CN115844957A - Traditional Chinese medicine composition for treating acute spinal cord injury in acute stage and using method thereof - Google Patents
Traditional Chinese medicine composition for treating acute spinal cord injury in acute stage and using method thereof Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicine composition for treating acute spinal cord injury in the acute stage and a using method thereof, relating to the technical field of traditional Chinese medicine preparations and comprising the following components: saviae Miltiorrhizae radix, rhizoma Polygoni Cuspidati, fel Ursi, and calculus bovis. The Chinese medicinal composition for clearing heat and activating blood circulation disclosed by the invention is used for treating spinal cord injury by comprehensively considering the medicinal properties of the Chinese medicaments and strictly controlling the treatment modes of the Chinese medicaments, is unique in use mode, is used for treating spinal cord injury by combining the unique use mode of the Chinese medicinal composition for clearing heat and activating blood circulation (consisting of salvia miltiorrhiza, polygonum cuspidatum, bear gall and bezoar), has the functions of improving the motor function after interventional therapy of spinal cord injury, promoting wound healing, reducing the area of colloid scars and protecting neurons in an injury area, thereby accelerating the repair after spinal cord injury and providing a basis for clinical treatment of spinal cord injury.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a traditional Chinese medicine composition for treating acute spinal cord injury in an acute stage and a using method thereof.
Background
Acute Spinal Cord Injury (ASCI) is a spinal cord that, following an external impact, results in complete or incomplete spinal cord movement, sensation, sphincter dysfunction, etc. Worldwide, the annual incidence of acute spinal cord injury is about 14-40/1000000, which is one of the most serious catastrophic injuries in humans. ASCI not only affects the patient's work and life, but also places a significant burden on the patient's home and even the entire society. Therefore, effective treatment of acute spinal cord injury is very important for the development of society.
The key to the early treatment of ASCI is to minimize the secondary damage to the spinal cord, including rapid and accurate diagnosis, early surgical decompression and fixation, and to maintain mean arterial pressure of 85-90 mmHg, improve the blood perfusion of the spinal cord and ensure effective respiration. In addition, methylprednisolone, ganglioside, neurotrophic factor, etc. are used for neuroprotection in clinic. Previous observations suggest that high-dose methylprednisolone impact therapy has a promoting effect on the recovery of injured spinal cord function after ASCI. However, clinical studies in recent years have found that the treatment method has no obvious benefits, but increases the occurrence of complications such as deep vein thrombosis, pneumonia, gastrointestinal diseases, hyperglycemia and the like. Clinical care is therefore recommended, particularly for elderly ASCI patients.
The traditional Chinese medicine considers that for acute spinal cord injury, external force can damage qi and blood channels to cause channel separation and blood vessel damage. Blood vessels are damaged, so blood overflows from the vessels and does not circulate in the channels, and blood stasis is formed; blood stasis and broken vessels can cause unsmooth qi movement of governor vessels, qi as commander of blood, blood as mother of qi, and qi stagnation to worsen blood stasis, which are causalities of the two, and finally lead to channel blockage, qi and blood obstruction and dysfunction after spinal cord injury. The early spinal cord injury belongs to blood stasis blockage and meridian obstruction, and the treatment is suitable for promoting qi circulation, removing blood stasis, purging, clearing heat and dredging governor vessels, the middle period belongs to qi deficiency and blood stasis, and the later period belongs to deficiency of the spinal cord, so the treatment is taken as tonifying kidney and replenishing vital essence, and the better curative effect can be obtained after the treatment according to stage differentiation. Therefore, the early treatment of acute spinal cord injury is very critical, and the early treatment is proper, so that the expansion of secondary spinal cord injury can be effectively prevented and treated, and the repair of the spinal cord injury is accelerated.
At present, the traditional Chinese medicine composition comprises the following components: astragalus root, morinda officinalis, curculigo orchioides, salvia miltiorrhiza, red peony root, angelica, spatholobus stem, ligusticum wallichii and prepared aconite root are used for treating the injury at the later stage of spinal cord injury by means of activating blood, removing blood stasis, tonifying kidney and the like, but for acute spinal cord injury, the acute stage of the injury is the key stage of treatment, and currently, research is rarely carried out. Therefore, there is a need to develop and research a Chinese medicinal composition for treating acute spinal cord injury in the acute stage.
Disclosure of Invention
The invention aims to solve the existing problems and provides a traditional Chinese medicine composition for treating acute spinal cord injury in an acute stage and a using method thereof.
The invention is realized by the following technical scheme:
a preparation method of a traditional Chinese medicine composition for treating acute spinal cord injury in an acute stage comprises the following steps:
(1) Weighing raw materials:
weighing 10-20 g of salvia miltiorrhiza, 10-20 g of giant knotweed, 0.3-0.7 g of bear gall and 0.1-0.2 g of bezoar with corresponding weights for later use;
(2) Cold plasma treatment:
respectively placing the salvia miltiorrhiza, the giant knotweed rhizome, the bear gall and the bezoar weighed in the step (1) into cold plasma equipment for cold plasma treatment;
by adopting the technical scheme, the cold plasma treatment is carried out on the traditional Chinese medicine raw materials, and the cold plasma enables a large amount of free radicals to be generated on the surface of the traditional Chinese medicine materials and can activate the surface of the traditional Chinese medicine materials to improve the surface sparks. Meanwhile, the cold plasma can also damage partial ray cells and enlarge the water transfer way, so that the dissolution of active ingredients during decoction can be promoted, the bioavailability of the raw materials of the traditional Chinese medicine is improved, and the effect of a finished product is improved.
(3) And (3) cryogenic crushing treatment:
respectively placing the bear gall and the bezoar subjected to the cold plasma treatment in the step (2) into a cryogenic pulverizer for pulverization treatment, and obtaining bear gall powder and bezoar powder for later use;
by adopting the technical scheme, the bear gall and the bezoar subjected to the cold plasma treatment are subjected to crushing treatment under the condition of deep cooling, so that the activity of the raw materials is not damaged, the raw materials can be refined, a good foundation is laid for subsequent homogeneous dissolution, and the bioavailability of the bear gall and the bezoar is effectively improved.
(4) Soaking treatment:
taking the salvia miltiorrhiza and the polygonum cuspidatum which are treated by the cold plasma in the step (2), adding purified water until the liquid level is 1-3 cm higher than that of the medicine, soaking for 1.5-2.5 h, and carrying out intermittent ultrasonic treatment while soaking;
by adopting the technical scheme, the intermittent ultrasonic treatment is carried out while soaking, and the soaking effect is improved by mainly utilizing the cavitation effect, the acoustic flow effect and the heat effect of ultrasonic waves.
(5) Decocting;
after soaking, firstly decocting the medicines with strong fire until the decoction is boiled, then converting the decoction into small fire for continuously decocting for 0.5 to 1.5 hours, pouring the medicines in the decoction into a beaker after the decoction is finished, and reserving the dregs for later use;
(6) And (3) vacuum freeze drying treatment:
putting the dregs obtained in the step (5) into a vacuum drying oven for low-temperature vacuum drying treatment, and taking out for later use after the treatment is finished;
by adopting the technical scheme, the decoction dregs obtained after decoction are put into a vacuum drier and dried under the low-temperature vacuum condition, the temperature of the decoction dregs is slowly increased from outside to inside to generate certain vapor pressure, and the permeability of the decoction dregs is well improved under the action of the vapor pressure, so that the leaching of active ingredients in the decoction dregs in the decoction process is facilitated, and the bioavailability of the raw materials is further improved.
(7) And (3) secondary decoction treatment:
adding purified water into the dried medicine dregs again, boiling the medicine dregs again by strong fire until the liquid level of the medicine dregs is 1.5-2.5 cm higher than that of the medicine dregs, turning to small fire for decocting for 30-50 min, and leading out decoction;
(8) And (3) filtering:
mixing the decoctions, covering the cup with clean gauze, and filtering to obtain medicinal liquid;
(9) And (3) centrifugal treatment:
placing the obtained liquid medicine in a centrifugal tube for centrifugal treatment;
(10) Concentration treatment:
centrifuging, removing the residue at the bottom, pouring the centrifuged medicinal liquid into a clean frying pan, boiling with strong fire, and continuously boiling and concentrating with slow fire;
(11) And (3) preparing a finished product:
and (3) standing at room temperature after the concentration is finished, adding the bear gall powder and the bezoar powder obtained in the step (3) when the temperature is reduced to 65-75 ℃, and stirring until the bear gall powder and the bezoar powder are dissolved.
Furthermore, the processing power is controlled to be 80-100W during the cold plasma processing in the step (2), and the processing time is 30-40 s.
Further, the temperature in the pulverizer is controlled to be-30 to-20 ℃ during the cryogenic grinding treatment in the step (3), the rotating speed of the pulverizer is 1000 to 1200rpm during the grinding treatment, and the grinding treatment time is 6 to 10min.
Further, a pot cover is needed to cover the decocting process in the step (4).
Further, the intermittent ultrasonic treatment in the step (4) is specifically: ultrasonic treatment is carried out every 4-6 min in the soaking process, the ultrasonic treatment time is 10-12 min each time, and the ultrasonic treatment frequency is 20-30 kHz.
Further, the drying temperature is controlled to be 27-33 ℃ and the vacuum degree is controlled to be 1-2 Pa during the low-temperature vacuum drying treatment in the step (6), and the drying is carried out until the water content is 2-5%.
Further, the rotation speed of the centrifugal machine during the centrifugation in the step (9) is 1300-1700 rpm, and the centrifugation time is 4-6 min.
Further, the pot cover is uncovered during the small fire treatment in the step (10), and the volume of the liquid medicine is repeatedly measured until 120-124 mL.
Compared with the prior art, the invention has the following advantages:
1. the salvia miltiorrhiza in the traditional Chinese medicine composition is the essential drug of blood system, enters heart, liver and kidney channels, is combined with the giant knotweed, can break blood stasis and dredge channels, activate blood and promote blood circulation, can internally remove stasis of internal organs, can dredge channels and promote articulation, and can remove pain caused by blood stasis and blood circulation. Bezoar and bear gall are bitter in taste, cold and cool in nature, harmonize the pungent and hot nature of other herbs, and have the effects of inducing resuscitation, relieving spasm and dredging brain and marrow.
2. The Chinese medicinal composition for clearing heat and activating blood circulation disclosed by the invention is used for treating spinal cord injury by comprehensively considering the medicinal properties of various Chinese medicaments and strictly controlling the treatment modes of the various Chinese medicaments, is unique in use mode, is used for treating spinal cord injury by combining the unique use mode of the Chinese medicinal composition for clearing heat and activating blood circulation (consisting of salvia miltiorrhiza, polygonum cuspidatum, bear gall and bezoar), has the functions of improving the motor function after intervening treatment of spinal cord injury, promoting wound healing, reducing the area of colloid scars and protecting neurons in an injury area, so that the repair after spinal cord injury is accelerated, and a basis is provided for clinical treatment of spinal cord injury.
3. Aiming at the problem that the spinal cord is difficult to repair after the spinal cord injury at present, the invention adopts the treatment means of promoting blood circulation and activating blood circulation, inducing resuscitation and relieving spasm and dredging brain and marrow for the acute stage of the acute spinal cord injury by taking the theory of traditional Chinese medicine as guidance, and has obvious repairing effect on the spinal cord after the acute spinal cord injury of the mice.
Drawings
FIG. 1 is a graph of footprint distribution maps, BBB motor score maps, 14-day longitudinal H & E staining patterns after injury, and Neisseria staining results for surviving neurons for different groups;
FIG. 2 is an analysis chart of immunofluorescence staining results of paraffin sections of spinal cord tissues.
Detailed Description
Example 1
A preparation method of a traditional Chinese medicine composition for treating acute spinal cord injury in the acute stage comprises the following steps:
(1) Weighing raw materials:
weighing 10g of salvia miltiorrhiza, 10g of giant knotweed, 0.3g of bear gall and 0.1g of bezoar with corresponding weights for later use;
(2) Cold plasma treatment:
respectively placing the salvia miltiorrhiza, the giant knotweed rhizome, the bear gall and the bezoar weighed in the step (1) into cold plasma equipment for cold plasma treatment, controlling the treatment power to be 80W, and controlling the treatment time to be 30s;
(3) And (3) cryogenic crushing treatment:
respectively placing the fel Ursi and calculus bovis processed by cold plasma in step (2) into a cryogenic pulverizer to perform pulverizing treatment, controlling the temperature in the pulverizer to be-30 deg.C, the rotation speed of the pulverizer during pulverizing treatment to be 1000rpm, and pulverizing for 6min to obtain fel Ursi powder and calculus bovis powder;
(4) Soaking treatment:
taking the salvia miltiorrhiza and the polygonum cuspidatum which are treated by the cold plasma in the step (2), adding purified water until the liquid level is 1cm higher than that of the medicine, soaking for 1.5h, and carrying out intermittent ultrasonic treatment while soaking, wherein the specific steps are as follows: carrying out ultrasonic treatment every 4min in the soaking process, wherein the ultrasonic treatment time is 10min each time, and the ultrasonic treatment frequency is 20kHz;
(5) Decocting;
after soaking, decocting the medicines with strong fire until the decoction is boiled, then turning to slow fire to continue decocting for 0.5h, pouring the decoction into a beaker after the decoction is boiled, and reserving the dregs for later use;
(6) Vacuum freeze drying treatment:
placing the dregs obtained in the step (5) in a vacuum drying oven for low-temperature vacuum drying treatment, controlling the drying temperature to be 27 ℃ and the vacuum degree to be 1Pa, drying until the water content is 2%, and taking out for later use;
(7) And (3) secondary decoction treatment:
adding purified water into the dried residue again until the liquid level of the purified water is 1.5cm higher than that of the residue, boiling with strong fire again, decocting with slow fire for 30min, and collecting decoction;
(8) And (3) filtering:
mixing the decoctions, covering the cup with clean gauze, and filtering to obtain medicinal liquid;
(9) And (3) centrifugal treatment:
placing the obtained medicinal liquid in a centrifugal tube for centrifugal treatment, wherein the rotation speed of the centrifugal machine is 1300rpm, and the centrifugal time is 4min;
(10) Concentration treatment:
centrifuging, removing the residue at the bottom, pouring the centrifuged liquid medicine into a clean frying pan, boiling with strong fire, converting to slow fire for continuous boiling and concentrating, removing the pan cover when treating with slow fire, and repeatedly measuring the volume of the liquid medicine until the volume reaches 120mL;
(11) Preparing a finished product:
and (3) standing at room temperature after the concentration is finished, adding the bear gall powder and the bezoar powder obtained in the step (3) when the temperature is reduced to 65 ℃, and stirring until the bear gall powder and the bezoar powder are dissolved.
Example 2
A preparation method of a traditional Chinese medicine composition for treating acute spinal cord injury in the acute stage comprises the following steps:
(1) Weighing raw materials:
weighing 15g of salvia miltiorrhiza, 15g of giant knotweed, 0.5g of bear gall and 0.15g of bezoar according to the corresponding weight for later use;
(2) Cold plasma treatment:
respectively placing the salvia miltiorrhiza, the giant knotweed rhizome, the bear gall and the bezoar weighed in the step (1) into cold plasma equipment for cold plasma treatment, controlling the treatment power to be 90W, and controlling the treatment time to be 35s;
(3) And (3) cryogenic crushing treatment:
respectively placing the fel Ursi and calculus bovis processed by cold plasma in step (2) into a cryogenic pulverizer to perform pulverizing treatment, controlling the temperature in the pulverizer to be-25 deg.C, the rotation speed of the pulverizer during pulverizing treatment to be 1100rpm, and pulverizing for 8min to obtain fel Ursi powder and calculus bovis powder;
(4) Soaking treatment:
taking the salvia miltiorrhiza and the polygonum cuspidatum which are subjected to cold plasma treatment in the step (2), adding purified water until the liquid level is 2cm higher than that of the medicines, soaking for 2 hours, and carrying out intermittent ultrasonic treatment while soaking, wherein the method specifically comprises the following steps: carrying out ultrasonic treatment every 5min in the soaking process, wherein the ultrasonic treatment time is 11min each time, and the ultrasonic treatment frequency is 25kHz;
(5) Decocting;
after soaking, firstly decocting the medicines with strong fire until the decoction is boiled, then turning to slow fire for continuously decocting for 1h, pouring the medicines in the decoction into a beaker after the decoction is boiled, and reserving the dregs for later use;
(6) Vacuum freeze drying treatment:
placing the dregs obtained in the step (5) in a vacuum drying oven for low-temperature vacuum drying treatment, controlling the drying temperature at 30 ℃ and the vacuum degree at 1.5Pa, drying until the water content is 3.5%, and taking out for later use;
(7) And (3) secondary decoction treatment:
adding purified water into the dried residue again until the liquid level of the residue is 2cm higher than that of the purified water, boiling with strong fire again, decocting with slow fire for 40min, and collecting decoction;
(8) And (3) filtering:
mixing the decoctions, covering the cup with clean gauze, and filtering to obtain medicinal liquid;
(9) And (3) centrifugal treatment:
placing the obtained medicinal liquid in a centrifugal tube for centrifugal treatment, wherein the rotation speed of the centrifugal machine is 1500rpm, and the centrifugal time is 5min;
(10) Concentration treatment:
centrifuging, removing the residue at the bottom, pouring the centrifuged liquid medicine into a clean frying pan, boiling with strong fire, converting to slow fire for continuous boiling and concentrating, removing the pan cover during slow fire treatment, and repeatedly measuring the volume of the liquid medicine until the volume reaches 122mL;
(11) And (3) preparing a finished product:
and (3) standing at room temperature after the concentration is finished, adding the bear gall powder and the bezoar powder obtained in the step (3) when the temperature is reduced to 70 ℃, and stirring until the bear gall powder and the bezoar powder are dissolved.
Example 3
A preparation method of a traditional Chinese medicine composition for treating acute spinal cord injury in the acute stage comprises the following steps:
(1) Weighing raw materials:
weighing 20g of salvia miltiorrhiza, 20g of giant knotweed, 0.7g of bear gall and 0.2g of bezoar with corresponding weights for later use;
(2) Cold plasma treatment:
respectively placing the salvia miltiorrhiza, the giant knotweed rhizome, the bear gall and the bezoar weighed in the step (1) into cold plasma equipment for cold plasma treatment, controlling the treatment power to be 100W, and controlling the treatment time to be 40s;
(3) And (3) cryogenic crushing treatment:
respectively placing the bear gall and the bezoar subjected to the cold plasma treatment in the step (2) into a cryogenic pulverizer to perform pulverization treatment, controlling the temperature in the pulverizer to be-20 ℃, controlling the rotation speed of the pulverizer to be 1200rpm during the pulverization treatment, and performing the pulverization treatment for 10min to obtain bear gall powder and bezoar powder for later use;
(4) Soaking treatment:
taking the salvia miltiorrhiza and the polygonum cuspidatum which are subjected to the cold plasma treatment in the step (2), adding purified water until the liquid level is 3cm higher than that of the medicine, soaking for 2.5h, and performing intermittent ultrasonic treatment while soaking, wherein the steps are as follows: carrying out ultrasonic treatment every 6min in the soaking process, wherein the ultrasonic treatment time is 12min each time, and the ultrasonic treatment frequency is 30kHz;
(5) Decocting;
after soaking, decocting the medicines with strong fire until the decoction is boiled, then decocting with slow fire for 1.5h, pouring the decoction into a beaker, and keeping the dregs for later use;
(6) Vacuum freeze drying treatment:
placing the dregs obtained in the step (5) in a vacuum drying oven for low-temperature vacuum drying treatment, controlling the drying temperature to be 33 ℃ and the vacuum degree to be 2Pa, drying until the water content is 5%, and taking out for later use;
(7) And (3) secondary decoction treatment:
adding purified water into the dried residue again until the liquid level of the residue is 2.5cm higher than that of the purified water, boiling with strong fire again, decocting with slow fire for 50min, and collecting decoction;
(8) And (3) filtering:
mixing the decoctions, covering the cup with clean gauze, and filtering to obtain medicinal liquid;
(9) And (3) centrifugal treatment:
placing the obtained liquid medicine in a centrifugal tube for centrifugal treatment, wherein the rotating speed of a centrifugal machine is 1700rpm, and the centrifugal time is 6min;
(10) Concentration treatment:
centrifuging, removing the residue at the bottom, pouring the centrifuged medicinal liquid into a clean frying pan, boiling with strong fire, converting to slow fire, continuously boiling and concentrating, removing the pan cover during slow fire treatment, and repeatedly measuring the volume of the medicinal liquid until the volume is 124mL;
(11) And (3) preparing a finished product:
and (3) standing at room temperature after the concentration is finished, adding the bear gall powder and the bezoar powder obtained in the step (3) when the temperature is reduced to 75 ℃, and stirring until the bear gall powder and the bezoar powder are dissolved.
In order to verify the technical effects of the present application, the experiment of the traditional Chinese medicine composition of example 2 was performed. The method specifically comprises the following steps:
1.establishment of Allen's damage model
60 male C57BL/6 mice at 8 weeks of age were selected, and after one week of laboratory rearing, body weights thereof were measured. Experimental mice were randomly divided into 3 groups of 20 mice each, namely a sham group (sham control group), an SCI group (no intervention group), and a traditional Chinese medicine group (traditional Chinese medicine composition intervention group). Weighing the mice, and injecting 1% sodium pentobarbital into the abdominal cavity for anesthesia according to the standard of 50 mg/kg; after the muscle of the mouse is relaxed, the corneal reaction is delayed and the skin pinching reaction disappears, the mouse is put on an operating table in a prone position, the head and the four limbs are respectively fixed, the hair around the operating area is removed, the T10 spinous process is positioned and marked by taking the spinous process high point and the rib as marks, the operating area is sterilized by iodophor, and a sterile operating sheet is laid on the operating area; the skin and subcutaneous tissue of the mouse dorsal area were cut longitudinally along the posterior midline, centered at T10, at a size of about 1.5cm. Obtusely separating the erector spinae muscle close to the bone surface and retracting to two sides, stopping bleeding by gauze and exposing a T9-T11 vertebral plate; the spinous process and the vertebral lamina of the T9-T11 are bitten off by using vertebral lamina forceps, the T10 section of spinal cord is fully exposed, and the operation process is careful to avoid extruding the spinal cord and damaging the dura mater; using PinPoint TM A precision spinal cord injury impactor (model: PCI 3000-2) strikes a T10 spinal cord after setting relevant parameters.
Wherein, the successful establishment of SCI group is marked by that the mouse body generates a transient tremor, the two lower limbs retract and swing rapidly, and the tail swings back and forth. Covering a layer of autologous fat on the surface of the spinal cord to reduce adhesion, suturing muscles, subcutaneous tissues and skin layer by layer after checking no bleeding, and disinfecting the skin by 75% ethanol; sham groups did not undergo shock after spinal cord exposure, and the rest of the procedure was as above. SCI group postoperative conventional anti-infection treatment to prevent operative area, urinary system infection, etc.; when defecation is difficult after operation, the bladder is massaged to help defecation. The grouping of mice and the method of administration are shown in table 1.
TABLE 1
3. The evaluation method comprises the following steps:
A.H & E staining
Fixation with 4% paraformaldehyde solution. The specimens were dehydrated and paraffin-embedded and then H & E stained with a thickness of 5um by serial sectioning with the lesion area as the center. H & E staining procedure: dewaxing xylene for 10min multiplied by 2 times, dewaxing absolute ethyl alcohol for 5min multiplied by 2 times, 5min of 95% ethyl alcohol, 5min of 80% ethyl alcohol, 5min of 70% ethyl alcohol, 5-10 min of hematoxylin, 2-5 s of hydrochloric acid ethyl alcohol, flushing with running water for 30min, 5min of 70% acetic acid, 5min of 80% acetic acid, redyeing eosin for 5-10min, 5min multiplied by 2 times of 95% acetic acid, and transparence of xylene for 5min multiplied by 3 times. And after H & E staining, observing the section of the damaged area and the adjacent part of the damaged area, and observing and taking a picture under a light mirror.
Bbb score
BBB scoring was performed 1 day, 3 days, 1 week, and weekly thereafter, respectively, after SCI of the mice up to week 4, to evaluate recovery of hindlimb motor function in the mice. The evaluation method comprises the following steps: the mice were placed in a spacious field and allowed to move freely for 4min. The two observers score according to the BBB scoring standard, respectively observe and score the hip, knee and ankle joints, the movement of limbs and trunk and the coordination condition thereof, and take an average value. Statistical analysis: BBB scores per group weekly, row mean T test, p <0.05.
C. Footprint test
Mice were subjected to a footprint experiment 14 days after surgery. Ordinary ink was applied to the hind paw of a mouse and allowed to pass through a 1m long, 10cm wide footprint experimental channel, leaving its footprint on channel white paper.
D. Nie's dyeing
(1) Slicing the dried paraffin, dewaxing conventionally to water (xylene I, xylene II each 15min, then gradient alcohol dehydration: 100% I, 100% II, 95%, 90%, 80%, 70%, 50% each 5 min), washing with distilled water for 2min;
(2) The tissue slices are put into 0.5 percent toluidine blue dye liquor for 2 to 5min, washed with water, slightly differentiated by 0.1 percent glacial acetic acid, washed with tap water to stop the reaction, the degree of differentiation is controlled under a microscope, and the tissue slices are placed in an oven to be baked after being washed with the tap water.
(3) Transparent sealing sheet: slicing, adding clean xylene, transparency for 10min, and sealing with neutral gum;
(4) And (5) observing the damaged area and the adjacent part by using a microscope, and observing and taking a picture under a light microscope.
In the figure 1, a graph A is a footprint analysis graph of different groups, a graph B is a BBB movement score graph of different groups, the intervention of the traditional Chinese medicine composition can promote the recovery of the function of hind limbs of a mouse, a graph C is a longitudinal section H & E staining graph after injury for 14 days, compared with a sham operation group, the injury part of a model group has obvious deformity and cavity after spinal cord injury for 14 days, the lesion area in an administration group is obviously reduced, the tissue injury is smaller, and a graph D is the Neisseria staining of the surviving neurons in each group, and the number of the neurons in the injury area of the administration group is obviously increased, so that the intervention of the traditional Chinese medicine composition can reduce the tissue injury, protect the neurons in the injury area and promote the recovery of the movement function of the mouse.
E. Immunofluorescence staining of paraffin section of spinal cord tissue
(1) Heat the slide in an oven at 49 ℃ for 20min (ensure slide dry before proceeding to the next step);
(2) Dewaxing to water conventionally, dewaxing xylene 5min × 3 times, gradually hydrating with ethanol (100%, 95%, 80%, 70%, tap water) (ensuring no liquid drop in xylene);
(3) Washing with PBS for 5min × 3 times;
(4) Antigen retrieval was performed with boiling water citric acid buffer (pH 6.0) in a microwave oven for 20min (3.82 g citric acid +2.412g sodium citrate +1L water, pH adjusted to 6.0 with 10N NaOH);
(5) Slowly cooling to room temperature;
(6) Washing with PBSx3 for 5min each time;
(7) Circling out the tissue part with a pap pen;
(8) Blocking the triton X-100 block solution containing 10% serum 1% at room temperature for 1h (20-30 deg.C);
(9) Diluting primary antibodies from two different species with a confining liquid, and incubating overnight at 4 ℃;
(10) Washing with PBS for 5min × 3 times;
(11) Secondary antibodies linked to Alexa Fluo 555 (1);
(12) PBS 5min x3 times;
(13) And (3) sealing by using a sealing liquid containing DAPI. Mounting solution PBS and glycerol (4) (containing 1.5. Mu.g/ml DAPI);
(14) And (4) observing under a fluorescence microscope.
The glial scar area of the injured spinal cord was studied by immunofluorescence assay, GFAP is a marker for astrocytes, and as can be seen from the a graph in fig. 2, the glial scar formed by astrocytes and the fibrous scar surrounded by the glial scar were larger after 14 days in the model group, while the scar area of the fibrous part surrounded by astrocytes in the administration group was significantly reduced.
Claims (8)
1.A preparation method of a traditional Chinese medicine composition for treating acute spinal cord injury in the acute stage is characterized by comprising the following steps:
(1) Weighing raw materials:
weighing 10-20 g of salvia miltiorrhiza, 10-20 g of giant knotweed, 0.3-0.7 g of bear gall and 0.1-0.2 g of bezoar with corresponding weights for later use;
(2) Cold plasma treatment:
respectively placing the salvia miltiorrhiza, the giant knotweed rhizome, the bear gall and the bezoar weighed in the step (1) into cold plasma equipment for cold plasma treatment;
(3) And (3) cryogenic crushing treatment:
respectively placing the bear gall and the bezoar subjected to the cold plasma treatment in the step (2) into a cryogenic grinder for grinding treatment, and obtaining bear gall powder and bezoar powder for later use;
(4) Soaking treatment:
taking the salvia miltiorrhiza and the polygonum cuspidatum which are treated by the cold plasma in the step (2), adding purified water until the liquid level is 1-3 cm higher than that of the medicines, soaking for 1.5-2.5 h, and carrying out intermittent ultrasonic treatment while soaking;
(5) Decocting;
after soaking, firstly decocting the medicines with strong fire until the decoction is boiled, then converting the decoction into small fire for continuously decocting for 0.5 to 1.5 hours, pouring the medicines in the decoction into a beaker after the decoction is finished, and reserving the dregs for later use;
(6) Vacuum freeze drying treatment:
putting the dregs obtained in the step (5) into a vacuum drying oven for low-temperature vacuum drying treatment, and taking out for later use after the treatment is finished;
(7) And (3) secondary decoction treatment:
adding purified water into the dried medicine dregs again, boiling the medicine dregs again by strong fire after the purified water level is 1.5-2.5 cm higher than the liquid level of the medicine dregs, turning to slow fire for decocting for 30-50 min, and leading out the decoction;
(8) And (3) filtering:
mixing the decoctions, covering the cup with clean gauze, and filtering to obtain medicinal liquid;
(9) And (3) centrifugal treatment:
placing the obtained liquid medicine in a centrifugal tube for centrifugal treatment;
(10) Concentration treatment:
centrifuging, removing the residue at the bottom, pouring the centrifuged liquid medicine into a clean frying pan, boiling with strong fire, and continuously boiling and concentrating with slow fire;
(11) And (3) preparing a finished product:
and (3) standing at room temperature after the concentration is finished, adding the bear gall powder and the bezoar powder obtained in the step (3) when the temperature is reduced to 65-75 ℃, and stirring until the bear gall powder and the bezoar powder are dissolved.
2. The method for preparing a Chinese medicinal composition for treating acute phase of acute spinal cord injury according to claim 1, wherein the treatment power is controlled to be 80-100W and the treatment time is controlled to be 30-40 s during the cold plasma treatment in the step (2).
3. The method for preparing a Chinese medicinal composition for treating acute stage of acute spinal cord injury according to claim 1, wherein the temperature in the pulverizer is controlled to be-30 to-20 ℃ during the cryogenic pulverization treatment in step (3), the rotation speed of the pulverizer is 1000 to 1200rpm during the pulverization treatment, and the pulverization treatment time is 6 to 10min.
4. The method for preparing a Chinese medicinal composition for treating acute stage of acute spinal cord injury as claimed in claim 1, wherein the decocting in step (4) is carried out by covering with pot cover.
5. The preparation method of the traditional Chinese medicine composition for treating acute phase of acute spinal cord injury according to claim 1, wherein the intermittent ultrasonic treatment in the step (4) is specifically: ultrasonic treatment is carried out every 4-6 min in the soaking process, the ultrasonic treatment time is 10-12 min each time, and the ultrasonic treatment frequency is 20-30 kHz.
6. The method for preparing a Chinese medicinal composition for treating acute stage of acute spinal cord injury according to claim 1, wherein the drying temperature is controlled to be 27-33 ℃ and the vacuum degree is controlled to be 1-2 Pa during the low-temperature vacuum drying treatment in step (6), and the drying is carried out until the water content is 2-5%.
7. The method for preparing a Chinese medicinal composition for treating acute stage of acute spinal cord injury according to claim 1, wherein the rotation speed of the centrifuge in the step (9) is 1300-1700 rpm, and the centrifugation time is 4-6 min.
8. The method for preparing a Chinese medicinal composition for treating acute stage of acute spinal cord injury according to claim 1, wherein the pot cover is opened during the mild fire treatment in step (10), and the volume of the liquid medicine is repeatedly measured until 120-124 mL.
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Citations (4)
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| CN101513510A (en) * | 2009-04-03 | 2009-08-26 | 南京中医药大学 | Traditional Chinese medicine compound preparation for treating spinal cord injury and method for preparing same |
| CN113908191A (en) * | 2021-10-09 | 2022-01-11 | 杨业 | Composition for treating chloasma and preparation method thereof |
| CN114010703A (en) * | 2021-11-22 | 2022-02-08 | 长沙医学院 | Chinese herbal medicine composition and preparation and application thereof |
| CN114042115A (en) * | 2021-09-30 | 2022-02-15 | 林定坤 | Traditional Chinese medicine composition for treating spinal cord injury and application and preparation method thereof |
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| CN101513510A (en) * | 2009-04-03 | 2009-08-26 | 南京中医药大学 | Traditional Chinese medicine compound preparation for treating spinal cord injury and method for preparing same |
| CN114042115A (en) * | 2021-09-30 | 2022-02-15 | 林定坤 | Traditional Chinese medicine composition for treating spinal cord injury and application and preparation method thereof |
| CN113908191A (en) * | 2021-10-09 | 2022-01-11 | 杨业 | Composition for treating chloasma and preparation method thereof |
| CN114010703A (en) * | 2021-11-22 | 2022-02-08 | 长沙医学院 | Chinese herbal medicine composition and preparation and application thereof |
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