtain a uniform dispersion, standing for 28 hours, and filtering to obtain a filtrate; adjusting pH of the filtrate to = 9 with ammonia water, extracting with chloroform until no alkaloid reaction occurs to obtain extract, mixing the extracts, concentrating, and drying to obtain extract. Subjecting the extract to silica gel (100-200 mesh) column chromatography, gradient eluting with petroleum ether-ethyl acetate mixed solution at volume ratio of 10:1 → 6:1 → 4:1 in sequence to obtain each eluting part, concentrating into soft extract, subjecting the petroleum ether-ethyl acetate eluting fraction at volume ratio of 6:1 to Sephadex LH-20 gel adsorption resin column chromatography, eluting with 90% methanol by mass percent, mixing the obtained eluting fractions, and concentrating and drying under reduced pressure to obtain herba Capsellae total alkaloid extract with extraction rate of 1%.
Example 3
Pulverizing the above-ground part of dried herba Capsellae to 1.0 cm, extracting with 95% ethanol under reflux for 3 times (2.5 hr each time), filtering, collecting filtrate, and concentrating under reduced pressure at 55 deg.C to obtain soft extract. Then adding 1.8% of HCl (extract: HCl =1:1, V/V) in percentage by mass, stirring to obtain a uniform dispersion, standing for 30 hours, and filtering to obtain a filtrate; adjusting pH of the filtrate to = 10 with ammonia water, performing alkaloid-free reaction with chloroform to obtain extract, mixing the extracts, concentrating, and drying to obtain extract. Subjecting the extract to silica gel (100-; separating petroleum ether-ethyl acetate eluate at volume ratio of 6:1 with Sephadex LH-20 gel adsorption resin column chromatography, eluting with 95% methanol by mass, mixing the eluates, concentrating under reduced pressure, and drying to obtain herba Capsellae total alkaloid extract with extraction rate of 0.6%.
Example 4
Pulverizing the above-ground part of dried herba Capsellae to 0.5 cm, extracting with 80% ethanol under reflux for 3 times (2.5 hr each time), filtering, collecting filtrate, and concentrating under reduced pressure at 45 deg.C to obtain soft extract. Then adding 1.0% of HCl (extract: HCl =1:1, V/V) in percentage by mass, stirring to obtain uniform dispersion, standing for 24 hours, and filtering to obtain filtrate; adjusting pH of the filtrate to = 10 with ammonia water, performing alkaloid-free reaction with chloroform to obtain extract, mixing the extracts, concentrating, and drying to obtain extract. Subjecting the extract to silica gel (100-; separating petroleum ether-ethyl acetate elution fraction with volume ratio of 6:1 on Sephadex LH-20 gel adsorption resin column chromatography, eluting with 90% methanol by mass, mixing the obtained elution fractions, and finally concentrating and drying under reduced pressure to obtain the shepherd's purse total alkaloid extract with extraction rate of 0.5%.
Example 5
Pulverizing the above-ground part of dried herba Capsellae to 0.8 cm, extracting with 85% ethanol under reflux for 3 times (2.0 hr each time), filtering, collecting filtrate, and concentrating under reduced pressure at 55 deg.C to obtain soft extract. Then adding 2.0% of HCl (extract: HCl =1:1, V/V) in percentage by mass, stirring to obtain uniform dispersion, standing for 28 hours, and filtering to obtain filtrate; adjusting pH of the filtrate to = 9 with ammonia water, performing alkaloid-free reaction with chloroform to obtain extractive solution, mixing the extractive solutions, concentrating, and drying to obtain extract. Subjecting the extract to silica gel (100-; separating petroleum ether-ethyl acetate elution fraction with a volume ratio of 6:1 on Sephadex LH-20 gel adsorption resin column chromatography, eluting with 85% methanol by mass, mixing the obtained elution fractions, and finally concentrating and drying under reduced pressure to obtain the shepherd's purse total alkaloid extract with an extraction rate of 0.85% -1%.
Example 6 application experiment of the Capsella bursa-pastoris total alkaloid extract of the invention
(ii) hepatoprotective Activity
(1) The influence of the shepherd's purse total alkaloid extract on the survival rate of HL-7702 cells is observed by an MTT method. The MTT method is called 3- (4, 5-dimethylthiazolidil-2-yl) -2,5-diphenyltetrazolium bromide, and is named as 3- (4, 5-dimethylthiazole-2) -2,5-diphenyl tetrazolium bromide, which is called as tetrazolium salt for short, and thiazole blue for short. MTT colorimetric assay is a common method for detecting cell growth and survival rate. It means that the amount of MTT crystal formed is directly proportional to the number of cells and metabolic activity within a certain range of cell number. Dimethyl sulfoxide (DMSO) was able to solubilize formazan crystals in cells, and the absorbance value A (OD value) was measured at 570nm by an enzyme-linked immunosorbent assay. And (4) judging the number of living cells according to the measured absorbance value, wherein the larger the OD value is, the stronger the cell activity is. Therefore, the change of the light absorption value of the tumor cells after the antitumor drug is added is measured, and the inhibition rate of the drug on the cells is calculated, so that the sensitivity degree of the tumor cells to the drug is presumed to know the action degree of the drug.
(2) Placing HL-7702 cells at 37 deg.C and containing 5% CO2The culture medium is DMEM (10% fetal bovine serum, 100U/mL penicillin and 100)μg/mL streptomycin, pH 7.2-7.4).
(3) Grouping experiments:
normal group: HL-7702 cells;
control group: D-GalN (D-galactosamine);
control group: a bicyclol;
sample group: the shepherd's purse total alkaloid extract prepared in embodiment 3 of the invention;
(3) dispersing the cells in a medium to a density of 1X 105Single cell per mLThe suspension was added to a 96-well plate at 0.1 mL per well. And changing a culture medium containing the test drug after 24 h of culture, wherein the concentration of the test drug in the culture medium is 50 mug/ml, and adding D-galactosamine (the final concentration is 50 mmol/L) for continuous culture 1 h after administration. After 24 hours after the administration, the medium containing MTT (0.5 mg/mL) in the serum-free medium was replaced, and after 4 hours of incubation, the medium was aspirated, dissolved by adding DMSO (150. mu.L/air) and the absorbance of each well was measured at 490 nm with shaking. Inhibition (%) = [ (OD)(sample set)- OD(control group))/(OD(Normal group)-OD(control group))]X 100. The results of the experiment are shown in Table 1.
TABLE 1 protective action of Capsella bursa-pastoris Total alkaloid extract on liver cell damage
Figure DEST_PATH_IMAGE002
Note: the sample group is shepherd's purse total alkaloid extract; the control group was bicyclic alcohol.ap<0.05。
The experimental results (table 1) show that the shepherd's purse total alkaloid extract has obvious liver protection effect, and the survival rate of the extract on injured liver cells is obviously higher than that of a positive control medicament bicyclol from the experimental data. Therefore, the total alkaloid extract has important guiding significance for developing a medicament with liver protection effect from ' dual-purpose medicine and food ' shepherd ' herb.
(II) lipid-lowering Activity
(1) Dissolving calcium chloride 0.294g in 0.2mol/L trihydroxymethyl aminomethane solution 40mL, adjusting pH to 8.1 with 1mol/L hydrochloric acid solution, and diluting with water to 100mL to obtain trihydroxymethyl aminomethane buffer solution (pH 8.1).
(2) Preparing the shepherd's purse total alkaloid extract into sample solutions with different concentrations by using a trihydroxymethyl aminomethane buffer solution; preparing 10mg of porcine pancreatic lipase into 1mg/mL enzyme solution by using a trihydroxymethyl aminomethane buffer solution; meanwhile, preparing 4-methylumbelliferone oleate into a 0.1mmol/L substrate solution by using a tris buffer solution; a 0.1mmol/L solution of sodium citrate was prepared using tris buffer and adjusted to pH =4.2 with HCl for use.
(3) Adding 30 mu L of the sample liquid obtained in the step (2) into 30 mu L of the enzyme solution, and fully and uniformly mixing; a further 60. mu.L of substrate solution was added, incubated at 25 ℃ for 20 minutes and the reaction was stopped with 110. mu.L of sodium citrate solution. The final concentrations of the samples to be tested are 600, 300, 150, 75, 37.5 and 18.8 mu g/mL respectively.
(4) The method comprises the steps of selecting a sample solution, an enzyme solution and a substrate solution to be mixed as a sample group, selecting the sample solution and the substrate solution to be mixed as a background group, selecting the enzyme solution and the substrate solution to be mixed as a control group, and selecting the substrate solution as a blank control group. The absorbance was measured at 320nm excitation light and 450nm emission light, respectively. Lipase inhibition of the compounds was then calculated according to the formula. The results are shown in Table 2. The lipase inhibition rate calculation formula is as follows: lipase inhibition% =1- (sample group-background group)/(control group-blank control group) × 100%. See table 2 for results.
TABLE 2 results of lipid-lowering Activity of Capsella bursa-pastoris Total alkaloid extract
Figure DEST_PATH_IMAGE004
The experimental data show that the shepherd's purse total alkaloid extract has an obvious inhibition effect on the porcine pancreatic lipase, and the lipase inhibition rate is as high as 90.24%. Therefore, the capsella bursa-pastoris total alkaloid extract has important significance for developing a medicament with lipid-lowering activity from 'medicinal and edible' capsella bursa-pastoris.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (6)

1. A preparation method of a shepherd's purse total alkaloid extract is characterized in that: the method comprises the following steps:
(1) pulverizing aerial parts of dried herba Capsellae, extracting with ethanol under reflux, collecting extractive solution, and concentrating under reduced pressure at low temperature to obtain extract;
(2) adding HCl into the extract obtained in the step (1), stirring to obtain uniform dispersion liquid, standing, and filtering to obtain filtrate;
(3) adjusting the pH of the filtrate obtained in the step (2) to be 8-10, extracting with chloroform until no alkaloid reaction exists, combining the extract solutions, and concentrating to obtain a chloroform extract;
(4) performing silica gel column chromatography on the chloroform extract obtained in the step (3), and performing gradient elution by using petroleum ether-ethyl acetate mixed liquor with the volume ratio of 10:1 → 6:1 → 4:1 in sequence to obtain elution parts;
(5) concentrating the petroleum ether-ethyl acetate elution fraction obtained in the step (4) in a volume ratio of 6:1, loading on Sephadex LH-20 gel adsorption resin column chromatography, eluting with 85-95% methanol by mass fraction, and collecting the elution fraction to obtain a shepherd's purse total alkaloid extract;
the ethanol reflux extraction in the step (1) is to heat and reflux extract for 2 to 3 times by using 80 to 95 mass percent of ethanol, wherein each time lasts for 1.5 to 2.5 hours;
the volume ratio of the extract to HCl in the step (2) is 1: 1; the mass percent of the HCl is 1.0-2.0%, and the standing time is 24-30 hours;
in the step (5), after the elution fraction is collected, the method further comprises the step of concentrating under reduced pressure and drying.
2. The shepherd's purse total alkaloid extract prepared by the method of claim 1.
3. The use of the shepherd's purse total alkaloid extract of claim 2 in preparing liver-protecting medicaments.
4. The application of the shepherd's purse total alkaloid extract of claim 2 in preparing a medicament for reducing blood fat.
5. A medicament, characterized by: the effective component of the shepherd's purse total alkaloid extract is the shepherd's purse total alkaloid extract of claim 2.
6. The medicament of claim 5, wherein: also comprises medically acceptable auxiliary materials.
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