阅读说明：本技术 一种降香组合物及其应用 (Dalbergia wood composition and application thereof ) 是由 赵月然 齐越 姚倚琦 鄢长余 李军 郭月秋 贾冬 于 2021-01-11 设计创作，主要内容包括：本发明涉及一种降香组合物及其应用,属于中药药物研发领域。本发明的降香组合物由中药降香及丹参组成,采用水蒸馏法提取降香挥发油,取回流提取法提取丹参药材水提物,将降香挥发油与丹参液混合,即得。采用气相色谱法对降香进行质量控制,液相色谱法对丹参药材进行质量控制。降香与丹参配伍后能够芳香开窍,引药归经,经动物实验证实本发明的降香组合物具有防治血管性痴呆的药效活性,可能机制为减少神经细胞的凋亡来保护血管性痴呆大鼠的大脑皮质及海马神经元细胞。(The invention relates to a dalbergia wood composition and application thereof, belonging to the field of traditional Chinese medicine research and development. The dalbergia wood composition is prepared by extracting dalbergia wood volatile oil by a water distillation method, extracting an aqueous extract of a red sage root medicinal material by a reflux extraction method, and mixing the dalbergia wood volatile oil with a red sage root liquid. The quality control of lignum Dalbergiae Odoriferae and the quality control of Saviae Miltiorrhizae radix by gas chromatography are performed. Animal experiments prove that the dalbergia wood composition has the efficacy activity of preventing and treating vascular dementia, and the possible mechanism is to reduce the apoptosis of nerve cells to protect cerebral cortex and hippocampal neuron cells of a rat with vascular dementia.)

1. A dalbergia wood composition is characterized in that: is prepared from the following raw materials in parts by weight: 10-30 g of dalbergia wood and 10-30 g of salvia miltiorrhiza.

2. A preparation method of a dalbergia wood composition is characterized by comprising the following steps: 10-30 g of dalbergia wood and 10-30 g of salvia miltiorrhiza, crushing dalbergia wood medicinal materials, and extracting dalbergia wood volatile oil by a water distillation method; crushing a salvia miltiorrhiza medicinal material, adding water which is 2-5 times of the weight of the salvia miltiorrhiza, soaking for 1-3 hours, performing reflux extraction for 1-3 times at 90-100 ℃, combining extracting solutions, concentrating, mixing the extracting solution, and mixing the dalbergia wood volatile oil and the salvia miltiorrhiza solution to obtain the traditional Chinese medicine composition, wherein each milliliter of the solution contains 1-3 g of crude drug of the dalbergia wood and 1-3 g of crude drug of the salvia miltiorrhiza.

3. The use of the dalbergia wood composition of claim 1 in the preparation of an anti-vascular dementia medicament.

Technical Field

The invention belongs to the technical field of preparation of traditional Chinese medicine formulas, and particularly relates to a composition of dalbergia wood and salvia miltiorrhiza and application thereof.

Background

Vascular Dementia (VaD) refers to the qualitative and functional changes of cerebral vessels caused by cerebral ischemia, cerebral hemorrhage or hemorrhagic brain tissue damage, acute and chronic ischemia and hypoxia, mainly the impairment of memory and cognitive function, and the accompanying speech, movement, visual space disorder and personality disorder. Epidemiological investigations show that: vascular dementia is the second most common cause of dementia following Alzheimer's Disease (AD), with a 3.9% incidence of dementia in older adults over 65 years of age in china, with vascular dementia as high as 68.5% occurring. The average life age of vascular dementia is only 4-6 years.

Vascular dementia is a type of dementia which can be prevented and treated, and the early treatment has certain reversibility. However, vascular dementia is a multifactorial process, the etiology is complex, the exact pathogenesis is not clear, and an effective therapeutic drug is lacked.

At present, fewer drugs are used for treating vascular dementia, galantamine and donepezil are common cholinesterase blockers, galantamine has no obvious improvement effect on patients with simple vascular dementia, and can be used for treating patients with vascular dementia complicated with Alzheimer disease, and donepezil can improve the cognition and the overall function of the patients to a certain extent. Therefore, it is necessary to develop a Chinese patent medicine for treating vascular dementia, to treat vascular dementia at an early stage, and to avoid the decline of cognitive function and memory function of a patient with vascular dementia, so as to achieve the purpose of treating vascular dementia. The red sage root and the dalbergia wood are a traditional medicine pair, the red sage root has the functions of activating blood circulation to dissipate blood stasis, stimulating the menstrual flow and relieving pain, and the red sage root enters heart channels and liver channels, and the dalbergia wood has the functions of dissipating blood stasis, stopping bleeding, regulating qi and relieving pain. After being combined with Danshen root, Dalbergia wood can induce resuscitation with aromatics and induce the drugs to enter meridians.

Disclosure of Invention

The invention aims to provide a rosewood heart wood composition and application thereof. The content of the application mainly comprises the weight ratio of the dalbergia wood and the salvia miltiorrhiza medicine and the application thereof.

The purpose of the invention is realized by the following technical scheme:

the invention provides a dalbergia wood composition, which comprises the following raw material medicines in parts by weight: 10-30 g of dalbergia wood and 10-30 g of salvia miltiorrhiza.

The invention provides a preparation method of a dalbergia wood composition, which comprises the steps of taking 10-30 g of dalbergia wood medicinal materials and 10-30 g of salvia miltiorrhiza, crushing the dalbergia wood medicinal materials, and extracting dalbergia wood volatile oil by a water distillation method; crushing a salvia miltiorrhiza medicinal material, adding water which is 2-5 times of the weight of the salvia miltiorrhiza, soaking for 1-3 hours, performing reflux extraction for 1-3 times at 90-100 ℃, combining extracting solutions, concentrating, mixing the dalbergia wood volatile oil and the salvia miltiorrhiza liquid, and adding polysorbate 80 with the volume ratio of 2-5% to obtain the traditional Chinese medicine composition, wherein each milliliter of the solution is equivalent to 1-3 g of the crude drug of the dalbergia wood, and 1-3 g of the crude drug of the salvia miltiorrhiza.

The rosewood heart wood; the main components of Saviae Miltiorrhizae radix include danshensu, protocatechualdehyde, rosmarinic acid and salvianolic acid B.

Animal experiments prove that the dalbergia wood composition has obvious prevention and treatment effects on a vascular dementia model of a rat. Pharmacodynamic experiment results show that by adopting a Morris water maze experimental method, the lignum dalbergiae odoriferae composition can promote the spatial learning and memory ability of rats with vascular dementia; shortening the swimming latency and total swimming distance of the vascular dementia rats, increasing the quadrant distance percentage of the platform, increasing the number of Nissl (Nissl) bodies and reducing the apoptosis of neurons.

Damage to neuronal cells in the brain of patients with VaD can lead to a loss of memory. Apoptosis of nerve cells is a major cause of neuronal cell damage in the brain. Apoptosis is the process of cell death, which is called programmed cell death, because some gene of cell is activated, and cell is actively and gradually killed according to a certain program. Apoptosis is also an essential process for the elimination of damaged cells in the process of maintaining cellular homeostasis and progression. Because of the limited ability of central nervous system neurons to proliferate and regenerate, reducing neuronal apoptosis is a major goal in protecting the VaD cerebral cortex and hippocampal neurons.Bcl-2(B-cell lymphoma2) Bcl-2 protein family is an indispensable system in the regulation program of apoptosis, and Bcl-2 regulates and controls the apoptosis of mitochondria, acts on the upstream of Caspase-3, inhibits the release of cytochrome C and the like from the mitochondria, and plays an anti-apoptosis effect. When Bcl-2 is highly expressed, the Bcl-2 can form heterodimer Bcl-2/Bax with apoptosis factor Bax, the heterodimer has the effect of inhibiting Bax/Bax homodimer from inducing apoptosis, a PI3K/Akt signal pathway is one of apoptosis pathways, the PI3K signal pathway can improve the cell viability, inhibit cell senescence and death and the like, after PI3K is activated, Akt activation can be induced, and the activated Akt is transferred from cytoplasm to a cell membrane and provides an anchoring site for recruiting PI3K translocation to the membrane to participate in signal transduction. Apoptosis can also occur via the mitochondrial pathway, the Caspase family^[68]Mainly involved in mitochondrial pathways, of which Caspase 3, Caspase-9 are one of the most important downstream effectors of apoptosis. In the VaD neurodegenerative process, Caspase 3 and Caspase-9 activity can be inhibited to generate a neuroprotective effect.

The dalbergia wood composition can reduce Bax expression and increase Bcl-2 expression, so that Bcl-2/Bax ratio is increased, apoptosis of cells is inhibited, the Bax expression level of a VaD model group is obviously up-regulated, the expression level of Bcl-2 factors is reduced, so that Bax/Bcl-2 ratio is higher, and neuronal cells are induced to undergo apoptosis. The protective effect of the dalbergia wood composition on VaD is realized by up-regulating Bcl-2 and down-regulating Bax expression to protect neuronal cells. The rosewood composition can increase the expression of PI3K and Akt protein and reduce the content of Caspase 3 and Caspase-9 protein. The suggestion shows that the rosewood heart wood composition can inhibit the expression of Caspase 3 and Caspase-9 by activating a PI3K/Akt signal channel, thereby reducing the apoptosis of neuron cells.

Drawings

FIG. 1 is a high performance liquid chromatogram of lignum Dalbergiae Odoriferae volatile oil in lignum Dalbergiae Odoriferae composition;

FIG. 2 is a high performance liquid chromatogram of an aqueous solution of Salvia miltiorrhiza Bunge in a composition of lignum Dalbergiae Odoriferae;

FIG. 3 shows the results of the Morris water maze test for testing the effect of the lignum Dalbergiae Odoriferae composition on the spatial learning and memory ability of rats with vascular dementia;

FIG. 4 is a graph showing the results of HE staining, 1. normal group, 2. model group, and 3. lignum Dalbergiae Odoriferae composition group;

FIG. 5 is a graph showing the results of Niger's staining, 1. normal group, 2. model group, and 3. Dalbergia Odorifera composition group;

FIG. 6 shows the effect of the Dalbergia Odorifera composition group on the expression of Caspase-3 and Caspase-9 proteins in the VaD model rat, 1. normal group, 2. model group, and 3. Dalbergia odorifera composition group;

FIG. 7 Effect of Dalbergia Odorifera composition group on the expression of Bax, Bcl-2 proteins in VaD model rats;

FIG. 8 Effect of Dalbergia Odorifera composition on VaD model rat PI 3K/Akt;

Detailed Description

The following examples further illustrate the invention but are not intended to limit the invention.

Example 1

Respectively crushing 10 g of dalbergia wood medicinal material and 10 g of salvia miltiorrhiza medicinal material, extracting dalbergia wood volatile oil by a water distillation method, and separating 1ml of volatile oil suspension; taking a salvia miltiorrhiza medicinal material, adding 50ml of water, soaking for 1 hour, carrying out reflux extraction for 2 times at 90 ℃, combining extracting solutions, concentrating to 9ml, mixing the dalbergia wood volatile oil and the salvia miltiorrhiza liquid, and adding 0.5 g of polysorbate 80 to obtain the traditional Chinese medicine, wherein each ml of solution is equivalent to 1g of crude dalbergia wood and 1g of crude salvia miltiorrhiza.

Example 2

Respectively crushing 20 g of dalbergia wood medicinal material and 30 g of salvia miltiorrhiza medicinal material, extracting dalbergia wood volatile oil by a water distillation method, and separating 1ml of volatile oil suspension; taking a salvia miltiorrhiza medicinal material, adding 90ml of water, soaking for 2 hours, carrying out reflux extraction for 1 time at 100 ℃, taking an extracting solution, concentrating to 9ml, mixing the dalbergia wood volatile oil and the salvia miltiorrhiza liquid, and adding 0.2 g of polysorbate 80 to obtain the traditional Chinese medicine, wherein each ml of the solution is equivalent to 2g of crude dalbergia wood and 3g of crude salvia miltiorrhiza.

Example 3

Respectively crushing 30 g of dalbergia wood medicinal material and 20 g of salvia miltiorrhiza medicinal material, extracting dalbergia wood volatile oil by a water distillation method, and separating 1ml of volatile oil suspension; taking salvia miltiorrhiza, adding 40ml of water, soaking for 3 hours, carrying out reflux extraction for 3 times at 100 ℃, combining extracting solutions, concentrating to 9ml, mixing the dalbergia wood volatile oil and the salvia miltiorrhiza solution, and adding 0.75 g of polysorbate 80 to obtain the traditional Chinese medicine, wherein each ml of solution is equivalent to 3g of crude drug of dalbergia wood and 2g of crude drug of salvia miltiorrhiza.

Example 4

Content determination of dalbergia wood in dalbergia wood composition

Agilent 7890A gas chromatograph (Agilent, usa); chromatography column HP-INNOWAX (column length 30m, internal diameter 0.32mm, membrane thickness 0.25 mm); a FID detector; carrier gas: nitrogen gas; temperature programming: the initial temperature was 100 ℃ for 5 minutes, the temperature was raised to 160 ℃ at a rate of 5 ℃ per minute for 7 minutes, and then to 230 ℃ at a rate of 5 ℃ per minute for 2 minutes. Sample inlet temperature: 230 ℃, carrier gas flow rate: 3 ml/min.

Control solutions were prepared by dissolving the inverted nerolidol control (88.3% pure, available from Sigma) in n-hexane.

Preparing a test solution, precisely measuring 2ml of the dalbergia wood composition solution, adding 20ml of n-hexane for extraction, taking n-hexane solution, evaporating to dryness at low temperature, and metering volume with n-hexane to 2ml of a measuring bottle to obtain the test solution shown in figure 1.

The results show that the content of the trans-nerolidol in 6 batches of the rosewood composition solution is 8.2-31.5 (mu g/ml).

Example 8

Content determination of dalbergia root in dalbergia wood composition

Ultra high performance liquid chromatography (Waters, usa), Empower workstation (Waters, usa); a chromatographic column: sigma Ascentis Express C₁₈(100 mm. times.2.1 mm, 2.7 μm); mobile phase: acetonitrile (A) -0.1% formic acid (B), and gradient elution is carried out for 0-3 min, wherein the concentration of A is 5%; 5-13% of A for 3-9 min; 13-25% of A for 9-18 min; column temperature: 35 ℃; detection wavelength: 280 nm; flow rate: 0.6 ml/min; sample introduction amount: 3 μ l.

Preparing a reference substance solution, namely purchasing a danshensu sodium reference substance (with the purity of 98.1%), a protocatechuic aldehyde reference substance (with the purity of 98.2%), a rosmarinic acid reference substance (with the purity of 99.8%) and a salvianolic acid B reference substance (with the purity of 95.4%) from a Chinese institute for testing and determining medicinal and biological products; the resulting solution was dissolved in 50% methanol to prepare a mixed control solution.

Preparing a test solution, precisely measuring 4ml of the dalbergia wood composition solution, placing the solution in a 50ml measuring flask, adding 50% methanol to the scale, and shaking up to obtain the test solution shown in figure 2.

The result shows that the content of the danshensu sodium in 6 batches of the dalbergia wood composition solution is 1.2-3.6 (mg/ml); the protocatechuic aldehyde content is 0.2-0.6 (mg/ml); the content result of the rosmarinic acid is 0.08-0.24 (mg/ml); the content of salvianolic acid B is 0.45-1.15 (mg/ml).

Example 9

Preparation of VaD rat model A VaD rat model was prepared by permanent ligation of bilateral common carotid arteries. The experiment adopts a two-blood vessel blocking method (2-VO) to establish a VaD rat model, and the principle is that after the bilateral common carotid arteries are permanently ligated, incomplete cerebral ischemia can be caused, chronic cerebral tissue hypoperfusion is caused, and ischemia and hypoxia injury can be caused to the brain and the like. 2-VO is an effective rat model making method for simulating a VaD pathogenesis, and the model is applied more at home and abroad and is one of the more recognized VaD models;

the VaD rats are divided into model groups, and randomly divided into a pseudo-operation group, a model group, a nimodipine group (3mg/kg), a gold group (12mg/kg) and a rosewood composition group (the dosage is 40g/kg, 32g/kg or 16g/kg), orally administered once a day for 21 days continuously until the behavioral test of the rats is finished.

The Morris water maze experiment of the rat, the positioning navigation experiment, and the experiment are carried out for 5 days, and the rat needs to be familiar with the environment on the first day and is allowed to swim freely for about 2 minutes. The platform of the Morris water maze is arranged at the middle point of the fourth quadrant, a rat is placed into water from the opposite quadrant at a fixed position, the rat needs to be back to the platform, the collection time is 120s in Total, the Escape Latency (Escape Latency) and the Total swimming Distance (Total Distance) of the rat are recorded by a video tracking analysis system, and data are subjected to statistical analysis. The experimental result shows that from the fourth day, the dalbergia wood composition group, the gold groups can obviously shorten the swimming latency and the total swimming route, and the nimodipine group can shorten the swimming latency and the total swimming route of the model rat at the fifth day (P is less than 0.01). And (3) a space exploration experiment, namely removing the platform on the sixth day of the Morris experiment, enabling the experimental rat to enter water at the same water inlet point, freely swimming for 120s, and recording the number of times that the experimental rat passes through the platform area in 120s and the residence time of the quadrant of the platform. The space exploration experiment result shows that the percentage of the route of the rats in the VaD model group in the quadrant of the original platform is obviously reduced (p is less than 0.05) compared with the rats in the sham operation group; the rosewood composition group, gold groups and nimodipine group significantly increased the percentage of swimming distance in the platform quadrant of rats (. about.p <0.05) compared to the model group.

Morris water maze experimental study finds that the dalbergia wood composition group has a promoting effect on the spatial learning and memory ability of VaD rat mice.

Example 10

Effect of lignum Dalbergiae Odoriferae composition on brain histopathology experiment of VaD rats

After the rat behavioral experiment was completed, 7 rats were taken out of each group, whole brains were removed, fixed in 4% paraformaldehyde at 4 ℃, dehydrated with alcohol, transparent with xylene, embedded in paraffin, and sliced to a thickness of 5 μm for histopathological examination. Paraffin sections were subjected to conventional HE staining and Nissl (Nissl) staining.

The HE staining result shows that the nerve cells of the visible under-mirror pseudo-operation group are orderly arranged, gaps are not abnormal, the tissue structure around the cells is complete, the cell nucleus is clear, the nucleolus is obvious, the cytoplasm is uniformly stained, and the cell membrane is complete; the model group had disorganized nerve cell arrangement, edema. Pale staining, the chromatin in the pycnotic nuclei agglutinates into clumps. The rosewood composition group has orderly arranged nerve cells, tightly arranged pyramidal cells, increased number of neuron cell layers and recovery of nerve synapses of part of neuron cells, as shown in figure 4.

The apoptosis condition of the neuron cells is reflected by the result of Niger staining, the neuron cells of the visible under-mirror pseudo-operation group are orderly arranged, the neuron cell structure is clear and complete, and the Niger body is in a blue granular shape and is relatively rich; the neuron cell structure of the model group is obviously changed, the karyotype is irregular, the chromatin in the nucleus is collected, and the nissl is obviously reduced, which indicates that the nerve cells are apoptotic. The number of hippocampal neuron cells and the number of nissl body of the rats are increased and the structure is clear in each administration group of the dalbergia wood composition, which shows that the dalbergia wood composition has the function of protecting the neuron cells of the rats of the VaD model, and the figure is 5; nissl staining statistical results show that compared with a sham operation group, the number of neuron cells in a model group is obviously reduced, and the neuron cells have significant difference (P < 0.01); compared with the model group, the rosewood heart wood composition group can improve the survival rate of the cells of the hippocampal neurons of the VaD rats and has a significant difference (P < 0.01).

Example 11

Effect of lignum Dalbergiae Odoriferae composition group on expression of Caspase-3 and Caspase-9 proteins in VaD model rat

(1) Extraction of proteins

After the behavioral experiments of rats are finished, 5 rats in each group are respectively taken, 10% chloral hydrate (350mg/kg) is injected into the abdominal cavity for anesthesia, the head is broken, the brain is taken out, the taken brain is placed on clean filter paper, the filter paper is placed on ice, the hippocampus is quickly separated, tissue lysate is added, and the weight ratio of the added tissue lysate to the added tissue lysate is 5: 1, freezing in a refrigerator at the temperature of 20 ℃ below zero, homogenating by a tissue homogenizer, and carrying out ice bath for 30min after homogenating. Centrifuging the sample at 14000g of revolution at 4 deg.C for 20min, collecting supernatant, quantifying, packaging, and storing in-80 deg.C refrigerator;

(2) determination of protein concentration

The BCA method was used for the assay. Adding BCA working solution into a protein standard BSA (bovine serum albumin) solution with the concentration of 0.5mg/mL to prepare a series of standard curve solutions, preparing a test sample by the same method, and measuring the absorbance of each hole at the wavelength of 490nm by using an enzyme-labeling instrument;

(3) preparation of sodium dodecyl sulfate-Polyacrylamide gel (SDS-PAGE)

The glue making procedure is as follows: and (5) installing the glass plate. Preparing 12% separating glue solution, mixing, immediately injecting into the gap of glass plate, sealing with deionized water for 60min, adding 5% concentrated glue solution, inserting into comb, sealing, and polymerizing for 60 min. Storing at 4 deg.C;

(4) conditions of electrophoresis

According to the result of protein quantification by the BCA method, adjusting the volume of each sample before loading to make the total amount of each group of proteins consistent, adding 5 xSDS-PAGE protein loading buffer solution, carrying out denaturation by boiling water bath, loading, wherein the starting voltage is 80V, when the sample enters the separation gel from the concentration gel, the voltage is increased to 180V, and when the indicator runs to the bottom of the separation gel, the electrophoresis is stopped;

(5) rotary die

The gel was removed from the concentrated gel, and the filter paper and PVDF membrane were activated with methanol and placed in the transfer buffer. Sequentially arranging filter paper, a PVDF film, gel and filter paper from the anode to the cathode, performing die rotation under the condition of constant current rotation for 1-2 h and current of 50-80 mA, and taking out the film for later use;

(6) blocking and immune response

The membrane was placed in 5% skim milk powder blocking solution (prepared with PBS buffer) and shaken gently for 2h at room temperature. Adding primary antibodies (Caspase-3, Caspase-9, I kappa B, PI3K, AKT, Bax, Bcl-2 and beta-actin), standing overnight at 4 deg.C, washing membrane, adding secondary antibody, incubating for 2h at room temperature on a shaker, and washing membrane;

(7) color development

The ECL luminescence solution was developed, the developer was developed for about 5min, and the fixing solution was fixed and then analyzed by Image J software. Carrying out quantitative analysis on target proteins, and correcting differences and changes of protein expression among groups by taking beta-actin as an internal reference;

(8) statistical method

Statistical analysis of experimental data was performed using SPSS17.0 software, data toShowing that data between two groups were statistically analyzed by T-test, comparison between groups was statistically analyzed by one-way analysis of variance, p<0.05 is statistically significant.

As a result: the protein expression of Caspase-3 and Caspase-9 is determined by using a Western Blot method, and the experimental result shows that compared with a sham operation group, the protein expression of the model group Caspase-3 and Caspase-9 is obviously increased (p is less than 0.01), which indicates that the Caspase-3 and Caspase-9 are activated; compared with the model group, the expression of Caspase-3 and Caspase-9 can be obviously reduced in each dosage group of the rosewood composition, which shows that the rosewood composition inhibits the activation of Caspase-3 and Caspase-9, and the result is shown in figure 6. V

Example 12

Effect of lignum Dalbergiae Odoriferae composition group on the expression of Bax and Bcl-2 proteins in VaD model rat

The Western Blot method is used for determining the expression of rat apoptosis factors Bax and Bcl-2 proteins in each group, and the experimental result shows that compared with a sham operation group, the expression of the model group Bax is obviously increased, and the expression of the Bcl-2 is obviously reduced (P is less than 0.01); compared with the model group, the dalbergia wood composition up-regulates the anti-apoptosis factor Bcl-2 and down-regulates the pro-apoptosis factor Bax, which indicates that the expression of Bax and Bcl-2 protein can be regulated (P is less than 0.05).

Example 13

Effect of Dalbergia Odorifera composition on VaD model rat PI3K/Akt

The Western Blot method is used for measuring the protein expression of rat apoptosis factor PI3K/Akt of each group, and research shows that after the activation of PI3K, Akt can be induced to be activated, so that the survival of neuron cells is promoted, the apoptosis of the neuron cells is inhibited, and the brain protection effect is exerted. Western Blot experiment results show that the expression levels of PI3K and Akt in the brains of rats in a model group are obviously lower than those of a sham operation group, the two groups of experiments have statistical significance (P is less than 0.001), and compared with the model group, the expression levels of PI3K and Akt in the brains of a rosewood composition group are obviously increased, which indicates that the rosewood composition can play an anti-apoptosis role through an activated PI3K/Akt signal channel.