CN112107653A - Application of dendrobe extract in preparation of drugs for regulating cholesterol metabolism - Google Patents
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Abstract
The invention belongs to the technical field of pharmacy, and relates to application of a dendrobium extract in preparing a medicine for regulating cholesterol metabolism. In particular to the regulation of cholesterol metabolism by a dry dendrobium water extract, a fresh dendrobium water extract and a dendrobium alcohol extract under the state of high blood fat. In vitro experiments prove that the dry dendrobium water extract, the fresh dendrobium water extract and the dendrobium alcohol extract prepared by the invention can increase the gene expression of atherosclerotic scavenger receptor type B I, proprotein convertase subtilisin/kexin 9, low-density lipoprotein receptor and lecithin cholesterol acyltransferase in HUH7 cells, so that the conclusion that the dendrobium extract can be used as a medicine for regulating cholesterol metabolism can be obtained.
Description
Technical Field
The invention relates to the technical field of pharmacy, in particular to application of a dendrobium extract in preparing a medicine for regulating cholesterol metabolism.
Background
Cholesterol is a steroid compound, is indispensable to the metabolism of the body, and plays an important role in the composition of cell membranes and the synthesis of hormones, in particular. Cholesterol is mainly derived from the body's own synthesis, partly from food. The human body can synthesize cholesterol in the liver, and also can ingest exogenous cholesterol through diet. In coronary heart disease and atherosclerosis, atheromatous plaques are formed by the deposition of lipids such as cholesterol. In the body, cholesterol is bound to proteins, mainly in the form of lipoproteins, and is transported in the body mainly in the form of both high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C). Among them, high density lipoprotein cholesterol (HDL-C) is called good cholesterol because existing studies indicate that elevated levels of HDL-C in serum have an effect of cleaning atherosclerosis and a protective effect on cardiovascular diseases. In contrast, if the level of LDL-C in the blood is elevated, it is likely to cause atherosclerosis, damage to the heart blood vessels, and lead to an increased incidence of coronary heart disease and other complications, and thus LDL-C is known as bad cholesterol relative to HDL-C.
In recent years, there have been many studies focusing on improving the cholesterol level of the body by diet regulation, thereby effectively preventing a series of complications caused by the increase of cholesterol. At present, it has been found that the plant components have the effects of regulating body fat levels and reducing cholesterol levels in the plasma and liver of the body, and it is indicated that the plant proteins have the effect of significantly reducing and regulating cholesterol levels in the serum and liver of experimental animals, compared to animal proteins.
Dendrobe is a common and rare traditional Chinese medicine, is sweet, light and slightly salty in taste and cold, and enters stomach, kidney and lung meridians. The dendrobium huoshanense, the dendrobium officinale, the dendrobium nobile and the dendrobium nobile are superior products in dendrobium nobile. Also called Linlan (Shennong Bencao Jing), Qiannian Ruan (Bencao gang mu), etc., it has higher ornamental value and medicinal function, recorded in Li Shizhen Bencao gang mu, dendrobe has the efficacies of nourishing five internal organs, fatigue slimming, nourishing yin and replenishing essence, taking thick intestines and stomach for a long time, calming stomach qi, growing muscle, fixing mind and removing fright, reducing weight and prolonging life, etc. According to the research of our literature, the dendrobium or the dendrobium extract has not been found to be applicable to the treatment of obesity diseases. According to the research of our literature, the application and report that the dendrobium or the dendrobium extract has the function of regulating the metabolism of cholesterol are not found.
Disclosure of Invention
The invention aims to provide application of a dendrobium extract in preparing a medicine for regulating cholesterol metabolism, and is a new application of the dendrobium extract. There are no reports at present that the dendrobe extract can be used for the regulation of cholesterol metabolism.
The application of the dendrobium extract in preparing the medicine for regulating cholesterol metabolism is characterized in that the dendrobium extract is obtained by extracting a dendrobium medicinal material, and the dendrobium extract is a dendrobium water extract or a dendrobium alcohol extract.
Further, the dendrobium stem is one or a combination of more than two of dendrobium huoshanense, dendrobium officinale, dendrobium nobile, dendrobium fimbriatum and dendrobium chrysotoxum.
Further, the dendrobium water extract comprises a dry dendrobium water extract and 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 preparation steps of the dendrobium alcohol extract are as follows: extracting herba Dendrobii dry powder with 18-20 times of 75% ethanol at 55-60 deg.C, repeatedly extracting, mixing extractive solutions, concentrating under reduced pressure to 1/6-1/8, and freeze drying to constant weight.
Furthermore, the dendrobium 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 beneficial effects that:
the dendrobe extract prepared by the invention can increase gene expression of atherosclerosis scavenger receptor B type I, proprotein convertase subtilisin/kexin 9, low-density lipoprotein receptor and lecithin cholesterol acyltransferase in HUH7 cells, so that the dendrobe extract can be used as a medicine for regulating cholesterol metabolism.
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FIG. 1 shows that water extract of dried dendrobii herba increases gene expression of atherosclerotic scavenger receptor class B type I, proprotein convertase subtilisin/kexin 9, low density lipoprotein receptor and lecithin cholesterol acyltransferase in HUH7 cells; wherein, the data are expressed as means + -SD, SR-BI: type I of atherosclerosis scavenger receptor type B, with P < 0.05; PCSK 9: proprotein convertase subtilisin/kexin type 9, P < 0.01; LDLR: a low density lipoprotein receptor, P < 0.05; LCAT: lecithin cholesterol acyltransferase; a administration group vs control group; DWE: dry Dendrobium Water extract; fold: multiple times.
FIG. 2 shows that fresh dendrobe water extract increases gene expression of atherosclerotic scavenger receptor type B I, proprotein convertase subtilisin/kexin 9, low density lipoprotein receptor and lecithin cholesterol acyltransferase in HUH7 cells; wherein, the data are expressed as means + -SD, SR-BI: type I of atherosclerosis scavenger receptor class B, with < 0.01 of < Lamin P; PCSK 9: proprotein convertase subtilisin/kexin type 9, P < 0.05; LDLR: a low density lipoprotein receptor, P < 0.05; LCAT: lecithin cholesterol acyltransferase, # # P < 0.01; a administration group vs control group; FWE: water extract of fresh herba Dendrobii of resh Dendrobium water Extraction; fold: multiple times.
FIG. 3 is a graph showing that an alcohol extract of Dendrobium nobile enhanced gene expression of atherosclerotic scavenger receptor class B type I, proprotein convertase subtilisin/kexin type 9, low density lipoprotein receptor and lecithin cholesterol acyltransferase in HUH7 cells; wherein, the data are expressed as means + -SD, SR-BI: type I of atherosclerosis scavenger receptor class B, < 0.05 of < Lamin P; PCSK 9: proprotein convertase subtilisin/kexin type 9, P < 0.01; LDLR: a low density lipoprotein receptor, P < 0.05; LCAT: lecithin cholesterol acyltransferase, < 0.05; a administration group vs control group; DEE: an alcohol extract of Dendrobium officinale extract from Dry Dendrobium ethanol; fold: multiple times.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
As an important organ of cholesterol metabolism, the expression level of mRNA, a metabolic gene related to cholesterol in the liver, plays a key role in the cholesterol level of the body. The invention researches the influence of the dendrobium extract as a medicament on the regulation of cholesterol metabolism by detecting the expression level of the cholesterol metabolism related genes.
High Density Lipoprotein (HDL) has the effect of reversing cholesterol transport back to the liver. In the process, Lecithin Cholesterol Acyltransferase (LCAT) plays a crucial role in the synthesis of HDL and reverse cholesterol transfer, LCAT is synthesized and released by liver into blood circulation and plays a role in catalyzing blood plasma, LCAT transfers C2 unsaturated fatty acid of HDL Lecithin to free cholesterol to generate lysolecithin and cholesterol ester, and plasma cholesterol is almost 70% -80% cholesterol ester and is all caused by the catalytic generation of LCAT.
Low Density Lipoprotein Receptor (LDLR) is one of the major members of the Low Density Lipoprotein Receptor family, and is capable of binding to Low Density Lipoprotein (LDL), playing an important role in the metabolism of cholesterol in vivo. LDLR is expressed in almost all cells, but in the largest number in the liver. LDL binds to LDLR on the surface of cell membrane, and is swallowed into cells, and LDLB is hydrolyzed by lysosomal enzyme to release free cholesterol and fatty acid for the cells to utilize. Cholesterol can regulate the transcription of LDLR through a negative feedback regulation mechanism, and when the level of cholesterol in cells is increased, the transcription of LDLR is reduced; when intracellular cholesterol is consumed, transcription of LDLR is activated. Proprotein convertase subtilisin/kexin type 9 (PCSK9), also known as neuronal apoptosis regulating convertase (neuralaApoptosis-regulated convertase1, NARC-1)), inhibitors of which have been approved by the regulatory agencies for the treatment of patients with inadequate LDL-C decline. PCSK9 is a serine protease encoded by the PCSK9 gene, produced mainly by the liver, and PCSK9 binds to LDL receptors (LDL-R) on the surface of hepatocytes, and is capable of degrading LDL-R and elevating LDL-C levels in the blood. Antibodies to PCSK9 interfere with their binding to LDL-R, allowing the liver to express more LDL-R and lower blood LDL-C levels. The atherosclerosis scavenger receptor class B type I (SR-BI) plays an important role in regulating High Density Lipoprotein (HDL) mediated cholesterol and cholesterol ester uptake, can promote reverse cholesterol transport, clear excessive cholesterol in blood and tissues, protect blood vessels, prevent the formation of atherosclerosis or improve the formed atherosclerosis. SRB1 is a well-known HDL receptor, a key receptor for mediating reverse cholesterol transport, and is capable of causing bidirectional cholesterol transport. On one hand, SR-BI can mediate excessive free cholesterol in peripheral cells to flow out, and prevent atherosclerosis caused by excessive accumulation of free cholesterol; on the other hand, SR-BI can be combined with HDL, promote the liver cells to take up cholesterol in HDL, and return the surplus cholesterol to the liver for further metabolism.
Because the types of the existing medicinal dendrobium are various, the embodiment of the invention takes dendrobium huoshanense as an example, and researches the effect of the dendrobium extract as a medicament applied to the treatment of regulating cholesterol. However, the dendrobium in the invention is not limited to dendrobium huoshanense, but also can be medicinal dendrobium of other same genus, such as dendrobium officinale, dendrobium nobile, dendrobium fimbriatum or dendrobium chrysotoxum.
The dendrobium huoshanense is originally produced in places such as Anhuoshan in China, is the first protective variety in more than 200 endangered rare varieties which are important special items for Chinese traditional medicines in China, 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 Hohan county, and is historically known as the first of nine major Chinese immortals and 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.
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.
(3) Preparing the dendrobe alcohol extract: weighing 20g of dendrobium huoshanense dry powder, extracting with 400ml of 75% ethanol at 55 ℃ for 2 times, extracting for 24h each time, combining the 2 times of extracting solutions, concentrating the combined extracting solutions under reduced pressure to 1/6-1/8 of the original volume, and freeze-drying to constant weight to obtain the dendrobium alcohol extract with the mass volume concentration of 100 mg/ml.
The dry dendrobium water extract, the fresh dendrobium water extract and the dendrobium alcohol extract with the mass concentration of 100mg/mL prepared in the embodiment 1 are filtered and sterilized by a filter membrane of 0.22 mu m before the test, and then are diluted to prepare a series of dry dendrobium water extract, fresh dendrobium water extract and dendrobium alcohol extract with the mass concentrations of 10 mu g/mL, 100 mu g/mL and 1000 mu g/mL in sequence to be used as medicines for standby.
(I) Detecting the expression grouping of the dendrobium extract on cholesterol metabolism related gene mRNA in HUH7 cells in vitro: after HUH7 cells were recovered, they were transferred to a petri dish in DMEM (high-sugar) medium containing 10% FBS, cultured at 37 ℃ under 5% CO2 conditions, synchronized for 12h, and randomly divided into 2 groups, namely: blank control group: no drug administration (0. mu.g/ml); (ii) administration group: (1) high dose group of dried herba Dendrobii water extract (DWE: 1000 μ g/mL), medium dose group of dried herba Dendrobii water extract (DWE: 100 μ g/mL), and low dose group of dried herba Dendrobii water extract (DWE: 10 μ g/mL); (2) high dose group of fresh herba Dendrobii water extract (FWE: 1000 μ g/mL), medium dose group of fresh herba Dendrobii water extract (FWE: 100 μ g/mL), and low dose group of fresh herba Dendrobii water extract (FWE: 10 μ g/mL); (3) and performing fluorescence quantitative PCR (polymerase chain reaction) after 12 hours of administration on the dendrobe alcohol extract medium-dose group (DEE: 100 mu g/mL) and dendrobe alcohol extract low-dose group (DEE: 10 mu g/mL): total RNA from HUH7 cells was extracted using RNAiso Plus, 1. mu.g of total RNA was reverse transcribed to cDNA by OligodT18, and primers were PCR amplified by quantitative PCR using Toyobo company ThunderBirdSYBR qPCR Mix reagent as per instructions. Each set of samples had 3 duplicate wells to ensure validity of experimental data. Using GAPDH as an internal reference, the expression changes of target genes Lecithin Cholesterol Acyltransferase (LCAT), scavenger receptor class B type I (SR-B1), Low Density Lipoprotein Receptor (LDLR) and proprotein convertase subtilisin kexin9 type (PCSK9) were measured and quantified relative to the internal reference Gene (GAPDH). Data (i.e., fold change in expression of the gene of interest in the experimental group relative to the blank control group) was analyzed using Graphpad 7. The primers used were: LCAT (hLCAT-f: ACGGGGCCCTACTGTAATAA; hLCAT-r: AGATGCAGCACTGGAAGGAG), SR-B1 (hSRBI-f: CTGTGGGTGAGATCATGTGG; hSRBI-r: GCTCAGCTGCAGTTTCACAG), LDLR (hLDLR-f-2: AGGAGACGTGCTTGTCTGTC; hLDLR-r-2: CTGAGCCGTTGTCGCAGT), and PCSK9(hPCSK 9-f: AGTTGCCCCATGTCGACTAC; hPCSK 9-r: GAGATACACCTCCACCAGGC). The experimental results shown in fig. 1 show that the dried dendrobium water extract prepared by the present invention can increase the mRNA expression levels of scavenger receptor class B type I (SR-B1) and Low Density Lipoprotein Receptor (LDLR) by administering high dose of dendrobium water extract and increase the mRNA expression levels of scavenger receptor class B type I (SR-B1), proprotein convertase subtilisin kexin9 type (PCSK9), Low Density Lipoprotein Receptor (LDLR) and Lecithin Cholesterol Acyltransferase (LCAT) by administering low dose of dendrobium water extract, compared with the blank control group (administration concentration of 0 μ g/mL), and thus, the dried dendrobium water extract has the effect of significantly regulating cholesterol metabolism as a drug. The experimental results shown in the attached figure 2 show that the administration of the low-dose fresh dendrobe water extract and the administration of the medium-dose fresh dendrobe water extract can both obviously increase the mRNA expression levels of SR-B1, PCSK9, LDLR and LCAT, and the administration of the high-dose fresh dendrobe water extract can increase the mRNA expression levels of SR-B1 and LDLR, namely the fresh dendrobe water extract has the effect of obviously regulating cholesterol metabolism when being used as a medicine, compared with a blank control group (administration concentration is 0 mu g/mL).
The experiment results shown in the attached figure 3 show that compared with a blank control group (administration concentration is 0 mug/mL), the administration of the low-dose dendrobe alcohol extract and the administration of the medium-dose dendrobe alcohol extract can both obviously increase the mRNA expression levels of SR-B1, PCSK9, LDLR and LCAT, namely the dendrobe alcohol extract as a medicament has the effect of obviously regulating cholesterol metabolism.
In conclusion, the dendrobium extract provided by the invention can effectively regulate cholesterol metabolism in the liver.
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 (7)
1. The application of the dendrobium extract in preparing the medicine for regulating cholesterol metabolism is characterized in that the dendrobium extract is obtained by extracting dendrobium medicinal materials, and the dendrobium extract is a dendrobium water extract or a dendrobium alcohol extract.
2. The application of the dendrobium extract in preparing the drugs for regulating cholesterol metabolism according to claim 1, wherein the dendrobium is one or the combination of more than two of dendrobium huoshanense, dendrobium officinale, dendrobium nobile, dendrobium fimbriatum and dendrobium chrysotoxum.
3. The use of the dendrobe extract of claim 1 in the preparation of a medicament for regulating cholesterol metabolism, wherein the dendrobe water extract comprises a dry dendrobe water extract and a fresh dendrobe water extract.
4. The application of the dendrobium extract in preparing the drugs for regulating cholesterol metabolism according to claim 3, wherein the preparation steps of the dried dendrobium water extract are as follows: 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.
5. The application of the dendrobium extract in preparing the drugs for regulating cholesterol metabolism according to claim 3, 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.
6. The application of the dendrobe extract in preparing a medicine for regulating cholesterol metabolism according to claim 1, wherein the preparation steps of the dendrobe alcohol extract are as follows: extracting herba Dendrobii dry powder with 18-20 times of 75% ethanol at 55-60 deg.C, repeatedly extracting, mixing extractive solutions, concentrating under reduced pressure to 1/6-1/8, and freeze drying to constant weight.
7. The use of the dendrobe extract of any one of claims 1 to 6 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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