CN113509496A - Ganoderma extract and preparation method and application thereof - Google Patents
Ganoderma extract and preparation method and application thereof Download PDFInfo
- Publication number
- CN113509496A CN113509496A CN202110861155.4A CN202110861155A CN113509496A CN 113509496 A CN113509496 A CN 113509496A CN 202110861155 A CN202110861155 A CN 202110861155A CN 113509496 A CN113509496 A CN 113509496A
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- Prior art keywords
- extract
- ganoderma
- ganoderma lucidum
- ethanol
- group
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- Granted
Links
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Abstract
The invention discloses a false ganoderma extract and a preparation method and application thereof. The preparation method comprises the preparation of water extract of Ganoderma and the preparation of ethanol extract of Ganoderma. The Ganoderma extract comprises water extract and ethanol extract of Ganoderma. The application is the application of the false ganoderma extract in preparing anti-gastric ulcer medicines and foods. The invention discloses a false ganoderma extract, and the pharmacological test proves the effectiveness of the false ganoderma extract in resisting gastric ulcer, and discloses that the anti-gastric ulcer action mechanism of the false ganoderma extract is as follows: the false ganoderma extract can inhibit NF-kappa B/NLRP3 through inhibiting nuclear migration of NF-kappa B P65, so that proinflammatory factors of gastric mucosa tissues are reduced, inflammatory reaction is inhibited, and gastric ulcer is prevented.
Description
Technical Field
The invention relates to the field of medicinal and edible fungi, and in particular relates to a false ganoderma extract and a preparation method and application thereof.
Background
Pseudo-ganoderma (Amauroderma rugosum) is a medicinal and edible fungus with rich nutritional value and remarkable medicinal effect activity, is considered as black ganoderma in six ancient ganoderma, and has a long application history in China. The fungus system classification of the ganoderma lucidum belongs to the genus ganoderma of the family ganoderma lucidum, and the ganoderma lucidum is commonly called as xuezhi because the white pileus edges of young fruiting bodies and the back of pileus are lightly touched to generate blood sample secretion. Ancient books and modern documents, namely Yucheng, Turkey, Rui Bao Kai, and the like, Chinese medicinal fungi, Zhi [ M ], Heilongjiang Harbin, university of northeast forestry, publishers 2013:38-41, all record that the pseudo-ganoderma has the effects of resisting tumor, resisting inflammation and promoting urination.
Gastric ulcer is a common clinical disease and frequently encountered disease in the digestive system, and refers to that mucosa is subjected to inflammatory reaction or defect necrosis caused by various pathogenic factors, and complications such as upper gastrointestinal bleeding, obstruction, perforation, canceration and the like often occur, so that the life quality and physical and psychological health of a patient are seriously affected. According to survey, the incidence rate of gastric ulcer in the world can reach 10% -12%, and with the acceleration of the modern society rhythm and the increase of work, family and mental pressure, the incidence rate of gastric ulcer still rises year by year. The etiology and pathogenesis of gastric ulcer are not clear at present, and modern medical personnel generally think that the pathogenesis of gastric ulcer is mainly related to the imbalance between self injury factors and defense factors, wherein the injury factors mainly comprise: abnormal gastric acid secretion, helicobacter pylori infection, abuse of non-steroidal anti-inflammatory drugs, bad habits and customs such as smoking, drinking, overeating and the like, and when the strength of defense factors such as epidermal growth factors, a mucous membrane barrier, a mucous membrane-bicarbonate barrier, endogenous prostaglandin and the like is reduced, the stomach mucous membrane is damaged by the above damage factors, so that ulcer is formed.
Clinically used frequently H2Receptor antagonists and proton pump inhibitors, which treat gastric ulcers by inhibiting gastric acid secretion, have the disadvantage of being susceptible to relapse, although the cure rate is high. In recent years, many studies have shown that inflammatory response is an important pathogenesis of acute gastric ulcer, and thus inhibition of gastric inflammatory response is an effective anti-gastric ulcer mechanism. The research shows that the ganoderma lucidum has better in-vitro anti-inflammatory and anti-oxidation effects, but no pharmacological research report about the application of the ganoderma lucidum in the aspect of resisting gastric ulcer exists.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to solve the technical problem of providing a false ganoderma extract and a preparation method and application thereof.
The technical scheme for solving the technical problems of the preparation method is to provide a preparation method of a ganoderma lucidum extract, which is characterized by comprising the steps of preparing a ganoderma lucidum water extract and preparing a ganoderma lucidum ethanol extract;
the preparation of the ganoderma lucidum water extract comprises the following steps: adding water into the pseudostellaria root fruiting body powder, heating and refluxing the pseudolarix root fruiting body powder at 90-100 ℃, extracting the pseudolarix root fruiting body powder for at least 1 time, extracting the pseudolarix root fruiting body powder for at least 30min each time, and filtering the pseudolarix root fruiting body powder after each extraction to obtain filtrate; after all the extractions are completed, all the obtained filtrates are combined; then decompressing and concentrating the merged filtrate to prepare 1mL of standard fluid extract which is equivalent to 1g of crude drug, wherein the fluid extract is the water extract of the ganoderma lucidum;
the preparation of the ganoderma lucidum ethanol extract comprises the following steps: extracting Ganoderma fruiting body powder with ethanol under reflux at extraction temperature for at least 1 time, each time for at least 30min, and filtering to obtain filtrate; after all the extractions are completed, all the obtained filtrates are combined; then decompressing and concentrating the merged filtrate to prepare 1mL of standard fluid extract which is equivalent to 1g of crude drug, namely the false ganoderma ethanol extract; the extraction temperature is between 5 ℃ below the boiling point of ethanol and the boiling point of ethanol.
The technical scheme for solving the technical problem of the ganoderma lucidum extract is to provide the ganoderma lucidum extract obtained by the preparation method of the ganoderma lucidum extract, and the ganoderma lucidum extract is characterized by comprising a ganoderma lucidum water extract and a ganoderma lucidum ethanol extract.
The technical scheme for solving the application technical problem is to provide the application of the ganoderma lucidum extract in preparing anti-gastric ulcer medicines and foods.
Compared with the prior art, the invention has the beneficial effects that: the invention discloses a false ganoderma extract, and the pharmacological test proves the effectiveness of the false ganoderma extract in resisting gastric ulcer, and discloses that the anti-gastric ulcer action mechanism of the false ganoderma extract is as follows: the Ganoderma extract can inhibit NF-kB/NLRP 3 of inflammation pathway by inhibiting nuclear migration of NF-k B P65 (nuclear factor-k B P65), so as to reduce proinflammatory factor of gastric mucosa tissue, inhibit inflammation reaction, and achieve gastric ulcer resisting effect.
Drawings
FIG. 1 is HPLC chromatogram for measuring nucleoside content of Ganoderma lucidum water extract prepared in example 1 of the present invention;
FIG. 2 is HPLC chromatogram for measuring the ergosterol content of the alcohol extract of Ganoderma lucidum prepared in example 2 of the present invention;
FIG. 3 is a photograph of the gastric mucosa of rats of each group according to example 6 of the present invention; in the figure, A is a normal group; b is a model group; c is a positive drug group; d is the high dose group of pseudoganoderma lucidum aqueous extract of example 1; e is the dose group of the water extract of Ganoderma lucidum of example 1; f is the low dose group of the water extract of Ganoderma lucidum of example 1;
FIG. 4 is a graph of data on the area of a gastric ulcer in rats from each group in example 6 of the present invention;
FIG. 5 is a graph showing the results of tests on the influence of various groups on the proinflammatory factor content of gastric mucosal tissue according to example 6 of the present invention; in the figure, A is TNF-alpha; b is IL-1 beta; c is IL-6;
FIG. 6 is a graph showing the results of tests on the effect of various groups on the expression level of proinflammatory related genes in the stomach according to example 6 of the present invention; in the figure, a is Nlrp 3; b is Asc; c is Caspase 1;
FIG. 7 is a graph showing the results of tests on the influence of various groups of example 6 of the present invention on the expression level of NF-. kappa. B P65 nuclear migration; in the figure, A is the level of NF-. kappa. B P65 in the cytoplasm; b is the level of NF-. kappa. B P65 in the nucleus.
FIG. 8 is a photograph of the gastric mucosa of rats in each group according to example 7 of the present invention; in the figure, A is a normal group; b is a model group; c is a positive drug group; d is the high dose group of pseudoganoderma lucidum aqueous extract of example 1; e is the dose group of the water extract of Ganoderma lucidum of example 1; f is the low dose group of the water extract of Ganoderma lucidum of example 1;
FIG. 9 is a graph of data on the area of a gastric ulcer in rats from each group in example 7 of the present invention;
FIG. 10 is a graph showing the results of tests on the influence of the groups on the expression level of the gastric mucosal defense and repair gene Cox-2 in example 7 of the present invention;
FIG. 11 is a photograph of the gastric mucosa of rats in each group according to example 8 of the present invention; in the figure, A is a normal group; b is a model group; c is a positive drug group; d is the alcohol extract high dose group of the pseudo-ganoderma of example 2; e is the ethanol extract medium dose group of example 2; f is the alcohol extract low dose group of the pseudo-ganoderma of example 2;
FIG. 12 is a graph showing data on the areas of gastric ulcers in rats of groups according to example 8 of the present invention.
Detailed Description
Specific examples of the present invention are given below. The specific examples are only intended to illustrate the invention in further detail and do not limit the scope of protection of the claims of the present application.
The invention provides a preparation method (method for short) of a ganoderma lucidum extract, which is characterized by comprising the steps of preparing a ganoderma lucidum water extract and preparing a ganoderma lucidum ethanol extract;
the preparation of the ganoderma lucidum water extract comprises the following steps: collecting Ganoderma fruiting body powder, precisely weighing, adding water, extracting under reflux at 90-100 deg.C for at least 1 time (preferably 3 times), each time for at least 30min (preferably 1 hr), and filtering after each extraction to obtain filtrate; after all the extractions are completed, all the obtained filtrates are combined; then decompressing and concentrating the merged filtrate to prepare 1mL of standard fluid extract which is equivalent to 1g of crude drug, wherein the fluid extract is the water extract of the ganoderma lucidum;
the preparation of the ganoderma lucidum ethanol extract comprises the following steps: collecting Ganoderma fruiting body powder, precisely weighing, adding ethanol, heating and reflux-extracting at least 1 time (preferably 3 times) at extraction temperature for at least 30min (preferably 1 hr) each time, and filtering after each extraction to obtain filtrate; after all the extractions are completed, all the obtained filtrates are combined; then decompressing and concentrating the merged filtrate to prepare 1mL of standard fluid extract which is equivalent to 1g of crude drug, namely the false ganoderma ethanol extract; the extraction temperature is between 5 ℃ below the boiling point of ethanol and the boiling point of ethanol.
Preferably, the ratio of the volume of water added to the pseudo-ganoderma lucidum fruit body powder per time is 10-40: 1, in the embodiment, the ratio of the volume of water added at the 1 st time to the mass of the pseudo-ganoderma lucidum fruit body powder is 20:1, the ratio of the volume of water added at the 2 nd time to the mass of the pseudo-ganoderma lucidum fruit body powder is 16:1, and the ratio of the volume of water added at the 3 rd time to the mass of the pseudo-ganoderma lucidum fruit body powder is 16: 1;
preferably, the ratio of the volume of ethanol added to the mass of the pseudo-ganoderma lucidum fruit body powder is 10-40: 1, in the embodiment, the ratio of the volume of ethanol added at the 1 st time to the mass of the pseudo-ganoderma lucidum fruit body powder is 20:1, the ratio of the volume of ethanol added at the 2 nd time to the mass of the pseudo-ganoderma lucidum fruit body powder is 16:1, and the ratio of the volume of ethanol added at the 3 rd time to the mass of the pseudo-ganoderma lucidum fruit body powder is 16: 1;
preferably, the ethanol concentration of the ganoderma lucidum is 75-95% (preferably 75%) in the preparation of the ethanol extract of ganoderma lucidum.
Preferably, the artificial ganoderma is artificially cultivated artificial ganoderma or wild artificial ganoderma.
The invention also provides a false ganoderma Extract (EA) obtained by the preparation method of the false ganoderma Extract, which is characterized by comprising a false ganoderma water Extract and a false ganoderma ethanol Extract.
The water extract of Ganoderma comprises polysaccharides and nucleosides;
the false ganoderma ethanol extract comprises triterpenes, sterol substances, ergosterol and the like;
the content and detection method of the polysaccharide substances are as follows: precisely measuring 2mL of pseudostellaria root water extract, slowly dropwise adding 30mL of ethanol while stirring, shaking, standing at 4 ℃ for 12h, taking out, centrifuging (3500r/min), pouring out supernatant, dissolving precipitate in hot water, transferring to a 50mL measuring flask, cooling, adding water to scale, shaking uniformly, taking an appropriate amount of solution, centrifuging (3500r/min), precisely measuring 3mL of supernatant, placing in a 25mL measuring flask, adding water to scale, and shaking uniformly to obtain a sample solution. The glucose control was dissolved in purified water to make a 0.1mg/mL control solution. And (3) determining the content of polysaccharide in the sample by using an anthrone sulfate method and an ultraviolet-visible spectrophotometer, wherein the detection wavelength is 625 nm.
The content and the detection method of the nucleoside substances are as follows: precisely measuring 0.6mL of Ganoderma extract, placing in a 20mL measuring flask, adding water to scale, and shaking to obtain test solution. Measuring cytidine, uridine, guanosine and adenosine contents in the sample by rapid HPLC, as shown in FIG. 1, with the chromatographic condition of GL Sciences InertSustain AQ-C18Chromatographic column (2.1 × 100mm, 1.9 μm) with methanol (A) -water (B) as mobile phase, and gradient eluting (0-2 min, 0% A; 2-2.5 min, 0% -2% A; 2.5-4 min, 2% A; 4-4.5 min, 2%10% of A; 4.5-8.5 min, 10% A; 8.5-9.0 min, 10% -30% A; 9.0-10.5 min, 30-90% A); flow rate 0.3 ml/min-1(ii) a The column temperature is 40 ℃; the sample injection volume is 1 mu l; the detection wavelength is 259 nm.
The content and detection method of the triterpene and the sterol substances are as follows: precisely measuring 0.5mL of Ganoderma ethanol extract, placing in a 25mL measuring flask, adding ethanol to the scale, and shaking to obtain test solution. Dissolving oleanolic acid control with ethanol to obtain 0.2mg/mL control solution. The method comprises the steps of performing a vanillin-perchloric acid color reaction, measuring the contents of triterpenoids and sterols in a sample by using an ultraviolet visible spectrophotometer, and detecting the wavelength of 546 nm.
The content and the detection method of the ergosterol are as follows: precisely measuring 1mL of Ganoderma ethanol extract, placing in a 20mL measuring flask, adding ethanol to the scale, and shaking to obtain test solution. Determining ergosterol content in the sample by HPLC, as shown in FIG. 2, and subjecting to Wondasil chromatographyTM C18Chromatography column (4.6X 250mm, 5 μm) isocratically eluting with acetonitrile-water (98: 2); flow rate 1.0 ml/min-1(ii) a The column temperature is 30 ℃; the injection volume is 20 mul; the detection wavelength was 282 nm.
The invention also provides application of the false ganoderma extract in preparation of anti-gastric ulcer medicines and foods.
The medicine comprises an effective amount of false ganoderma extract and a pharmaceutically acceptable carrier.
Example 1
Collecting Ganoderma fruiting body powder 800g, precisely weighing, heating and reflux-extracting at 98-100 deg.C for 3 times, each time for 1 hr, adding 16L water for 1 st time, 12.8L water for 2 nd time, and 12.8L water for 3 rd time, filtering after each extraction to obtain filtrate; after all the extractions are completed, all the obtained filtrates are combined; and then concentrating the merged filtrate under reduced pressure to 800mL to prepare 1mL of standard fluid extract which is equivalent to 1g of crude drug, namely the ganoderma lucidum water extract.
The physical and chemical indexes of the nucleoside substances in the example 1 are shown in the table 1; the polysaccharide content is 7.20mg/ml, and the RSD value is 2.92%.
TABLE 1
| Nucleoside species | Cytidine | Uridine (uridine) | Guanosine | Adenosine (I) | Total up to |
| Sample content (mg/ml) | 0.2465 | 0.6301 | 0.4064 | 0.4989 | 1.7819 |
| RSD(％) | 3.87 | 3.00 | 4.40 | 3.99 | 1.53 |
Example 2
Collecting Ganoderma fruiting body powder 800g, precisely weighing, heating and reflux extracting at 75 deg.C for 3 times, each for 1 hr, adding 75% ethanol 16L for 1 time, adding 75% ethanol 12.8L for 2 times, adding 75% ethanol 12.8L for 3 times, and filtering after each extraction to obtain filtrate; after all the extractions are completed, all the obtained filtrates are combined; and then concentrating the combined filtrate under reduced pressure to 800mL to prepare 1mL of standard fluid extract which is equivalent to 1g of crude drug, namely the false ganoderma ethanol extract.
Through tests, the content of the triterpenoids and the sterols in the example 2 is 3.60mg/ml, and the RSD value is 2.51%; the ergosterol content is 0.387mg/ml, and the RSD value is 2.99%.
Example 3
Collecting Ganoderma fruiting body powder 800g, precisely weighing, heating and reflux-extracting at 80 deg.C for 3 times, each for 1 hr, adding 50% ethanol 16L for 1 time, adding 50% ethanol 12.8L for 2 times, adding 50% ethanol 12.8L for 3 times, and filtering after each extraction to obtain filtrate; after all the extractions are completed, all the obtained filtrates are combined; and then concentrating the combined filtrate under reduced pressure to 800mL to prepare 1mL of standard fluid extract which is equivalent to 1g of crude drug, namely the false ganoderma ethanol extract.
Example 4
Collecting Ganoderma fruiting body powder 800g, precisely weighing, heating and reflux-extracting at 70 deg.C for 3 times, each time for 1 hr, adding 80% ethanol 16L for 1 time, adding 80% ethanol 12.8L for 2 times, adding 80% ethanol 12.8L for 3 times, and filtering after each extraction to obtain filtrate; after all the extractions are completed, all the obtained filtrates are combined; and then concentrating the combined filtrate under reduced pressure to 800mL to prepare 1mL of standard fluid extract which is equivalent to 1g of crude drug, namely the false ganoderma ethanol extract.
Example 5
Collecting Ganoderma fruiting body powder 800g, precisely weighing, heating and reflux-extracting at 70 deg.C for 3 times, each time for 1 hr, adding 90% ethanol 16L for 1 time, adding 90% ethanol 12.8L for 2 times, adding 90% ethanol 12.8L for 3 times, and filtering after each extraction to obtain filtrate; after all the extractions are completed, all the obtained filtrates are combined; and then concentrating the combined filtrate under reduced pressure to 800mL to prepare 1mL of standard fluid extract which is equivalent to 1g of crude drug, namely the false ganoderma ethanol extract.
Example 6
Pharmacological test scheme: the effect of the ganoderma lucidum water extract on gastric mucosa in a rat gastric ulcer model caused by absolute ethyl alcohol;
1. drugs and animals:
1) the Ganoderma extract is the Ganoderma extract prepared in example 1. The positive drug is Lansoprazole (Lansoprazole).
2) Animals: SPF grade male SD rats (6-7 weeks old, 200-.
2. Grouping and administration:
the rats were randomly divided into 6 groups, namely a normal group (Control), a Model group (Model) and an administration group, wherein the administration group comprises a positive drug group, a high-dose false ganoderma extract (H-EA) group, a medium-dose false ganoderma extract group (M-EA) and a low-dose false ganoderma extract group (L-EA). The number of rats in the model group is 8, and the other groups are 6.
Normal group: the normal saline is taken continuously for 7 days, and the last day of the 7 days is fasted for 24h and free water can be drunk.
Model group: continuously taking normal saline for 7 days, and fasting for 24h on the last day of 7 days and allowing free water drinking; after the last drenching for 1h, 1ml of absolute ethyl alcohol is given to the stomach.
A positive drug group: continuously filling lansoprazole for 7 days at a dose of 30mg/kg, and fasting for 24h on the last day of 7 days and allowing free water drinking; after the last drenching for 1h, 1ml of absolute ethyl alcohol is given to the stomach.
Group H-EA: continuously administering the water extract of Ganoderma of example 1 at a dose of 200mg/kg for 7 days, wherein the last day of the 7 days is fasted for 24h and water can be freely drunk; after the last drenching for 1h, 1ml of absolute ethyl alcohol is given to the stomach.
Group M-EA: the water extract of Ganoderma lucidum of example 1 was continuously administered at a dose of 100mg/kg for 7 days, with 24h fasting on the last of the 7 days and free access to water; after the last drenching for 1h, 1ml of absolute ethyl alcohol is given to the stomach.
Group L-EA: continuously administering the water extract of Ganoderma of example 1 at a dose of 50mg/kg for 7 days, wherein the last day of the 7 days is fasted for 24h and water can be freely drunk; after the last drenching for 1h, 1ml of absolute ethyl alcohol is given to the stomach.
(II) detection results:
1. and (3) data analysis: all measurements were mean ± sem.
In fig. 4, 5, 6, 7, 9, 10 and 12, the comparison of the model group with the normal group employs the paired sample t test. Comparison of the dosing group with the model group, probability was expressed using one-way analysis of variance, Probasic (P). When P <0.05, the model group shows that the data of the two groups have significant difference compared with the normal group, and the figure shows that the data of the two groups are marked by "#"; when P <0.01, it indicates a very significant difference between the two sets of data, indicated by "##" in the figure. When P <0.05, the administration group showed significant difference between the two groups of data, as indicated by "+" in the figure; when P <0.01, it indicates a very significant difference between the two sets of data, indicated by "×", in the figure. The analytical software was SPSS 20.0.
2. And (3) observing characters:
(1) after 1 hour of intragastric administration of 1ml of absolute ethanol, 10% chloral hydrate 4ml/kg was intraperitoneally injected to anesthetized rats, the rats were dissected, the stomach was cut along the greater curvature of the stomach, rinsed with ice-cold physiological saline, unfolded and photographed with an instrument applanation, and the ulcer area (mm) was measured with Image J software (National Insti)2). As can be seen from fig. 3, the deeper color of the inner wall of the gastric mucosa is the ulcer part, the ulcer does not occur in the normal group, the ulcer area of the model group is widely distributed, and the gastric ulcer degree of the administration group is lighter than that of the model group.
(2) As can be seen from FIG. 4, the ulcer area of the gastric mucosa is significantly increased in the model group compared with the normal group, indicating that the modeling is successful. Compared with the model group, the ulcer area of the administration group is obviously reduced, which indicates that the water extract of the ganoderma lucidum has the effect of resisting ulcer.
3. Measurement of proinflammatory factors of gastric mucosal tissue: the contents of tumor necrosis factor (tumor factor-alpha, TNF-alpha), Interleukin (IL) -1 beta (IL-1 beta), and interleukin-6 (IL-6) were measured by homogenizing the tissue of the stomach wall of each group according to the kit instructions. As can be seen from FIG. 5, the proinflammatory factors of the three gastric mucosal tissues, namely TNF-alpha, IL-1 beta and IL-6, are remarkably increased in the model group and reduced in the administration group to different degrees, and the effects of the H-EA group, the M-EA group and the L-EA group on the IL-1 beta down-regulation are most remarkable, which indicates that the water extract of the pseudostellaria root has an anti-inflammatory effect.
4. Real-time quantitative polymerase chain reaction (Q-PCR): extracting total RNA from the homogenate tissue sample, and designing corresponding primers to perform determination according to a Q-PCR process. Genes determined by a rat gastric ulcer model caused by absolute ethyl alcohol comprise proinflammatory related genes Nlrp3, Caspase-1 and Asc. As can be seen from FIG. 6, in the absolute ethanol rat gastric ulcer model, the expression level of the model group is increased, and the expression level of the administration group is reduced by three proinflammatory related genes, namely Nlrp3, Asc and Caspase 1, so that the water extract of Ganoderma lucidum has the effect of inhibiting the generation of proinflammatory factors in gastric mucosal tissues by regulating the expression of the proinflammatory genes.
5. Western blotting (Western blot, WB): 4 rats are selected in each group, and the level of an important regulatory factor NF-kappa B P65 of an inflammatory pathway NF-kappa B/NLRP3 in cytoplasm and nucleus is detected according to a WB test operation flow. As can be seen from fig. 7, the important regulatory factor P65 of the inflammatory pathway NF- κ B/NLRP3 significantly reduces the cytoplasmic content and significantly increases the nuclear content in the model group, i.e., P65 significantly migrates in the model group to activate the inflammatory pathway NF- κ B/NLRP3, thereby promoting the expression of inflammatory genes and increasing the generation of proinflammatory factors; in contrast, in the administered group, the migration of P65 nucleus was significantly reduced, so that the inflammatory response was reduced by inhibiting NF-. kappa.B/NLRP 3 pathway, and finally the anti-gastric ulcer effect was achieved.
Example 7
Pharmacological test scheme: the effect of the ganoderma lucidum water extract on gastric mucosa in a rat gastric ulcer model caused by indomethacin;
1. drugs and animals:
1) the Ganoderma extract is the Ganoderma extract prepared in example 1. The positive drug is lansoprazole.
2) Animals: SPF grade male SD rats (6-7 weeks old, 200-.
2. Grouping and administration:
the rats were randomly divided into 6 groups, namely a normal group, a model group and an administration group, wherein the administration group comprises a positive drug group, a high-dose pseudoganoderma extract (H-EA) group, a medium-dose pseudoganoderma extract (M-EA) group and a low-dose pseudoganoderma extract (L-EA) group. The number of rats in the model group is 8, and the other groups are 6.
Normal group: the normal saline is taken continuously for 7 days, and the last day of the 7 days is fasted for 24h and free water can be drunk.
Model group: continuously taking normal saline for 7 days, and fasting for 24h on the last day of 7 days and allowing free water drinking; after the last gastric lavage for 30min, indomethacin is administered with a dose of 100 mg/kg.
A positive drug group: continuously filling lansoprazole for 7 days at a dose of 30mg/kg, and fasting for 24h on the last day of 7 days and allowing free water drinking; after the last gastric lavage for 30min, indomethacin is administered with a dose of 100 mg/kg.
Group H-EA: continuously administering the water extract of Ganoderma of example 1 at a dose of 200mg/kg for 7 days, wherein the last day of the 7 days is fasted for 24h and water can be freely drunk; after the last gastric lavage for 30min, indomethacin is administered with a dose of 100 mg/kg.
Group M-EA: the water extract of Ganoderma lucidum of example 1 was continuously administered at a dose of 100mg/kg for 7 days, with 24h fasting on the last of the 7 days and free access to water; after the last gastric lavage for 30min, indomethacin is administered with a dose of 100 mg/kg.
Group L-EA: continuously administering the water extract of Ganoderma of example 1 at a dose of 50mg/kg for 7 days, wherein the last day of the 7 days is fasted for 24h and water can be freely drunk; after the last gastric lavage for 30min, indomethacin is administered with a dose of 100 mg/kg.
(II) detection results:
1. and (3) observing characters:
(1) after 5h of gavage with a dose of 100mg/kg indomethacin, 10% chloral hydrate 4ml/kg anesthetized rats were intraperitoneally injected, the rats were dissected, the stomach was cut open along the greater curvature, rinsed with ice cold normal saline, unfolded and photographed with an instrument applanation, and the ulcer area (mm) was measured using Image J software (National Insti)2). As can be seen from FIG. 8, the deeper color of the inner wall of the gastric mucosa is the ulcer part, no ulcer occurs in the normal group, the ulcer area distribution of the model group is wider, and the gastric ulcer degree of the administration group is lighter than that of the model group.
(2) As can be seen from FIG. 9, the ulcer area of the gastric mucosa is significantly increased in the model group compared with the normal group, indicating that the modeling is successful. The ulcer area was reduced in the M-EA group compared with the model group; compared with the model group, the positive medicine group, the H-EA group and the L-EA group have obviously reduced ulcer areas, which indicates that the ganoderma lucidum water extract has the effect of resisting ulcer.
2. Real-time quantitative polymerase chain reaction (Q-PCR): each group selected 5 rats, total RNA was extracted from the homogenate samples, and corresponding primers were designed for determination according to the Q-PCR protocol. The gene measured by the indometacin-induced rat gastric ulcer model is a gastric mucosa defense and repair gene Cox-2. As can be seen from FIG. 10, in the indomethacin-induced gastric ulcer model of rats, the expression level of the gastric mucosa defense and repair gene Cox-2 in the model group is reduced, and the expression level in the administration group is increased, which indicates that the water extract of Ganoderma lucidum has the effect of enhancing the gastric protection.
Example 8
Pharmacological test scheme: the effect of the alcohol extract of Ganoderma on gastric mucosa in a rat gastric ulcer model caused by absolute alcohol;
1. drugs and animals:
1) the Ganoderma extract is ethanol extract of Ganoderma prepared in example 2. The positive drug is lansoprazole.
2) Animals: SPF grade male SD rats (6-7 weeks old, 200-.
2. Grouping and administration:
the rats were randomly divided into 6 groups, namely a normal group, a model group and an administration group, wherein the administration group comprises a positive drug group, a high-dose pseudoganoderma extract (H-EA) group, a medium-dose pseudoganoderma extract (M-EA) group and a low-dose pseudoganoderma extract (L-EA) group. The number of rats in each group was n-7.
Normal group: the normal saline is taken continuously for 7 days, and the last day of the 7 days is fasted for 24h and free water can be drunk.
Model group: continuously taking normal saline for 7 days, and fasting for 24h on the last day of 7 days and allowing free water drinking; after the last drenching for 1h, 1ml of absolute ethyl alcohol is given to the stomach.
A positive drug group: continuously filling lansoprazole for 7 days at a dose of 30mg/kg, and fasting for 24h on the last day of 7 days and allowing free water drinking; after the last drenching for 1h, 1ml of absolute ethyl alcohol is given to the stomach.
Group H-EA: the ethanol extract of Ganoderma of example 2 was administered continuously at a dose of 200mg/kg for 7 days, with 24h fasting on the last of the 7 days and free access to water; after the last drenching for 1h, 1ml of absolute ethyl alcohol is given to the stomach.
Group M-EA: the ethanol extract of Ganoderma of example 2 was administered continuously at a dose of 100mg/kg for 7 days, with 24h fasting on the last of the 7 days and free access to water; after the last drenching for 1h, 1ml of absolute ethyl alcohol is given to the stomach.
Group L-EA: the ethanol extract of Ganoderma of example 2 was administered continuously at a dose of 50mg/kg for 7 days, with 24h fasting on the last of the 7 days and free access to water; after the last drenching for 1h, 1ml of absolute ethyl alcohol is given to the stomach.
(II) detection results:
1. and (3) observing characters:
(1) after 1 hour of intragastric administration of 1ml of absolute ethanol, 10% chloral hydrate 4ml/kg was intraperitoneally injected to anesthetized rats, the rats were dissected, the stomach was cut along the greater curvature of the stomach, rinsed with ice-cold physiological saline, unfolded and photographed with an instrument applanation, and the ulcer area (mm) was measured with Image J software (National Insti)2). As can be seen from FIG. 11, the deeper color of the inner wall of the gastric mucosa is the ulcer part, no ulcer occurs in the normal group, the ulcer area of the model group is widely distributed, and the gastric ulcer degree of the administration group is lighter than that of the model group.
(2) As can be seen from FIG. 12, the ulcer area of the gastric mucosa is significantly increased in the model group compared with the normal group, indicating that the modeling is successful. Compared with the model group, the ulcer areas of the positive medicine and the ganoderma lucidum water extract are both obviously reduced, which indicates that the ganoderma lucidum ethanol extract has the effect of resisting ulcer.
Nothing in this specification is said to apply to the prior art.
Claims (8)
1. A preparation method of a false ganoderma extract is characterized in that the method comprises the preparation of a false ganoderma water extract and the preparation of a false ganoderma ethanol extract;
the preparation of the ganoderma lucidum water extract comprises the following steps: adding water into the pseudostellaria root fruiting body powder, heating and refluxing the pseudolarix root fruiting body powder at 90-100 ℃, extracting the pseudolarix root fruiting body powder for at least 1 time, extracting the pseudolarix root fruiting body powder for at least 30min each time, and filtering the pseudolarix root fruiting body powder after each extraction to obtain filtrate; after all the extractions are completed, all the obtained filtrates are combined; then decompressing and concentrating the merged filtrate to prepare 1mL of standard fluid extract which is equivalent to 1g of crude drug, wherein the fluid extract is the water extract of the ganoderma lucidum;
the preparation of the ganoderma lucidum ethanol extract comprises the following steps: extracting Ganoderma fruiting body powder with ethanol under reflux at extraction temperature for at least 1 time, each time for at least 30min, and filtering to obtain filtrate; after all the extractions are completed, all the obtained filtrates are combined; then decompressing and concentrating the merged filtrate to prepare 1mL of standard fluid extract which is equivalent to 1g of crude drug, namely the false ganoderma ethanol extract; the extraction temperature is between 5 ℃ below the boiling point of ethanol and the boiling point of ethanol.
2. The method for preparing a Ganoderma lucidum extract according to claim 1, wherein the ratio of the volume of water added to the mass of the Ganoderma lucidum fruiting body powder per time is 10-40: 1.
3. The method for preparing the Ganoderma lucidum extract according to claim 1, wherein the ratio of the volume of ethanol added to the mass of the Ganoderma lucidum fruiting body powder is 10-40: 1.
4. The method for preparing an ethanol extract of Ganoderma lucidum as claimed in claim 1, wherein the ethanol is added to the ethanol extract of Ganoderma lucidum at a concentration of 75-95%.
5. The method for preparing the ganoderma lucidum extract according to claim 1, wherein the ganoderma lucidum is artificially cultivated ganoderma lucidum or wild ganoderma lucidum.
6. A Ganoderma lucidum extract obtained by the method for producing a Ganoderma lucidum extract according to any one of claims 1 to 5, wherein the Ganoderma lucidum extract comprises a water extract and an ethanol extract of Ganoderma lucidum.
7. Use of the Ganoderma extract of claim 6 in the preparation of anti-gastric ulcer drugs and foods.
8. The use of claim 7, wherein said medicament comprises an effective amount of an extract of Ganoderma lucidum and a pharmaceutically acceptable carrier.
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