CN1284561C - Drugs that promote peripheral nerve repair - Google Patents

Abstract

The present invention relates to a traditional Chinese medicine compound formula for promoting nerve regeneration. The compound formula is prepared by that traditional Chinese medicines of manyinflorescenced sweetvetch roots, epimedium herbs, earthworms, etc. are used as principal raw materials. Medicinal theory researches prove that the local administration and the whole body administration of each prescription of the traditional Chinese medicines of the present invention can both promote the regeneration of the peripheral nerves, and the administration of the whole body has protective action on the spinal marrow neuron. The traditional Chinese medicine compound formula of the present invention also has the function of promoting the proliferation and the differentiation of schwann cells.

Description

Drugs that promote peripheral nerve repair

Technical field:

The invention relates to a traditional Chinese medicine compound prescription for promoting nerve regeneration.

Background technique:

In modern trauma surgery, peripheral nerve injury is difficult to repair and regenerate due to its high disability rate. Although the current peripheral nerve repair technology has made great progress, even if the fresh and clean peripheral nerve rupture can be repaired in time using microsurgical techniques, it often cannot be completely regenerated, resulting in the disability of many nerve injury patients. Therefore, how to promote the regeneration of peripheral nerve after injury and restore its function has increasingly become the focus of research.

In the study of factors that promote peripheral nerve regeneration, the earliest report and the only nerve cell regulatory factor that elucidated its molecular structure was nerve growth factor (NGF). Studies have confirmed its receptors and biological effect pathways, and observed its promotion of axon regeneration, myelination and neuron protection. At the same time, gangliosides and basic fibroblast growth factor (bFGF) have been studied more intensively, which have been proved to have different degrees of neurogrowth-promoting effects. However, the curative effect of the above-mentioned factors is uncertain, and the research and application of single factors are currently the main ones, which are expensive and difficult to promote and apply in clinic.

The repair and regeneration of peripheral nerve after injury is a multi-factor, multi-factor involved physiological process (Berger-A, Uierner-R; Rokde-H, shen-EL, Diagnosis and Treatment of Opertpheral Nerve Injury, the integrated therapy concept. Unfallcaitrurg.1999 :102(1):59-68). The microenvironment required for peripheral nerve regeneration should be a biological network in which multiple factors participate together, which is called a multi-factor mutual aid ring. Therefore, supplementation of a single factor has very limited effects on nerve regeneration. The research and application of nerve growth factor and neurotrophic factor substitutes and inducers in foreign countries are progressing slowly, and so far there is no reliable drug for promoting nerve regeneration.

The traditional Chinese medicine compound prescription of motherland medicine is multi-factor composite, the medicament that complements each other. Exploration and development of single and compound preparations in Chinese herbal medicine may provide more environments with a ratio closer to neurophysiological needs and growth active factors, which may be involved in the activation of Schwann cells in the process of nerve regeneration, regeneration of axon growth, and degeneration of substances. Absorption, promotion of local blood circulation, enhancement of immunity and other aspects all play a role.

Traditional prescriptions for peripheral nerve injury repair include:

1. Buyang Huanwu Decoction, which was originally recorded in "Yilin Gaicuo" written by Wang Qingren in the Qing Dynasty. The whole prescription is composed of Astragalus, Angelica, Radix Paeoniae Rubra, Rhizoma Chuanxiong, Safflower, Peach Kernel, and Earthworm (Edited by Xu Jiqun. Prescription Science Shanghai Science and Technology Press, 1990: 150). It has the effect of invigorating qi, promoting blood circulation and stimulating menstruation.

Researchers have conducted a large number of experimental and clinical studies on the mechanism of Buyang Huanwu Decoction and on the repair and regeneration of peripheral nerve injuries. For example, it is believed that Buyang Huanwu Decoction has the effect of promoting axoplasmic transport after nerve injury (Shi Guantong et al., Effect of Buyang Huanwu Tang on axoplasmic transport of sciatic nerve in clamped rats. Chinese Orthopedic Trauma. 1996.9 (1): 3- 4. It can improve microcirculation early and promote local inflammatory edema to subside (Shi Guantong et al. Experimental research on the effect of Buyang Huanwu Decoction on gastrocnemius muscle and blood viscosity after peripheral nerve injury. Chinese Journal of Orthopedics and Traumatology. 1996.4 (2): 4-7). It is confirmed that Buyang Huanwu Decoction can significantly promote the regeneration of nerve fibers, the active proliferation of Schwann cells, the formation of myelin sheath and the integrity of the structure, and has the effect of improving denervated muscular atrophy (Zhao Cuiping, Wang Jianzhi, etc. The morphological experimental study of Buyang Huanwu Decoction on peripheral nerve regeneration. Traditional Chinese Medicine Zhenggu. 1993.5 (2): 6-8). Modern pharmacological studies have found that this prescription has the effects of strengthening the heart, increasing cerebral blood flow, improving circulation, and It can promote the phagocytic ability of phagocytic cells (Xu Qingai, Experimental Research on the Effects of Buyang Huanwu Decoction on Animal Cerebral Circulation and Hemorheology. Chinese Patent Medicine: 1990, 3:25), and can also promote the growth of blood vessels in regenerated nerves, improve Blood supply, promoting neuron repair and regeneration (Liu Meiyu et al. The main study on the effect of Buyang Huanwu Decoction on the morphology of neuron cell bodies in the posterior frontal cortex of mice. Journal of Kunming Medical College. 1995.16(2): 43).

2. Huangqi Guizhi Wuwu Decoction, created by Zhang Zhongjing, is composed of Astragalus membranaceus, white peony root, cassia twig, ginger, and jujube. The main prescription for stagnation, disharmony between the battalion and the defense, and dystrophy of the muscles and arteries.

At present, it is believed that after nerve injury, the sensory and motor dysfunction of the limbs and the neurotrophic changes caused by nerve injury are mostly due to post-traumatic blockage of Qi and blood, and the disharmony between camp and health, which is very similar to the pathogenesis of blood paralysis. Make the blood vessels unblocked, qi and blood filled, camping and defending, promoting blood circulation and strengthening tendons (Nie Xiaopu Peripheral Nerve Injury Treated from Blood Arthralgia Hubei Journal of Traditional Chinese Medicine. 1997.19 (6): 37). Peripheral nerve injury was treated with this prescription, and the function was cured or the function was significantly improved (Sui Xiuzhi Traditional Chinese Medicine cured 5 cases of peripheral nerve injury. Shandong Medicine 2001.41 (16): 72; Zhang Tianjian Modified Huangqi Guizhi Wuwu Decoction Treated 98 Cases of Spinal Nerve Injury Report of Traditional Chinese Medicine Orthopedics 1994 6(2): 18).

Although there are above-mentioned studies on traditional Chinese medicine for promoting peripheral nerve regeneration, these studies and results generally have the following characteristics and shortcomings:

1. The main ingredients of various compound prescriptions are Astragalus, which shows that Astragalus is a traditional Chinese medicine that is generally recognized as having a positive effect on peripheral nerve injury.

2. According to the theory of traditional Chinese medicine, the main principle of promoting peripheral nerve regeneration is to replenish blood and qi, activate blood circulation and remove stasis, promote muscle regeneration and dredge collaterals. The therapeutic effect on peripheral nerve injury is not specifically described.

3. The research methods are not scientific enough. Although the studies involved histological, electrophysiological, and neurological evaluation methods, many were indirect observations. No substance that actually promotes nerve regeneration, such as NGF (nerve growth factor), was used as a control. Therefore, it is difficult to fully prove the effectiveness of each traditional Chinese medicine compound.

4. Oral dosage forms are mainly used, and there are many types of drugs in the formula; because oral dosage forms are used for the whole body, the dosage is large and the range of action is wide, and side effects are inevitable.

Radix Hedysari (Radix Hedysari) is a plant of the genus Astragalus in the leguminous family, which is a different genus from the same family as Astragalus, and is mainly produced in Gansu, my country. The currently known pharmacological effects of Radix Astragali mainly include: 1) enhancing the immune function of the body; 2) promoting growth and anti-aging; 3) strengthening the heart and lowering blood pressure; 4) resisting hypoxia; 5) antibacterial and antiviral; 6) promoting muscle growth wound healing. But there is no clinical application of the compound traditional Chinese medicine preparation for promoting peripheral nerve regeneration.

Invention content:

The purpose of the present invention is to develop a medicine for promoting nerve regeneration with traditional Chinese medicine as an active ingredient.

Combining with the basis of peripheral nerve research, this study investigated the effects of Radix Radix Radix Radix and its compatible traditional Chinese medicine compound from the aspects of histology, immunohistochemistry and electrophysiology, providing scientific experimental basis for further clinical application and drug development, and Further validation of the multifactorial commensal ring hypothesis for neurogenesis.

The present invention has carried out following research:

1. Screen out a compound that is reasonable and effective for peripheral nerve repair;

2. Study the optimal way or route of administration of compound preparations, including systemic administration and local administration;

3. Carry out more in-depth research on the theory of the compound on promoting peripheral nerve regeneration, and clarify the immune regulation mechanism of the compound.

The traditional Chinese medicine compound prescription for promoting peripheral nerve repair developed by the invention is prepared from the following crude drugs in parts by weight: 6-16 parts of Radix Hemorrhoids, 4-10 parts of Epimedium, and 2-6 parts of Dilong.

The preferred proportioning ratio of the raw materials is as follows: 8-14 parts of Radix Radix Radix, 5-9 parts of Epimedium, and 3-5 parts of Dilong.

The best proportioning ratio is: 9-13 parts of red qi, 6-8 parts of epimedium, and 3-4 parts of earthworm.

The monarch drug in the above-mentioned compound recipe remains unchanged, and other medicines can also be added to form multiple prescriptions to exert different degrees of efficacy.

Can add 1-6 parts of Angelica sinensis in addition as above-mentioned compound recipe.

In the compound formula with Radix Esperigo, Epimedium, Earthworm and Angelica, 1-6 parts of Chuanxiong and/or 1-6 parts of Radix Paeoniae Rubra can be added;

Alternatively, add 1-4 parts of safflower and/or 1-4 parts of walnut kernel to the compound formula with red qi, epimedium, earthworm and angelica.

It is also possible to add one or more of the following medicines on the basis of the three basic ingredients of Radix Radix Sinensis, Epimedium, and Earthworm: 1-6 parts of Rhizoma Chuanxiong, 1-4 parts of Safflower, and 1 part of peach kernel. -4 parts, 1-6 parts of Radix Paeoniae Rubra.

Alternatively, use only the extract of the herba radix.

The medicine of the present invention can be prepared into any conventional preparation by conventional methods of traditional Chinese medicine preparations, for example, decocting the raw material drug in water, or extracting it with alcohol or a mixture of alcohol and water, then concentrating the decoction or extract, and refining it into a Chinese medicine extract. The extract is compatible with various pharmaceutically acceptable carriers and adjuvants, and prepared into various preparations by conventional pharmaceutical methods of those skilled in the art.

The dosage forms of the medicine of the present invention can be various oral preparations, such as traditional water decoction, concentrated oral liquid, powder, granule, capsule, tablet, granule, etc., and can also be made into injections, such as for local injection or intraperitoneal injection. injection etc.

From the theory of traditional Chinese medicine, Radix Astragali has the functions of nourishing qi, activating blood circulation, detoxifying and promoting muscle growth; from the theory of Western medicine, Radix Astragali contains polysaccharides, which can significantly promote the activity of macrophages, improve immune function, and significantly reduce plasma lipid peroxide. content, increase the white globulin ratio, and promote RNA synthesis. In addition, red qi can still improve the oxygen uptake function of the lungs. The present inventor has conducted in vitro cell culture experiments on the extract of Radix Hegali, which has proved that it has a better growth-promoting effect on Schwann cells. It shows that only single medicine of Radix Hegali can also repair and regenerate peripheral nerves after injury.

Among the other medicines in the formula of the present invention, angelica nourishes blood and activates blood circulation, red spoon clears away heat and blood stasis, earthworm unblocks collaterals, Chuanxiong activates blood and promotes Qi, safflower and peach kernel promotes blood circulation and eliminates blood stasis, epimedium tonifies kidney and strengthens yang. Taken together, the compound preparation has the effects of nourishing qi, promoting blood circulation, dredging collaterals and tonifying kidney. Chishao has the functions of inhibiting platelet aggregation, activating fibrinolysis, and antithrombosis; ferulic acid, one of the active ingredients of Angelica sinensis, can promote the function of phagocytes and inhibit thrombus formation, and its other active ingredient, immune active polysaccharide, can enhance the body's hematopoietic function, Significantly increase the activity of mononuclear macrophages; Earthworm extract lowers blood pressure, and Earthworm also contains thrombolysin, which can directly catalyze fibrinolytic proteases and inhibit thrombus formation; Chuanxiong also contains ferulic acid, which can improve microcirculation and reduce blood clots. Compress blood vessels and inhibit platelet aggregation; peach kernel and safflower can expand blood vessels and inhibit thrombosis; Epimedium can expand blood vessels, inhibit platelet aggregation, promote nucleic acid and protein synthesis, increase sodium pump activity, and improve energy metabolism .

In the composition of this prescription, Heqi has similar effects with safflower, peach kernel, red peony root, angelica, and Chuanxiong. Therefore, in the present invention, the different combinations of Radix Radix Radix Astragali as the main herbal medicine, such as the prescriptions of Radix Radix Radix Radix, Epimedium Herba, and Earthworm, all can expand the peripheral and internal blood vessels of injured nerves, and inhibit local nerve damage after injury. Vascular thrombosis can improve the microcirculation of damaged nerves, increase the blood flow of arterioles, and increase the blood supply of damaged nerves; at the same time, it can enhance immune function, increase the activity of macrophages, and accelerate the removal of degenerated and necrotic substances of damaged nerves; regeneration.

The present invention adopts sciatic nerve function index, nerve conduction velocity, and myelinated nerve fiber count as pharmacodynamic experiment observation indexes, and nerve conduction velocity comprehensively reflects the composite action potential of sensory conduction velocity and motor conduction velocity in nerve trunk, and is used in detecting nerve trunk trauma It is simple, quick and feasible when there is a sexual injury. The count of myelinated nerve fibers can intuitively reflect the number of regenerated myelinated nerve fibers after nerve injury, and accurately reflect the situation of nerve regeneration. The sciatic nerve function index is an index to detect the general functional recovery after sciatic nerve injury in rats. It can reflect the degree of recovery of nerve function after sciatic nerve injury. Compared with the first two indicators, it has more clinical significance. These three indicators were used in this experiment to comprehensively reflect the sciatic nerve regeneration and the degree of functional recovery of rats in each group from microcosmic to macrocosmic. They are comparable and can describe the treatment group and the treatment group, the treatment group and the treatment group more accurately. The difference between the control group results and its magnitude. Experimental result shows, each index of medicine of the present invention is all better than blank control group. And the tissue slices were observed under the light microscope. The rats in the medication group had significantly less ulcerated myelin sheaths than those in the blank control group at 2 weeks, 4 weeks and 6 weeks after nerve clamping.

The present invention has also carried out other pharmacological experiments on the above-mentioned various formula combinations, including: cytotoxicity test, research on the mechanism of action of traditional Chinese medicine combined with nerve regeneration chamber, and induction of human and animal bone marrow hematopoietic stem cells to differentiate into nerve tissue cells. Optimize the developed marine biological casing to have good nerve tissue compatibility and proper degradation and absorption cycle. The nerve tissue cells induced to differentiate, the effective components of the traditional Chinese medicine compound of the present invention and the marine biological casing are jointly constructed into a nerve regeneration chamber. Its repairing effect on peripheral nerve injury was verified by animal experiments.

In addition to the various formula groups of the present invention and the single Radix Radix Radix Radix Extract group, the above experiments also included a positive drug control NGF group and a blank control group without drugs.

Pharmacological research found that:

1. The recovery of the electrophysiological function of the local administration of the Chinese medicine compound of the present invention and NGF is significantly better than that of the control group, and in terms of the recovery of the SFI motor function index, the Chinese medicine compound group of the present invention has shown an effect better than that of the NGF group, illustrating that this The role of group compound preparations in promoting peripheral nerve regeneration.

2. Systemic administration of the traditional Chinese medicine compound extract of the present invention has a protective effect on spinal cord neurons.

3. The traditional Chinese medicine compound of the present invention has the function of promoting the proliferation and differentiation of Schwann cells, and it is proved that the differentiated cells are newly-proliferated cells after the action of the drug.

Therefore, it can be considered that the Chinese medicine compound extract of the present invention plays a positive role in the repair of peripheral nerves. Its mechanism of action may be that after peripheral nerve injury, it can fully expand the peripheral and internal blood vessels of the nerve, improve the microcirculation of the injured nerve, increase the blood flow of the arterioles, enhance immune function, increase the activity of macrophages, and promote nucleic acid , Protein synthesis and metabolism, accelerate the removal of damaged nerve degeneration and necrosis substances, increase the supply of nerve regeneration substances, promote the recovery of axoplasmic flow, and then prevent the degeneration and necrosis of neurons and promote their early recovery.

Because the Radix Radix Polysaccharide is the main component of Radix Radix Radix, the present invention adopts the improved phenol-concentrated sulfuric acid method to measure the content of Radix Radix Radix Radix Radix Radix Polysaccharide in the medicinal extract of the present invention.

Detailed ways

The proportioning of many groups of compound prescriptions of embodiment 1

Use red qi single herb, or use red qi, epimedium, and earthworm as the monarch drug in the compound, and add one or more of angelica, chuanxiong, safflower, peach kernel, and red peony. The composition is shown in the table 1.

Table 1 The composition ratio of compound raw materials in each group formula API name and dosage (parts by weight) Red Qi Epimedium Earthworm angelica peach kernel Paeoniae Rubra Chuanxiong safflower 1 1 - - - - - - - 2 6 4 2 - - - - - 3 9 2 3 - - - - - 4 12 6 4 - - - - - 5 16 8 6 - - - - - 6 12 6 4 1 - - - - 7 13 10 4 4 - - 1 - 8 11 6 3 6 3 - - - 9 8 6 4 4 - 4 - - 10 8 4 4 3 - - - 3 11 2 7 2 4 - 6 6 - 12 14 4 4 - 4 - - 1 13 12 6 5 - 1 1 - 4 14 11 5 2 - - 4 4 -

Embodiment 2 crude drug decocting and extracting

Equipment: rotary evaporator (including vacuum pump), electric decoction device, condenser tube, eggplant-shaped bottle and other glass instruments.

Red Qi was purchased from Gansu, the place of origin, and the rest of the medicinal materials were purchased from Tongrentang Pharmacy in Beijing.

step:

1. Weigh each drug according to the formula, wherein, cut the Radix Radix Radix Radix Radix into small sections of about 2 cm, weigh after rolling.

2. Put the medicines of each formula group in a round-bottomed flask, decoct each group of medicines three times, add 10 times, 8 times, and 6 times the amount of water, decoct for two hours for the first time, and decoct for two times after each Decoct for one hour, filter the decoction from each time into the container, dewater the decoction through a rotary evaporator, and then melt it into a liquid containing 2g/ml of crude drug with distilled water, transfer it to a sterile bottle and freeze it for storage.

The ethanol extraction of embodiment 3 crude drug

Each medical material is extracted three times with 75% ethanol, and then the alcohol is recovered by rotary evaporation, and the obtained liquid is mainly polysaccharide and concentrated.

Example 4 Chinese medicine compound extraction cytotoxicity test

The purified Chinese medicine compound extract of the present invention was filtered through a 0.22 μm filter, cultured with PRMI1640 (Gibco BRL, USA) containing 15% fetal bovine serum and diluted in multiples, and placed in 24-well plates. For the SP2/0 cells in the logarithmic growth phase, adjust the cell concentration to 10 ⁶ /ml, add 10 μl to the 24-well plate containing the doubling-diluted Radix Astragali culture solution, and culture at 37°C. Cell growth was observed every day.

After 3 days of observation, the results showed that the extract of compound Radix Hegali could promote the proliferation and differentiation of Schwann cells at 1:16000 and 1:32000 (that is, the effective dose of NGF). Description can promote the proliferation of Schwann cells. Medication safety.

Embodiment 5 Each group of drug extracts and NGF contrasts SD rat sciatic nerve culture

The formula extract NGF of each group was diluted 1:16000 as a positive reference, and the normal culture medium was used as a negative control. Put freshly cut SD rat sciatic nerves respectively. Incubate at 37°C. 3 parallel samples. The sciatic nerves were removed at 48h, 72h and 96h after culture, and stored in a -20°C refrigerator. Tyrosine protein kinase test system: product of Gibco, USA (NO: 13154-018). Containing substrate solution and control solution without RR-SRC. Before use, add [γ- ³² P]ATP (>5000Ci/mmol) (Beijing Yahui Biomedical Engineering Company) at 100uCi/ml, and prepare the extract according to the recommended formula of the reagent system. Before the test, the samples were prepared according to the instructions of the kit. Add 200ul of extracting solution to each nerve, homogenate the glass homogenate, place it on ice for 20min, centrifuge at 12000g for 2min, and measure the protein concentration by Coomassie Brilliant Blue method ^[7] . Take samples according to protein concentration and dilute to 0.5mg/ml with extract. Add 10ul of substrate solution or control solution respectively, incubate on ice for 10min, and then centrifuge at 12000g for 10min (4°C). 20ul of the supernatant of the reaction solution was drawn, dropped on the marked phosphocellulose paper, washed twice with 1% (v/v) acetic acid and tap water, each time for 5min. Add to liquid scintillation cup for detection. The samples were counted by Wallac 1410 liquid scintillation counter (Finland), and each sample was counted for 1 min.

Result analysis: ①The cpm per pmol of phosphorus is calculated according to the formula: cpm value of 10ul containing ³² P substrate (a) and control (b)/1200; 2/① _a - control cpm value × 2/① _b ; ③ Kinase activity is calculated according to the formula: ②/30. The results of samples at different concentrations and at different times were statistically processed by the rank sum test of multiple sample comparisons. The variance analysis was used to analyze the mean difference among the formulas, NGF and the control in each group at 48h.

Embodiment 6 animal pharmacodynamics experiment

Model and grouping: SD male rats were randomly divided into two groups. One group was injured by bilateral sciatic nerves by clamping method. They were randomly divided into medication group and control group. , and three doses of high doses were randomly selected for feeding. The control group used normal saline, and the other group cut off the bilateral sciatic nerves of the rats and left a gap of 2 mm at the ends, which were sutured with our self-made marine biological cannula. Prescriptions were given, and NGF+gangliosides were applied locally in the control group.

Observation time: 3d, 1W, 2w, 3w, 6w, 9w.

Early Observation Project:

1. SC value-added form, quantity

2. Time to regenerate new shoots, walk

3. The time for the fiber to pass through the casing

4. Myelin formation time and layers (light microscope and electron microscope)

5. Changes in the aggregation of macrophages at the nerve injury site in each group

6. Changes in intracellular submicrostructure (DIC).

Late Observation Project:

1. Observe the number of nerve fiber regeneration

2. Nerve conduction velocity and activity potential

3. Measure muscle tension and muscle dry and wet weight.

Histological staining methods: solid blue myelin staining and HE staining; SY-38 growth cone staining; S-100 Schwann cell staining; SMI-31 axonal staining.

The research of the mechanism of action of embodiment 7 compound medicine

During the in vivo experiments in rats, before local or systemic administration and after a period of administration, samples or blood were collected to detect the changes in the number and function of macrophages and the changes in MHC antigens in the body to detect the traditional Chinese medicine Hongqi. Effects on the immune status of rats.

Example 8 Observation experiment on drug-induced differentiation of human and mouse hematopoietic stem cells to neural tissue cells

1. Purpose of the experiment

Studying the effect of the compound recipe of the present invention on the growth of neural stem cells is the basis for carrying out research on artificial nerves.

2. Method

Isolation and purification of hematopoietic stem cells by different methods: Directed differentiation of hematopoietic stem cells and bone marrow stromal cells sorted by different methods: Under the action of nerve growth-stimulating factors (EGF, FGF-2, PDGF), the above cells were induced to differentiate Neuroblasts; identified by monoclonal antibodies, the antibodies used include: anti-neuroepithelial stem cell protein (nestin) to identify neural stem cells, anti-neuron-specific enolase to identify neuronal cells, anti-S100 to identify Schwann cells; The induced nerve tissue cells were used for nerve potential measurement.

3. Results

It is proved that the compound can effectively promote the proliferation and differentiation of neural stem cells, which will be beneficial to the research and application of artificial nerves.

Example 9 The active ingredients of the Chinese medicine compound and the marine biological casing are jointly constructed into a nerve regeneration chamber

1. Preparation and performance optimization of artificial marine biological tube bridge:

Research on the biological properties of the material (absorption phase and tissue compatibility in the animal body); research on the compatibility of the material with Schwann cells and the characteristics of inhibiting the growth of fibroblasts: the details are as follows: take out the sciatic nerve by aseptic surgery, cut it into pieces, and use The culture medium is used for in vitro Schwann cell culture in a petri dish, and a biological material film is placed in the petri dish, and a control is set up, and the inverted microscope, light microscope section, histological and immunohistochemical staining, scanning electron microscope and other means are used in different periods. Observe the morphology, activity and density of Schwann cells and fibroblasts, count per unit area, and observe the internal structure and functional activity of Schwann cells.

2. The neural tissue cell suspension or stem cells induced to differentiate and the active ingredient of the Chinese medicine compound of the present invention are inoculated on the marine biological material support developed by this subject, and then the two are added together in the rotating wall bioreactor to make Cells and materials are uniformly combined to construct a nerve regeneration chamber for rat sciatic nerve repair experiments.

① The experimental animals were randomly divided into two groups. The first group was further divided into group A: taking the sciatic nerve from the left leg of the rat and performing in situ suture of the adventitia; group B: taking the sciatic nerve from the right leg of the rat and performing in situ in situ suturing with a small gap in the cannula. The second group was further divided into group C: taking the sciatic nerve from the left leg of the rat and performing 180-degree rotation of the adventitia; group D: taking the sciatic nerve from the right leg of the rat and performing 180-degree rotation on the distal end of the small gap of the cannula. Another group of rats were taken as normal neurological controls. ②Experimental method: SD rats were anesthetized by intraperitoneal injection of 2.5% pentobarbital sodium (50mg/kg), and the bilateral sciatic nerves were exposed through the vastus lateralis muscle space, and the sciatic nerves were cut sharply at 1.0cm below the ischial tubercle, and then Treat each group accordingly. ③Bone marrow puncture was performed on the animals in each experimental group in advance, and bone marrow stromal cells and hematopoietic stem cells were isolated and cultured into neural tissue cells. The individual neural tissue cells of the experimental group were used for the preparation of biological tissue bridges, and the neural tissue cells of the other groups were used in vitro Observation of activity experiment; ④ In the first, second, third and fourth weeks after operation, rats were randomly selected from the first group and the second group each time, and the samples were observed under the operating microscope, and the myelinated nerve fibers were counted under the light microscope. Osmic acid stained histological section observation, electrophysiological examination and functional examination (sciatic nerve function index).

The experimental result of above embodiment 1～9 shows:

1. The recovery of the electrophysiological function of the local administration of the Chinese medicine compound of the present invention and NGF is significantly better than that of the control group, and in terms of the recovery of the SFI motor function index, the Chinese medicine compound group of the present invention has shown an effect better than that of the NGF group, fully illustrating This group of compound preparations can promote the regeneration of peripheral nerves.

2. Systemic administration of the traditional Chinese medicine compound extract of the present invention has a protective effect on spinal cord neurons. The research on the mechanism of action of the preparation proves that it has the function of promoting the proliferation and differentiation of Schwann cells which play an important role in the nerve regeneration process. At the same time, it is proved that the differentiated cells are new growth cells after drug action. Therefore, it can be considered that the Chinese medicine compound extract of the present invention plays a positive role in the repair of peripheral nerves. Its mechanism of action may be that after peripheral nerve injury, it can fully expand the peripheral and internal blood vessels of the nerve, improve the microcirculation of the injured nerve, increase the blood flow of the arterioles, enhance immune function, increase the activity of macrophages, and promote nucleic acid , Protein synthesis and metabolism, accelerate the removal of damaged nerve degeneration and necrosis substances, increase the supply of nerve regeneration substances, promote the recovery of axoplasmic flow, and then prevent the degeneration and necrosis of neurons and promote their early recovery.

Example 10 Observation of the promoting effect of Chinese medicine compound extract of the present invention on peripheral nerve regeneration (1)

In this experiment, the method of clamping the sciatic nerve of rats is used to make the model of peripheral nerve injury, and the Chinese medicine compound extract of the present invention is used as the therapeutic drug and the nerve growth factor and the blank group as contrasts, through the sciatic nerve function index (SFI), nerve conduction Speed, the measurement of regenerated myelin sheath count to ascertain whether the traditional Chinese medicine compound extract of the present invention has promoting effect on peripheral nerve regeneration.

1. Experimental method

Experimental animals and grouping: The experiment used 40 adult healthy SD male rats, provided by the Department of Animal Experiments, Beijing Medical University, weighing about 250g, and randomly divided into 4 groups, with 10 rats in each group. Each group is numbered N, Q, S, B.

2. Experimental drugs:

1) Contains 50 parts of red qi, 10 parts of angelica, 8 parts of red peony, 5 parts of earthworm, 5 parts of chuanxiong, 5 parts of safflower, 5 parts of peach kernel, and 15 parts of epimedium, concentrated to 1.125g/ml.

2) Containing Radix Radix Radix, Epimedium, Earthworm, concentrated to 0.774g/ml.

3) Ingredients of Buyang Huanwu Decoction: Contains astragalus, angelica, red peony, chuanxiong, peach kernel, safflower, earthworm, concentrated to 2.6g/ml.

3. Experimental methods and observation indicators:

SD rats were anesthetized by intraperitoneal injection of 2.5% pentobarbital sodium (50 mg/kg), and the bilateral sciatic nerves were exposed through the vastus lateralis muscle space, and the sciatic nerves were clamped with toothless forceps (3 teeth, 30 cm) at 1.0 cm below the sciatic tubercle. second species) to cause a 2mm wide lesion area, this method has been confirmed by histology to make the epineurium intact, and the axon is completely broken. The incision is then closed. Group Q: Experimental drug 1) The dosage of extract solution is 2ml/body/day, Group S: Experimental drug 2) The dosage of irrigation is 2ml/body/day; Group B: Experimental drug 3) Buyang Huanwu Decoction, administered Dose 2ml/bird/day; N group: normal saline irrigation dose 2ml/bird/day; once a day. Three animals died under anesthesia during the operation (1 in S group, 2 in Q group). In each group, 5 rats took out the bilateral sciatic nerves for histological observation in 2 weeks (drug administration once a day, 14 days in total), and 5 rats in 4 weeks (drug administration once a day, 28 days in total). The functional index of sciatic nerve was measured before operation and 2 and 4 weeks before the rats were collected, and the bilateral sciatic nerves were taken to measure the nerve conduction velocity before the rats were collected 2 weeks and 4 weeks ago.

Observation indicators: sciatic nerve function index (SFI), nerve conduction velocity, histomorphology and myelinated nerve fiber count.

SFI measurement: self-made rat footprint walking box, 15cm high, 15cm wide, and 50cm long, placed a 20cm long, 15cm wide, and 15cm high mouse box with a door on one side at the far end of the passage. Place white paper of the same length and width as the walking box at the bottom of the walking box. Put a little cotton in the petri dish, pour an appropriate amount of carbon ink to soak the cotton, dip the hind feet of the rat in the petri dish, put it into one end of the walking box, and walk to the other end of the box by itself. 4-5 rear paw prints on both sides of the rat were left on the marking paper. 10 rats in each group left hind footprints on the marked paper before the clamp operation, and then left the rear footprints on the marked paper 2 and 4 weeks after the clamp operation. Both sides of the footprints were taken, and the former was the normal group (N), the latter is the experimental group (E). Three variables are measured each, and the measurements are accurate to the millimeter.

Footprint length (print length factor, PLF): the longest distance of the footprint, that is, from the heel to the toe, the longest PLF value is selected each time.

Toe spread factor (TSF): the distance between the 1st toe 5th toe, the longest TSF value is selected each time.

Intermediary toe spread (ITF): the distance between the 2nd and 4th toes, and the longest TSF value is selected each time.

The SFI can be calculated by substituting the above three variables into the Bain formula.

SFI＝-38.3(EPL-NPL/NPL)+109.5(ETS-NTS/NTS)+13.3(EIT-NIT/NIT)-8.8

EPL: footprint length of the experimental group; NPL: footprint length of the normal group; ETS: span width of the experimental group; NTS: span width of the normal group; EIT: middle toe distance of the experimental group; NIT: middle toe distance of the normal group.

SFI takes ±10 as the normal value, and -100 as the index of complete nerve disconnection.

Nerve conduction velocity (NCV): After performing SFI detection, the bilateral sciatic nerves were exposed according to the original route, and electrodes were placed about 1.5 cm on both sides of the clamped injury. The central end was the stimulating electrode, and the peripheral end was the guiding electrode. A plotter measures nerve conduction velocity. Formula: speed = distance between two electrodes / difference in action potential latency

Histological examination:

Materials collection and staining: Rats in each group were subjected to the clamp operation for 2 weeks, and the sciatic nerve was exposed by the original surgical approach 4 weeks later. The nerve segment 0.5 cm away from the clamp was removed, placed in 10% formalin, and fixed for 24 hours. Osmium acid staining.

Osmium acid staining:

1% osmic acid post-fixation and staining for 72 hours

After rinsing with running water for 10 minutes, soak in distilled water for 10 minutes, a total of 3 times

Dehydration: alcohol 50% → 70% → 75% → 80% → 85% → 90% → 95% → 100% (1 time), → 100% (2 times) for 15 minutes each → xylene (1 time) → two Toluene (2 times) 15 minutes each

Dip wax: 60 minutes → 30 minutes → 30 minutes → 15 minutes

embedding

Sectioning: 3-5 μm transected sections of the nerve

Baked slices: 12-24 hours at 38°C

Xylene 5 minutes 2 times

Mount

Microscopic examination: 1-2 slices were selected for each specimen, and the osmium-stained slices were used for the counting of the myelinated nerve fibers of the common peroneal nerve on the basis of observing the differences in the staining and morphology of the myelin sheath in each group of slices.

Counting of myelinated nerve fibers: There were 75 slices in each experimental group and the control group. Under the field of view magnified 400 times (10*40) by an optical microscope, the number of myelinated nerve fibers in each visual area of the common peroneal nerve was measured for each slice, and the number was taken as mean. Perform statistical analysis.

4. Experimental results

1) Sciatic nerve function index (SFI): X+SD group 2 weeks after surgery 4 weeks after surgery Blank control group -50.3405±20.5918 -22.1106±10.3532 Group S -32.9944±20.0971 -11.9985±5.4533 Q group -27.4385±13.5411 -16.8660±8.6201 Group B -58.3396±12.3949 -21.1303±7.0933

At 2 weeks, there were 3 rats with 5 legs in group N, 3 rats with 5 legs in group Q, and 2 rats with 3 legs in group B due to different degrees of toe ulcers and contractures. There was no significant difference between the Buyang Huanwu Decoction group and the experimental drug 2) group and the control group (P=0.086>0.05; P=0.530>0.05 respectively); but there was a significant difference between the Buyang Huanwu Decoction group and the experimental drug 2) group , and the experimental drug 2) group is better than the Buyang Huanwu Decoction group (P=0.01<0.05), the experimental drug 1) group is significantly different from the control group, and is better than the control group (P=0.046>0.05 ), the experimental drug 1) group was also better than the Buyang Huanwu Decoction group, and the difference was significant (P=0.002<0.010). At 4 weeks, 1 dog in group N had 1 side, 2 dogs in group S had 2 sides, and 1 dog in group B had toe ulcers and contractures of different degrees on both sides. There was no significant difference between the groups (P=0.827>0.05, P=0.246>0.05), but the difference between the experimental drug 2) group and the control group was significant, and it was better than the control group (P=0.048<0.05), and better than the experimental drug 2) group. Drug 1) group, and the difference was significant (P=0.023<0.05), but there was no significant difference with Buyang Huanwu Decoction group.

2) Nerve conduction velocity (NCV) (m/s) X+SD group postoperative time 2 weeks 4 weeks N group 17.0000±14.4855 26.2900±11.3439 Group S 20.5100±8.1175 40.7857±14.4462 Group B 22.5700±16.2037 39.1516±10.1192 Q group 27.7540±3.9825 40.4375±13.9800

At 2 weeks, there were four animals with four sides in group Q, two animals with two sides in group B, and three animals with five sides in group N, which could not be detected. The control group was better than the control group, but the difference was not statistically significant. At 4 weeks, 1 animal in group S had 1 side, 1 mouse in group B had 1 side, and 1 mouse in group Q had not been detected on 2 sides, including experimental drug 1) group, experimental drug 2) group, Buyang Huanwu decoction group and control group The ratios were better than those of the control group, and the difference was significant (P=0.005<0.01, P=0.02<0.05, P=0.003<0.05), but there was no significant difference among the three groups.

Histological observation

Morphological observation under the light microscope: osmic acid stained sections, 2 weeks later, regenerated myelinated nerve fibers began to appear, thinner, with thinner walls, and the regenerated myelinated nerve fibers in the treatment groups were significantly more than those in the control group, and some of the myelin sheaths were degenerated After being disintegrated and absorbed, the blank group still had more denatured myelin sheaths and fewer regenerated myelinated nerve fibers. At 4 weeks, the regenerated myelinated nerve fibers increased significantly, and the regenerated myelinated nerve fibers in each treatment group were significantly more than those in the blank group, and the degenerated myelin sheath had basically disintegrated and disappeared, while the blank group still had more degenerated myelinated sheaths.

3) Count of myelinated nerve fibers per field of view (bars) X+SD group 2 weeks after surgery 4 weeks after surgery N group 11.3±1.06 19.0±2.50 Group S 17.3±1.99 29.0±3.67 Group B 17.6±2.85 23.5±2.31 Q group 17.6±3.87 22.9±2.94

At 2 weeks, each group was significantly better than the control group, (p=0.001<0.01), but the difference between each medication group was meaningless, and at 4 weeks, each group was significantly better than the control group (p<0.05), and the experiment Drug 2) group was significantly better than the whole formula group and Buyang Huanwu Decoction group (p=0.002<0.01, p=0.004<0.01). However, there was no significant difference between the Quanfang group and the Buyang Huanwu Decoction group.

Example 11 Observation of the promoting effect of Chinese medicine compound extract of the present invention on peripheral nerve regeneration (two)

1. Experimental animals and grouping: The experiment used 30 adult healthy SD male rats, provided by the Animal Experiment Department of Beijing Medical University, with a body weight of about 250 g, and were randomly divided into 3 groups, with 10 rats in each group.

2. Experimental drugs:

1) The formulas in Example 1 included 60 parts of Radix Astragali, 20 parts of Epimedium, 6 parts of Dilong, 15 parts of Angelica, and 10 parts of Radix Paeoniae Rubra, and each dose of the extract was concentrated to 0.963 g/mL. (please rewrite)

2) Containing 70 parts of Radix Radix Radix, 25 parts of Epimedium, and 5 parts of Dilong (write out the proportioning relationship). Each dose is concentrated to 0.774g/mL.

3. Experimental methods and observation indicators:

SD rats were anesthetized by intraperitoneal injection of 2.5% pentobarbital sodium (50mg/kg), and the bilateral sciatic nerves were exposed through the vastus lateralis muscle space, and a small incision was made in the posterior femur to expose the sciatic nerves until the distal tibial nerve and common peroneal nerve At 5 mm above the bifurcation of the nerve, the nerve was clamped with toothless forceps with a head width of 2 mm, and the forceps were uniformly occluded with 3 teeth for 1 minute. From the second day after operation, group S and group Q were given medicine once a day at 5ml/kg each time, and the control group was given normal saline 5ml/kg every day. After operation, the three groups were housed in separate cages under the same conditions for 6 weeks.

The bilateral sciatic nerves were taken out for histological observation at 6 weeks (administration once a day, 42 days in total), and the functional index of sciatic nerves was measured before operation and 6 weeks before the rats in each group were collected.

OUTCOME MEASURES: Sciatic nerve function index (SFI), histomorphology and myelinated nerve fiber count.

General status: including appetite, mental status, aggression, local infection rate and mortality.

SFI measurement: self-made rat footprint walking box, 15cm high, 15cm wide, and 50cm long, placed a 20cm long, 15cm wide, and 15cm high rat box with one side of the door at the far end of the passage. Place white paper of the same length and width as the walking box at the bottom of the walking box. Put a little cotton in the petri dish, pour an appropriate amount of carbon ink to soak the cotton, dip the hind feet of the rat in the petri dish, put it into one end of the walking box, and walk to the other end of the box by itself. 4-5 rear paw prints on both sides of the rat were left on the marking paper. 10 rats in each group left hind footprints on the marked paper before the clamp operation, and then left the rear footprints on the marked paper 6 weeks after the clamp operation. Both sides of the footprints were taken, and the former was the normal group (N ), the latter is the experimental group (E). Three variables are measured each, and the measurements are accurate to the millimeter.

Footprint length (print length factor, PLF): the longest distance of the footprint, that is, from heel to toe, choose the longest PLF value each time.

Toe spread factor (TSF): the distance between the 1st toe 5th toe, the longest TSF value is selected each time.

Intermediary toe spread (ITF): the distance between the 2nd and 4th toes, and the longest TSF value is selected each time. The SFI can be calculated by substituting the above three variables into the Bain formula.

SFI＝-38.3(EPL-NPL/NPL)+109.5(ETS-NTS/NTS)+13.3(EIT-NIT/NIT)-8.8

EPL: footprint length of the experimental group; NPL: footprint length of the normal group; ETS: span width of the experimental group; NTS: span width of the normal group; EIT: middle toe distance of the experimental group; NIT: middle toe distance of the normal group.

SFI takes ±10 as the normal value, and -100 as the indicator of complete nerve disconnection.

Histological examination:

Materials collection and staining: Rats in each group were exposed to the sciatic nerve by the original surgical approach 6 weeks after the clamping operation, and the nerve segment 0.5 cm away from the clamping place was taken out, placed in 10% formalin, fixed for 24 hours, and then stained with osmic acid .

Osmium acid staining:

1% osmic acid post-fixation and staining for 72 hours

After rinsing with running water for 10 minutes, soak in distilled water for 10 minutes, a total of 3 times

Dehydration: alcohol 50% → 70% → 75% → 80% → 85% → 90% → 95% → 100% (1 time), → 100% (2 times) for 15 minutes each → xylene (1 time) → two Toluene (2 times) 15 minutes each

Dip wax: 60 minutes → 30 minutes → 30 minutes → 15 minutes

embedding

Sectioning: 3-5 μm transected sections of the nerve

Baked slices: 12-24 hours at 38°C

Xylene 5 minutes 2 times

Mount

Microscopic examination: 1-2 slices were selected for each specimen, and the osmium-stained slices were used for the counting of the myelinated nerve fibers of the common peroneal nerve on the basis of observing the differences in the staining and morphology of the myelin sheath in each group of slices.

Counting of myelinated nerve fibers: a total of 75 slices in each experimental group and control group, under the field of view magnified 400 times (10*40) by an optical microscope, as shown in the figure, the myelinated nerve fibers in each visual area of the common peroneal nerve were measured for each slice The number of nerve fibers was taken as the mean value. Perform statistical analysis.

4. Experimental results

1) General status Except for 6 rats who died under anesthesia during the operation (3 rats in group Q, 2 rats in group S, and 1 rat in group N), all rats in each group had no obvious abnormal behavior after operation. There was no significant change in sleep and appetite since 24 hours after operation, and no wound infection or death occurred in any case. The surface of the bilateral sciatic nerves of the rats in the treatment group was generally smoother than that of the control group, easy to separate and expose from the tissue, and the adventitial blood vessels were abundant.

2) Sciatic nerve function index (SFI): +SD group Postoperative time (6 weeks) N -25.1729±15.3830 Q -30.2700±10.8001 S -19.1105±9.1048

The results showed that the experimental drug 1) group and the experimental drug 2) group had no significant difference from the control group (P=0.262, >0.05; P=0.479>0.05, respectively). However, there is a significant difference in the ratio between the experimental drug 1) group and the experimental drug 2) group, and the experimental drug 2) group is better than the experimental drug 1) group (P=0.03, <0.05).

3) Histological observation

Morphological observation under light microscope:

Osmic acid stained sections, at 6 weeks, the regenerated myelinated nerve fibers increased significantly, and the regenerated myelinated nerve fibers in the Chinese medicine group and the NGF group were more than those in the blank group. myelin sheath. It is suggested that the traditional Chinese medicine compound extract of the present invention can promote the regeneration of myelinated nerve fibers and the decomposition and absorption of degenerated myelin sheath.

4) Count of myelinated nerve fibers per field of view (bar)+SD group Postoperative time (6 weeks) N 75.77±14.36 Q 112.33±17.99 S 109.26±15.73

The average number of myelinated nerve fibers per visual field in the traditional Chinese medicine compound group of the present invention is 112.33±17.99, which is better than 75.77±14.36 in the control group, and there is a statistically significant difference (P=0.000, <0.01). 109.26±15.73 in the experimental drug 2) group was significantly better than that in the control group (P=000, <0.01), but there was no significant difference between the experimental drug 1) group and the experimental drug 2) group.

Embodiment 10～11 result:

(1) Sciatic nerve function index (SFI): At 2 weeks, there was no significant difference between the Buyang Huanwu Decoction group and the experimental drug 2) group and the control group, but there was a significant difference between the Buyang Huanwu Decoction group and the experimental drug 2) group, And the experimental drug 2) group is better than the Buyang Huanwu Decoction group, the experimental drug 1) group is significantly different from the control group, and is better than the control group, and the experimental drug 1) group is also better than the Buyang Huanwu Decoction group , and the difference is significant. At 4 weeks, there was no significant difference between the experimental drug 1) group and the Buyang Huanwu Decoction group and the control group, but there was a significant difference between the experimental drug 2) group and the control group, and it was better than the control group and at the same time better than the experimental drug 1) group. And the difference is significant, but the difference with Buyang Huanwu Decoction group is meaningless. At 6 weeks, there was no significant difference between the experimental drug 1) group and the experimental drug 2) group and the control group; however, there was a significant difference between the experimental drug 1) group and the experimental drug 2) group, and the experimental drug 2) group was better than the experimental drug Group 1.

(2) Nerve conduction velocity (NCV): The experimental drug 1) group, experimental drug 2) group, Buyang Huanwu Decoction group and the control group were all better than the control group at 2 weeks, but the difference was not statistically significant. At 4 weeks, the experimental drug 1) group, the experimental drug 2) group, and the Buyang Huanwu Decoction group were all better than the control group, and the difference was significant, but there was no significant difference among the three drug groups.

(3) Histological observation: all medication groups were significantly better than the control group at 2 weeks, but the difference between the medication groups was meaningless. In the Quanfang group and the Buyang Huanwu Decoction group, there was no significant difference between the Quanfang group and the Buyang Huanwu Decoction group. At 6 weeks, the Chinese medicine compound group of the present invention was superior to the control group and had statistically significant differences. The experimental drug 2) group was significantly better than the control group, but there was no significant difference between the experimental drug 1) group and the experimental drug 2) group.

Example 12 Determination of Radix Astragali Polysaccharide Content in the Compound Chinese Medicine Extract of the Present Invention

On the basis that the extract formula containing 70 parts of Radix Radix, 25 parts of Epimedium, and 5 parts of Earthworm can effectively promote the regeneration of injured sciatic nerve in rats, the main material component of the prescription, Radix Polysaccharides, was prepared by the phenol-concentrated sulfuric acid method. Assay.

Materials and methods

1.1 Materials: phenol (analytical pure), re-distilled to make 50g/l aqueous solution; concentrated sulfuric acid (analytical pure, d=1.84g/cm ^-3 ); absorbance was measured with a 721-type spectrophotometer, using a 10mm optical path compared to quartz Add 50g/l phenol and concentrated sulfuric acid to the color dish with an Optifix dosing device. Pure glucose. Purified Chinese medicine compound prescription extract of the present invention (concentration 86mg/ml)

1.2 Method: Preparation of standard solution: Accurately weigh 10 mg of dried pure glucose, dissolve it in 100 ml of water, and then dilute it to a concentration of 2, 4, 8, 10, 20 ug/ml, extract the traditional Chinese medicine compound recipe of the present invention Liquid dilution is 4.3ug/ml. First take 0.4ml of each standard glucose solution that has been diluted, put it in a 10ml test tube, add 0.8ml of 50g/l phenol solution and mix, then quickly add 4ml of concentrated sulfuric acid, after mixing evenly, place it at room temperature for 30min, measure the absorbance at a wavelength of 490nm, blank control Substitute distilled water for the sugar solution. After the standard curve is to be measured, get the diluted Chinese medicine compound prescription extract 0.4ml of the present invention, place it in a 10ml test tube, add 50g/l phenol solution 0.8ml after mixing, add 4ml vitriol oil rapidly, after mixing uniformly, Leave it at room temperature for 30 minutes, measure the absorbance at a wavelength of 490nm, and substitute the absorbance into the standard curve equation to obtain the amount of polysaccharides contained in the formula, so as to obtain the content of polysaccharides in the prescription.

2. Results and Discussion

2.1 Obtain the standard curve from the sample absorbance values shown in the table below, and obtain the linear equation as Y=14.73X-0.0343Chi-Square: 0.00091

Where Y is the sugar content, X is the absorbance at 490nm

The diluted Chinese medicine compound prescription extract of the present invention records that the absorbance is 0.076, and it is 1.0908ug to find that the contained sugar content is substituted into the equation, and the dilution contains 1.72ug of the prescription. 63.41%. sample number Standard glucose content (ug) Absorbance at 490nm 1 0.8 0.051 2 1.6 0.110 3 3.2 0.225 4 4.0 0.280 5 8.0 0.541

In the reduced formula, because Epimedium and Earthworm contain almost no sugar in the formula, the measured sugar can be considered to be red qi polysaccharide. The measured result is 63.41% of the total prescription

The pharmacodynamics experiment of embodiment 13 single medicine Radix Radix Radix

1. The purpose of the experiment: To observe the effect of Hongqi single drug on promoting the regeneration of peripheral nerves after injury

2. Experimental method

The single drug extract of Radix Radix Radix and NGF was diluted 1:16000 as a positive reference, and the normal culture medium was used as a negative control. Put freshly cut SD rat sciatic nerves respectively. Incubate at 37°C. 3 parallel samples. The sciatic nerves were removed at 48h, 72h and 96h after culture, and stored in a -20°C refrigerator. Tyrosine protein kinase test system: product of Gibco, USA (NO: 13154-018). Containing substrate solution and control solution without RR-SRC. Before use, add [γ- ³² P]ATP (>5000Ci/mmol) (Beijing Yahui Biomedical Engineering Company) at 100uCi/ml, and prepare the extract according to the recommended formula of the reagent system. Before the test, the samples were prepared according to the instructions of the kit. Add 200ul of extracting solution to each nerve, homogenate the glass homogenate, place it on ice for 20min, centrifuge at 12000g for 2min, and measure the protein concentration by Coomassie Brilliant Blue method ^[7] . Take samples according to protein concentration and dilute to 0.5mg/ml with extract. Add 10ul of substrate solution or control solution respectively, incubate on ice for 10min, and then centrifuge at 12000g for 10min (4°C). 20ul of the supernatant of the reaction solution was drawn, dropped on the marked phosphocellulose paper, washed twice with 1% (v/v) acetic acid and tap water, each time for 5min. Add to liquid scintillation cup for detection. The samples were counted by Wallac 1410 liquid scintillation counter (Finland), and each sample was counted for 1 min.

3. Result analysis:

①The cpm of phosphorus per pmol is calculated according to the formula: cpm value of 10ul containing ³² P substrate (a) and control (b)/1200; _a - Control cpm value × 2/① _b ; ③ Kinase activity is calculated according to the formula: ②/30. The results of samples at different concentrations and at different times were statistically processed by the rank sum test of multiple sample comparisons. The variance analysis was used to analyze the mean difference between Radix Radix Radix, NGF and the control at the 48th hour.

4. Conclusion: Hongqi monotherapy can promote the proliferation and differentiation of Schwann cells, and the effect is similar to that of NGF. It shows that single red qi also has the effect of promoting the regeneration of peripheral nerves after injury.

The preparation of embodiment 14 injection

Preparation of injections: Purify the water decoction of the prescription with a rotary evaporator, then extract the water contained in a vacuum machine, and then dry it into powder through a water bath. When using it, accurately weigh the required amount, and use an injection After diluting with water, it is applied by local injection to rats.

The preparation of embodiment 15 oral agent

The water decoction of the prescription was first purified with a rotary steamer, concentrated to the required dosage, and administered to rats for a gavage experiment.

Claims (9)

1. A medicine for promoting the repair of peripheral nerves, the raw materials of which are made into active ingredients are: 6-16 parts by weight of Radix Astragali, 4-10 parts by weight of Epimedium, and 2-6 parts by weight of Dilong. 2. The medicine for promoting the repair of peripheral nerves according to claim 1, wherein the raw materials are composed of: 8-14 parts by weight of Hegalysus, 5-9 parts by weight of Epimedium, and 3-5 parts by weight of Dilong. 3. The medicine for promoting the repair of peripheral nerves according to claim 1, wherein the raw materials are composed of: 9-13 parts by weight of Radix Astragali, 6-8 parts by weight of Epimedium, and 3-4 parts by weight of Dilong. 4. The medicine for promoting peripheral nerve repair described in claim 1, said raw material also has 1-6 parts by weight of Angelica sinensis. 5. The medicine for promoting peripheral nerve repair described in claim 4, said raw material also has 1-6 parts by weight of Rhizoma Chuanxiong and/or 1-6 parts by weight of Radix Paeoniae Rubra. 6. The medicine for promoting peripheral nerve repair described in claim 4, said raw material also has 1-4 parts by weight of safflower and/or 1-4 parts by weight of peach kernel. 7. The use of single-herb Radix Hegali in the preparation of medicines for promoting peripheral nerve repair. 8. The medicine for promoting peripheral nerve repair according to any one of claims 1-7, the dosage form is an oral agent. 9. The medicine for promoting peripheral nerve repair according to any one of claims 1-7, the dosage form is injection.