CN113999272A - Preparation method and application of melissoside - Google Patents
Preparation method and application of melissoside Download PDFInfo
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- CN113999272A CN113999272A CN202111274490.0A CN202111274490A CN113999272A CN 113999272 A CN113999272 A CN 113999272A CN 202111274490 A CN202111274490 A CN 202111274490A CN 113999272 A CN113999272 A CN 113999272A
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- C—CHEMISTRY; METALLURGY
- 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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- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
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- C07H17/07—Benzo[b]pyran-4-ones
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Abstract
The invention discloses a preparation method and application of melissoside. The method comprises the following steps: 1) decocting herba Clinopodii with 9-11 times of water for 1.5-2.5 hr for 1-3 times, concentrating the extractive solution, adding 95% ethanol, stirring, standing overnight, filtering, introducing macroporous resin, washing the column with 6-10 times of column volume of water, eluting with 50% ethanol, collecting eluate, and recovering ethanol to obtain extract; 2) separating the extract with silica gel column chromatography, gradient eluting with chloroform-methanol, sequentially separating and purifying with Sephadex LH-20 chromatographic column and MCI macroporous resin for 2-4 times. The application is the application of the melissoside in preparing the medicine for treating the non-alcoholic fatty liver disease; the preparation has the specific expression of protecting liver pathological injury, and can relieve lipid drop formation and lipid accumulation. The invention provides a novel compound with clear structure and high efficiency for treating the nonalcoholic fatty liver disease and develops a new medication way.
Description
Technical Field
The invention relates to medical application of a plant extract, in particular to a preparation method and application of melissoside.
Background
Non-alcoholic fatty liver disease (non-alcoholic fatty liver disease) is one of the most common chronic liver diseases in the world, and is a fat accumulation disease caused by various factors. The pathogenesis of nonalcoholic fatty liver disease is extremely complex and still in the hypothesis stage. At present, the second hit hypothesis is a widely accepted theory, the first hit is caused by abnormal accumulation of lipid, and the second hit is mainly caused by inflammatory reaction and lipid peroxidation stress.
Clinopodium chinensis (Benth.) O.Kuntze) is the dry aerial parts of Clinopodium chinensis (L.) DC. of Clinopodium, and mainly contains flavonoids, triterpenes, saponins, volatile oil, phenylpropanoids, steroids and other compounds, and melissoside (Didymin) belongs to nonvolatile substances. The melissoside is a typical dietary glucoside, is widely present in citrus fruits and bellflower, comprises citrus, bergamot, tangerine, oregano and the like besides the Clinopodium chinense, is considered to be a high-cost-performance, safe and effective oral drug, and does not generate toxicity to normal tissues. The melissoside has the characteristics of oxidation resistance, tumor resistance, inflammation resistance, cell protection, cardiovascular protection and the like, and is proved to be a potential treatment drug for tumors such as lung cancer, breast cancer, brain tumor and the like, and diseases such as neurodegenerative diseases, insomnia, cardiovascular complications and the like; furthermore, it has been reported that it has a liver-protecting effect. However, no report on the application of melissoside to the treatment of non-alcoholic fatty liver disease has been found yet.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method and application of melicitrin, providing a new method for preparing the melicitrin and providing new medical application of the melicitrin.
In order to solve the technical problems, the invention adopts the following technical scheme:
on one hand, the invention provides a preparation method of melissoside, which comprises the following steps:
1) decocting herba Clinopodii with 9-11 times volume of water for 1.5-2.5 hr for 1-3 times, concentrating the extractive solution, adding 95% ethanol, stirring, standing overnight, filtering, introducing macroporous resin, washing column with 6-10 times column volume of water, eluting with 50% ethanol, collecting eluate, and recovering ethanol to obtain extract;
2) separating the extract with silica gel column chromatography, gradient eluting with chloroform-methanol at a volume ratio of chloroform to methanol of 100:0-0:100, sequentially separating and purifying with Sephadex LH-20 chromatographic column and MCI macroporous resin for 2-4 times to obtain the final product.
On the other hand, the invention also provides the application of the melissoside in preparing the medicine for treating the non-alcoholic fatty liver disease.
Specifically, the expression of the medicine in the process of treating the nonalcoholic fatty liver disease comprises the following steps:
a. reducing liver damage;
b. reducing lipid droplet formation;
c. relieving lipid accumulation;
d. reduce ALT, AST, TC, TG and LDL contents, and increase HDL content.
The melissoside can be used alone, or used together with other medicines, or made into compound preparation together with other medicines when used for treating non-alcoholic fatty liver disease. When used alone, the medicine is a medicine containing the melissoside and pharmaceutically acceptable auxiliary materials. The pharmaceutically acceptable auxiliary materials refer to various conventional auxiliary materials required by the preparation of different dosage forms, such as diluents, adhesives, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, adsorbing materials and the like. The dosage form of the medicine can be granules, powder, tablets, capsules, pills, oral liquid, injection and the like.
The invention has the beneficial effects that:
the invention provides a preparation method of melissoside with high yield and high purity by taking Clinopodium chinense as a raw material, and further experiments show that the action mechanism of the melissoside is clear, the purpose of the melissoside in preparing a medicine for treating non-alcoholic fatty liver diseases is provided, a novel compound with clear chemical structure and high efficiency is provided for treating the non-alcoholic fatty liver diseases, and a novel medicine for treating the non-alcoholic fatty liver diseases is developed.
Drawings
FIG. 1 shows the effect of melissoside on the histopathology of the liver of rats with nonalcoholic fatty liver (oil red O, 200X).
In the figure: a1 is normal control group, A2 is model group, A3 is silibinin group, A4 is low-dose group of melissoside, A5 is medium-dose group of melissoside, and A6 is high-dose group of melissoside.
Detailed Description
The present invention will now be described with reference to the accompanying drawings, wherein the specific embodiments described herein are merely illustrative and explanatory of the invention and are not restrictive thereof.
Preparation of melissoside
Decocting herba Clinopodii 10kg with 10 times volume of water for 2 hr, repeating once, concentrating the extractive solution, adding 95% ethanol, stirring, standing overnight, filtering, introducing into macroporous resin, washing with 6-10 times column volume of water, eluting with 50% ethanol, collecting eluate, and recovering ethanol to obtain extract. Separating the extract with silica gel column chromatography, gradient eluting with chloroform-methanol at a volume ratio of chloroform to methanol of 100:0-0:100, sequentially separating and purifying with Sephadex LH-20 chromatographic column and MCI macroporous resin for 3 times to obtain white needle crystal compound (50 mg).
And performing spectral detection on the obtained white needle crystal compound, wherein the spectral detection data are as follows:
ESI-MS m/z：581[M+H]+。1H-NMR(600MHZ,DMSO-d6)δ：12.01(1H,s,5-OH),7.46(2H,d,J＝9.0Hz,H-2＇,6＇),6.98(2H,d,J＝9.0Hz,H-3＇,5＇),6.14(1H,d,J＝1.8Hz,H-8),6.13(1H,d,J＝1.8Hz,H-6),5.57(1H,dd,J＝12.6,2.4Hz,H-2),3.10(1H,dd,J＝17.4,12.6Hz,H-3b),2.78(1H,dd,17.4,2.4Hz,H-3a),3.77(3H,s,OMe),4.98(1H,t,J＝7.8Hz,H-1″),4.52(1H,br s,H-1″),1.09(3H,m,rha-Me)。13C-NMR(125MHz,DMSO-d6)δ:78.3(C-2)、41.8(C-3)、197.01(C-4)、163.0(C-5)、96.4(C-6)、165.1(C-7), 95.4(C-8), 162.5(C-9), 103.3(C-10), 130.4(C-1 '), 128.3(C-2 '), 113.9(C-3 '), 159.4(C-4 '), 113.9(C-5 '), 128.3(C-6 '), 55.2(OMe), 100.6(C-1 '), 72.9(C-2 '), 75.5(C-3 '), 70.7(C-4 '), 76.2(C-5 '), 66.0(C-6 '), 99.3(C-1 '), 69.6(C-2 '), 70.2(C-3 '), 68.3(C-5 '), 17.8(C-6 '). The above data indicate that the compound is melissoside.
Second, protection effect of melissoside on non-alcoholic fatty liver
1. Preparation, grouping and processing of animal models
Rat model 76 male SPF-grade rats (120. + -.20 g) were randomly divided into 2 groups, 14 in group I and 62 in group II after adaptive feeding for 1 week, the rats in group I were fed with basal diet, and the rats in group II were fed with high fat diet to prepare non-alcoholic fatty liver disease model and fed for 8 weeks. At the end of 8 weeks, 2 of the groups I and II were randomly selected and used as oil red O pathological staining to observe whether the molding was successful. After the success of model building is confirmed, 12 rats in group I are divided into normal groups, 60 rats in group II are divided into model groups, positive control groups (silibinin groups) and melissoside treatment groups (low, medium and high dose groups). Starting administration from successful modeling, continuing high fat diet to prevent reversion for other control groups except for normal group with basic diet, killing rats after 8 weeks, taking blood, centrifuging at 4000r/min for 5min, and collecting serum; taking out the liver and cleaning. The serum and liver tissue were stored in a freezer at-80 ℃ for further use.
2. Detecting the index
Firstly, oil red dyeing is carried out to observe the formation condition of lipid droplets;
② detecting the level of glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), total Triglyceride (TG), Total Cholesterol (TC), Low Density Lipoprotein (LDL) and High Density Lipoprotein (HDL).
3. Results of the experiment
(1) Melissoside reduces pathological changes in the liver
As shown in FIG. 1, the liver pathology of the normal rat showed only a small amount of red lipid droplets, while the liver cells of the model rat showed a large amount of red lipid droplets, indicating that the lipid level in the liver of the model rat was significantly higher than that of the normal control. After the melissa officinalis glycoside is taken for treating the liver, the formation of lipid droplets in liver cells of each administration group is obviously reduced, and the lipid level is obviously reduced.
(2) The melissoside can obviously reduce ALT, AST, TC, TG and LDL contents, and increase HDL content
The experimental results show that the serum contents of ALT, AST, TC, TG and LDL in the rat of the model group are obviously reduced, and the content of HDL is reduced (P <0.05) compared with the normal group. The dry melissa officinalis glycoside can obviously relieve the abnormal increase of ALT, AST, TC, TG and LDL contents caused by the stimulation of high fat feed, and improve the abnormal decrease of HDL. The results are shown in Table 1.
TABLE 1 Effect of Melissside on serum ALT, AST, TC, TG, LDL and HDL levels in non-alcoholic fatty liver rats
Note: compared with the normal control group, the composition has the advantages that,#P<0.05; compared with the model control group,*P<0.05。
4. conclusion
The melissa officinalis glycoside can obviously improve the non-alcoholic fatty liver induced by high-fat feed, relieve liver injury, reduce the formation of lipid droplets, relieve the accumulation of lipid and have obvious protective effect on the non-alcoholic fatty liver.
Claims (5)
1. The preparation method of the melissoside is characterized by comprising the following steps:
1) decocting herba Clinopodii with 9-11 times volume of water for 1.5-2.5 hr for 1-3 times, concentrating the extractive solution, adding 95% ethanol, stirring, standing overnight, filtering, introducing macroporous resin, washing column with 6-10 times column volume of water, eluting with 50% ethanol, collecting eluate, and recovering ethanol to obtain extract;
2) separating the extract with silica gel column chromatography, gradient eluting with chloroform-methanol at a volume ratio of chloroform to methanol of 100:0-0:100, sequentially separating and purifying with Sephadex LH-20 chromatographic column and MCI macroporous resin for 2-4 times to obtain the final product.
2. Application of melissoside in preparing medicine for treating non-alcoholic fatty liver disease is provided.
3. The use according to claim 2, wherein the manifestations of said medicament in the treatment of non-alcoholic fatty liver disease comprise:
a. reducing liver damage;
b. reducing lipid droplet formation;
c. relieving lipid accumulation;
d. reduce ALT, AST, TC, TG and LDL contents, and increase HDL content.
4. Use according to claim 2, characterized in that: the medicine contains melissoside and pharmaceutically acceptable auxiliary materials.
5. Use according to claim 2, characterized in that: the dosage form of the medicine comprises granules, powder, tablets, capsules, pills, oral liquid or injection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112851729A (en) * | 2020-12-31 | 2021-05-28 | 涟源康麓生物科技有限公司 | Method for separating trifolioside from hesperidin waste liquid |
| CN116726034A (en) * | 2023-07-20 | 2023-09-12 | 山东大学齐鲁医院 | Application of compound as SIRT1 agonist and in preparation of medicament for improving metabolic-related fatty liver disease |
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2021
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112851729A (en) * | 2020-12-31 | 2021-05-28 | 涟源康麓生物科技有限公司 | Method for separating trifolioside from hesperidin waste liquid |
| CN116726034A (en) * | 2023-07-20 | 2023-09-12 | 山东大学齐鲁医院 | Application of compound as SIRT1 agonist and in preparation of medicament for improving metabolic-related fatty liver disease |
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