CN110964126A - Radix ophiopogonis degradation extract and application thereof in preparation of cardiovascular disease prevention and treatment medicines - Google Patents
Radix ophiopogonis degradation extract and application thereof in preparation of cardiovascular disease prevention and treatment medicines Download PDFInfo
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Abstract
The invention discloses a radix ophiopogonis degradation extract with higher hypoglycemic activity. The preparation method comprises the following steps: adding water into the medicinal materials according to the liquid-material ratio of 5-20, extracting for 1-3 times, extracting for 0.5-2 hours each time, filtering, concentrating the filtrate to obtain an extract with the raw material content of 0.5-2 g/ml, adding ethanol until the alcohol content reaches 75-90%, precipitating and evaporating to dryness to obtain the crude ophiopogon japonicus polysaccharide; adopting Sevage method to remove protein and purify; adding water after purification, fully dissolving to a transparent state, adding 2mol/L acid according to the material-liquid ratio of 10:1, degrading for 4-6 h, adding an alkali solution to neutrality, performing ultrafiltration interception to obtain a degradation product with the molecular weight range of 500-2500, concentrating, and freeze-drying to obtain the product. Compared with the prior art, the extract has higher activity in the aspect of treating cardiovascular diseases, and is obviously superior to the ophiopogon japonicus crude polysaccharide and other ophiopogon japonicus small molecular polysaccharides obtained by the existing methods. The yield can reach more than 95 percent, and the method is simple and feasible and can be used for large-scale production.
Description
Technical Field
The invention relates to a radix ophiopogonis degradation extract and application thereof in preparing medicines for preventing and treating cardiovascular diseases.
Background
Radix Ophiopogonis is dried root tuber of Ophiopogon japonica (L.f) Ker-Gawl belonging to Liliaceae. The dwarf lilyturf tuber is one of the traditional yin-nourishing traditional Chinese medicines, has sweet and slightly bitter taste and slightly cold nature, has the efficacies of nourishing yin, promoting the production of body fluid, moistening lung and clearing heart, and is used for treating lung dryness, dry cough, consumptive disease, cough, body fluid injury, thirst, vexation, insomnia, internal heat, diabetes and constipation due to intestinal dryness. The active ingredients of radix Ophiopogonis mainly include steroid saponin, polysaccharide, homoisoflavonoids, etc.
Pharmacological and clinical trial researches show that the crude polysaccharide extracted from radix Ophiopogonis can effectively reduce the blood sugar level of experimental diabetic mice, reduce fasting blood sugar and postprandial 2h blood sugar level of type II diabetes patients, and improve insulin resistance.
The activity of the polysaccharide is mainly determined by molecular mass, and the polysaccharide with smaller molecular mass has higher activity and is easier to be absorbed and utilized by human bodies. The conventional extraction method of the ophiopogonpolysaccharide is a water extraction and alcohol precipitation method, and the obtained ophiopogonpolysaccharide has high molecular weight. The preparation of the small molecular ophiopogonpolysaccharide in the prior art adopts an ultrafiltration method, the polysaccharide with the molecular weight more than 10000 is filtered and removed, and the degradation of the polysaccharide changes the macromolecular polysaccharide into the small molecular polysaccharide, so that the polysaccharide can be fully utilized, and the activity is increased.
In order to obtain low-molecular-weight oligomeric ophiopogon japonicus polysaccharide with low molecular weight by a simple and easy method, a chemical degradation method is adopted to degrade polysaccharide molecules to obtain small-molecular polysaccharide with higher activity.
Disclosure of Invention
The invention aims to provide a radix ophiopogonis degradation extract with higher activity, and the preparation method is simple and easy to implement.
The radix ophiopogonis degradation extract is prepared by the following method, and comprises the following steps:
1. extracting the crude polysaccharide of the dwarf lilyturf tuber: adding water into the ophiopogon japonicus according to a liquid-material ratio of 5-20, extracting for 1-3 times, extracting for 0.5-2 hours each time, filtering, concentrating the filtrate to obtain an extract with the raw material content of 0.5-2 g/ml, adding ethanol to enable the alcohol content to reach 75% -90%, standing, taking the precipitate, and evaporating to dryness to obtain crude ophiopogon japonicus polysaccharide;
2. deproteinization: deproteinizing and purifying the ophiopogon japonicus crude polysaccharide by a Sevage method;
3. and (3) degradation: adding water into the ophiopogonpolysaccharide obtained in the step (2) to fully dissolve the ophiopogonpolysaccharide to a transparent state, adding 2mol/L acid according to the material-liquid ratio of 10:1 based on the mass of the ophiopogon root medicinal material, degrading for 4-6 h, adding NaOH solution to neutralize to neutrality, performing ultrafiltration to obtain degradation products with the molecular weight range of 500-2500, concentrating, and freeze-drying to obtain the ophiopogon root degradation extract.
Wherein, the process of the step 1 is preferably as follows: extracting radix Ophiopogonis with water for 2 times at a liquid-to-material ratio of 10 for 2 hr, filtering, mixing filtrates, concentrating the filtrate to obtain extract with raw material content of 1g/ml, adding ethanol to ethanol content of 80%, and standing overnight; filtering to remove supernatant, evaporating the precipitate to dryness to obtain crude radix Ophiopogonis polysaccharide.
The acid in the step 3 is preferably hydrochloric acid or sulfuric acid, and the degradation temperature is preferably 50-100 ℃, and the optimal degradation temperature is 50 ℃.
The preferable process in the step 2 is as follows: dissolving the crude ophiopogon root polysaccharide in distilled water, adding trichloromethane: and n-butanol (4: 1), stirring to fully dissolve, standing by a separating funnel, centrifuging the lower layer liquid for 10 minutes at 5000r/min by a high-speed centrifuge, removing precipitates, precipitating the clear liquid by ethanol, and drying to obtain the purified ophiopogonpolysaccharide.
Compared with the prior art, the invention has the following beneficial effects: the degradation extract has higher activity in the aspect of preventing and treating cardiovascular diseases, and experiments prove that: the radix ophiopogonis degradation extract has strong functions of reducing blood fat, clearing free radicals in vivo, preventing cell lipid from being oxidized and resisting atherosclerosis; is obviously superior to the ophiopogon japonicus polysaccharide and other ophiopogon japonicus polysaccharide with small molecules obtained by the prior method. The yield can reach more than 98 percent, and the method is simple and feasible and can be used for large-scale production.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1:
taking 5000 g of ophiopogon japonicus, adding water, decocting and extracting for 1 time, adding 20 times of water, decocting for 2 hours, filtering, combining the filtrates, concentrating the filtrate to obtain an extract with the raw material content of 1g/ml, adding ethanol until the ethanol content is 80%, standing overnight, precipitating, filtering and drying to obtain the ophiopogon japonicus crude polysaccharide. Deproteinizing the radix ophiopogonis crude polysaccharide solution by a Sevage method, precipitating with ethanol, and drying to obtain purified radix ophiopogonis polysaccharide; adding water into the purified ophiopogonpolysaccharide, fully dissolving to a transparent state, adding 500ml of 2mol/L sulfuric acid, adding NaOH solution for neutralizing to be neutral when degrading for 4 at 50 ℃, ultrafiltering degradation liquid to intercept degradation products with molecular weight less than 10000, concentrating ultrafiltrate by adopting a nanofiltration technology, and freeze-drying the concentrated solution to obtain the ophiopogon degraded extract.
Example 2:
taking 5000 g of ophiopogon japonicus, adding water, decocting and extracting for 2 times, adding 10 times of water, decocting for 2 hours, filtering, combining the filtrates, concentrating the filtrate to obtain an extract with the raw material content of 1.5g/ml, adding ethanol until the ethanol content is 85%, standing overnight, precipitating, filtering and drying to obtain the ophiopogon japonicus crude polysaccharide. Deproteinizing the radix ophiopogonis crude polysaccharide solution by a Sevage method, precipitating with ethanol, and drying to obtain purified radix ophiopogonis polysaccharide; adding water into the purified ophiopogonpolysaccharide, fully dissolving to a transparent state, adding 500ml of 2mol/L hydrochloric acid, adding NaOH solution for neutralizing to be neutral when degrading for 5 times at 70 ℃, ultrafiltering degradation liquid to intercept degradation products with molecular weight less than 10000, concentrating ultrafiltrate by adopting a nanofiltration technology, and freeze-drying the concentrated solution to obtain the ophiopogon degradation extract.
Example 3:
taking 5000 g of ophiopogon japonicus, adding water, decocting and extracting for 1 time, adding 20 times of water, decocting for 2 hours, filtering, combining the filtrates, concentrating the filtrate to obtain an extract with the raw material content of 1g/ml, adding ethanol until the ethanol content is 80%, standing overnight, precipitating, filtering and drying to obtain the ophiopogon japonicus crude polysaccharide. Deproteinizing the radix ophiopogonis crude polysaccharide solution by a Sevage method, precipitating with ethanol, and drying to obtain purified radix ophiopogonis polysaccharide; adding water into the purified ophiopogonpolysaccharide, fully dissolving to a transparent state, adding 500ml of 2mol/L sulfuric acid, adding NaOH solution for neutralizing to be neutral when degrading for 3 times at 80 ℃, ultrafiltering degradation liquid to intercept degradation products with molecular weight less than 10000, concentrating ultrafiltrate by adopting a nanofiltration technology, and freeze-drying the concentrated solution to obtain the ophiopogon degradation extract.
Example 4:
taking 5000 g of ophiopogon japonicus, adding water, decocting and extracting for 2 times, adding 5 times of water, decocting for 1 hour, filtering, combining the filtrates, concentrating the filtrate to obtain an extract with the raw material content of 0.5g/ml, adding ethanol until the ethanol content is 75%, standing overnight, precipitating, filtering and drying to obtain the ophiopogon japonicus crude polysaccharide. Deproteinizing the radix ophiopogonis crude polysaccharide solution by a Sevage method, precipitating with ethanol, and drying to obtain purified radix ophiopogonis polysaccharide; adding water into the purified ophiopogonpolysaccharide, fully dissolving to a transparent state, adding 500ml of 2mol/L hydrochloric acid, adding NaOH solution for neutralizing to be neutral when degrading for 1 at 100 ℃, ultrafiltering degradation liquid to intercept degradation products with molecular weight less than 10000, concentrating ultrafiltrate by adopting a nanofiltration technology, and freeze-drying the concentrated solution to obtain the ophiopogon degradation extract.
Example 5:
the polysaccharide content of radix Ophiopogonis degraded extract is determined by measuring polysaccharide content with sulfuric acid anthrone method
(1) Preparation of a standard solution:
taking 33mg of anhydrous glucose reference substance dried to constant weight at 105 ℃, precisely weighing, placing in a 100ml measuring flask, adding water to dissolve and dilute to scale, and shaking up to obtain the final product.
(2) Preparation of the Standard Curve
Preparation of a standard curve A turbid solution of 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml and 0.6ml is precisely measured and respectively placed in 10ml test tubes with scales and plugs, water is added to 2.0ml respectively, the test tubes are shaken evenly, 0.2% anthrone-sulfuric acid solution is slowly dripped into an ice water bath to the scales, the mixture is uniformly mixed, placed in the water bath for heat preservation for 10 minutes after being cooled, taken out, immediately placed in the ice water bath for cooling for 10 minutes, and taken out, and corresponding reagents are used as blanks. The absorbance was measured at a wavelength of 582nm by UV-visible spectrophotometry. And drawing a standard curve by taking the absorbance as a vertical coordinate and the concentration as a horizontal coordinate.
(3) Preparation of sample solution
Precisely weighing about 10mg of the dwarf lilyturf tuber degraded extract, adding water to dissolve the dwarf lilyturf tuber degraded extract, transferring the dwarf lilyturf tuber degraded extract into a 50ml volumetric flask, adding water to the scale mark, and shaking up.
(4) Sample assay
Precisely absorbing 0.1ml of sample solution, operating under the item of 'standard curve preparation', measuring the absorbance value of the sample, and calculating the content.
The content of sugar in the ophiopogon root degraded extract is measured, wherein the sugar content of example 1 is more than 98%, the sugar content of example 2 is 97%, and the sugar content of examples 3 and 4 is 95%.
Example 6:
and (3) measuring the molecular weight range of the radix ophiopogonis degradation extract: preparing 2 mu g/ml solution of each radix ophiopogonis degradation extract, taking 0.5ul sample solution and 5mg/ml matrix 2, 5-dihydroxy benzoic acid (DHB) to mutually dissolve and volatilize the solution, and performing MALDI-TOF-MASS sample injection detection, wherein the molecular weight range of the radix ophiopogonis degradation extract is between 500 and 2500.
Example 7:
effect of Ophiopogon japonicus degradation extract (example 4) on blood lipids of mice in each group
(1) Grouping animals
Healthy Kunming mice (SPF grade), male, 18-20 g (provided by southern medical university laboratory animal center), are randomly divided into a normal control group and a model building group, the model building group is established according to the following method, after the model building is successful, the model group animals are randomly divided into the model control group and a positive drug group, and a radix ophiopogonis crude polysaccharide group and a radix ophiopogonis degraded extract group with corresponding doses are arranged, and the administration mode is the same as that of the traditional Chinese medicine. The administration was continued for 4 weeks.
(2) The mouse model is prepared by adopting a high-fat feed feeding method, and an experimental mouse hyperlipidemia model is established. The normal group was fed with normal diet, and the other groups were fed with high-fat diet. All mice were fed freely and were weighed after fasting for 12h on days 14 and 28 of molding, blood was taken from the tail vein, and the TC content of fasting serum of the mice was determined, and the success of molding was regarded as the TC value of the mice being greater than 7 mmol.L-1.
(3) Compared with a model group, the influence on the blood fat of the mice with hyperlipidaemia is that TC, TG and LDL-C of the dwarf lilyturf tuber degraded extract are obviously reduced, and HDL-C is obviously increased (P is less than 0.01). The effect of the radix ophiopogonis degradation extract on reducing blood fat is superior to that of the radix ophiopogonis crude polysaccharide group. See table 1.
TABLE 1 Effect on blood lipids of hyperlipidemic mice
Note:*P<0.05vs model set**P<0.01vs model set
(4) Influence on oxidation resistance and arteriosclerosis index of hyperlipidemic mice
Compared with the model group, the radix ophiopogonis degraded extract can obviously reduce the AI value and the MDA content, the HDL-C/TC ratio and the SOD activity are obviously improved (P is less than 0.01), and the radix ophiopogonis degraded extract group is superior to the radix ophiopogonis crude polysaccharide group. The results are shown in Table 2.
TABLE 2 Effect on antioxidant capacity and arteriosclerosis index in hyperlipidemic mice
Note: model set P <0.05 vs.. model set P <0.01 vs. model set
After continuous administration for 4 weeks, the mouse serum TC, TG and LDL-C of the radix ophiopogonis degraded extract are obviously reduced compared with the model group, and HDL-C is obviously increased, which indicates that the radix ophiopogonis degraded extract has better blood fat reducing effect, and the radix ophiopogonis degraded extract has more obvious effects on reduction of TC, TG, LDL-C and increase of HDL-C than the radix ophiopogonis crude polysaccharide group, which indicates that the radix ophiopogonis degraded extract has stronger blood fat reducing effect.
SOD can effectively eliminate free radicals in vivo, and has effects of preventing lipid peroxidation activity on cell biomembrane. MDA is the end product of peroxidation of free radicals on lipids. The SOD activity of the radix ophiopogonis degraded extract group is obviously improved compared with that of the model group, and the MDA content is obviously reduced, which shows that the radix ophiopogonis degraded extract has obvious antioxidation. The effect of the radix Ophiopogonis degrading extract group on increasing SOD activity and reducing MDA content is stronger than that of the radix Ophiopogonis crude polysaccharide group. An increase in HDL-C/TC is indicative of a reduced atherosclerotic condition and protective effects on the arterial wall, while higher AI's increase the incidence of atherosclerosis and coronary heart disease. The HDL-C/TC value of the radix ophiopogonis degraded extract group in the experiment is obviously increased compared with that of a model group, and the AI value is obviously reduced, so that the radix ophiopogonis degraded extract has better relieving and preventing effects on related diseases caused by hyperlipidemia, and the effect of the radix ophiopogonis degraded extract is better than that of the radix ophiopogonis crude polysaccharide group.
In conclusion, the experiment proves that the ophiopogon root degraded extract has better prevention and treatment effects on experimental hyperlipidemia, and the ophiopogon root degraded extract group is superior to the ophiopogon root crude polysaccharide group.
Example 8:
preparing an oral preparation of the dwarf lilyturf tuber degraded extract:
(1) and (3) capsule preparation: 200g of the dwarf lilyturf tuber degraded extract prepared in the embodiment 1 is added with 100g of starch and 5g of micro silica gel powder, evenly mixed and encapsulated to obtain the compound preparation.
(2) General tablet preparation:
200g of the dwarf lilyturf tuber degraded extract prepared according to the example 2, 100g of starch, 50g of powdered sugar and 50g of dextrin are mixed uniformly, sieved by a 80-mesh sieve, added with 80% ethanol for mixing uniformly, granulated, dried and granulated, added with 1% magnesium stearate for mixing uniformly and tabletting to obtain the compound ophiopogon tuber degraded extract.
(3) Chewable tablet preparation:
200g of the ophiopogon root degradation extract prepared in the example 5, 100g of mannitol and 100g of microcrystalline cellulose are mixed uniformly and sieved by a 80-mesh sieve. Adding 10% PVPK30 ethanol solution, granulating, drying, grading, adding 4g of silica gel micropowder and 4g of orange essence powder, mixing, and tabletting.
(4) Granules: firstly, respectively crushing auxiliary materials, sieving the auxiliary materials by a 100-mesh sieve, uniformly mixing 200g of the radix ophiopogonis degraded extract prepared in the embodiment 6, 100g of sodium carboxymethyl starch, 150g of superfine silica gel powder and 400g of superfine cellulose powder, adding 100g of starch to prepare a soft material, sieving the soft material by a 20-mesh sieve, drying, and sieving the soft material by a 20-mesh sieve to obtain the radix ophiopogonis degradable soft capsule.
(5) Oral liquid: 200g of the dwarf lilyturf tuber degraded extract prepared in the embodiment 4, 801 g of tween-801 g, 0.2g of potassium sorbate and 0.5g of lemon essence are added with water to 1L, and the mixture is sterilized and subpackaged.
Claims (8)
1. The ophiopogon root degradation extract is characterized by being prepared by the following steps:
(1) extracting the crude polysaccharide of the dwarf lilyturf tuber: adding water into the ophiopogon japonicus according to a liquid-material ratio of 5-20, extracting for 1-3 times, extracting for 0.5-2 hours each time, filtering, concentrating the filtrate to obtain an extract with the raw material content of 0.5-2 g/ml, adding ethanol to enable the alcohol content to reach 75% -90%, standing, taking the precipitate, and evaporating to dryness to obtain crude ophiopogon japonicus polysaccharide;
(2) deproteinization: deproteinizing and purifying the ophiopogon japonicus crude polysaccharide by a Sevage method;
(3) and (3) degradation: adding water into the ophiopogonpolysaccharide obtained in the step (2) to fully dissolve the ophiopogonpolysaccharide to a transparent state, adding 2mol/L acid according to the material-liquid ratio of 10:1 based on the mass of the ophiopogon root medicinal material, degrading for 4-6 h, adding NaOH solution to neutralize to neutrality, performing ultrafiltration to obtain degradation products with the molecular weight range of 500-2500, concentrating, and freeze-drying to obtain the ophiopogon root degradation extract.
2. Extract according to claim 1, characterized in that step (1) is in particular: extracting radix Ophiopogonis with water for 2 times at a liquid-to-material ratio of 10 for 2 hr, filtering, mixing filtrates, concentrating the filtrate to obtain extract with raw material content of 1g/ml, adding ethanol to ethanol content of 80%, and standing overnight; filtering to remove supernatant, evaporating the precipitate to dryness to obtain crude radix Ophiopogonis polysaccharide.
3. Extract according to claim 1, characterized in that step (3) is in particular: the acid is hydrochloric acid or sulfuric acid, and the degradation temperature is 50-100 ℃.
4. Extract according to claim 3, characterized in that step (3) is in particular: the acid degradation temperature is 50 ℃, and the degradation time is 4 hours.
5. Extract according to claim 1, characterized in that step (2) is in particular: dissolving the crude ophiopogon root polysaccharide in distilled water, adding trichloromethane: and n-butanol (4: 1), stirring to fully dissolve, standing by a separating funnel, centrifuging the lower layer liquid for 10 minutes at 5000r/min by a high-speed centrifuge, removing precipitates, precipitating the clear liquid by ethanol, and drying to obtain the purified ophiopogonpolysaccharide.
6. Use of an extract according to any one of claims 1 to 5 in the manufacture of a medicament for lowering blood lipid levels.
7. Use of the extract of any one of claims 1 to 5 for the preparation of a medicament for scavenging free radicals in vivo and preventing lipid peroxidation in cells.
8. Use of an extract according to any one of claims 1 to 5 in the manufacture of an anti-atherosclerotic medicament.
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| PCT/CN2019/128992 WO2021128234A1 (en) | 2019-12-24 | 2019-12-27 | Radix ophiopogonis degradation extract and application thereof in preparation of drugs for prevention and treatment of cardiovascular diseases |
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| CN112830977A (en) * | 2021-02-26 | 2021-05-25 | 西南科技大学 | Method for extracting and purifying polysaccharide from fibrous root residues of radix ophiopogonis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112830977A (en) * | 2021-02-26 | 2021-05-25 | 西南科技大学 | Method for extracting and purifying polysaccharide from fibrous root residues of radix ophiopogonis |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2021128234A1 (en) | 2021-07-01 |
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