CN111329963A - Gastrodia elata composition and application thereof - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/32—Burseraceae (Frankincense family)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
Abstract
The invention discloses a gastrodia elata composition, which comprises 0.1-0.3 g of gastrodia elata and 0.5-1.5 mL of olive oil; according to the invention, enzyme-linked immunosorbent assay and real-time fluorescent quantitative PCR related technical research prove that the combination of the gastrodia elata and the olive oil can promote the release of a neurotransmitter 5-hydroxytryptamine (5-HT) in the brain of a mouse, and is obviously superior to the independent use of the gastrodia elata and the olive oil in the aspects of reducing the level of an inflammatory factor in the brain, protecting the liver and the kidney and the like; the combination of the gastrodia elata and the olive oil is perfused into a stomach mouse, and experiments show that: the combination of Gastrodia elata Blume and olive oil can promote the release of mouse neurotransmitter 5-HT, reduce the level of inflammatory factors in brain, and protect liver and kidney, while Gastrodia elata Blume alone and olive oil alone can not protect liver and kidney.
Description
Technical Field
The invention belongs to the technical field of medicinal diet and molecular biology, and particularly relates to a composition of gastrodia elata and olive oil, application of the composition in improving the 5-HT content of a neurotransmitter in a mouse brain and reducing the expression level of an inflammatory factor in the mouse brain, and application of the composition in protecting the liver and the kidney.
Background
The medicated diet is food prepared by matching medicinal materials and food materials, which is characterized in that the medicine is taken as food, the food is endowed with medicine, the medicine is aided by eating power, and the food is aided by medicine, the medicine and the food are supplemented with each other, and the medicine and the food complement each other; it has high nutritive value, and can prevent and treat diseases, promote health, strengthen body constitution, and prolong life. Rhizoma Gastrodiae is used as a traditional food material with homology of medicine and food, and has effects of treating headache, endogenous liver wind, convulsive epilepsy, convulsion and giddiness. The olive oil is prepared by directly cold pressing fresh olive fruits, is not subjected to heating and chemical treatment, retains natural nutritional ingredients, and has natural health promotion effect.
5-hydroxytryptamine (5-HT) is synthesized from the amino acid precursor tryptophan, which is derived from the diet, in which about 1% of tryptophan is converted to 5-HT. This enzyme is present in enterochromaffin cells of the small intestine, and 5-HT is present in the nervous system as an important central neurotransmitter and is distributed mainly in hypothalamic, mesencephalic, pineal and brainstem mesenteric nuclear neurons. 5-HT is two independent systems in the central nervous system and the periphery, and the blood brain barrier makes it difficult for 5-HT to penetrate centrally from the peripheral blood, but the pathways for biosynthesis of 5-HT are consistent in the two independent systems. The central nervous system 5-HT is used as an important signal molecule and neurotransmitter, is involved in a plurality of nerve regulations and is closely related to a plurality of neuropsychiatric disorders or diseases, such as cognitive disorder, insomnia, mania, depression and the like, and the peripheral tissues 5-HT is involved in physiological processes of intestinal tract movement, visceral sensation and epithelial secretion and the like. 5-HT acts primarily through the serotonin receptor, 5-HT being a typical G-protein coupled receptor, a family of at least 14 members of which are divided into 7 groups (5-HT)1–5-HT7) Each 5-HT receptor subtype may be involved in the regulation of different physiological functions.
5-HT1AIs closely related to mood regulation; 5-HT1B/5-HT1DAs one of the targets of the action of the migraine-resistant drug, the agonist (such as sumatriptan) has good curative effect in the treatment of migraine; 5-HT2AMay be involved in pain-related affective changes, and 5-HT has also been shown2ADown regulation ofIs associated with the progression of Parkinson's disease. 5-HT2BMainly distributed in liver, kidney, fundus and intestinal tract of mammals, and 5-HT2BThe antagonist is expected to be used for treating chronic heart disease. 5-HT2CHigher expression in amygdala for modulating anxiety, blocking 5-HT2CHas anxiolytic and antidepressant effects. 5-HT3The antagonist has effects in improving abnormal behavior and resisting anxiety after withdrawal from diazepam, nicotine, cocaine and alcohol, and can be used for treating 5-HT3The antagonist has certain curative effect on improving memory and cognitive function and relieving fibromyalgia. 5-HT4The agonist has an effect of improving learning and memory, and thus is expected to improve part of symptoms of patients with Alzheimer's Disease (AD). 5-HT5Has 5-HT5AAnd 5-HT5BSubtype, 5-HT5AMay play an important role in 5-HT mediated cerebellar function. Furthermore, 5-HT5AMay also be associated with circadian rhythm, mood and cognitive function. 5-HT6Closely related to cognitive function, 5-HT in recent years6Ligands have been the subject of research for improving cognitive disorders. 5-HT7Associated with anxiety depression, furthermore, 5-HT7Partial antagonists block 5-HT mediated hypothermia in guinea pigs and rats, and thus 5-HT is considered7May be involved in thermoregulation.
Inflammation of the central nervous system is characterized by activation of nerve cells and release of inflammatory cytokines and chemokines, the inflammatory factors of the central nervous system are risk factors of many diseases, the over-expression of the inflammatory factors relates to neurodegenerative diseases such as AD and Parkinson's disease, mental diseases such as schizophrenia and depression, and the inflammation is also related to cerebral function deterioration, memory deterioration, insomnia and the like; the liver inflammation refers to inflammation change of the liver caused by injuries such as virus, medicament, alcohol or metabolism abnormality, the occurrence of the liver inflammation can cause the expression of a plurality of inflammation factors to be increased, and the anti-inflammatory medicament can inhibit the expression of the related inflammation factors and play a role in improving the liver function; the nephritis is divided into primary glomerulonephritis and secondary glomerulonephritis, and the pathological characteristics of the nephritis are mainly inflammatory cell invasion in kidneys, mesangial cells are proliferated, various inflammatory factors and polypeptide growth factors are secreted, so that vascular damage in the kidneys is caused, extracellular matrix synthesis is increased, and a large amount of extracellular matrix is accumulated, so that glomerular sclerosis and kidney fibrosis are finally caused.
The inflammatory factor refers to various cytokines involved in inflammatory response, and among various inflammatory cytokines, interleukin-6 (IL-6), TNF- α, IL-1 β and the like play a main role, IL-6 is a cytokine in a chemokine family, is produced by various cells, can widely induce B cell differentiation and produce immunoglobulin, promote T cell proliferation and growth, promote bone marrow hematopoietic stem cell proliferation, enhance blood cell differentiation and resist tumors and the like, IL-6 is clinically associated with many diseases, IL-6 is related to AD and renal diseases, AD is an inflammatory neurodegenerative disease caused by neuronal cell death and progressive dementia, in addition, IL-6 is also increased in children with acute lymphocytic leukemia, TNF- α is the earliest and most important inflammatory mediator in the inflammatory response process, can activate neutrophils and lymphocytes, increases vascular endothelial cell permeability, regulates other tissue metabolic activity and promotes synthesis and release of other cytokines, in addition, the inflammatory response of nerve cells is related to the excitable nerve response, the inflammatory response is increased, and the inflammatory response of nerve damage of nerve cells can be closely related to the nerve cell injury of a nerve system, and the nerve damage can be caused by the chronic nerve injury of Foc, and the nerve cell injury can be caused by the chronic nerve.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a gastrodia elata composition which comprises 0.1-0.3 g of gastrodia elata and 0.5-1.5 mL of olive oil.
When the composition is used, the composition is diluted by normal saline, and 2mL-4mL of normal saline is added.
The invention also aims to apply the gastrodia elata composition to the preparation of the medicine for promoting the release of neurotransmitter 5-HT in the brain and reducing the expression level of inflammatory factors in the brain;
the gastrodia elata composition can reduce the side effect of gastrodia elata or olive oil on the liver and reduce the side effect of gastrodia elata or olive oil on the kidney while reducing the expression level of inflammatory factors in the brain.
According to the invention, the gastrodia elata composition is administrated to a mouse in a continuous gastric perfusion mode, then the 5-HT content in the brain of the mouse is determined by an enzyme-linked immunosorbent assay (ELISA) method, the expression of serotonin receptors and inflammatory factors in the brain, the liver and the kidney is determined by real-time fluorescence quantitative PCR (RT-qPCR), and the experimental result shows that the gastrodia elata and olive oil composition is obviously superior to the gastrodia elata or the olive oil in the aspects of improving the 5-HT content of neurotransmitter in the brain of the mouse, reducing the expression level of the inflammatory factors in the brain of the mouse, protecting the liver and the kidney and the like.
The gastrodia elata-olive oil composition accords with the medical food definition of 'medicine is taken by livelihood', can remarkably up-regulate 5-HT of neurotransmitter in brain, and has a remarkably better up-regulation effect than the gastrodia elata or olive oil, the gastrodia elata-olive oil composition can hardly up-regulate the level of injury marker inflammatory factors in brain, liver and kidney while up-regulating 5-HT, and the gastrodia elata and the olive oil up-regulate the level of the inflammatory factors in brain, liver and kidney while up-regulating 5-HT, so that the gastrodia elata and the olive oil are reacted to be accompanied by side effects when acting independently, and the stress on the kidney and the liver is higher.
The intracerebral inflammatory factor is a risk factor of a plurality of diseases, including neurodegenerative diseases such as AD and Parkinson's disease, mental diseases such as schizophrenia and depression, and the like, and also has the functions of brain deterioration, memory decline, insomnia and the like; inflammatory factors in the liver are risk factors for many diseases, including hepatitis, liver cancer, etc.; inflammatory factors in the kidney are risk factors for many diseases, including nephritis, kidney cancer, and the like.
The elevated brain neurotransmitter 5-HT of the gastrodia elata composition has the effects of resisting depression, improving cognitive impairment of AD patients, enhancing memory of the AD patients and the like, and provides a new way for treating related diseases caused by brain inflammatory factors; the gastrodia elata composition of the invention provides a new way for treating related diseases caused by inflammatory factors in liver and kidney by reducing the inflammatory factors in liver and kidney. The invention provides possibility for the development of related medicaments based on the gastrodia elata composition in the future, and has great application value and prospect.
Drawings
FIG. 1 shows the results of the measurement of 5-HT content in the brain of a mouse according to the present invention, wherein: NS is normal saline group (control); gastrodia is a rhizoma gastrodiae experimental group; oil is olive Oil experimental group; gastrodia + Oil is a Gastrodia elata-olive Oil combined experimental group;
FIG. 2 shows the results of the expression test of 5-HT receptor 5-HT1B in the mouse brain of the present invention, in which: NS is normal saline group (control); gastrodia is a rhizoma gastrodiae experimental group; oil is olive Oil experimental group; gastrodia + Oil is a Gastrodia elata-olive Oil combined experimental group;
FIG. 3 shows the results of the expression test of 5-HT receptor 5-HT6 in the mouse brain of the present invention, in which: NS is normal saline group (control); gastrodia is a rhizoma gastrodiae experimental group; oil is olive Oil experimental group; gastrodia + Oil is a Gastrodia elata-olive Oil combined experimental group;
FIG. 4 is a schematic diagram showing the results of detecting the expression of the inflammatory factors IL-2 (A) and IL-6 (B) in the mouse brain according to the present invention; in the figure: NS is normal saline group (control); gastrodia is a rhizoma gastrodiae experimental group; oil is olive Oil experimental group; gastrodia + Oil is a Gastrodia elata-olive Oil combined experimental group;
FIG. 5 shows the results of the detection of the expression of the cell activation marker Fos in the brain of a mouse according to the present invention, in which: NS is normal saline group (control); gastrodia is a rhizoma gastrodiae experimental group; oil is olive Oil experimental group; gastrodia + Oil is a Gastrodia elata-olive Oil combined experimental group;
FIG. 6 shows the results of the expression tests of IL-1 β (A) and IL-6 (B) as the mouse liver inflammatory factors of the present invention, wherein NS is normal saline group (control), Gastrodia is Gastrodia elata experimental group, Oil is olive Oil experimental group, and Gastrodia + Oil is Gastrodia elata-olive Oil combined experimental group;
FIG. 7 shows the results of the expression tests of the mouse kidney inflammatory factors IL-1 β (A) and TGF- β (B), wherein NS is a normal saline solution group (control), Gastrodia is a Gastrodia elata experimental group, Oil is an olive Oil experimental group, and Gastrodia + Oil is a Gastrodia elata-olive Oil combined experimental group.
Detailed Description
The invention is explained in more detail below with reference to the figures and examples, without limiting the scope of the invention.
Example 1: drug formulation and animal administration
Gastrodia elata-olive oil combined experimental group: taking 0.2 g of gastrodia elata, 1mL of olive oil and 3 mL of physiological saline; gastrodia elata experimental group: taking 0.2 g of gastrodia elata and 4mL of physiological saline; olive oil experimental group: taking 1mL of olive oil and 3 mL of physiological saline; saline control group: 4mL of physiological saline. The medicines are heated and mixed into suspension at 37 ℃, the medicines in each group are continuously perfused into the stomach of mice for 20 days, 10 mice in each group and 5 mice in each group are respectively perfused into the stomach, the total dose of each group is 4 mL/day, and 5 parallel tests are carried out in each experimental group. The mice used in the test are SPF-level Kunming mice purchased from the center of SPF-level laboratory animals of Kunming medical university, and the experimental animals produce license number SCXK (Dian) K2015-0002, 4 weeks old and weight (20 +/-2) g.
Example 2: detection of neurotransmitter 5-HT content in mouse brain
The ELISA kit is used for detecting the 5-HT content of the mouse brain, and the operation process is as follows:
1. after each group of drugs is continuously infused into the stomach of a mouse for 20 days, the mouse is killed by a cervical dislocation method, the whole brain of the mouse is taken, the following operations are all carried out at 4 ℃, bloodstains are washed off in normal saline, the whole brain of the mouse is placed into a centrifuge tube, the whole brain of the mouse is ground by using a handheld homogenizer to obtain the whole brain homogenate of the mouse, 0.1g of the whole brain homogenate of the mouse is weighed and quickly mixed in 0.8 mL of 1 × PBS, the mixture is fully vortexed to dissolve out a transmitter, 5000 g of the mixture is centrifuged for 10min, and the supernatant is taken for carrying out an ELISA experiment;
2. the ELISA test is carried out according to the instructions of an ELISA kit (Mlbio company), the result is shown in figure 1, and the result shows that compared with the normal saline group, the mouse intracerebral neurotransmitter content of the gastrodia elata-olive oil combined test group, the gastrodia elata test group and the olive oil test group is increased, but compared with the gastrodia elata-olive oil, the 5-HT increasing effect of the gastrodia elata-olive oil combined test group and the olive oil test group is not as good as that of the gastrodia elata-olive oil combined test group, the experiment result shows that the gastrodia elata-olive oil combined test group can be used for increasing the 5-HT content in the mouse brain to the greatest extent, and the reaction regulation of inducing high-level participation of the 5-HT by the gastrodia elata-olive oil combined test group is stronger than that of the gastrodia elata-olive oil combined.
Example 3: detection of 5-HT1B, 5-HT6 receptor expression in mouse brain
Extracting total RNA of mouse whole brain with Trizol reagent (Saimenfiel technologies), and detecting 5-HT1B and 5-HT6 expression in mouse brain with RT-qPCR kit (Saimenfiel technologies)
1. Respectively weighing 0.1g of whole brain homogenate of each group in an EP (the EP is marked with RNase-Free), adding 1mL of Trizol for cracking, blowing with an inverse multiplexing gun or shaking with an oscillator to fully crack tissues, adding 0.2 mL of chloroform according to the amount of each 1mL of Trizol, reversing and uniformly mixing for 10 times, standing for 15 min at normal temperature, and then centrifuging for 15 min at 4 ℃ and 12000 g, wherein the layering phenomenon can occur at the moment, the upper layer is colorless, and the RNA is near transparent; the middle layer is white (very thin) is DNA; taking the upper layer as a red protein, putting the upper layer into another EP tube (the EP tube is labeled with RNase-Free), adding isopropanol with the same volume into the EP tube only containing the upper layer, reversing and uniformly mixing for 10 times, standing at room temperature for 15 min, centrifuging at 4 ℃ and 12000 g for 10min, removing the supernatant, adding 1mL of precooled 75% ethanol for washing, carrying out vortex mixing, centrifuging at 4 ℃ and 7500 g for 5min, removing the supernatant, naturally drying the precipitated RNA at room temperature for 5-10 min, and adding 20 mu L of DEPC water to dissolve the RNA precipitate;
2. first strand cDNA was synthesized using a reverse transcription kit (Seimer Feishhl. Co.) using total RNA as a template
The Reaction system and the operation process are that 2 mu L of each group of RNA dissolving solution in the step 1 is respectively taken and put into a PCR tube, 1 mu of LOoligo (dt) 18 primer is added, then 9 mu L of nuclease-free high-purity water is added, the total volume is 12 mu L, the mixture is centrifuged for a short time, after the mixture is heated and denatured for 5min at 65 ℃, the mixture is rapidly cooled on ice for 3 min to 5min, then the mixture is rapidly cooled on ice, 4 mu L of 5 × Reaction Buffer and 1 mu of RNA dissolving solution are sequentially added into the PCR tubeL RiboLock RNase inhibitor, 2. mu.L 10mM dNTP, 1. mu.L reverse AidTMM-MULV reverse transcriptase with a total volume of 20 μ L, mixing, centrifuging briefly, incubating at 42 deg.C for 60 min, and heating at 75 deg.C for 10min to terminate the reaction; the reaction product can be directly used for PCR reaction;
3. detection of 5-HT1B expression in mouse brain Using RT-qPCR kit (Saimer Feishell science Co., Ltd.)
Designing and synthesizing RT-qPCR primers, an upstream primer 5'-TGTACGTGAACCAAGTCAAAGTG-3' and a downstream primer 5'-GTAGATGATGGGGTTGATGAGAG-3'; sequentially adding 4 mu L of MasterMix, 10 mu L of nuclease-free high-purity water, 2 mu L of each group of cDNA synthesized in the step 2 of the embodiment 3, 1 mu L of 10pM 5-HT1B upstream primer, 1 mu L of 10pM 5-HT1B downstream primer and 20 mu L of total volume into a PCR tube special for RT-qPCR; reaction conditions are as follows: denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 10s, and 45 cycles; the size of the RT-qPCR product of 5-HT1B is 200 bp; the detection results are shown in fig. 2, and it can be seen from the figure that compared with the normal saline group, the 5-HT1B expressions in the brains of mice in the gastrodia elata-olive oil combination experimental group, the gastrodia elata experimental group and the olive oil experimental group are all up-regulated, but compared with the gastrodia elata experimental group and the olive oil experimental group, the gastrodia elata-olive oil combination experimental group has no statistical difference in the up-regulation effect on the 5-HT1B expression, and the experimental results show that the gastrodia elata-olive oil combination has no significant effect on the up-regulation of the 5-HT1B expression in the brains of the mice.
4. Detection of 5-HT6 expression in mouse brain Using RT-qPCR kit (Saimer Feishell science Co., Ltd.)
Designing and synthesizing RT-qPCR primer, upstream primer 5'-CTGGGAATGTTCTTTGTCACCT-3' downstream primer, 5'-GAAGCGGAGTCTGAATCTGAGTT-3'; sequentially adding 4 mu L of MasterMix, 10 mu L of nuclease-free high-purity water, 2 mu L of each group of cDNA, 1 mu L of 10pM 5-HT6 upstream primer, 1 mu L of 10pM 5-HT6 downstream primer and 20 mu L of total volume into a special RT-qPCR tube; reaction conditions are as follows: denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 10s, and 45 cycles; the RT-qPCR product size of 5-HT6 is 190 bp; the results are shown in fig. 3, and it can be seen from the figure that compared with the normal saline group, the 5-HT6 expressions in the mice of the gastrodia elata-olive oil combined experimental group, the gastrodia elata experimental group and the olive oil experimental group are all up-regulated, but the gastrodia elata-olive oil combined experimental group has a weaker up-regulation effect on the 5-HT6 expressions than the gastrodia elata experimental group and the olive oil experimental group, and the experimental results show that the gastrodia elata-olive oil has no significant effect on the 5-HT6 expression in the mice.
Example 4: detection of mouse intracerebral inflammatory factors IL-2, IL-6, nerve cell excitation marker Fos expression
1. The extraction and reverse transcription method of total RNA of mouse whole brain refers to steps 1 and 2 in example 3;
2. detection of IL-2 in mouse brain Using RT-qPCR kit (Saimer Feishell science Co., Ltd.)
Designing and synthesizing RT-qPCR primers, an upstream primer 5'-TGAGCAGGAGGAGAATTACAGG-3' and a downstream primer 5'-GTCCAAGTTCATCTTCTAGGCAC-3'; sequentially adding 4 muL of MasterMix, 10 muL of nuclease-free high-purity water, 2 muL of each group of cDNA, 1 muL of 10pM IL-2 upstream primer, 1 muL of 10pM IL-2 downstream primer and 20 muL of total volume into a special RT-qPCR tube; reaction conditions are as follows: denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 10s, and 45 cycles; the size of the RT-qPCR product of the IL-2 is 120 bp; the results are shown in fig. 4A, and it can be seen from the figure that compared with the normal saline group, the gastrodia elata-olive oil combined experimental group, the gastrodia elata experimental group and the olive oil experimental group can all up-regulate the expression of the inflammation factor IL-2 in the brain of the mice, but the gastrodia elata-olive oil combined experimental group up-regulates the expression level of the inflammation factor IL-2 to be far lower than that of the gastrodia elata experimental group and the olive oil experimental group, and the experimental results show that the gastrodia elata-olive oil combination can reduce the risk of diseases induced by the gastrodia elata and the olive oil and caused by the inflammation factor IL-2 in the brain.
3. Detection of IL-6 in mouse brain Using RT-qPCR kit (Saimer Feishell science Co., Ltd.)
Designing and synthesizing RT-qPCR primers, an upstream primer 5'-TAGTCCTTCCTACCCCAATTTCC-3' and a downstream primer 5'-TTGGTCCTTAGCCACTCCTTC-3'; sequentially adding 4 muL of MasterMix, 10 muL of nuclease-free high-purity water, 2 muL of each group of cDNA, 1 muL of 10pM IL-6 upstream primer, 1 muL of 10pM IL-6 downstream primer and 20 muL of total volume into a special RT-qPCR tube; reaction conditions are as follows: denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 10s, and 45 cycles; the size of the RT-qPCR product of the IL-6 is 76 bp; the results are shown in fig. 4B, and it can be seen from the figure that compared with the normal saline group, the gastrodia elata-olive oil combined experimental group, the gastrodia elata experimental group and the olive oil experimental group can all up-regulate the expression of the mouse intracerebral inflammatory factor IL-6, but the gastrodia elata-olive oil combined experimental group up-regulates the expression level of the inflammatory factor IL-6 to be far lower than that of the gastrodia elata experimental group and the olive oil experimental group, and the experimental results show that the gastrodia elata-olive oil combination can reduce the risk of diseases induced by gastrodia elata and olive oil and induced by the intracerebral inflammatory factor IL-6.
4. Detection of Fos in mouse brain by RT-qPCR kit (Saimer Feishell science Co.)
Designing and synthesizing RT-qPCR primers, an upstream primer 5'-CGGGTTTCAACGCCGACTA-3' and a downstream primer 5'-TTGGCACTAGAGACGGACAGA-3'; sequentially adding 4 mu L of MasterMix, 10 mu L of nuclease-free high-purity water, 2 mu L of each group of cDNA, 1 mu L of 10pM Fos upstream primer and 20 mu L of total volume into a RT-qPCR special-purpose PCR tube; reaction conditions are as follows: denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 10s, and 45 cycles; the RT-qPCR product size for Fos was 166 bp. The results are shown in fig. 5, and it can be seen from the figure that compared with the normal saline group, the gastrodia elata-olive oil combined experimental group, the gastrodia elata experimental group and the olive oil experimental group can reduce the nerve cell activation marker Fos, and compared with the gastrodia elata experimental group and the olive oil experimental group, the gastrodia elata-olive oil group has a more significant effect of reducing the nerve cell activation marker Fos, and the experimental results show that the gastrodia elata-olive oil can relieve the excitability of nerve cells, so that irreversible damage of the nerve cells caused by the inflammatory reaction of the nerve cells is reduced.
Example 5 detection of IL-1 β and IL-6 inflammatory factor expression in mouse liver
1. The mouse used in the step is the mouse in the step 1 of the embodiment 2, the mouse is killed by cervical dislocation, the whole brain of the mouse is taken, then the liver of the mouse is taken, the following operations are all carried out at 4 ℃, the blood stain of the liver of the mouse is washed out in normal saline, the liver of the mouse is placed in a centrifuge tube, a hand-held homogenizer is used for grinding the liver of the mouse to obtain liver homogenate, 0.1g of the liver homogenate of each group is respectively weighed in an EP tube (the EP tube is marked with RNase-Free), and the extraction and reverse transcription method of the total RNA of the liver of the mouse refers to the steps 1 and 2 in the embodiment 3;
2. the detection method is the same as the steps 3 and 5 of the embodiment 4;
the detection result of IL-1 β is shown in figure 6A, and the expression of inflammatory factor IL-1 β in the liver induced by the gastrodia elata-olive oil combined experimental group is far lower than the expression of inflammatory factor IL-1 β in the liver induced by the gastrodia elata experimental group and the olive oil experimental group, and the experimental result shows that the gastrodia elata-olive oil can reduce the risk of related diseases caused by the inflammatory factor IL-1 β in the liver induced by the gastrodia elata and the olive oil when the gastrodia elata and the olive oil are used alone, and reduce the toxic and side effects of the gastrodia elata or the olive oil on the liver when the gastrodia elata or the;
the IL-6 detection result is shown in figure 6B, and it can be seen from the figure that there is no significant difference (no statistical difference) in the expression of the inflammatory factor IL-6 among the three groups of the gastrodia elata-olive oil combined experimental group, the olive oil experimental group and the normal saline group, but compared with the gastrodia elata-olive oil, the gastrodia elata up-regulates the expression of the inflammatory factor IL-6 in the liver of a mouse, and the experimental result shows that the gastrodia elata-olive oil can reduce the risk of related diseases caused by induction of the gastrodia elata and the inflammatory factor IL-6 in the liver, and reduce the toxic and side effects of the gastrodia elata on the liver.
Example 6 detection of IL-1 β and TGF- β inflammatory factor expression in mouse Kidney
1. Mouse kidney sample preparation method refer to example 5, step 1;
2. the detection method is the same as the step 5 of the embodiment 4;
the detection results are shown in fig. 7A, and it can be seen from the figure that the gastrodia elata experimental group and the olive oil experimental group can both up-regulate the expression of IL-1 β in the kidney of the mouse compared with the gastrodia elata-olive oil combined experimental group, and the experimental results show that the gastrodia elata-olive oil can reduce the risk of related diseases caused by the induction of inflammatory factors IL-1 β in the kidney when the gastrodia elata and the olive oil are used alone, and reduce the toxic and side effects of the gastrodia elata or the olive oil on the kidney when the gastrodia elata or the olive oil;
RT-qPCR upstream primer 5'-GTGTGGAGCAACATGTGGAACTCTA-3' and downstream primer 5'-TTGGTTCAGCCACTGCCGTA-3' of TGF- β, RT-qPCR product size 238 bp of TGF- β, and detection results are shown in figure 7B, and it can be seen from the figure that a gastrodia elata experimental group and an olive oil experimental group can both up-regulate TGF- β expression in mouse kidney compared with a gastrodia elata-olive oil combined experimental group, and the experimental results show that the gastrodia elata-olive oil can reduce the risk of related diseases caused by inflammatory factor TGF- β in kidney when the gastrodia elata and the olive oil are used alone, and reduce the toxic and side effects of the gastrodia elata or the olive oil on kidney when the gastrodia elata or the olive oil are used alone.
Sequence listing
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Claims (4)
1. A gastrodia elata composition is characterized in that: the composition comprises 0.1-0.3 g rhizoma Gastrodiae and 0.5-1.5 mL oleum Olivarum.
2. The use of the composition of gastrodia elata as claimed in claim 1 in the preparation of a medicament for promoting the release of neurotransmitter 5-HT in the brain and reducing the expression level of inflammatory factors in the brain.
3. Use according to claim 2, characterized in that: the gastrodia elata composition can reduce the side effect of gastrodia elata or olive oil on the liver while reducing the expression level of inflammatory factors in the brain.
4. Use according to claim 2, characterized in that: the gastrodia elata composition can reduce the side effect of gastrodia elata or olive oil on the kidney while reducing the expression level of inflammatory factors in the brain.
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| CN116735514A (en) * | 2023-08-11 | 2023-09-12 | 昆明理工大学 | Method for rapidly detecting gastrodia elata sulfuration markers by nano-enzyme combined liquid-liquid microextraction |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105395899A (en) * | 2015-11-18 | 2016-03-16 | 中国医学科学院医药生物技术研究所 | External use dosage form of gastrodia elata |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105395899A (en) * | 2015-11-18 | 2016-03-16 | 中国医学科学院医药生物技术研究所 | External use dosage form of gastrodia elata |
Non-Patent Citations (3)
| Title |
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| PEI-JU CHEN,ET.: "Rhizomes of Gastrodia elata BL Possess", 《THE AMERICAN JOURNAL OF CHINESE MEDICINE》 * |
| 祝洪艳等: ""天麻苷元药理作用研究进展"", 《上海中医药杂志》 * |
| 胡鹏程等: ""天麻改善小鼠睡眠作用及其机制研究"", 《中草药》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116735514A (en) * | 2023-08-11 | 2023-09-12 | 昆明理工大学 | Method for rapidly detecting gastrodia elata sulfuration markers by nano-enzyme combined liquid-liquid microextraction |
| CN116735514B (en) * | 2023-08-11 | 2023-11-03 | 昆明理工大学 | Method for rapidly detecting gastrodia elata sulfuration markers by nano-enzyme combined liquid-liquid microextraction |
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