CN111773323A - Application of dendrobium water extract in treating type 2 diabetes - Google Patents
Application of dendrobium water extract in treating type 2 diabetes Download PDFInfo
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- A61K2236/30—Extraction of the material
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Abstract
The invention belongs to the technical field of pharmacy, and relates to application of a dendrobium water extract in treating type 2 diabetes. In particular to the regulation of insulin resistance and triglyceride synthesis by a dry dendrobium water extract and a fresh dendrobium water extract under a high-sugar state. Experimental results prove that the dendrobe water extract prepared by the invention can obviously inhibit the expression level of mRNA of fatty acid binding protein, improve peripheral insulin resistance, protect pancreatic beta cell function and improve insulin sensitivity, can obviously inhibit the expression level of mRNA of diacylglycerol acyltransferase, reduce biosynthesis of TG, improve sensitivity of an organism to insulin and leptin, increase the expression level of mRNA of sterol regulatory element binding protein 1 and effectively regulate the biosynthesis of triglyceride, thereby realizing the inhibition of type 2 diabetes from multiple aspects. Therefore, the dendrobium water extract can be used for treating type 2 diabetes and has good development prospect in the aspect of treating type 2 diabetes.
Description
Technical Field
The invention relates to the technical field of pharmacy, in particular to application of a dendrobium water extract in treating type 2 diabetes.
Background
Diabetes (diabetes) is a series of metabolic disorder syndromes of sugar, protein, fat, water and electrolyte, etc. caused by hypofunction of pancreatic islets, Insulin Resistance (IR), etc. due to the action of various pathogenic factors such as genetic factors, immune dysfunction, microbial infection and toxins thereof, free radical toxins, mental factors, etc. on the body. Common diabetes mellitus includes mainly type 1 diabetes and type 2 diabetes. Among them, type 1 diabetes mostly occurs in adolescents, and depends on exogenous insulin supplementation to maintain life due to lack of insulin secretion. Type 2 diabetes is most seen in the middle-aged and the elderly, and its insulin secretion is not low or even high, and clinically it is manifested as insufficient insulin sensitivity, i.e. Insulin Resistance (IR). Among diabetic patients, type 2 diabetes accounts for about 95%, and has become the "leading force" of diabetic patients. The disease may be aggravated with the age, and various complications are accompanied, so that the physical health of the patient is seriously affected.
Currently, there are three main categories of type 2 diabetes treatment drugs: biological drugs such as insulin, chemical drugs such as sulfonylureas, biguanides, and Chinese patent drugs. The insulin preparation is expensive and difficult to popularize; although the chemical medicine has obvious effect of reducing blood sugar, the chemical medicine has large toxic and side effects on liver, kidney and the like, and is easy to cause aggravation of cardiovascular complications and the like. The Chinese patent medicine has certain treatment effect, but the modern Chinese medicine with obvious efficacy, clear effective components and action mechanism, controllable quality and advanced preparation is absent at present.
Dendrobium huoshanense (the name of Dendrobiumhuoshanense) is wild Dendrobium huoshanense called as panda in the pharmaceutical field internationally, also called as dendrobe, and has been in history for more than 200 years from now. The dendrobium huoshanense is originally produced in places such as Anhui Huoshan in China, is the first protective variety in more than 200 endangered rare varieties which are important special items of national modern traditional Chinese medicines, is an extreme product in medicinal dendrobium, is also a unique and famous traditional Chinese medicine material with geographical marks of original production places in Huoshan county, and is historically known as the first of nine major Chinese immortals and the healthy soft gold, and is recorded in Shen nong Ben Cao and Ben Cao gang mu. The polysaccharide contained in the dendrobium huoshanense can greatly improve SOD (main substance for delaying senility) in a human body, and has obvious effects on enhancing immunity, resisting fatigue, delaying senility, promoting cancer cell apoptosis and the like, thereby achieving the effects of health care and life prolonging. Besides dendrobium huoshanense, other medicinal dendrobium (dendrobium officinale, dendrobium cupreum and the like) also have similar health care and medicinal values. According to the research of our literature, the application and research of dendrobium huoshanense or dendrobium huoshanense extract in the aspect of treating type 2 diabetes mellitus are not found.
Disclosure of Invention
The invention aims to provide application of a dendrobe water extract in treating type 2 diabetes, and is new application of a dendrobe extract. At present, no report indicates that the dendrobium extract can be applied to the treatment of type 2 diabetes.
The application of the dendrobium water extract in treating type 2 diabetes is characterized in that the dendrobium water extract is obtained by purifying and extracting dendrobium medicinal materials.
Further, the dendrobium water extract is a dry dendrobium water extract or a fresh dendrobium water extract.
Further, the preparation method of the dried dendrobium water extract comprises the following steps: extracting herba Dendrobii dry powder with 18-20 times of distilled water in 55-65 deg.C water bath, filtering, collecting filtrate, repeatedly extracting, mixing extractive solutions, concentrating under reduced pressure to 1/6-1/8 of original total volume, and freeze drying to constant weight.
Further, the preparation method of the fresh dendrobium water extract comprises the following steps: removing roots and leaves of fresh dendrobium, keeping stems, crushing, adding distilled water with the weight 5-8 times of that of the fresh dendrobium, immersing, extracting in water bath at 75-85 ℃, filtering, collecting filtrate, repeatedly extracting, combining extracting solutions, then concentrating under reduced pressure to 1/6-1/8 of the original volume, and freeze-drying to constant weight.
Further, the dendrobium water extract can be prepared into any pharmaceutically acceptable oral and non-oral preparation forms, such as one of granules, pills, capsules, tablets, powder, paste and oral liquid;
the invention has the following beneficial effects:
(1) the dendrobe water extract prepared by the invention can obviously inhibit the expression level of mRNA (messenger ribonucleic acid) of FABP4, further improve peripheral insulin resistance, protect the function of islet beta cells and improve insulin sensitivity, so that diseases related to FABP4, such as type 2 diabetes, are inhibited;
(2) the dendrobe water extract prepared by the invention can obviously inhibit the mRNA expression level of diacylglycerol acyltransferase (DGAT1), reduce the biosynthesis of TG, and improve the sensitivity of an organism to insulin and leptin, thereby inhibiting the occurrence of type 2 diabetes;
(3) the dendrobe water extract prepared by the invention can increase the mRNA expression level of sterol regulatory element binding protein 1(SREBP1) and effectively regulate the biosynthesis of TG, thereby inhibiting the occurrence of type 2 diabetes.
Therefore, the dendrobe water extract prepared by the invention can be used for treating type 2 diabetes and has a good development prospect in the aspect of treating type 2 diabetes.
Drawings
FIG. 1 shows that water extracts of dried and fresh dendrobe inhibit the gene expression of fatty acid binding protein;
wherein, the data are expressed as means ± SD, FABP 4: a fatty acid binding protein which is capable of binding to a fatty acid,**P＜0.01，***P<0.05; a administration group vs blank group; DWE: dry Dendrobium Water ExtractionTaking the object; FWE: fresh dendrobium Water extract of FreshDendrobium Water Extraction; fold: multiple times.
FIG. 2 is a water extract of Dendrobium nobile for increasing gene expression of sterol regulatory element binding protein 1 in HuH7 cells;
where data are expressed as means ± SD, SREBP 1: the sterol regulatory element binds to protein 1 and,*P＜0.05，**P＜0.01，###P<0.05，##p is less than 0.01; a administration group vs blank group; DWE: dry Dendrobium Water extract; FWE: fresh Dendrobium extract of Fresh dendrobe of Fresh Dendrobium tinctoria; fold: multiple times.
FIG. 3 is a graph of water extract of Dendrobium nobile for inhibiting gene expression of diacylglycerol acyltransferase in HuH7 cells;
where data are expressed as means ± SD, DGAT: (ii) a diacylglycerol acyltransferase enzyme,**P＜0.01，***P<0.05，####p is less than 0.01; a administration group vs control group; DWE: dry Dendrobium Water extract; FWE: fresh Dendrobium extract of Fresh dendrobe of Fresh Dendrobium tinctoria; fold: multiple times.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
Fatty acid binding proteins (FABP4) are a family of endogenous low molecular weight proteins that are widely distributed in various cells of the small intestine, liver, fat, heart, brain, and skeletal muscle of mammals and have tissue specificity. FABP4 plays an important role in the regulation of the uptake, transport and metabolism of long chain fatty acids. The research shows that the polypeptide is used as a fatty acid receptor in cells, and the affinity of the polypeptide to fatty acids and derivatives thereof promotes the uptake of the fatty acids. Researches on mice with gene defects of FABP4 (fatty acid binding protein) show that the lipid carrier has close relationship with the occurrence of diseases such as metabolic syndrome, inflammation and atherosclerosis, the reduction of the expression of FABP4 can improve the peripheral insulin resistance, protect the function of islet beta cells and improve the insulin sensitivity, so the lipid carrier can be used as a potential target for treating type 2 diabetes.
Triglyceride (abbreviated as TG) is the main form of biological energy storage, when the energy taken in by human body exceeds the energy consumed by human body, the surplus energy is stored in the form of T G in fat tissue, and simultaneously, the excessive TG can be deposited in tissues such as islet beta cells, and finally, diseases such as diabetes mellitus are caused. Therefore, inhibition of TG synthesis in the above tissues can be a new strategy for the treatment of diabetes.
Diacylglycerol acyltransferase (DGAT) is a key enzyme catalyzing TG synthesis pathway, and the level of expression directly affects the final TG content. DGAT is an endoplasmic reticulum membrane integrin and has two subtypes: the two types of DGAT1 and DGAT2 show that the research shows that the inhibition of the activity of DGAT1 can reduce the synthesis of TG, improve the sensitivity of the body to insulin and leptin, increase the glucose uptake rate and energy consumption, inhibit the occurrence of type 2 diabetes and can be used as a target for treating type 2 diabetes.
Sterol regulatory element-binding protein 1(SREBP1) is an important transcriptional regulator that regulates lipid synthesis, primarily regulating fatty acid, triglyceride, and cholesterol biosynthesis. Abnormal expression of SREBP-1 and its target gene can cause a series of metabolic diseases such as insulin resistance, diabetes and fatty liver.
According to the invention, the expression level of the related mRNA is detected to research the effect of the dendrobe aqueous extract as a medicine applied to the treatment of type 2 diabetes.
Example 1
Respectively preparing a dry dendrobium water extract, a fresh dendrobium water extract and a dendrobium alcohol extract, which are as follows:
(1) preparing a dry dendrobium water extract: weighing 20g of dendrobium huoshanense dry powder, extracting with 400ml of distilled water in a water bath at 55 ℃, filtering, collecting filtrate, repeatedly extracting for 3 times, extracting for 1 hour each time, and combining the extracting solutions for 3 times. Concentrating the combined extractive solutions at 55 deg.C under reduced pressure to 1/6-1/8 of the original liquid volume, and freeze drying to constant weight to obtain dried herba Dendrobii water extract with mass volume concentration of 100 mg/ml.
(2) Preparing a fresh dendrobium water extract: weighing 100g of freshly picked dendrobium huoshanense, removing roots and leaves, keeping stems, crushing, adding 500ml of distilled water to immerse the dendrobium huoshanense medicinal material, extracting in a water bath at 80 ℃, filtering and collecting filtrate, repeatedly extracting for 3 times, extracting for 1 hour each time, and combining the extracting solutions for 3 times. Concentrating the mixed extractive solutions at 55 deg.C under reduced pressure to 1/6-1/8 of original volume, and freeze drying to constant weight to obtain fresh herba Dendrobii water extract with mass volume concentration of 100 mg/ml.
The dry dendrobium water extract and the fresh dendrobium water extract which are prepared in the embodiment 1 and have the mass concentration of 100 mg/mL are filtered and sterilized by using a filter membrane of 0.22 mu m before the test, and then are diluted to prepare a series of working solutions of the dry dendrobium water extract and the fresh dendrobium water extract which have the mass concentrations of 10 mu g/mL, 100 mu g/mL and 1000 mu g/mL in sequence and are used as medicines for standby.
(I) detecting the expression of the mRNA of an insulin resistance related gene FABP4 in RAW264.7 cells by the dendrobium water extract
RAW264.7 (mouse mononuclear macrophage) DMEM complete medium (with 10% fetal calf serum, 100U/mL penicillin, 100. mu.g/mL streptomycin added) at 37 ℃ with 5% CO by volume fraction2The cells were synchronized for 12h and randomly divided into 2 groups, ① blank control group, no administration (0. mu.g/mL), ② administration group, (1) dried dendrobe water extract high dose group (DWE: 1000. mu.g/mL), dried dendrobe water extract medium dose group (DWE: 100. mu.g/mL), dried dendrobe water extract low dose group (DWE: 10. mu.g/mL) (2) fresh dendrobe water extract high dose group (1000. mu.g/mL; ⑥ fresh dendrobe water extract medium dose group (100. mu.g/mL), ⑦ fresh dendrobe water extract low dose group (10. mu.g/mL), administration for 12h, fluorescence quantitative PCR, total RNA of RAW264.7 cells was extracted using RNAi Plus, 1. mu.g total RNA was reverse transcribed to cDNA by OlidTGo 18, PCR was performed using Toyobo thundergardBirdBR amplification reagent according to the instructions, PCR was performed using 3 PCR primers to detect the validity of the internal fatty acid binding protein of the sample, 4 protein binding to the internal fatty acid binding protein of the reference sampleThe change in expression of the Gene (GAPDH) was quantified. By using 2-ΔΔCtThe method analyzes data (i.e., fold change of expression amount of target gene of experimental group relative to blank control group). The primers used were: FABP4 (mFABP 4-F: AAATCACCGCAGACGACAGG; mFABP 4-R: AACTCTTGTGGAAGTCACGCC).
The experimental results shown in the attached figure 1 show that the administration of the dry dendrobium water extract and the fresh dendrobium water extract prepared by the invention can obviously inhibit the mRNA expression level of fatty acid binding protein (FABP4) compared with the blank control group (administration concentration is 0 mu g/mL) through the administration of the high-dose and medium-dose dendrobium water extracts. Therefore, the dry dendrobium water extract and the fresh dendrobium water extract can obviously inhibit the mRNA expression level of fatty acid binding protein (FABP4), further improve the peripheral insulin resistance, protect the function of islet beta cells, improve the sensitivity of insulin and inhibit the occurrence of type 2 diabetes, so the dendrobium water extract can be used as a medicine for treating type 2 diabetes.
(II) detecting the expression of the dendrobium water extract on mRNA of TG synthesis related genes DGAT1 and SREBP1 in vitro HuH7 cells
Grouping: after the HuH7 cells were recovered, they were cultured in DMEM complete culture dish at 37 ℃ with 5% CO2Culturing under conditions, synchronizing cells for 12h, randomly dividing into 2 groups, that is, ① blank control group, no administration (0. mu.g/mL), ② administration group, (1) dried dendrobe water extract high dose group (DWE: 1000. mu.g/mL), dried dendrobe water extract medium dose group (DWE: 100. mu.g/mL), dried dendrobe water extract low dose group (DWE: 10. mu.g/mL), (2) fresh dendrobe water extract high dose group (1000. mu.g/mL; ⑥ fresh dendrobe water extract medium dose group (100. mu.g/mL), ⑦ fresh dendrobe water extract low dose group (10. mu.g/mL), administration for 12h, fluorescence quantitative PCR, total RNA of HuH7 cells is extracted using RNAi Plus, 1. mu.g total RNA is reverse transcribed into cDNA by Oligo 18, PCR is performed by quantitative PCR technique using Toyoord SYSTEN Mix reagent according to amplification instructions, PCR is performed using 3 PCR primers, and the two-fold acyl transferase is used to detect the validity of glycerol acyltransferase in the target gene (DGAT) data of 3. 1)The mRNA of the control element binding protein 1(SREBP1) was quantified relative to the change in expression of the reference Gene (GAPDH), respectively. By using 2-ΔΔCtThe method analyzes data (i.e., fold change of expression amount of target gene of experimental group relative to blank control group). The primers used were: SREBP1 (hSREBP 1-f: GGATTGCACTTTCGAAGACATG; hSREBP 1-r: AGCATAGGGTGGGTCAAATAGG), DGAT1 (hDGAT 1-f: GGTGCCCTGACGGAGCAGGC; hDGAT 1-r: CAGTAAGACCACAGCCGCTG).
The experimental results shown in the attached figure 2 show that the dried dendrobium water extract and the fresh dendrobium water extract prepared by the invention can increase the mRNA expression level of the sterol regulatory element binding protein 1(SREBP1) by administration of the low-dose and medium-dose dried dendrobium water extracts, and can obviously increase the mRNA expression level of the sterol regulatory element binding protein 1(SREBP1) by administration of the low-dose fresh dendrobium water extract, compared with a blank control group (administration concentration is 0 mu g/mL). Therefore, the dry dendrobium water extract and the fresh dendrobium water extract can increase the mRNA expression level of sterol regulatory element binding protein 1(SREBP 1)), so that the biosynthesis of TG can be effectively regulated, and the occurrence of type 2 diabetes is inhibited, therefore, the dendrobium water extract can be used as a medicine for treating type 2 diabetes.
The experimental results shown in fig. 3 show that the dry dendrobium water extract and the fresh dendrobium water extract prepared by the method can obviously inhibit the mRNA expression level of diacylglycerol acyltransferase (DGAT1) through administration of medium-dose and high-dose dry dendrobium water extracts and can obviously inhibit the mRNA expression level of diacylglycerol acyltransferase (DGAT1) through administration of high-dose fresh dendrobium water extracts, so that the dry dendrobium water extract and the fresh dendrobium water extract can obviously inhibit the mRNA expression level of diacylglycerol acyltransferase (DGAT1), reduce biosynthesis of TG, and improve the sensitivity of an organism to insulin and leptin, thereby inhibiting the occurrence of type 2 diabetes, and therefore the dendrobium water extract can be used as a medicament for treating type 2 diabetes.
In conclusion, the dendrobium water extract prepared by the invention not only can obviously inhibit the mRNA expression level of diacylglycerol acyltransferase (DGAT1), reduce the biosynthesis of TG and improve the sensitivity of an organism to insulin and leptin, but also can increase the mRNA expression level of sterol regulatory element binding protein 1(SREBP1) and effectively regulate the biosynthesis of TG, thereby inhibiting the occurrence of type 2 diabetes. Therefore, the dendrobium water extract prepared by the invention can be applied to the treatment of type 2 diabetes.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. The application of the dendrobium water extract in treating type 2 diabetes is characterized in that the dendrobium water extract is obtained by purifying and extracting dendrobium medicinal materials.
2. The use of the aqueous extract of dendrobe of claim 1 for the treatment of type 2 diabetes, wherein the aqueous extract of dendrobe is an aqueous extract of dry dendrobe or an aqueous extract of fresh dendrobe.
3. The use of the aqueous extract of dendrobii herba as claimed in claim 2 for the treatment of type 2 diabetes, wherein the aqueous extract of dry dendrobii herba is prepared by the steps of: extracting herba Dendrobii dry powder with 18-20 times of distilled water in 55-65 deg.C water bath, filtering, collecting filtrate, repeatedly extracting, mixing extractive solutions, concentrating under reduced pressure to 1/6-1/8 of original total volume, and freeze drying to constant weight.
4. The application of the dendrobium extract in preparing the drug for regulating cholesterol metabolism according to claim 2, wherein the preparation steps of the fresh dendrobium water extract are as follows: removing roots and leaves of fresh dendrobium, keeping stems, crushing, adding distilled water with the weight 5-8 times of that of the fresh dendrobium, immersing, extracting in water bath at 75-85 ℃, filtering, collecting filtrate, repeatedly extracting, combining extracting solutions, then concentrating under reduced pressure to 1/6-1/8 of the original volume, and freeze-drying to constant weight.
5. The use of the dendrobe extract of any one of claims 1 to 4 in the preparation of a medicament for regulating cholesterol metabolism, wherein the dendrobe extract can be prepared into any pharmaceutically acceptable oral and non-oral dosage forms.
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