CN115844957B - Traditional Chinese medicine composition for treating acute spinal cord injury in acute stage and application method thereof - Google Patents
Traditional Chinese medicine composition for treating acute spinal cord injury in acute stage and application method thereof Download PDFInfo
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Abstract
The application discloses a traditional Chinese medicine composition for treating acute spinal cord injury in an acute stage and a use method thereof, relating to the technical field of traditional Chinese medicine preparations, and comprising the following components: radix Salviae Miltiorrhizae, rhizoma Polygoni Cuspidati, fel Ursi, and calculus bovis. The traditional Chinese medicine composition for clearing heat and activating blood circulation is used for treating spinal cord injury by comprehensively considering the medicine properties of the traditional Chinese medicines, strictly controlling the treatment mode of the traditional Chinese medicines, has unique use mode, combines the unique use mode of the traditional Chinese medicine composition for clearing heat and activating blood circulation (comprising red sage root, giant knotweed rhizome, bear gall and bezoar), has the effects of improving the exercise function, promoting wound healing, reducing the area of colloid scar and protecting neurons in an injury area after the intervention treatment of spinal cord injury, thereby accelerating the repair of the spinal cord injury and providing a basic basis for clinical treatment of spinal cord injury.
Description
Technical Field
The application 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 use method thereof.
Background
Acute spinal cord injury (acute spinal cord injury, ASCI) results in complete or incomplete spinal cord movement, sensation, sphincter dysfunction, etc., after the spinal cord is subjected to external force blows. Worldwide, the incidence of acute spinal cord injury is about 14-40/1000000 per year, which is one of the most serious disastrous injuries in humans. ASCI not only affects the work and life of the patient, but also places a great burden on the patient's home or even the whole society. Therefore, effective treatment of acute spinal cord injury is very important to the development of society.
The key point of ASCIs early treatment is to lighten secondary injuries of spinal cord as far as possible, including rapid and accurate diagnosis, early operation decompression and fixation, maintenance of average arterial pressure of 85-90 mmHg, improvement of blood perfusion of spinal cord, guarantee of effective respiration and the like. In addition, methylprednisolone, ganglioside, neurotrophic factor, etc. are used clinically for neuroprotection. The prior art believes that high doses of methylprednisolone impact therapy have a promoting effect on recovery of impaired spinal cord function following ASCI. However, clinical studies in recent years have found that the treatment method has no obvious benefit, but rather increases the occurrence of complications such as deep vein thrombosis, pneumonia, gastrointestinal diseases, hyperglycemia and the like. Thus, clinical cautious applications are suggested, especially for elderly ASCII patients.
Traditional Chinese medicine holds that for acute spinal cord injury, external force can damage qi and blood channels and collaterals to cause meridian separation and blood vessel damage. Broken blood vessels cause excessive blood flow, blood does not flow through the channels, and blood stasis is formed; the blood stasis and the pulse interruption can cause the qi movement of the governor vessel to be unsmooth, qi is the general purpose of blood, blood is the mother of qi, qi stagnation causes blood stasis to be even worse, and the qi stagnation and the blood stasis are causal, and malignant circulation is formed after spinal cord injury, so that the qi and the blood are obstructed, and dysfunction is finally caused. The early spinal cord injury belongs to blood stasis blockage, meridian obstruction, qi circulation and blood stasis removal, purgation and heat release, governor vessel dredging, qi deficiency and blood stasis in the middle stage and spinal cord deficiency in the later stage, and kidney tonifying and essence benefiting are treated according to the stage dialectical treatment, so that a better curative effect can be obtained. Therefore, the early treatment of the acute spinal cord injury is very critical, the early treatment is proper, the expansion of the secondary spinal cord injury can be effectively prevented and controlled, and the repair of the spinal cord injury is accelerated.
At present, the compound composition comprises the following components: astragalus root, morinda root, curculigo rhizome, red sage root, red paeony root, angelica, suberect spatholobus stem, szechuan lovage rhizome and prepared aconite root treat the injury in the later stage of spinal cord injury by means of activating blood circulation, removing blood stasis, tonifying kidney and the like, but for acute spinal cord injury, the acute stage of injury is a key period of treatment, and at present, fresh researches are carried out. Therefore, development and research of a traditional Chinese medicine composition for treating acute spinal cord injury in the acute stage are needed.
Disclosure of Invention
The application aims at solving 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 application 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 the raw materials:
weighing 10-20 g of red sage root, 10-20 g of giant knotweed, 0.3-0.7 g of bear gall and 0.1-0.2 g of bezoar for standby;
(2) Cold plasma treatment:
respectively placing the red sage root, the giant knotweed rhizome, the bear gall and the bezoar weighed in the step (1) into cold plasma equipment for cold plasma treatment, wherein the treatment power is controlled to be 80-100W during the cold plasma treatment, and the treatment time is 30-40 s;
by adopting the technical scheme, the Chinese medicinal materials are subjected to cold plasma treatment, and the cold plasma can enable the surfaces of the Chinese medicinal materials to generate a large amount of free radicals and activate the surfaces of the Chinese medicinal materials to improve the surface mars. Meanwhile, part of ray cells can be damaged by cold plasma, and the transmission path of moisture is enlarged, so that the dissolution of active ingredients during decoction can be promoted, the bioavailability of traditional Chinese medicine raw materials is further improved, and the effect of a finished product is improved.
(3) Cryogenic grinding treatment:
respectively placing the bear gall and the bezoar subjected to cold plasma treatment in the step (2) in a cryogenic pulverizer to perform pulverizing treatment, obtaining bear gall powder and bezoar powder for standby, controlling the temperature in the pulverizer to be minus 30 to minus 20 ℃ during the cryogenic pulverizing treatment, controlling the rotating speed of the pulverizer to be 1000-1200 rpm during the pulverizing treatment, and performing the pulverizing treatment for 6-10 min;
by adopting the technical scheme, bear gall and bezoar subjected to cold plasma treatment are crushed under the cryogenic condition, so that the activity of the raw materials is not damaged, the raw materials can be refined, a good foundation is laid for subsequent homogenization and dissolution, and the bioavailability of the raw materials can be effectively improved.
(4) Soaking treatment:
taking the red sage root and the giant knotweed rhizome subjected to cold plasma treatment in the step (2), adding purified water to a liquid level which is 1-3 cm higher than the medicine, soaking for 1.5-2.5 h, and carrying out intermittent ultrasonic treatment while soaking;
by adopting the technical scheme, intermittent ultrasonic treatment is carried out while soaking, and mainly cavitation effect, acoustic flow effect and thermal effect of ultrasonic waves are utilized to improve the soaking effect.
(5) Decocting;
after soaking, decocting the medicinal herbs with strong fire until decoction is boiled, then turning into small fire, continuously decocting for 0.5-1.5 h, and pouring the medicinal herbs into a beaker after the completion of the decoction, wherein residues are reserved;
(6) Vacuum freeze drying treatment:
placing the dregs obtained in the step (5) in a vacuum drying oven for low-temperature vacuum drying treatment, and taking out for standby after the completion;
by adopting the technical scheme, the decoction dregs obtained after decoction are put into a vacuum dryer, are dried under the low-temperature vacuum condition, are slowly heated 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 raw materials is further improved.
(7) And (3) secondary decoction treatment:
adding purified water again into the dried residue, wherein the liquid level of purified water is 1.5-2.5 cm higher than that of the residue, boiling again with strong fire, decocting with small fire for 30-50 min, and guiding out decoction;
(8) And (3) filtering:
mixing the two decoctions, covering the cup mouth with clean gauze, and filtering to obtain medicinal liquid;
(9) And (3) centrifugal treatment:
placing the obtained liquid medicine into a centrifuge tube for centrifugal treatment;
(10) Concentration treatment:
centrifuging, removing residue at bottom, pouring the liquid medicine obtained by centrifuging into a clean frying pan, boiling with strong fire, and continuously boiling with small fire for concentration;
(11) Preparing a finished product:
and (3) standing at room temperature after concentration is finished, and 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.
Further, the batch ultrasonic treatment in the step (4) specifically comprises: and carrying out ultrasonic treatment every 4-6 min in the soaking process, wherein the time of each ultrasonic treatment is 10-12 min, and the frequency of each ultrasonic treatment is 20-30 kHz.
Further, the pot cover is required to be covered in the decocting process in the step (5).
Further, in the low-temperature vacuum drying treatment in the step (6), the drying temperature is controlled to be 27-33 ℃, the vacuum degree is 1-2 Pa, and the water content is 2-5%.
Further, the rotational speed of the centrifugal machine in the step (9) is 1300-1700 rpm, and the centrifugal 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 application has the following advantages:
1. the red sage root in the traditional Chinese medicine composition is the key medicine of blood system, enters heart, liver and kidney channels, is combined with giant knotweed, and can break blood stasis and remove meridian obstruction, activate blood and remove blood stasis of viscera in the interior, and can remove meridian obstruction to promote the circulation of qi and joints in the exterior, so that the blood stasis is removed and pain is removed. Niu Huang and bear gall are bitter in flavor and cold in nature, harmonize the pungent and warm nature of other herbs, and induce resuscitation and relieve spasm and unblock brain marrow.
2. The traditional Chinese medicine composition for clearing heat and activating blood circulation is used for treating spinal cord injury by comprehensively considering the medicine properties of the traditional Chinese medicines, strictly controlling the treatment mode of the traditional Chinese medicines, has unique use mode, combines the unique use mode of the traditional Chinese medicine composition for clearing heat and activating blood circulation (comprising red sage root, giant knotweed rhizome, bear gall and bezoar), has the effects of improving the exercise function, promoting wound healing, reducing the area of colloid scar and protecting neurons in an injury area after the intervention treatment of spinal cord injury, thereby accelerating the repair of the spinal cord injury and providing a basic basis for clinical treatment of spinal cord injury.
3. Aiming at the problem that the spinal cord is difficult to repair after the current spinal cord injury, the application takes the traditional Chinese medicine theory as guidance, adopts the treatment means of promoting blood circulation, inducing resuscitation, relieving spasm and dredging brain marrow to the acute stage of the acute spinal cord injury, and has obvious repairing effect on the spinal cord of mice after the acute spinal cord injury.
Drawings
FIG. 1 is a graph of footprint profile, BBB movement score, H & E staining of 14 day longitudinal sections after injury, and Nib staining of surviving neurons for different groups;
FIG. 2 is a graph of the results of immunofluorescent staining of paraffin sections of spinal cord tissue.
Description of the embodiments
Examples
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 the raw materials:
weighing 10g of red sage root, 10g of giant knotweed, 0.3g of bear gall and 0.1g of bezoar for standby;
(2) Cold plasma treatment:
respectively placing the red sage root, 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 the treatment time to be 30s;
(3) Cryogenic grinding treatment:
respectively placing the bear gall and bezoar subjected to cold plasma treatment in the step (2) in a cryogenic pulverizer to carry out pulverization treatment, controlling the temperature in the pulverizer to be minus 30 ℃, controlling the rotation speed of the pulverizer to be 1000rpm during the pulverization treatment, and obtaining bear gall powder and bezoar powder for standby after the pulverization treatment for 6min;
(4) Soaking treatment:
taking the red sage root and the giant knotweed rhizome which are subjected to cold plasma treatment 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 method specifically comprises the following steps: carrying out ultrasonic treatment every 4min in the soaking process, wherein the time of each ultrasonic treatment is 10min, and the frequency of the ultrasonic treatment is 20kHz;
(5) Decocting;
decocting the medicinal materials with strong fire until the decoction is boiled, then converting into small fire, continuously decocting for 0.5h, pouring the medicinal materials into a beaker after the decoction is completed, and reserving the medicinal residues 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 ℃, controlling 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 again into the dried residue, and decocting with strong fire for 30min after the liquid level of purified water is 1.5cm higher than the residue, and discharging decoction;
(8) And (3) filtering:
mixing the two decoctions, covering the cup mouth with clean gauze, and filtering to obtain medicinal liquid;
(9) And (3) centrifugal treatment:
placing the obtained liquid medicine into a centrifuge tube for centrifugal treatment, wherein the rotation speed of the centrifuge is 1300rpm, and the centrifugal time is 4min;
(10) Concentration treatment:
centrifuging, discarding residue at bottom, pouring the liquid medicine obtained by centrifuging into a clean frying pan, boiling with strong fire, continuously boiling with small fire, concentrating, uncovering the cover during the treatment with small fire, and repeatedly measuring the volume of the liquid medicine until 120mL;
(11) Preparing a finished product:
standing at room temperature after concentration is finished, and 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.
Examples
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 the raw materials:
weighing 15g of red sage root, 15g of giant knotweed, 0.5g of bear gall and 0.15g of bezoar for standby;
(2) Cold plasma treatment:
respectively placing the red sage root, 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 the treatment time to be 35s;
(3) Cryogenic grinding treatment:
respectively placing the bear gall and bezoar subjected to cold plasma treatment in the step (2) in a cryogenic pulverizer to carry out pulverization treatment, controlling the temperature in the pulverizer to be-25 ℃, controlling the rotation speed of the pulverizer to be 1100rpm during the pulverization treatment, and obtaining bear gall powder and bezoar powder for standby after 8 minutes of pulverization treatment;
(4) Soaking treatment:
taking the red sage root and the giant knotweed rhizome subjected to cold plasma treatment in the step (2), adding purified water until the liquid level is 2cm higher than the medicine, 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 time of each ultrasonic treatment is 11min, and the frequency of the ultrasonic treatment is 25kHz;
(5) Decocting;
decocting the medicinal materials with strong fire until the decoction is boiled, then converting into small fire, decocting for 1h, pouring the medicinal materials into a beaker, and keeping the residues 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 30 ℃, controlling the vacuum degree to be 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 again to the dried residue, standing 2cm above the residue, boiling again with strong fire, decocting with slow fire for 40min, and guiding out decoction;
(8) And (3) filtering:
mixing the two decoctions, covering the cup mouth with clean gauze, and filtering to obtain medicinal liquid;
(9) And (3) centrifugal treatment:
placing the obtained liquid medicine into a centrifuge tube for centrifugal treatment, wherein the rotation speed of the centrifuge is 1500rpm, and the centrifugal time is 5min;
(10) Concentration treatment:
centrifuging, discarding residue at bottom, pouring the liquid medicine obtained by centrifuging into a clean frying pan, boiling with strong fire, continuously boiling with small fire, concentrating, uncovering the cover during the treatment with small fire, and repeatedly measuring the volume of the liquid medicine until reaching 122mL;
(11) Preparing a finished product:
standing at room temperature after concentration is completed, and 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.
Examples
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 the raw materials:
weighing 20g of red sage root, 20g of giant knotweed, 0.7g of bear gall and 0.2g of bezoar for standby;
(2) Cold plasma treatment:
respectively placing the red sage root, 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 the treatment time to be 40s;
(3) Cryogenic grinding treatment:
respectively placing the bear gall and bezoar subjected to cold plasma treatment in the step (2) in a cryogenic pulverizer to carry out 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 obtaining bear gall powder and bezoar powder for standby after 10 minutes of pulverization treatment;
(4) Soaking treatment:
taking the red sage root and the giant knotweed rhizome which are subjected to cold plasma treatment in the step (2), adding purified water until the liquid level is 3cm higher than the medicine, soaking for 2.5h, and carrying out intermittent ultrasonic treatment while soaking, wherein the method specifically comprises the following steps: carrying out ultrasonic treatment every 6min in the soaking process, wherein the time of each ultrasonic treatment is 12min, and the frequency of the ultrasonic treatment is 30kHz;
(5) Decocting;
decocting the medicinal materials with strong fire until the decoction is boiled, then converting into small fire, continuously decocting for 1.5h, pouring the medicinal materials into a beaker after the decoction is completed, and reserving the medicinal residues 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 ℃, controlling 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 again to the dried residue, allowing the liquid level to exceed 2.5cm of the residue, boiling again with strong fire, decocting with small fire for 50min, and guiding out decoction;
(8) And (3) filtering:
mixing the two decoctions, covering the cup mouth with clean gauze, and filtering to obtain medicinal liquid;
(9) And (3) centrifugal treatment:
placing the obtained liquid medicine into a centrifuge tube for centrifugal treatment, wherein the rotation speed of the centrifuge is 1700rpm, and the centrifugal time is 6min;
(10) Concentration treatment:
centrifuging, discarding residue at bottom, pouring the liquid medicine obtained by centrifuging into a clean frying pan, boiling with strong fire, continuously boiling with small fire, concentrating, uncovering the cover during the treatment with small fire, and repeatedly measuring the volume of the liquid medicine until the volume reaches 124mL;
(11) Preparing a finished product:
standing at room temperature after concentration is completed, and 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, experimental verification was performed on the traditional Chinese medicine composition of example 2. The method comprises the following steps:
1. establishment of Allen's injury model
60 male C57BL/6 mice of 8 weeks old were selected, and after one week of laboratory feeding, their body weights were measured. The experimental mice were randomly divided into 3 groups of 20, each of which was sham group (sham control group), SCI group (sham pre-group), and traditional Chinese medicine group (traditional Chinese medicine composition intervention group). Weighing the mice, and performing anesthesia by intraperitoneal injection of 1% sodium pentobarbital according to the standard of 50 mg/kg; after the muscle relaxation, cornea reaction retardation and skin pinching reaction of the mice disappear, placing the mice on an operation table in a prone position, respectively fixing the head and the limbs, removing hairs around an operation area, positioning the T10 spinous processes by taking the spinous process high points and ribs as marks, marking, sterilizing the operation area by using iodophors, and paving a sterile operation sheet; the skin and subcutaneous tissue of the back of the mouse were cut longitudinally along the posterior midline, centered on T10, with a cut size of about 1.5cm. The vertical spines are separated passively and retracted towards two sides, gauze is used for stopping bleeding, and the T9-T11 vertebral plates are exposed; using the vertebral lamina forceps to finely bite the spinous processes and vertebral laminas of the T9-T11, fully exposing the spinal cord of the T10 section, and avoiding squeezing the spinal cord and damaging the dura mater in the operation process; the PinPoint ™ precision spinal cord injury impactor (model: PCI 3000-2) was used to strike the T10 spinal cord after setting the relevant parameters.
Wherein, the sign of successful SCI group establishment is that the body of the mouse generates transient tremor, the two lower limbs are rapidly retracted and swung, and the tail swings back and forth. The surface of the spinal cord is covered with a layer of autologous fat to reduce adhesion, after no bleeding is checked, the muscle, subcutaneous tissue and skin are sewn layer by layer, and the skin is sterilized by 75% ethanol; sham groups did not strike after spinal cord exposure, and the rest were the same. Conventional anti-infective treatment after SCI group operation to prevent operation area, urinary infection, etc.; after the operation, the patients have difficulty in defecation, and then the patients can perform bladder massage to help defecation. The grouping and administration methods of mice are shown in Table 1.
TABLE 1
| Group of | Quantity (only) | Dosage of usage |
| Sham | 10 | Only the spinal cord is exposed and not hit. Each day is filled with 0.4mL physiological saline |
| SCI | 15 | A striking instrument strikes after exposing the spinal cord. Each day is filled with 0.4mL physiological saline |
| Traditional Chinese medicine formula (example 2) | 15 | A striking instrument strikes after exposing the spinal cord. Decoction of 0.4mL of traditional Chinese medicine for each day of gastric lavage |
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 serial sections were H & E stained with 5um thick centered on the lesion. H & E staining step: xylene is dewaxed for 10min x 2 times, absolute ethanol for 5min x 2 times, 95% ethanol for 5min,80% ethanol for 5min,70% ethanol for 5min, hematoxylin for 5-10 min, hydrochloric acid for 2-5 s, running water is flushed for 30min,70% acetic acid for 5min,80% acetic acid for 5min, eosin counterstain for 5-10 min,95% acetic acid for 5min x 2 times, and xylene is transparent for 5min x3 times. After H & E staining, the damaged area and the section of the adjacent part are observed, and the image is observed and photographed under a light microscope.
BBB scoring
BBB scoring was performed 1 day, 3 days, 1 week and weekly thereafter, respectively, after SCI of mice, until week 4 to evaluate recovery of hindlimb motor function in mice. The evaluation method comprises the following steps: the mice were placed in a wide field and allowed to freely move for 4 min. Two observers were scored according to the BBB scoring criteria, and each observed and scored for hip, knee, ankle, and limb, trunk movements and coordination thereof, and averaged. Statistical analysis: BBB score per group weekly, row mean T test, p <0.05.
C. Footprint testing
The mice were subjected to footprint experiments 14 days post-surgery. The common ink was smeared on the hind paw of the mouse, and passed through a footprint experiment channel 1m long and 10cm wide, leaving its footprint on the channel white paper.
D. Nib staining
(1) Slicing the dried paraffin, conventionally dewaxing to water (xylene I, xylene II for 15min each, then gradient alcohol dehydration: 100% I, 100% II, 95%, 90%, 80%, 70%, 50% for 5min each), and washing with distilled water for 2min;
(2) The fabric slice is put into 0.5% toluidine blue dye liquor for 2-5 min, washed with water, and slightly differentiated by 0.1% glacial acetic acid, the reaction is stopped by running water, the differentiation degree is controlled under a microscope, and the slice is baked in an oven after running water.
(3) Transparent sealing piece: slicing into clean xylene, transparent for 10min, and sealing with neutral resin;
(4) And observing the damaged area and the adjacent parts by a microscope, and observing and photographing under a light microscope.
In fig. 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 functions of the hind limbs of the mice, a graph C is a longitudinal section H & E staining graph of 14 days after injury, obvious malformations and hollows appear at the injured part of 14 days after spinal cord injury in a model group compared with a sham operation group, the lesion area is obviously reduced and the tissue injury is smaller in a dosing group, and a graph D is a Nib staining of the surviving neurons in each group, and the number of neurons in the injured area of the dosing group is obviously increased.
E. Immunofluorescent staining of paraffin sections of spinal cord tissue
(1) Heating the slide in an oven at 49 ℃ for 20min (ensuring that the slide is dried and then subjected to the next step);
(2) Conventional dewaxing to water, xylene dewaxing 5min x3 times, progressive hydration with ethanol (100%, 95%, 80%, 70%, tap water) (ensuring no droplets in xylene);
(3) PBS film washing for 5min multiplied by 3 times;
(4) Antigen retrieval was performed in a microwave oven with boiling water citrate buffer (pH 6.0) for 20min (3.82 g citric acid+2.412 g sodium citrate+1L water, 10N NaOH to adjust pH to 6.0);
(5) Slowly cooling to room temperature;
(6) Washing with PBSx3 for 5min each time;
(7) Circling the tissue portion with a pap pen;
(8) Blocking for 1h (20-30 ℃) at room temperature with a triton X-100 blocking solution containing 10% serum and 1%;
(9) Diluting the primary antibodies of two different species sources with a blocking solution, and incubating overnight at 4 ℃;
(10) PBS film washing for 5min multiplied by 3 times;
(11) Incubating the secondary antibodies connected by Alexa Fluo 555 (1:300) and Alexa Fluo 488 (1:100) for 1h at normal temperature (20-30 ℃);
(12) PBS 5min x3 times;
(13) And adopting a sealing liquid containing DAPI to seal the chip. Sealing liquid PBS and glycerol (4:1) (containing 1.5. Mu.g/ml DAPI);
(14) And observing under a fluorescence microscope.
The gliosis area of the injured spinal cord was studied by immunofluorescence detection, GFAP was an index of astrocytes, and it can be seen from graph a in fig. 2 that after 14 days in the model group, the gliosis formed by astrocytes and the fibrous scar surrounded by gliosis were larger, while the fibrous part scar area surrounded by astrocytes in the administration group was significantly reduced.
Claims (6)
1. The preparation method of the traditional Chinese medicine composition for treating acute spinal cord injury in the acute stage is characterized by comprising the following steps:
(1) Weighing the raw materials:
weighing 10-20 g of red sage root, 10-20 g of giant knotweed, 0.3-0.7 g of bear gall and 0.1-0.2 g of bezoar for standby;
(2) Cold plasma treatment:
respectively placing the red sage root, the giant knotweed rhizome, the bear gall and the bezoar weighed in the step (1) into cold plasma equipment for cold plasma treatment, wherein the treatment power is controlled to be 80-100W during the cold plasma treatment, and the treatment time is 30-40 s;
(3) Cryogenic grinding treatment:
respectively placing the bear gall and the bezoar subjected to cold plasma treatment in the step (2) in a cryogenic pulverizer to perform pulverizing treatment, obtaining bear gall powder and bezoar powder for standby, controlling the temperature in the pulverizer to be minus 30 to minus 20 ℃ during the cryogenic pulverizing treatment, controlling the rotating speed of the pulverizer to be 1000-1200 rpm during the pulverizing treatment, and performing the pulverizing treatment for 6-10 min;
(4) Soaking treatment:
taking the red sage root and the giant knotweed rhizome subjected to cold plasma treatment in the step (2), adding purified water to a liquid level which is 1-3 cm higher than the medicine, soaking for 1.5-2.5 h, and carrying out intermittent ultrasonic treatment while soaking;
(5) Decocting;
after soaking, decocting the medicinal herbs with strong fire until decoction is boiled, then turning into small fire, continuously decocting for 0.5-1.5 h, and pouring the medicinal herbs into a beaker after the completion of the decoction, wherein residues are reserved;
(6) Vacuum freeze drying treatment:
placing the dregs obtained in the step (5) in a vacuum drying oven for low-temperature vacuum drying treatment, and taking out for standby after the completion;
(7) And (3) secondary decoction treatment:
adding purified water again into the dried residue, wherein the liquid level of purified water is 1.5-2.5 cm higher than that of the residue, boiling again with strong fire, decocting with small fire for 30-50 min, and guiding out decoction;
(8) And (3) filtering:
mixing the two decoctions, covering the cup mouth with clean gauze, and filtering to obtain medicinal liquid;
(9) And (3) centrifugal treatment:
placing the obtained liquid medicine into a centrifuge tube for centrifugal treatment;
(10) Concentration treatment:
centrifuging, removing residue at bottom, pouring the liquid medicine obtained by centrifuging into a clean frying pan, boiling with strong fire, and continuously boiling with small fire for concentration;
(11) Preparing a finished product:
and (3) standing at room temperature after concentration is finished, and 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 traditional Chinese medicine composition for treating acute spinal cord injury in the acute phase according to claim 1, wherein the intermittent ultrasonic treatment in the step (4) is specifically: and carrying out ultrasonic treatment every 4-6 min in the soaking process, wherein the time of each ultrasonic treatment is 10-12 min, and the frequency of each ultrasonic treatment is 20-30 kHz.
3. The method for preparing a traditional Chinese medicine composition for treating acute spinal cord injury in an acute phase according to claim 1, wherein a pot cover is required to be covered in the decoction process in the step (5).
4. The method for preparing a traditional Chinese medicine composition for treating acute spinal cord injury according to claim 1, wherein the low-temperature vacuum drying treatment in the step (6) is performed at a controlled drying temperature of 27-33 ℃, a vacuum degree of 1-2 Pa and a water content of 2-5%.
5. The method for preparing a traditional Chinese medicine composition for treating acute spinal cord injury in an acute phase according to claim 1, wherein the rotational speed of a centrifugal machine in the step (9) is 1300-1700 rpm, and the centrifugal time is 4-6 min.
6. The method for preparing a traditional Chinese medicine composition for treating acute spinal cord injury in an acute stage according to claim 1, wherein in the step (10), a pot cover is uncovered during the treatment with small fire, and the volume of the liquid medicine is repeatedly measured until 120-124 mL.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| 丹参注射液对急性脊髓损伤大鼠脊髓灰质GDNF mRNA的提高作用及其机制;韦理;张俐;;中国中西医结合杂志(07);933-937 * |
| 天然药物神经保护作用的研究进展;杨春艳, 邹坤;中草药(03);461-464 * |
| 现代中药加工技术研究进展;薛峰;黄剑宇;吴浩;李俊松;狄留庆;乔宏志;;南京中医药大学学报(05);727-735 * |
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