CN110693873B - Preparation and application of rabdosia rubescens active ingredient composition - Google Patents
Preparation and application of rabdosia rubescens active ingredient composition Download PDFInfo
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Abstract
The invention provides a preparation and application of a rabdosia rubescens active ingredient composition, wherein the composition consists of oridonin, rubescensin B, rosmarinic acid, ferulic acid and quercetin; the weight ratio of the oridonin to the rubescensine B to the rosmarinic acid to the ferulic acid to the quercetin is (3-10) to (1-3) to (1-4) to (2-6). The active ingredients in the composition can produce synergistic effect, thereby effectively treating NAFLD.
Description
Technical Field
The invention relates to a preparation method and application of a rabdosia rubescens active ingredient composition, in particular to a preparation method and application of the rabdosia rubescens active ingredient composition in treating non-alcoholic fatty liver disease.
Background
Non-alcoholic fatty liver disease (NAFLD) is a clinical pathological syndrome mainly including liver cell steatosis caused by genetic-environmental-metabolic stress related factors but without history of excessive drinking. The investigation shows that the incidence rate of NAFLD in adults in developed countries of Europe and America is up to 23%. Although there is no definite statistical data, most data show that the incidence of NAFLD is also obviously increased in recent years in China, and the population suffering from NAFLD is gradually aged. Therefore, NAFLD is also becoming a public health problem of general concern in our country.
In NAFLD, non-alcoholic steatohepatitis (NASH) is a very critical link, NASH is now well-established as a chronic invasive disease, 25% of patients can develop liver fibrosis, about 1.5-8.0% of patients develop cirrhosis, which is considered as one of the main causes of cryptogenic cirrhosis, and NAFLD is closely related to the occurrence of hepatocellular carcinoma. Unfortunately, to date, there are no good therapeutic approaches to treat NAFLD. Zhejiang is one of developed provinces in the southeast coast of China, and because of high-quality living conditions, Zhejiang has a large number of obese and fatty liver crowds, and is one of potential risk factors influencing the development of socioeconomic development of our province. Therefore, the intensive research on the pathogenesis and development mechanism of NAFLD and the finding of a safe, reasonable and effective blocking treatment method are the problems to be urgently solved in the development of the medical and health industry of our province.
NAFLD is an important component of the metabolic syndrome, for which Insulin Resistance (IR), Triglyceride (TG) and Total Cholesterol (TC) transport disorders, oxygen stress and lipid peroxidation damage may be primary factors. Since intrahepatic fat accumulation induces and aggravates IR, leading to IR/glucose toxicity, fatty liver/lipid toxicity, and a vicious cycle between fatty liver/IR, the therapeutic effects of NAFLD require intensive studies on the mechanisms of action of therapeutic drugs used in IR, Triglyceride (TG) and Total Cholesterol (TC) transport disorders, oxygen stress, and lipid peroxidation injury.
Rabdosia rubescens, also called as the Rabdosia rubescens, is dried overground part of the plant of Labiatae, namely Rabdosia rubescens (Hemsl.) Hara (Plectranthus rubesens Hemsl.), is Labiatae shrub 30-100 cm high, usually grows in hillside, valley, shrub, woodland and the like with the elevation of 100-1000 m, and is mainly produced in the Henan, and the southern area of the yellow river basin. According to related researches, rabdosia contains various terpenoids, wherein the diterpenoid structure is diversified, and volatile oil, alkaloid, amino acid, flavone, organic acid ester and some small molecular compounds are also contained in the rabdosia. At present, the main pharmacological action of rabdosia rubescens is anti-tumor, and diterpene anti-tumor drugs represented by oridonin are widely applied to the research of various tumor cell mechanisms. In addition, pharmacological activity studies are also conducted at the level of some animals. Including anti-inflammatory and hepatoprotective effects. Specifically, research shows that the rabdosia rubescens extract with 100% ethanol is an anti-inflammatory effective part of rabdosia rubescens; oridonin has effect in treating RAW264.7 cell inflammation induced by palmitic acid. The oridonin powder inhalation has the prospect of treating acute lung injury; rabdosia rubescens has the effect of treating liver injury.
The rabdosia rubescens alcohol extract and the rubescensine A not only have the function of reducing blood fat, but also can reduce liver damage and protect liver function, and the pathogenesis of the non-alcoholic fatty liver is mainly caused by liver fat accumulation caused by long-term hyperlipemia, so that the great speculation that the rabdosia rubescens extract and the rubescensine A have the treatment effect on NAFLD is made.
On the other hand, according to previous studies, we found that the activity of Rabdosia rubescens extract is better than that of oridonin under the same experimental conditions. This means that the active ingredients in the rabdosia rubescens extract act synergistically. In order to clarify the active ingredients and improve the therapeutic effect, intensive research is carried out on the ingredients which can synergistically treat NAFLD in the active ingredients in rabdosia rubescens.
Therefore, the research can comprehensively and deeply research the treatment mechanism of the rabdosia rubescens on NAFLD, prove the effectiveness of the rabdosia rubescens in NAFLD treatment and research the active ingredients in the rabdosia rubescens extract for treating the NAFLD.
Disclosure of Invention
The present invention provides the following:
a composition of active ingredients of Rabdosia rubescens for treating NAFLD is characterized by comprising oridonin, rubescensin B, rosmarinic acid, ferulic acid and quercetin.
The rabdosia rubescens active ingredient composition for treating NAFLD is characterized in that the weight ratio of the rabdosia rubescens A to the rabdosia rubescens B to the rosmarinic acid to the ferulic acid to the quercetin is (3-10), (1-3), (1-4) and (2-6).
The rabdosia rubescens active ingredient composition for treating NAFLD is characterized in that the weight ratio of the rabdosia rubescens A, the rabdosia rubescens B, the rosmarinic acid, the ferulic acid and the quercetin is (4-8), (1-2), (2-3) and (3-4).
The composition of the active ingredients of the rabdosia rubescens for treating NAFLD is characterized in that the weight ratio of the rabdosia rubescens A to the rabdosia rubescens B to the rosmarinic acid to the ferulic acid to the quercetin is 6:1:3:3: 4.
A medicament of rubescensine active ingredient for the treatment of NAFLD, it includes active ingredient and supplementary product, the said active ingredient is rubescensine A, rubescensine B, rosmarinic acid, ferulic acid and quercetin; the auxiliary material is pharmaceutically acceptable.
The medicament for treating the rabdosia rubescens active ingredient of NAFLD comprises 3-10 parts by weight of oridonin, 1-3 parts by weight of rubescensin, 1-4 parts by weight of rosmarinic acid, 1-4 parts by weight of ferulic acid and 2-6 parts by weight of quercetin.
The pharmaceutically acceptable auxiliary materials of the medicament of the rabdosia rubescens active ingredient for treating NAFLD comprise one or more solid or liquid auxiliary materials.
The medicament of the rabdosia rubescens active ingredient for treating NAFLD is in the dosage form of tablets, dispersible tablets, capsules, soft capsules, powder injection, pills or dripping pills.
The invention further provides application of the composition in preparing a medicine for treating NAFLD.
The invention further provides application of the composition in preparation of a medicine or health-care product with an auxiliary protection effect on NAFLD.
The invention further provides application of the medicine in preparing a medicine for treating NAFLD.
The invention has the beneficial effects that:
1. it is firstly proposed that the rabdosia rubescens extract has the therapeutic effect on NAFLD.
2. The rabdosia rubescens extract is found to have activity on NAFLD superior to that of rubescensine A, active ingredients which have synergistic therapeutic effect on NAFLD in the rabdosia rubescens extract are obtained through experimental screening, and the weight ratio of the active ingredients (the composition consisting of the active ingredients with the specific ratio, hereinafter referred to as the rabdosia rubescens composition) is determined by adjusting the experimental results according to the ratio of the active ingredients in the actual extract.
3. The oridonin composition and the oridonin are used for treating NAFLD, the histological changes of hepatic cell steatosis, inflammation, cell necrosis and the like and the correlation of insulin sensitivity, cytokine level, lipid peroxide, tumor necrosis factor-alpha (TNF-alpha) and IR/inflammation are discussed, and the possible mechanism of the oridonin for treating NAFLD is clarified.
Drawings
FIG. 1 HE staining results (. times.400) of liver tissues of rats in the normal group
FIG. 2 HE staining results (. times.400) of rat liver tissues in the model group
FIG. 3 Rabdosia rubescens A group rat liver tissue HE staining result (X400)
FIG. 4 HE staining results (. times.400) of liver tissues of rats in the low dose group
FIG. 5 HE staining results (. times.400) of liver tissues of rats in the high dose group
FIG. 6 HE staining results of liver tissues of rats in acetylcysteine group (. times.400)
FIG. 7 HE staining results (X400) of liver tissues of rats in taurine group
FIG. 8 HE staining results (X400) of liver tissues of rats in the Yikinao group
FIG. 9 expression of PPAR α protein in rat liver tissue of Normal group (. times.400)
FIG. 10 model group rat liver tissue PPAR α protein expression (. times.400)
FIG. 11 Rabdosia rubescens A group rat liver tissue PPAR alpha protein expression (x 400)
FIG. 12 PPAR α protein expression (. times.400) in rat liver tissue of Low dose group
FIG. 13 PPAR α protein expression (. times.400) in rat liver tissue in high dose group
FIG. 14 expression of PPAR α protein (. times.400) in liver tissue of rats in the acetylcysteine group
FIG. 15 PPAR α protein expression (. times.400) in rat liver tissue of taurine group
FIG. 16 PPAR α protein expression (. times.400) in liver tissue of rats in the easy recovery group
FIG. 17 PPAR γ protein expression (. times.400) in rat liver tissue of normal group
FIG. 18 model group rat liver tissue PPAR γ protein expression (x 400)
FIG. 19 Rabdosia rubescens A group rat liver tissue PPAR gamma protein expression (x 400)
FIG. 20 PPAR γ protein expression (. times.400) in rat liver tissue of Low dose group
FIG. 21 PPAR γ protein expression (. times.400) in rat liver tissue of high dose group
FIG. 22 acetylcysteine group rat liver tissue PPAR γ protein expression (X400)
FIG. 23 PPAR γ protein expression (X400) in rat liver tissue of taurine group
FIG. 24 expression of PPAR γ protein in rat liver tissue of the easy recovery group (. times.400)
FIG. 25 expression of PPAR α mRNA in liver tissue
FIG. 26 expression of liver tissue PPAR γ mRNA
FIG. 27 expression of SREBP-1c mRNA in liver tissue
Note: in FIGS. 25-27, M represents Marker, A-Normal group, B-model group, C-acetylcysteine group, D-taurine group, E-easy recovery group, F-Oridonin group, G-Low dose group, H-high dose group
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and should not be taken to be limiting.
EXAMPLE 1 tablet
6mg of oridonin, 1mg of rubescensine B, 3mg of rosmarinic acid, 3mg of ferulic acid, 4mg of quercetin, 80g of starch and 5g of magnesium stearate.
The preparation process comprises the following steps: mixing rubescensin A6 mg, rubescensin B1 mg, rosmarinic acid 3mg, ferulic acid 3mg and quercetin 4mg, adding appropriate amount of starch and magnesium stearate, mixing, granulating, drying, and tabletting.
EXAMPLE 2 capsules
Rubescensin A6 mg, rubescensin B1 mg, rosmarinic acid 3mg, ferulic acid 3mg, quercetin 4mg, starch 80g, and magnesium stearate 5 g.
The preparation process comprises the following steps: mixing rubescensin A6 mg, rubescensine B1 mg, rosmarinic acid 3mg, ferulic acid 3mg and quercetin 4mg, adding appropriate amount of starch and magnesium stearate, granulating, drying, and making into capsule.
Example 3 animal experiments
1. Purpose of study
Along with the improvement of the living standard of people, the incidence rate of non-alcoholic fatty liver diseases is increasing day by day, the research on the pathogenesis and development mechanism of NAFLD is deeply researched, and a safe, reasonable and effective blocking treatment method is found out, which is a problem to be urgently solved in the field of liver diseases. The research researches on treating NAFLD by the rabdosia rubescens composition and the oridonin, discusses the histological changes of hepatic cell steatosis, inflammation, cell necrosis and the like and the correlation of insulin sensitivity, cytokine level, lipid peroxide, tumor necrosis factor-alpha (TNF-alpha) and IR/inflammation of the NAFLD, and clarifies the possible mechanism of treating NAFLD by the rabdosia rubescens composition and the oridonin.
2. Content of research
2.1 establishing a rat model of simple steatohepatitis by adopting continuous simple high-fat diet;
2.2 observing the expression level of PPAR alpha and PPAR gamma protein in NAFLD liver tissue from protein level;
2.3 observing the influence of the composition and the oridonin on the indexes such as the indexes, pathology, blood biochemistry, protein and cell factor transcription expression level and the like after the treatment of rat NAFLD.
3. Research method
3.1 establishment of rat non-alcoholic steatohepatitis model: SD rats are fed continuously with high-fat feed (supplemented with 85% standard feed, 9% lard, 3% cholesterol, 1% bile salt, 2% egg yolk powder) for 20 weeks.
3.2 preparation of the medicament
(1) Oridonin: preparing a test drug temporarily according to the dosage converted by a rat body surface area method;
(2) rabdosia rubescens composition: the test drugs were the oridonin, rubescensin, rosmarinic acid, ferulic acid and quercetin compositions in a weight ratio of 6:1:3:3:4, the same as the above preparation method.
(3) Easy recovery suspension: preparing 6.92mg/mL suspension by using distilled water;
3.3 grouping and administration of Experimental animals: after being bred adaptively for 1 week, 80 SD rats are randomly divided into a normal group, a building group, an oridonin group, an rabdosia composition high dose group (short for a high dose group), an rabdosia composition low dose group (short for a low dose group), an acetylcysteine group, a taurine group and an easily recovered group, wherein 10 rats are selected. Feeding normal group with standard feed every day, feeding other groups with high-fat feed every day, and infusing normal group and model group with normal saline solution with equal volume every day; easy-to-restore-group daily irrigation100 mg/kg of easy rehabilitation suspension-1·d-1The low dose group, the high dose group and the oridonin group are respectively administered at 50, 150 mg.kg daily-1·d-1Rubescensine A, rubescensine B, rosmarinic acid, ferulic acid and quercetin composition (low dose group and high dose group) and rubescensine A (rubescensine A group) at ratio of 6:1:3:3: 4; acetylcysteine group 150mg kg-1·d-1(ii) a Taurine 150 mg/kg-1·d-1For 20 consecutive weeks; after 20 weeks, rats were fasted for 16 hours without water deprivation, anesthetized with 3% pentobarbital sodium (0.15mL/100g) by intraperitoneal injection on an empty stomach the next morning, subjected to blood sampling in the abdominal cavity, serum was separated, and stored at-20 ℃ for measuring blood biochemical indicators and cytokines; whole livers were quickly isolated and weighed. Then cutting a plurality of liver tissues in the middle of the right lobe of the liver, immediately putting into liquid nitrogen for freezing, transferring into a refrigerator at the temperature of-80 ℃ for storage after 3 hours, and extracting total RNA to detect the expression of SREBP-1c, PPAR alpha and PPAR gamma mRNA in the liver cells; another liver tissue of right lobe with a size of 3X 6mm is fixed with 10% formaldehyde (prepared with PBS (pH7.4)), dehydrated with alcohol step by step, and embedded with conventional paraffin for pathological examination and immunohistochemical staining.
3.4 Observation indicators and methods
(1) The serological index detection method comprises the following steps: serum ALT, AST, FBG, CHO, TG, HDL and LDL-C were measured using Hitachi 7020 full-automatic biochemical analyzer. TNF-alpha, INS and Leptin adopt an radioimmunoassay; FFA adopts a ketone reagent colorimetric method.
(2) Liver homogenate lipid and protein content determination: collecting 300mg liver tissue, cutting, adding ice normal saline, grinding into 10% liver tissue homogenate with homogenizer, centrifuging at 4000r/min for 10 min, and extracting supernatant. The liver tissue homogenate cholesterol is measured by adopting an isopropanol extraction and alumina adsorption ferric-acetic acid-sulfuric acid color development method; the triglyceride is measured by isopropanol extraction, phospholipid adsorption by alumina, and acetylacetone color development. The content of the liver tissue homogenate protein is measured by adopting a Coomassie brilliant blue method; measuring the MDA content of the liver tissue by adopting a TBA method; liver tissue SOD activity is measured by xanthine oxidase; hepatic tissue GSH-PXThe activity was measured by spectrophotometry.
(3) Histopathological examination of liver: and (4) performing conventional HE staining for pathological diagnosis, and calculating the degree of fat change.
(4) Immunohistochemistry: the SP method is adopted to detect the expression levels of the proteins of PPAR alpha and PPAR gamma in liver tissues.
(5) RT-PCR: GenBank searches for the primer sequences of rat SREBP-1c, PPAR α and PPAR γ, and the GenBank entrusts the synthesis of Shanghai Dingan Biotech Co., Ltd. The expression levels of SREBP-1c, PPAR α and PPAR γ mRNA in rat liver tissues were determined by RT-PCR.
4. Results of the study
Through the research on the mechanism of rabdosia rubescens for resisting non-alcoholic steatohepatitis, the rabdosia rubescens composition and oridonin have obvious curative effect on NAFLD, and are obviously superior to NAC, taurine and easy recovery. The rabdosia rubescens composition and the rubescensine A can obviously reduce the liver index of fatty liver rats and serum ALT and AST, and have obvious improvement effect on the fat indexes of serum and liver, and the pathological detection shows that compared with a model group, the rabdosia rubescens composition and the rubescensine A can obviously reduce the fat change and the inflammation necrosis degree of the fatty liver rats, and the specific results are as follows:
4.1 general conditions in rats: in the whole experimental process, rats in each group have no casualty, and rats in a normal control group have white hair, are relatively glossy, are quick to move, have more food intake and have granular excrement. The model group had greasy, rough and sluggish coat, manifesting as abdominal obesity with little food intake. The general conditions of the rats in each drug intervention group, such as food intake, stool character and the like, are improved to different degrees compared with the model control group. As can be seen from Table 1, there was no significant difference in initial body weight (p >0.05) among rats in each group, the body weight and liver index of rats in the model group at the end of the experiment were significantly increased (p <0.05), the body weight and liver index of rats in the high dose group, low dose group, taurine group, acetylcysteine group and easy recovery group were decreased (p <0.05), while the body weight decrease of oridonin group was not significant (p >0.05), but the liver index was significantly decreased compared with the model group, and the body weight change of rats in each group was shown in Table 1.
4.2 Effect on liver weight and liver index of rats with non-alcoholic fatty liver disease
The liver index is calculated as wet liver weight/rat body weight x 100, and may reflect the degree of liver injury. The liver index of the model group rat is 6.83 which is higher than that of the normal rat by 4.23, the significant difference exists, the liver index of the fatty liver rat can be significantly reduced by the drug group, and the liver index is in a descending trend along with the increase of the dosage of the therapeutic drug, and the result is shown in table 1.
TABLE 1 Effect of liver weight and liver index in groups of rats
Note: comparison with the normal group:ap<0.05; comparison with model groups:bp<0.05。
4.3 Effect on serum ALT, AST of rats in each group
Compared with a normal control group, the serum ALT and AST activity of the rats in the model group is obviously improved, and the difference has statistical significance (p is less than 0.05); each therapeutic drug can obviously reduce serological indexes of NAFLD rats, and the difference has statistical significance (p is less than 0.05); the Rabdosia rubescens (Hemsl.) Hara treatment prognosis is that the serum ALT and AST of the rat are obviously reduced compared with a model group, and the treatment effect is dose-dependent; the Rabdosia rubescens composition has better therapeutic effect compared with the rubescensin A, NAC and taurine group with equal dosage, and the result is shown in Table 2.
TABLE 2 Effect on serological indices in rats
Note: comparison with the normal group:ap<0.05; comparison with model groups:bp<0.05; comparison with NAC group:cp<0.05; comparison with taurine group:dp<0.05。
4.4 comparison of blood lipids in rats of each group
After the rats are fed with high-fat feed, the serum CHO, TG and LDL-C of the rats in the model group are obviously increased compared with those in the normal control group, the HDL content is obviously reduced, and the difference has statistical significance (p is less than 0.05); the high and low dose groups, the oridonin group, the easy recovery group and the taurine have the treatment effect on the NAFLD rat, all indexes of the rat blood fat are improved to a certain degree, and the difference has statistical significance (p is less than 0.05); the acetylcysteine has poor improvement on NAFLD rat serum lipid, and the result has no significant difference (p is more than 0.05); compared with the oridonin, NAC and taurine with equal dosage, the oridonin composition has better treatment effect on serum CHO and TG, the difference has statistical significance (p is less than 0.05), and the HDL-C treatment medicament has no significant difference with a control group, and the result is shown in a table 3.
TABLE 3 Effect on serum blood lipids in rats
Note: units are mmol/L, compared to normal group:ap<0.05; comparison with model group:bp<0.05; comparison with NAC group:cp<0.05; comparison with the taurine group:dp<0.05。
4.5 comparison of FFA and FBG indexes of rats in each group
The serum FFA and FBG of the rat in the model group are obviously increased (p is less than 0.05) compared with those of the normal control group, the indexes of the serum FFA and FBG of the rat in the high and low dose groups of the rabdosia composition are improved to a certain extent, the difference has statistical significance (p is less than 0.05), acetylcysteine is obviously improved on the serum FFA, taurine can obviously reduce the serum FBG, the treatment effect of the rabdosia composition with equal dose is obviously better than that of the control drug, and the result is shown in table 4.
4.6 Effect on TNF-alpha, INS, Leptin indices in rats of each group
The serum TNF-alpha, INS and Leptin of the rats in the model group are obviously increased compared with the serum TNF-alpha, INS and Leptin of the rats in the normal group (p is less than 0.05); compared with the model group, the Rabdosia rubescens high and low dose groups and the oridonin group have obvious reduction of TNF-alpha, INS and Leptin (p is less than 0.05); NAC improved TNF-alpha significantly, while taurine significantly reduced NAFLD rat serum INS, the results are shown in Table 5.
TABLE 4 Effect on rat serum FFA, FBG
Note: comparison with the normal group:ap<0.05; comparison with model groups:bp<0.05; comparison with NAC group:cp<0.05; comparison with the taurine group:dp<0.05。
TABLE 5 Effect on rat serum TNF-. alpha.INS, Leptin
Note: comparison with the normal group:ap<0.05; comparison with model groups:bp<0.05; comparison with NAC group:cp<0.05; comparison with the taurine group:dp<0.05。
4.7 Effect on the hepatic tissue GSH-Px, SOD and MDA indexes of various groups of rats
SOD and GSH-Px activities in liver tissues of rats in the model group are obviously reduced, MDA content is obviously increased, and the model group has statistical significance (p is less than 0.05) compared with a normal group; the oridonin and the rabdosia rubescens composition can obviously improve SOD, GSH-Px and MDA of liver tissues of NAFLD rats in different dosages; compared with the model group, taurine can not improve the liver tissue oxidation index of NAFLD rats, NAC has better treatment effect, and the result is shown in Table 6.
TABLE 6 Effect on rat liver tissue oxygenation index
Note: comparison with the normal group:ap<0.05; comparison with model groups:bp<0.05; comparison with NAC group:cp<0.05; comparison with the taurine group:dp<0.05。
4.8 Effect on the indices of hepatic tissue TG and TC of various groups of rats
The TC and TG contents of rat liver tissues in the rat high-fat feed feeding model group are obviously increased compared with those in the normal group, and the difference is significant (p is less than 0.05); after the rabdosia rubescens is treated, the contents of TC and TG are obviously reduced (p is less than 0.05) compared with a model group; NAC can not reduce the TC and TG contents of liver tissues of NAFLD rats, and taurine has a good treatment effect and is easy to recover; rabdosia rubescens composition group had better therapeutic effect compared to equal dose NAC, and the results are shown in Table 7.
TABLE 7 Effect on hepatic TG, TC
Note: comparison with the normal group:ap<0.05; comparison with model groups:bp<0.05; comparison with NAC group:cp<0.05; comparison with the taurine group:dp<0.05。
4.9 histopathological results of liver
The normal group of rats has complete and clear liver tissue structure under a light microscope, normal liver lobule structure and orderly arranged liver cell cables; the rat liver cells of the model group are seriously injured, diffuse liver cell steatosis and liver cell balloon-like degeneration can be seen, inflammatory cell infiltration is accompanied in a sink region, partial punctate liver cells are necrotic, clastic necrosis is accompanied, and a small amount of fiber deposition is accompanied; the damage degree of the rat liver in the high-dose group is obviously reduced compared with that in the model group, the fatty degeneration of liver cells is obviously reduced, only a few punctate necroses appear, a few fat drops vacuole, and no obvious inflammatory cell infiltration exists in a sink region; the liver tissues of rats in the low-dose group and the oridonin group also have diffuse liver cell steatosis, the degree is lighter than that of a model group, and inflammation is obviously improved; the liver lobules of the easily recovered group are clearer, obvious degeneration and fatty degeneration are not seen, vesicular fat drop vacuoles are occasionally seen, compared with a model group, the liver lobules are obviously reduced, the degree of fatty degeneration and inflammatory necrosis is obviously improved, and the pathological results are shown in figures 1-8.
4.10 immunohistochemical results of liver tissues of rats in each group
In the normal group, positive expression of PPAR alpha is seen in that brownish yellow particles are mainly distributed in the nucleus of the liver cell around the junction area, the cytoplasm is occasionally seen, positive expression of the liver cell around the central vein is less, and positive expression of Kunpen cells, endothelial cells and other cells is not seen; compared with the normal group, the positive expression of PPAR alpha cell nucleus in the model group is obviously reduced (p < 0.05); the low and high dose groups have increased PPAR alpha nucleus positive expression and increased dose-dependency compared with the model group; the high dose group and the model group have significant increase in PPAR alpha cell positive expression (p < 0.05);
similarly, PPAR γ positive expression is mainly localized in the hepatocyte nucleus, and brownish yellow granules are mainly distributed in the hepatocyte nucleus around the zone of the junction; compared with a normal group, the positive expression of the model group PPAR gamma cells is obviously reduced (p is less than 0.05); the positive expression of PPAR γ cells was increased in the low and high dose groups compared to the model group, and was dose-dependent, with a significant increase in positive expression of PPAR γ cells in the high dose group and the model group (p < 0.05). As shown in table 8, fig. 7-24.
TABLE 8 Effect on rat liver tissue PPAR α and PPAR γ protein expression
Note: comparison with the normal group:ap<0.05; comparison with model groups:bp<0.05；
4.11 Effect on rat liver tissue PPAR α, PPAR γ and SREBP-1c mRNA expression levels
Compared with the normal group, the rat liver tissue PPAR alpha mRNA expression of the model group is obviously reduced, the difference has statistical significance (p is less than 0.05), compared with the model group, the low-dose group and the high-dose group are obviously increased and are dose-dependent (p is less than 0.05); the PPAR gamma mRNA expression of rat liver tissues in the model group is obviously reduced compared with that in the normal group, the difference between the two groups is obvious (p is less than 0.05), and after the rat liver tissues are treated by the low-dose group and the high-dose group, the PPAR gamma mRNA expression of the rat liver tissues is obviously increased and is dose-dependent (p is less than 0.05). The expression quantity of SREBP-1c mRNA in liver tissue of a normal group of rats is less, the transcription of SREBP-1c mRNA is gradually increased along with the gradual increase of the degree of fatty liver, and compared with the normal group, the expression of SREBP-1c mRNA in a model group is more obvious, and the two have significant difference (p < 0.05). The Rabdosia rubescens composition group SREBP-1c mRNA expression was significantly reduced (p <0.05) compared to the model group, and the results are shown in FIGS. 25-27 and Table 9.
TABLE 9 Effect on the expression of PPAR α, PPAR γ and SREBP-1c mRNA in NAFLD rat liver tissue
Note: comparison with the normal group:ap<0.05; comparison with model groups:bp<0.05; comparison with NAC group:cp<0.05; comparison with the taurine group:dp<0.05。
5. summary of the invention
With the change of life style and eating habits of people, the incidence of NAFLD is increasing year by year and becomes one of the most common causes of chronic liver diseases worldwide. Nearly 1/3 adults in the United states suffer from NAFLD, obesity, type 2 diabetes, hypertriglyceridemia, excessive weight gain, and rapid weight loss (especially in obese subjects), and the special syndromes associated with insulin resistance (such as lipodystrophy diabetes and Mauriac syndrome) are at high risk for NAFLD.
The occurrence and development of NAFLD involve abnormal lipid metabolism, increased ROS production, liver lipid peroxidation, hepatic stellate cell activation, abnormal cytokine production and the like. In addition, researches show that triglyceride and cholesterol of fatty liver patients are obviously increased, and triglyceride is increased as a main characteristic, and the proportion of fatty liver caused by hyperlipidemia is increased along with the increase of the incidence rate of hyperlipidemia.
In the experiment, a high-fat feed is adopted to establish a NAFLD animal model, and the method is also a more and more ideal molding method at present; research on the use of the rabdosia rubescens composition and the rubescensine A for preventing and treating NAFLD shows that the composition can obviously improve the levels of ALT, AST, TC, TG, LDL-C and HDL-C in rat serum of NAFLD, particularly the composition with high dose of the rabdosia rubescens composition is most obvious, and the result also shows that the composition consisting of the rabdosia rubescens A, the rubescensine B, the rosmarinic acid, the ferulic acid and the quercetin in the weight ratio of 6:1:3:3:4 provided by the invention has the effect of synergistically treating NAFLD. The results of this study preliminarily confirm that: the protein level expressions of PPAR alpha mRNA and PPAR alpha in a NAFLD model group are obviously reduced, which indicates that the PPAR alpha expression is reduced to participate in the molecular basis of NAFLD disease, and the PPAR alpha is mainly expressed in the hepatocyte nucleus around a junction area, thereby indicating that the PPAR alpha shows an activated form to adapt to the hepatic portal vein high nutrition perfusion state in normal time. PPAR γ protein level expression is significantly increased in NAFLD rats, and gene knockout of PPAR γ results in reduction and hypertrophy of mouse adipocytes, elevation of blood FFA and TG, and increased occurrence of liver IR and fatty liver. Research results show that the rabdosia rubescens composition can obviously improve the expression of PPAR gamma of liver tissues of NAFLD rats, and can improve fat tissue IR by activating PPAR gamma genes, so that the rabdosia rubescens composition can prevent lipid peroxidation. Rabdosia rubescens composition develops a new way for clinically treating NAFLD by enhancing the expression of PPAR alpha and PPAR gamma and improving insulin resistance and lipid metabolism disorder. SREBPs are a class of membrane associated proteins, which are located on the endoplasmic reticulum. SREBP-1 is also a nuclear transcription factor, and SREBP-1c is one of the subtypes, is mainly involved in sugar metabolism and fatty acid metabolism, is a main regulator of fat synthesis gene transcription, and is closely related to steatosis. This item shows from the mRNA level results: SREBP-1c and PPAR gamma have good consistency relation with the degree of the liver cell lesion, and the SREBP-1c and the PPAR gamma are probably cooperated to participate in the generation and development of NAFLD. Further proves that the rabdosia rubescens composition achieves the purpose of preventing and treating NAFLD by regulating the expression of PPAR gamma and SREBP-1 c.
In addition, in the research, the liver tissue PPAR gamma mRNA expression of a high-dose group of the rabdosia rubescens composition has an increased trend compared with that of a model group after 10 weeks of the intervention treatment of the rabdosia rubescens composition, but the amplitude is not large in immunohistochemical staining result, and the change of the liver tissue PPAR gamma mRNA expression and the immunohistochemical staining result is inconsistent, so that the reason for the expression is presumed that on one hand, the PPAR gamma binding activity is enhanced after the intervention treatment of the rabdosia rubescens composition is administered, and the PPAR gamma mRNA expression with partial compensation in feedback inhibition is enhanced; on the other hand, it is also possible that competitive inhibition of endogenous ligands by exogenous ligands results in altered structural function of PPAR γ, resulting in a decrease in the detected PPAR γ mRNA levels. The specific reasons for this have yet to be further investigated.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.
Claims (10)
1. A composition containing Rabdosia rubescens active ingredients for treating NAFLD is characterized by comprising oridonin, rubescensin B, rosmarinic acid, ferulic acid and quercetin; the weight ratio of the oridonin, the rubescensine B, the rosmarinic acid, the ferulic acid and the quercetin is (3-10): (1-3): 1-4): 2-6.
2. The composition of rabdosia rubescens active ingredient for treating NAFLD of claim 1 wherein the weight ratio of rubescensine A, rubescensine B, rosmarinic acid, ferulic acid and quercetin is (4-8): 1-2): (2-3): (3-4).
3. The composition of rabdosia rubescens active ingredient for the treatment of NAFLD according to claim 1 or 2, wherein the weight ratio of rubescensine a, rubescensine b, rosmarinic acid, ferulic acid and quercetin is 6:1:3:3: 4.
4. A medicament of rubescensine active ingredient for the treatment of NAFLD, it includes active ingredient and supplementary product, the said active ingredient is rubescensine A, rubescensine B, rosmarinic acid, ferulic acid and quercetin; the weight ratio of the oridonin to the rubescensin B to the rosmarinic acid to the ferulic acid to the quercetin is (3-10): 1-3): 1-4): 2-6); the auxiliary materials are pharmaceutically acceptable auxiliary materials.
5. The medicament of rubescensine active ingredient for the treatment of NAFLD according to claim 4, wherein the weight ratio of rubescensine A, rubescensine B, rosmarinic acid, ferulic acid and quercetin is 6:1:3:3: 4.
6. A medicament of rabdosia rubescens active ingredient for the treatment of NAFLD as claimed in claim 4 or 5, wherein pharmaceutically acceptable excipients comprise one or more solid or liquid excipients.
7. The medicament of rabdosia rubescens active ingredient for treating NAFLD as claimed in claim 4 or 5, which is in the form of tablet, capsule, powder injection or pill.
8. The medicament of rabdosia rubescens active ingredient for the treatment of NAFLD according to claim 7, wherein the dosage form is a dispersible tablet, a soft capsule or a drop pill.
9. Use of a composition according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of NAFLD.
10. Use of a medicament according to any one of claims 4 to 8 in the manufacture of a medicament for the treatment of NAFLD.
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