CN1284561C - Chinese medicine compound for promoting nerve regeneration - 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

Promote the medicine that peripheral nerve is repaired

Technical field:

The present invention relates to promote the Chinese medicine compound of neuranagenesis.

Background technology:

In modern the traumatology department, peripheral nerve injury is because of its disability rate height, and repair and the comparatively difficulty of regenerating the damage back.Although at present the peripheral nerve recovery technique has had long-range progress, even fresh, the peripheral nerve rupture of cleaning can in time use microsurgical technique reparation, also often can not holomorphosis, cause many injured nerves patients' maimed person.Therefore, how to promote the regeneration behind the peripheral nerve injury, recover the emphasis that its function becomes research day by day.

In the research that promotes the peripheral nerve regeneration factor, report the earliest, unique neurocyte regulatory factor of illustrating molecular structure is nerve growth factor (NGF).Studies confirm that its receptor and biological effect approach, observed it and promote neural axon regeneration, myelinization and neuronic protective effect.Simultaneously, the ganglioside in addition that research is comparatively concentrated, basic fibroblast growth factor (bFGF) is proved to be short nerve growth effect in various degree respectively.But above-mentioned factor uncertain therapeutic efficacy is fixed, and at present with monofactorial research be applied as the master, costs an arm and a leg, and is difficult at clinical application.

Reparation behind the peripheral nerve injury and regeneration are multiple-factors, the physiological process of multifactor participation (Berger-A, Uierner-R; Rokde-H, shen-EL, Diagnosis and Treatment of Opertpheral Nerve Injury, the integrated therapyconcept.Unfallcaitrurg.1999:102 (1): 59-68).The required microenvironment of peripheral nerve regeneration should be the bio-networks of a multifactor fellowship, is referred to as multifactor mutual aid ring.Therefore, the effect of additional single factor pair neuranagenesis is very limited.The nerve growth factor of external research clinically at present and application and substitute that neurocele is supported the factor and inducer etc. are made slow progress, and do not have a kind of medicine of reliable promotion neuranagenesis so far.

The Chinese medicine compound of motherland's medical science is multifactor compound, complementary medicament.Seek and develop folk prescription and compound preparation in the Chinese herbal medicine, might provide more, ratio more near the environment of the nervous physiology demand and the growth activity factor, may all play effect at the activation of the Schwann cell in the process of neuranagenesis, the growth of regeneration axon, degeneration material absorbing and the aspects such as local blood circulation, enhancing immunity that promote.

Traditional prescription of peripheral nerve injury reparation comprises:

1. BUYANG HUANWU TANG, the former errors in Medicine Corrected that is stated from Qing Dynasty's Wang Qingren of this side.Full side is by the Radix Astragali, Radix Angelicae Sinensis, Radix Paeoniae Rubra, Rhizoma Chuanxiong, Flos Carthami, Semen Persicae, Pheretima form (the Xu Jiqun chief editor. pharmacology of Chinese medical formulae Shanghai science tech publishing house, 1990:150).Has the effect that inrigorating qi and promoting blood circulation stimulates the menstrual flow.

Research worker has been carried out a large amount of experimentation and clinical research to the BUYANG HUANWU TANG mechanism of action with to peripheral nerve injury reparation and regeneration.As think BUYANG HUANWU TANG have after nerve injury the effect that promotes the axoplasm transportation (Shiguan paulownias etc., BUYANG HUANWU TANG is to the influence of clamp rat sciatic nerve axoplasm transportation. Chinese bone injury .1996.9 (1): 3-4.And microcirculation improvement in early days, promote the local inflammation edema extinction (Shiguan paulownia etc., BUYANG HUANWU TANG to peripheral nerve injury after the experimentation of gastrocnemius and blood viscosity influence. Chinese orthopedics and traumatology of Chinese medicine magazine .1996.4 (2): 4-7).Confirm that BUYANG HUANWU TANG is to nerve fiber regeneration, the active propagation of Schwann cell, myelin forms and structural integrity has obvious facilitation, to muscle denervation atrophy have change general effect (Huanwu detoction for supplementing Yang such as Zhao Cuiping, Wang Jianzhi are to the morphology experimentation of peripheral nerve regeneration. traditional Chinese medical science bonesetting .1993.5 (2): 6-8).Modern pharmacological research is found, this side has heart tonifying, the cerebral blood flow increasing amount, improve the circulation effect, and can promote cytophagous phagocytic activity (Xu Qingai, BUYANG HUANWU TANG is learned the experimentation of influence to animal brain circulation and hemorheology. Chinese patent medicine: 1990,3:25), can also promote the growth of regenerating nerve medium vessels, improve the blood confession, promote neuron reparation and regeneration (BUYANG HUANWU TANG such as Liu Meiyu to the mice cortex of frontal lobe after the main research that influences of pericaryon form. the journal .1995.16 of unming Medical College (2): 43).

2, Huangqi Guizhi Wuwu Tang, this side is the Zhang Zhongjing initiative, is made up of the Radix Astragali, the Radix Paeoniae Alba, Ramulus Cinnamomi, Rhizoma Zingiberis Recens, Fructus Jujubae, and this side has supplementing QI to normalize nutrient QI, and the effect of warming YANG migratory arthralgia is the retardance of treatment QI and blood, and battalion defends and becomes estranged, and the muscle arteries and veins loses the arthralgia due to stagnation of blood disease main formula of supporting.

Think at present after the nerve injury, limbs sensation and dyskinesia and sexually revise the QI and blood retardances after the wounds that belong to because of the neuratrophia that causes after the nerve injury more, battalion defends and becomes estranged, quite similar with the pathogenesis of arthralgia due to stagnation of blood, this can make blood vessels logical, QI and blood is filled, the battalion defend and, (the little garden of Nie peripheral nerve injury is controlled Hubei Journal of Traditional Chinese Medicine .1997.19 (6) from the arthralgia due to stagnation of blood opinion to the strong muscle of invigorating blood circulation: 37).With this side treatment peripheral nerve injury, obtain recovery from illness or function be improved significantly (the Sui Xiu sesame traditional Chinese medical science is cured peripheral nerve injury 5 examples. Shandong medicine 2001.41 (16): 72; Zhang Tianjian adds flavor Huangqi Guizhi Wuwu Tang treatment and scratches the nerve injury 98 example report traditional Chinese medical science and boneset 1,994 6 (2): 18).

Although the Chinese medicine research of above-mentioned promotion peripheral nerve regeneration is arranged, these researchs and result have following characteristics and shortcoming generally:

1. the main component of various compound recipes is the Radix Astragali, illustrate relatively generally acknowledge peripheral nerve injury is had the Chinese medicine of sure curative effect is the Radix Astragali.

2. by the theory of the traditional Chinese medical science, promote that the peripheral nerve regeneration cardinal principle is blood-enriching qi-invigorating, activating blood circulation to dissipate blood stasis, granulation promoting collateral dredging.Do not specifically describe therapeutic effect to peripheral nerve injury.

3. the still not enough science of research means.Learning evaluation methodology though relate to histology, electrophysiology and function of nervous system in the research, much is indirect observation.Not truly having the material that promotes neuranagenesis, in contrast as NGF (nerve growth factor) etc.Therefore, the difficult effectiveness that fully proves each Chinese medicine compound.

4. based on peroral dosage form, and the formulation of drug kind is more; Because peroral dosage form is a systemic administration, consumption is big, sphere of action is wide, and side effect is arranged unavoidably.

Radix Hedysari (Radix Hedysari) is a pulse family sweetvetch platymiscium, does not belong to together with the Radix Astragali is equal, and main product is in China Gansu.Present known Radix Hedysari pharmacological action mainly contains: 1) enhancing body's immunological function; 2) promote the growth defying age; 3) heart tonifying blood pressure lowering; 4) anoxia enduring; 5) anti-bacteria and anti-virus; 6) granulation promoting promotes wound healing.But do not see the clinical practice of the Chinese traditional compound medicine that promotes peripheral nerve regeneration as yet.

Summary of the invention:

The objective of the invention is to develop a kind of is the medicine of the promotion neuranagenesis of active component with Chinese medicine.

This research combines with peripheral nerve research basis, from histology, immunohistochemistry and electrophysiology aspects investigated Radix Hedysari and with the effect of the Chinese medicine compound of its compatibility, for further clinical practice and patent medicine exploitation provides the experimental basis of science, the multifactor mutual aid ring hypothesis of the step of going forward side by side card neuranagenesis.

The present invention has carried out following research:

1. filter out peripheral nerve is repaired the rational and effective compound recipe;

2. the administering mode or the approach of research compound preparation optimum comprise the whole body administration, topical;

3. carry out more deep research at compound recipe to promoting the peripheral nerve regeneration theoretical side, illustrate the immune regulation mechanism of compound recipe.

The Chinese medicine compound of the promotion peripheral nerve reparation of the present invention's exploitation is made by following bulk drugs: Radix Hedysari 6-16 part, Herba Epimedii 4-10 part, Pheretima 2-6 part.

The optimum ratio of above-mentioned raw materials medicine is: Radix Hedysari 8-14 part, Herba Epimedii 5-9 part, Pheretima 3-5 part.

Best proportioning is: Radix Hedysari 9-13 part, Herba Epimedii 6-8 part part, Pheretima 3-4 part.

Monarch drug is constant in the above-mentioned compound recipe, can also add other medicines, is combined into multiple prescription, performance effect in various degree.

As adding Radix Angelicae Sinensis 1-6 part in addition in the above-mentioned compound recipe.

In the compound recipe that Radix Hedysari, Herba Epimedii, Pheretima and Radix Angelicae Sinensis are arranged, can also add Rhizoma Chuanxiong 1-6 part and/or Radix Paeoniae Rubra 1-6 part again;

Perhaps, have in the compound recipe of Radix Hedysari, Herba Epimedii, Pheretima and Radix Angelicae Sinensis, add Flos Carthami 1-4 part and/or Semen Persicae 1-4 part again.

Can also be on the basis of Radix Hedysari, Herba Epimedii, three kinds of fundamental components of Pheretima, without Radix Angelicae Sinensis, other adds in the following medicine one or more: Rhizoma Chuanxiong 1-6 part, Flos Carthami 1-4 part, Semen Persicae 1-4 part, Radix Paeoniae Rubra 1-6 part.

Perhaps, only use the extract of Radix Hedysari Herba indigoferae Pseudotinctoriae.

Medicine of the present invention can adopt the conventional method of Chinese medicine preparation to be prepared into the preparation of any routine, for example material medicine is carried out decocting, or extracts with alcohol or alcohol-water mixture, condensed water decocting liquid or extracting solution then, the refining Chinese medicine extract that becomes.With extract and the acceptable various carriers of medicine, adjuvant compatibility, the pharmaceutical methods with those skilled in the art's routine is prepared into various preparations.

The dosage form of medicine of the present invention can be various oral agents, as traditional water decoction, concentrated oral liquid, powder, granule, capsule, tablet, electuary etc., also can make injection, as is used to damage local injection or lumbar injection etc.

From theory of Chinese medical science, Radix Hedysari has the merit of benefiting QI for activating blood circulation, promoting pus discharge and tissue regeneration strengthening; From the doctor trained in Western medicine theory, Radix Hedysari contains Hedysamn polysaccharide, can significantly promote macrophage activity, improves immunologic function, obviously reduces the content of plasma lipid peroxide, promotes Archon albumen ratio, promotes RNA synthetic, and in addition, what Radix Hedysari still can improve lung takes the photograph the oxygen function.The inventor has carried out cell culture experiments in vitro to the extract of single medicine Radix Hedysari, confirms that it has growth promoting function preferably to Schwann cell.Only illustrate with the single simply medicine of Radix Hedysari reparation and regeneration behind the peripheral nerve injury are also arranged.

In several medicines in addition of the present invention's prescription, Chinese angelica blood supplementing is invigorated blood circulation, Radix Paeoniae Rubra heat clearing away blood stasis dispelling, Pheretima collateral dredging, Rhizoma Chuanxiong blood-activating and qi-promoting, Flos Carthami, Semen Persicae blood circulation promoting and blood stasis dispelling, Herba Epimedii kidney invigorating and YANG supporting.Integrate, this compound preparation has QI invigorating, invigorates blood circulation, the effect of collateral dredging, the kidney invigorating.Radix Paeoniae Rubra has anticoagulant, activates fibrinolytic, antithrombotic effect; One of the effective ingredient of Radix Angelicae Sinensis ferulic acid can promote phagocytic function, suppresses thrombosis, and its another effective ingredient immunocompetence polysaccharide can enhancing body hematopoietic function, significantly improves the activity of mononuclear phagocyte; Pheretima extract blood pressure lowering, Pheretima also contain thrombokatalysin, and directly catalysis fibrinolytic protein enzyme suppresses thrombosis; Rhizoma Chuanxiong also contains ferulic acid, has microcirculation improvement, and blood vessel, the function of anticoagulant are expanded in blood pressure lowering; Semen Persicae and Flos Carthami energy blood vessel dilating, and can suppress thrombosis; Herba Epimedii energy blood vessel dilating, anticoagulant promotes nucleic acid, protein anabolism, improves the sodium pump activity, improves energy metabolism.

In this prescription was formed, Radix Hedysari had similar effect to Flos Carthami, Semen Persicae, Radix Paeoniae Rubra, Radix Angelicae Sinensis, Rhizoma Chuanxiong, all enriches blood and invigorates blood circulation, the stasis-dispelling and pain-killing effect.Therefore, the present invention is the various combination of main several herbal medicines with the Radix Hedysari, as the prescription of Radix Hedysari, Herba Epimedii, Pheretima three flavor medicines etc., local vascular thrombosis after all can damaging by expansion injured nerve periphery and inner blood vessel, inhibition is improved the microcirculation of injured nerve, improve arteriolar blood flow, increase injured nerve blood and supply; Simultaneously can raise immunity, improve macrophage activity, accelerate the removing of the degeneration necrosis material of injured nerve; Be beneficial to injured nerve regeneration.

The present invention adopts sciatic nerve function index, nerve conduction velocity, myelinated nerve fiber to count as the pharmacodynamic experiment observation index, the sensation conduction velocity in the nerve conduction velocity concentrated expression nerve trunk and the compound action potential of motor conduction velocity, when detecting the nerve trunk traumatic injury, simple and fast is feasible.Myelinated nerve fiber counting can reflect regenerated myelinated nerve fiber number after the nerve injury intuitively, reflects the neuranagenesis situation exactly.The sciatic nerve function index is to detect the index that gross function recovers after the Rats'Sciatic Nerve Injury, and it can reflect the degree of neurological functional recovery after the injury of sciatic nerve, compares preceding two indexs, and it has more clinical meaning.These three indexs are adopted in this experiment, from the microcosmic to the macroscopic view, reflected more all sidedly and respectively organized rat sciatic nerve regeneration situation and functional rehabilitation degree thereof, and the tool comparability, difference and size thereof between treatment group and treatment group, treatment group and the matched group result can be described more exactly.Experimental result shows that every index of medicine of the present invention all is better than the blank group.And when 2 weeks, 4 weeks and 6 weeks, the degeneration myelin all obviously is less than the blank group behind neural clamp for tissues observed section under the light microscopic, medication group rat.

The present invention has also carried out other pharmacological evaluation to above-described various formula combination, comprising: cell toxicity test, carry out the Chinese medicine Its Mechanisms, induce people and animal bone marrow hemopoietic tissue stem cell directional to be divided into nervous tissue's cell in conjunction with the neuranagenesis chamber.Optimize the marine organisms sleeve pipe of having developed, make it to have the compatibility of good nervous tissue, the suitable degraded and absorbed cycle.Nervous tissue's cell of directional induction differentiation, the active princlple and the marine organisms sleeve pipe of Chinese medicine compound of the present invention are built into the neuranagenesis chamber jointly.Verify that by zoopery it is to the Repair of Peripheral Nerve Injury effect.

More than experiment removes and to comprise various prescription group of the present invention, single Radix Hedysari extract group, also is provided with positive drug contrast NGF group in addition, and without the blank group of medicine.

Pharmacological research is found:

1. the recovery of the electrophysiological function of topical administration Chinese medicine compound of the present invention and NGF is all significantly better than matched group, and the exponential recovery of SFI motor function aspect, Chinese medicine compound group of the present invention has shown the effect that is better than the NGF group, illustrates that this group compound preparation promotes the effect of peripheral nerve regeneration.

2. traditional Chinese medicine compound extract whole body of the present invention administration has protective effect to spinal neuron.

3. Chinese medicine compound of the present invention has the propagation that promotes Schwann cell, the function of differentiation, and the cell of proof differentiation is a new outgrowth cell behind the drug effect.

Therefore, can think that traditional Chinese medicine compound extract of the present invention plays a part sure to perineural reparation.Its mechanism of action may be for after the nerve injury around, give full play to its expand neural periphery and internal blood vessel, improve injured nerve microcirculation, improve arteriolar blood flow, and energy raise immunity, improve the activity of macrophage, promote nucleic acid, protein anabolism, accelerate the removing of injured nerve degeneration necrosis material, increase supply the neuranagenesis material, promote the recovery of axoplasmic flow, and then prevent neuronic degeneration necrosis and promote its early recovery.

Because Hedysamn polysaccharide is the Main Ingredients and Appearance of Radix Hedysari, the present invention adopts improved phenol-concentrated sulphuric acid method to measure the content of Hedysamn polysaccharide in the drug extract of the present invention.

The specific embodiment

The proportioning of embodiment more than 1 group compound recipes

Use the Radix Hedysari single medicinal material, or with Radix Hedysari, Herba Epimedii, Pheretima as the monarch drug in the compound recipe, other adds in Radix Angelicae Sinensis, Rhizoma Chuanxiong, Flos Carthami, Semen Persicae, the Radix Paeoniae Rubra one or more, prescription sees Table 1.

Table 1 is respectively organized compound recipe crude drug proportioning components

Prescription	Crude drug title and consumption (weight portion)
									Radix Hedysari	Herba Epimedii	Pheretima	Radix Angelicae Sinensis	Semen Persicae	Radix Paeoniae Rubra	Rhizoma Chuanxiong	Flos Carthami
	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 decoctings extract

Equipment: Rotary Evaporators (containing vacuum pump), electric heating decoction vessel, condensing tube, glass apparatus such as eggplant-shape bottle.

Radix Hedysari is available from Gansu, original producton location, and all the other medical materials are purchased in Beijing pharmacy of Tongrentang.

Step:

1. take by weighing every kind of medicine by prescription, wherein, Radix Hedysari is cut the segment of hand hay cutter into about 2cm, roll the back weighing.

2. each prescription group medicine is placed in the round-bottomed flask, every group of medicine fried in shallow oil three times, each is with 10 times of dose, and 8 times, 6 times of water add, first pass decocted two hours, respectively fried in shallow oil one hour for twice the back, and every time fried liquid is filtered in the container, and fried liquid is dewatered by revolving the steaming instrument, be the liquid that contains crude drug amount 2g/ml with the distillation aqueous fusion then, go to freezing preservation in the aseptic bottle.

The ethanol extraction of embodiment 3 crude drug

Each medical material with 75% ethanol extraction three times, is reclaimed alcohol by revolving to steam then, and gained liquid concentrates based on polysaccharide.

Embodiment 4 Chinese medicine compound are extracted cell toxicity test

With the traditional Chinese medicine compound extract of the present invention 0.22 μ m strainer filtering of purifying, cultivate (Gibco BRL, the U.S.) doubling dilution with the PRMI1640 that contains 15% hyclone, split in 24 orifice plates.The SP2/0 cell of exponential phase, adjusting cell concentration is 10 ⁶/ ml adds 10 μ l respectively, 37 ℃ of cultivations in 24 orifice plates of the Radix Hedysari culture fluid that contains doubling dilution.Every day the observation of cell upgrowth situation.

Observe that the result shows the compound recipe Radix Hedysari extract at 1: 16000 after 3 days, 1: 32000 o'clock (being that NGF acts on dosage effectively) all has the effect that promotes hypertrophy and differentiation to Schwann cell.Explanation can promote the hypertrophy of Schwann cell.Drug safety.

Embodiment 5 respectively organizes drug extract and NGF cultivates contrast to the SD rat sciatic nerve

Each assembly side extracting solution NGF was by dilution in 1: 16000, and as positive reference, normal culture fluid is made negative control.Put into the fresh SD rat sciatic nerve that cuts respectively.37 ℃ of cultivations.3 in parallel sample.Take out sciatic nerve respectively at cultivating back 48h, 72h and 96h ,-20 ℃ of refrigerators are preserved.Tyrosine protein kinase test macro: U.S. Gibco company product (NO:13154-018).Contain substrate solution and do not contain the contrast liquid of RR-SRC.With preceding by 100uCi/ml add [γ- ³²P] ATP (＞5000Ci/mmol) (Beijing inferior brightness biomedical engineering company), press the reagent system recommended formula and prepare extract.Sample preparation reference reagent box explanation before detecting, every nerve adds extract 200ul, and 20min is placed in the homogenate of glass homogenate product on ice, the centrifugal 2min of 12000g, the Coomassie brilliant blue method is surveyed protein concentration ^[7]Press the protein concentration sampling, be diluted to 0.5mg/ml with extract.Add substrate solution or contrast liquid 10ul respectively, hatch the centrifugal 10min of 12000g (4 ℃) behind the 10min on ice.Draw reactant liquor supernatant 20ul, drip on the cellulose phosphate paper of labelling, 1% (v/v) acetic acid and tap water respectively wash twice, at every turn 5min.Add liquid and dodge detection in the cup.Pattern detection adopts Wallac 1410 liquid scintillation counters (Finland) counting, every sample counting 1min.

Interpretation of result: the cpm of 1. every pmol phosphorus presses formula and calculates: 10ul contains ³²Cpm value/1200 of P substrate (a) and contrast (b); 2. the peptide pmol number that participates in phosphorus is pressed formula calculating: sample cpm value * 2/ 1. _a-contrast cpm value * 2/ 1. _b3. kinase activity is pressed formula calculating: 2./30.The sample result of variable concentrations and different time is carried out statistical procedures with the rank test of multisample comparison.The difference of average between each prescription agent of variance analysis 48h, NGF and contrast.

Pharmacodynamic experiment in embodiment 6 animal bodies

Model and grouping: get the SD male rat and be divided into two groups at random, one group is caused the bilateral injury of sciatic nerve with the clamp method, be divided into take medicine group and matched group at random, prescription is low preferably with the curative effect behind the in-vitro screening, in, Senior Three kind dosage is selected Mus at random and is gavaged, matched group adopts normal saline, another group is cut off the back broken ends of fractured bone with rat bilateral sciatic nerve and is left and taken two millimeters gaps, adopt us homemade marine organisms casing pipe sleeve seam to close, medication group topical administration prescription, matched group topical application NGF+ ganglioside.

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

Early stage observation item:

1.SC the increment form, quantity

2. the regeneration sprouting sends the time, traveling

3. fiber is by the telescopic time

4. the myelin formation time and the number of plies (light microscopic and Electronic Speculum)

5. respectively organize nerve injury place macrophage and assemble the variation of quantity

6. the variation (DIC) of submicroscopic structure in the cell.

Late period observation item:

1. observe nerve fiber regeneration quantity

2. nerve conduction velocity and active electrical potential

3. measure muscular tension and the dried wet weight of muscle.

Histological stain method: Gu blue myelin staining and HE dye; The dyeing of SY-38 vegetative cone; The dyeing of S-100 Schwann cell; The SMI-31 axon dyes.

Embodiment 7 compound medicine Its Mechanisms

In carrying out the rat body in the experimentation, respectively before part or whole body administration and after administration a period of time, draw materials or get blood, detect the variation of macrophage quantity and function, the antigenic variation of MHC in the body detects the influence of Chinese medicine Radix Hedysari to the rat immunity state.

The observation experiment of 8 couples of drug-induced people of embodiment and Mus hemopoietic tissue stem cell neurad histiocyte directed differentiation

1. experiment purpose

Study the influence of compound recipe of the present invention, for the basis is done in the research of carrying out artificial neuron to the neural stem cell growth.

2. method

Separate and the purification of hematopoietic tissue stem cell with distinct methods: the hemopoietic tissue stem cell and the marrow stromal cell of distinct methods sorting are carried out the directional induction differentiation: under short nerve growth factor (EGF, FGF-2, PDGF) effect, above cell induction is divided into nervous tissue's cell; Utilize monoclonal antibody to identify, used antibody comprises: anti-neuroepithelial stem cell albumen (nestin) is differentiated neural stem cell, and anti-neuronspecific enolase is differentiated neuronal cell, and anti-S100 differentiates Schwann cell; The nervous tissue's cell that induces is carried out neuropotential to be measured.

3. result

Prove that this compound recipe can effectively promote proliferation of neural stem cells and differentiation, will help the research and the application of artificial neuron.

The active ingredient of embodiment 9 Chinese medicine compound and marine organisms sleeve pipe are built into the research of neuranagenesis chamber jointly

1. artificial marine organisms pipe bridge preparation and performance optimization:

The biological property of material (reaching histocompatibility when absorbing in the animal body mutually) research; The material and the Schwann cell compatibility and be suppressed to the fibroblast growth The Characteristic Study: specific as follows, aseptic operation takes out sciatic nerve, shred, carrying out external Schwann cell with culture fluid in culture dish cultivates, pad is freeed the thing material membrane in the culture dish, and contrast is set, divide different times to observe Schwann cell and fibroblastic form, activity and density with means such as inverted microscope, light microscopic section, histology and immunohistochemical staining, scanning electron microscopies, carry out the unit are counting, and observe the internal structure and the functional activity of Schwann cell.

2. will induce the nervous tissue's cell suspension that differentiates or stem cell and Chinese medicine compound effective ingredient of the present invention to be seeded on the marine organism material support that this problem develops, then the two together being added changes in the wall type bioreactor, make that cell and material are uniform to be combined, make up the neuranagenesis chamber, carry out the rat sciatic nerve repairing test.

1. laboratory animal is divided into two groups greatly at random, and first group is further divided into A group: get the capable adventitia suture in situ of rat left side lower limb sciatic nerve; B group: get the right capable sleeve pipe of the lower limb sciatic nerve little gap original position socket of rat.Second group is further divided into the C group: get the capable adventitia Rotate 180 of rat left side lower limb sciatic nerve degree and sew up; D group: get the right little gap of the capable sleeve pipe of the lower limb sciatic nerve distally broken ends of fractured bone Rotate 180 degree socket of rat.Other gets a group rat as normal neural contrast.2. experimental technique: the SD rat with 2.5% pentobarbital sodium (50mg/kg) intraperitoneal injection of anesthesia after, enter by the vastus lateralis gap and to expose the bilateral sciatic nerve, the sharp property in 1.0cm place cut-out sciatic nerve carries out respective handling according to each group then below ischial tuberosity.3. test each treated animal bone marrow aspiration in advance in advance, separate marrow stromal cell and hemopoietic tissue stem cell and train into nervous tissue's cell for a certain area, nervous tissue's cell of experimental group individuality is used for biological tissue's pipe bridge preparation, and all the other are respectively organized nervous tissue's cell and are used for the external activity laboratory observation; 4. distinguish first, second, third and fourth week after surgery, get rat at random, the back row operating microscope observation down of drawing materials, myelinated nerve fiber counting under the light microscopic from first group and second group at every turn, electrophysiological examination and functional check (sciatic nerve function index) are observed by osmic acid dyeing Histological section.

The experimental result of above embodiment 1～9 shows:

1. the recovery of the electrophysiological function of topical administration Chinese medicine compound of the present invention and NGF is all significantly better than matched group, and the exponential recovery of SFI motor function aspect, Chinese medicine compound group of the present invention has shown the effect that is better than the NGF group, has proved absolutely that this group compound preparation promotes the effect of peripheral nerve regeneration.

2. traditional Chinese medicine compound extract whole body of the present invention administration has protective effect to spinal neuron.Confirm aspect the said preparation Its Mechanisms: it has the propagation of the Schwann cell that plays a significant role in the promotion neuranagenesis process, the function of differentiation.The cell of proof differentiation simultaneously is a new outgrowth cell behind the drug effect.Therefore, can think that traditional Chinese medicine compound extract of the present invention plays a part sure to perineural reparation.Its mechanism of action may be for after the nerve injury around, give full play to its expand neural periphery and internal blood vessel, improve injured nerve microcirculation, improve arteriolar blood flow, and energy raise immunity, improve the activity of macrophage, promote nucleic acid, protein anabolism, accelerate the removing of injured nerve degeneration necrosis material, increase supply the neuranagenesis material, promote the recovery of axoplasmic flow, and then prevent neuronic degeneration necrosis and promote its early recovery.

Embodiment 10 traditional Chinese medicine compound extract of the present invention are observed (one) to the peripheral nerve regeneration facilitation

This experiment makes the model of peripheral nerve injury with the sciatic method of clamp rat, with traditional Chinese medicine compound extract of the present invention in contrast as medicine and nerve growth factor and blank group, by to sciatic nerve function index (SFI), nerve conduction velocity, whether traditional Chinese medicine compound extract of the present invention is verified in the measurement of regeneration myelin counting has facilitation to peripheral nerve regeneration.

1. experimental technique

Laboratory animal and grouping: the SD male rat of 40 adult healthy is adopted in experiment, is provided by zoopery portion of Beijing Medical University, about body weight 250g, is divided into 4 groups at random, every group of 10 rats.Each group # is N, Q, S, B.

2. experiment medicine:

1) contains 50 parts of Radix Hedysari, 10 parts of Radix Angelicae Sinensis, 8 parts of Radix Paeoniae Rubra, 5 parts of Pheretimas, 5 parts of Rhizoma Chuanxiongs, 5 parts on Flos Carthami, 5 parts in Semen Persicae, 15 parts of Herba Epimedii, simmer down to 1.125g/ml.

2) contain Radix Hedysari, Herba Epimedii, Pheretima, simmer down to 0.774g/ml.

3) BUYANG HUANWU TANG composition: contain the Radix Astragali, Radix Angelicae Sinensis, Radix Paeoniae Rubra, Rhizoma Chuanxiong, Semen Persicae, Flos Carthami, Pheretima, simmer down to 2.6g/ml.

3. experimental technique and observation index:

The SD rat with 2.5% pentobarbital sodium (50mg/kg) intraperitoneal injection of anesthesia after, enter exposure bilateral sciatic nerve by the vastus lateralis gap, 1.0cm sentences smooth forceps clamp sciatic nerve (30 seconds kinds of 3 teeth) and causes wide 2mm damage zone below ischial tuberosity, this kind method confirms to make under the complete situation of epineurium the axon complete rupture through the histology.Close otch then.Q group: test medicine 1) extracting solution perfusion dosage is 2ml//day, the S group: perfusion dosage 2ml//sky experiment medicine 2); B group: BUYANG HUANWU TANG experiment medicine 3), perfusion dosage 2ml//day; N group: normal saline perfusion dosage 2ml//day; Once a day.3 of anesthetic deaths (1 of S group, 2 of Q groups) in the art.In every group, 5 (perfusion is for once a day when 2 weeks, totally 14 days), 5 (perfusion every day once when 4 weeks, totally 28 days), take out the capable histological observation of bilateral sciatic nerve, measure with the sciatic nerve function index that moves ahead of drawing materials in 2,4 weeks before every group of rat art, every group of 2 week of rat with drew materials in 4 weeks before get the capable nerve conduction velocity of bilateral sciatic nerve and measure.

Observation index: sciatic nerve function index (SFI), nerve conduction velocity, tectology and myelinated nerve fiber counting.

SFI measures: self-control rat footmark walking case, and high 15cm, wide 15cm, long 50cm, the passage far-end is placed a long 20cm, wide 15cm, the one-sided Mus case that opens the door of high 15cm.Place at the bottom of the walking case and isometric, the wide blank sheet of paper of walking case.In culture dish, put into a little Cotton Gossypii, pour an amount of burnt black ink into Cotton Gossypii is soaked, rat hindleg is dipped in culture dish, put into an end of walking case, it moves towards the other end of case voluntarily, in this process, stays behind the rat bilateral each 4-5 of footmark on the mark sheet.Each stays the back footmark before organizing 10 rat Clampings on mark sheet, stay the back footmark behind the Clamping 2,4 weeks again on mark sheet, all gets the bilateral footmark, and the former is normal group (N), and the latter is experimental group (E).Each measures 3 variablees, measures to be accurate to millimeter.

Footmark length (print length factor, PLF): the longest distance of footmark, promptly from the heel to the toe, select the longest PLF value for use at every turn.

The foot apart from width (toe spread factor, TSF): 1-5 toe line distance, select the longest TSF value for use at every turn.

Middle toes distance (intermediary toe spread, ITF): 2-4 toe line distance, select the longest TSF value for use at every turn.

Above-mentioned 3 variable substitution Bain formula just can be calculated SFI.

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

EPL: experimental group footmark length; NPL: normal group footmark length; ETS: the experimental group foot is apart from width; NTS: the normal group foot is apart from width; EIT: toes distance in the middle of the experimental group; NIT: toes distance in the middle of the normal group.

SFI is a normal value with ± 10, and-100 is neural fully from disconnected index.

Nerve conduction velocity (NCV): after row SFI detects, expose the bilateral sciatic nerve by former pathway, place electrode at about 1.5cm place, clamp injury region both sides, proximal end is a stimulating electrode, and outer circumference end is a leading electrode, measures nerve conduction velocity with the Oxford electromyograph(EMG.Formula: the difference when speed=two interelectrode distances/action potential is dived

Histological examination:

Draw materials and dye: each organizes rat respectively at 2 weeks of Clamping, and the former surgical approach in 4 week backs exposes sciatic nerves, takes out the nerve segment that clamp is sentenced 0.5cm far away, inserts 10% formalin, fix 24 hours after, the dyeing of row osmic acid.

Osmic acid dyeing:

Fixing dyeing 72 hours that reach behind 1% osmic acid

Flowing water washed after 10 minutes, distilled water immersion 10 minutes totally 3 times

Dehydration: ethanol 50% → 70% → 75% → 80% → 85% → 90% → 95% → 100% (1 time), each 15 minutes → dimethylbenzene (1 time) → dimethylbenzene (2 times) of → 100% (2 times) each 15 minutes

Waxdip: 60 minutes → 30 minutes → 30 minutes → 15 minutes

Embedding

Section: the nerves transected section of 3-5 micron

Roasting sheet: 38 ℃ 12-24 hour

Dimethylbenzene 5 minutes 2 times

Mounting

Microscopy: each specimen selects 1-2 to open section, and the painted section of osmic acid is done the research of common peroneal nerve myelinated nerve fiber counting on the basis of the dyeing of observing each group section myelin and number of modalities difference.

The myelinated nerve fiber counting: experiment each group and matched group totally 75 sections, to amplify at optical microscope under the visual field of 400 times (10*40), the myelinated nerve fiber number of each vision area of common peroneal nerve is measured in every section, gets average.The row statistical analysis.

4. experimental result

1) sciatic nerve function index (SFI): X+SD

Group	2 weeks of postoperative	4 weeks of postoperative
			The blank group	-50.3405±20.5918	-22.1106±10.3532
The S group	-32.9944±20.0971	-11.9985±5.4533
			The Q group	-27.4385±13.5411	-16.8660±8.6201
The B group	-58.3396±12.3949	-21.1303±7.0933

35 sides of 2 when week N groups wherein, Q organize 35 sides, and B organizes 23 sides and does not measure because of in various degree toes ulcer and contracture.BUYANG HUANWU TANG group and experiment medicine 2) group do not have significant difference and (is respectively P=0.086＞0.05 with matched group; P=0.530＞0.05); But BUYANG HUANWU TANG group and experiment medicine 2) there were significant differences for group, and experiment medicine 2) group is better than the BUYANG HUANWU TANG group, (P=0.01＜0.05), experiment medicine 1) group has the significance meaning with matched group than difference, and be better than matched group (P=0.046＞0.05), experiment medicine 1) group also is better than the BUYANG HUANWU TANG group, and difference has significance (P=0.002＜0.010).11 side of N group during 4 weeks, S organizes 22 sides, 12 side of B group is not measured because of in various degree toes ulcer and contracture, experiment medicine 1) group and BUYANG HUANWU TANG group and matched group difference meaningless (being respectively P=0.827＞0.05, P=0.246＞0.05), experiment medicine 2) group and matched group difference significance, and be better than matched group (P=0.048＜0.05), be better than testing medicine 1 simultaneously) group, and difference meaningful (P=0.023＜0.05), but meaningless with BUYANG HUANWU TANG group difference.

2) (m/s) X+SD of nerve conduction velocity (NCV)

Group	The postoperative time
			2 weeks	4 weeks
	The N group	17.0000±14.4855			26.2900±11.3439
The S group			20.5100±8.1175	40.7857±14.4462
	The B group	22.5700±16.2037			39.1516±10.1192
The Q group			27.7540±3.9825	40.4375±13.9800

2 when week the Q group four four sides are arranged, the B group has 22 sides, N organize 35 sides, fails to measure experiment medicine 1) group, experiment medicine 2) group, BUYANG HUANWU TANG group all are better than matched group with the matched group ratio, but difference not statistically significant.11 side of S group during 4 weeks, B organizes 11 side, 12 side of Q group fails to measure, wherein test medicine 1) group, experiment medicine 2) group, BUYANG HUANWU TANG group and matched group be than all being better than matched group, and difference has remarkable meaning and (is respectively P=0.005＜0.01, P=0.02＜0.05, P=0.003＜0.05), difference nonsignificance and between three groups of medication groups.

Histological observation

Morphological observation under the light microscopic: osmic acid stained, during 2 weeks, begun to occur the regeneration myelinated nerve fiber, thinner, wall is thin, and each group regeneration myelinated nerve fiber of medication is obviously more than matched group, and the disintegrate absorption of partial denaturation myelin, blank group still has than the polytropy myelin, and the regeneration myelinated nerve fiber is less.4 whens week, regeneration myelinated nerve fiber showed increased, each group regeneration myelinated nerve fiber of medication is obviously more than the blank group, and the degeneration myelin substantially disintegrate disappear, and blank the group still has than the polytropy myelin.

3) unit visual field myelinated nerve fiber counting (bar) X+SD

Group	2 weeks of postoperative	4 weeks of postoperative
			The N group	11.3±1.06	19.0±2.50
The S group	17.3±1.99	29.0±3.67
			The B group	17.6±2.85	23.5±2.31
The Q group	17.6±3.87	22.9±2.94

Each group of medication is all significantly better than matched group during 2 weeks, (p=0.001＜0.01), but each medication group difference is meaningless, each group of medication is all significantly better than matched group (p＜0.05) during 4 weeks, and experiment medicine 2) group is significantly better than full side's group and BUYANG HUANWU TANG group (p=0.002＜0.01, p=0.004＜0.01).And full side group is meaningless with the BUYANG HUANWU TANG group difference.

Embodiment 11 traditional Chinese medicine compound extract of the present invention are observed (two) to the peripheral nerve regeneration facilitation

1. laboratory animal and grouping: the SD male rat of 30 adult healthy is adopted in experiment, is provided by zoopery portion of Beijing Medical University, about body weight 250g, is divided into 3 groups at random, every group of 10 rats.

2. experiment medicine:

1) 60 parts of Radix Hedysari of prescription, 20 parts of Herba Epimedii, 6 parts of Pheretimas among the embodiment 1,15 parts of Radix Angelicae Sinensis, 10 parts of Radix Paeoniae Rubra, every dose of extracting solution simmer down to 0.963g/mL.(please rewrite)

2) contain 70 parts of Radix Hedysari, 25 parts of Herba Epimedii, 5 parts of Pheretimas (writing out proportion relation).Every dose of simmer down to simmer down to 0.774g/mL.

3. experimental technique and observation index:

The SD rat with 2.5% pentobarbital sodium (50mg/kg) intraperitoneal injection of anesthesia after, enter exposure bilateral sciatic nerve by the vastus lateralis gap, behind the bilateral thigh, make little otch exposure sciatic nerve and stick the 5mm place until far-end tibial nerve and common peroneal nerve branch, with head is the wide anodontia pliers clamp nerve of 2mm, clamp intensity is unified interlock 3 teeth, and the persistent period is 1 minute.Postoperative played S group and Q group on the 1st respectively by each 5ml/kg perfusion every day once, and matched group gavages normal saline 5ml/kg every day.3 groups of 6 weeks of sub-cage rearing under identical conditions of postoperative.

All (perfusion every day once, perfusion is 42 days altogether) takes out the capable histological observation of bilateral sciatic nerve in 6 whens week, measures with the sciatic nerve function index that moves ahead of drawing materials in 6 weeks before every group of rat art.

Observation index: sciatic nerve function index (SFI), tectology and myelinated nerve fiber counting.

General state: comprise appetite, the mental status, aggressivity, local infection rate and mortality rate.

SFI measures: self-control rat footmark walking case, and high 15cm, wide 15cm, long 50cm, the passage far-end is placed a long 20cm, wide 15cm, the one-sided Mus case that opens the door of high 15cm.Place at the bottom of the walking case and isometric, the wide blank sheet of paper of walking case.In culture dish, put into a little Cotton Gossypii, pour an amount of burnt black ink into Cotton Gossypii is soaked, rat hindleg is dipped in culture dish, put into an end of walking case, it moves towards the other end of case voluntarily, in this process, stays behind the rat bilateral each 4-5 of footmark on the mark sheet.Each stays the back footmark before organizing 10 rat Clampings on mark sheet, stay the back footmark behind the Clamping 6 weeks again on mark sheet, all gets the bilateral footmark, and the former is normal group (N), and the latter is experimental group (E).Each measures 3 variablees, measures to be accurate to millimeter.

Footmark length (print length factor, PLF): the longest distance of footmark, promptly from the heel to the toe, select the longest PLF value for use at every turn.

The foot apart from width (toe spread factor, TSF): 1-5 toe line distance, select the longest TSF value for use at every turn.

Middle toes distance (intermediary toe spread, ITF): 2-4 toe line distance, select the longest TSF value for use at every turn.Above-mentioned 3 variable substitution Bain formula just can be calculated SFI.

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

EPL: experimental group footmark length; NPL: normal group footmark length; ETS: the experimental group foot is apart from width; NTS: the normal group foot is apart from width; EIT: toes distance in the middle of the experimental group; NIT: toes distance in the middle of the normal group.

SFI is a normal value with ± 10, and-100 is neural fully from disconnected index.

Histological examination:

Draw materials and dye: each is organized rat and exposes sciatic nerve respectively at the former surgical approach in Clamping 6 week back, takes out the nerve segment that clamp is sentenced 0.5cm far away, inserts 10% formalin, fix 24 hours after, the dyeing of row osmic acid.

Osmic acid dyeing:

Fixing dyeing 72 hours that reach behind 1% osmic acid

Flowing water washed after 10 minutes, distilled water immersion 10 minutes totally 3 times

Dehydration: ethanol 50% → 70% → 75% → 80% → 85% → 90% → 95% → 100% (1 time), each 15 minutes → dimethylbenzene (1 time) → dimethylbenzene (2 times) of → 100% (2 times) each 15 minutes

Waxdip: 60 minutes → 30 minutes → 30 minutes → 15 minutes

Embedding

Section: the nerves transected section of 3-5 micron

Roasting sheet: 38 ℃ 12-24 hour

Dimethylbenzene 5 minutes 2 times

Mounting

Microscopy: each specimen selects 1-2 to open section, and the painted section of osmic acid is done the research of common peroneal nerve myelinated nerve fiber counting on the basis of the dyeing of observing each group section myelin and number of modalities difference.

The myelinated nerve fiber counting: experiment each group and matched group totally 75 sections, amplify at optical microscope under the visual field of 400 times (10*40), as shown in the figure, the myelinated nerve fiber number of each vision area of common peroneal nerve is measured in every section, gets average.The row statistical analysis.

4. experimental result

1) general state is except that outside 6 of the anesthetic deaths in the art (3 of Q groups, 2 of S groups, 1 of N group), and postoperative is respectively organized the equal Non Apparent Abnormality behavior of rat.Postoperative 24h plays sleep and appetite does not have significant change, does not have 1 example and wound infection or death occur.The bilateral sciatic nerve surface of administration group rat is generally smooth than matched group, separates from tissue easily and appears the adventitia rich blood vessel.

2) sciatic nerve function index (SFI) :+SD

Group	The postoperative time (6 week)
		N	-25.1729±15.3830
Q	-30.2700±10.8001
		S	-19.1105±9.1048

The result shows experiment medicine 1) group and experiment medicine 2) group do not have significant difference and (is respectively P=0.262,＞0.05 with matched group; P=0.479＞0.05).And experiment medicine 1) group and experiment medicine 2) group has the significance meaning than difference, and experiment medicine 2) group is better than testing medicine 1) group (P=0.03,＜0.05).

3) histological observation

Morphological observation under the light microscopic:

The osmic acid stained, during 6 weeks, regeneration myelinated nerve fiber showed increased, Chinese drug-treated group and NGF group regeneration myelinated nerve fiber are organized more than blank, and Chinese drug-treated group degeneration myelin disintegrate substantially disappears, and blank group still has than the polytropy myelin.Prompting, traditional Chinese medicine compound extract of the present invention can promote the regeneration of myelinated nerve fiber and the decomposition of degeneration myelin to absorb.

4) unit visual field myelinated nerve fiber counting (bar)+SD

Group	The postoperative time (6 week)
		N	75.77±14.36
Q	112.33±17.99
		S	109.26±15.73

The average unit of Chinese medicine compound group of the present invention visual field myelinated nerve fiber number is 112.33 ± 17.99 to be better than matched group 75.77 ± 14.36 and statistics has notable difference (P=0.000,＜0.01).Experiment medicine 2) group 109.26 ± 15.73 obviously is better than matched group (P=000,＜0.01), and experiment medicine 1) group and experiment medicine 2) organize between there was no significant difference.

Embodiment 10～11 results:

(1) sciatic nerve function index (SFI): during 2 weeks, the BUYANG HUANWU TANG group and the experiment medicine 2) group with matched group do not have significant difference, but BUYANG HUANWU TANG group and experiment medicine 2) there were significant differences for group, and experiment medicine 2) group is better than the BUYANG HUANWU TANG group, experiment medicine 1) group has the significance meaning with matched group than difference, and being better than matched group, experiment medicine 1) group also is better than the BUYANG HUANWU TANG group, and difference has significance.Experiment medicine 1 during 4 weeks) group and BUYANG HUANWU TANG group and matched group difference are meaningless, experiment medicine 2) group and matched group difference significance, and be better than matched group, be better than testing medicine 1 simultaneously) group, and difference is meaningful, but meaningless with BUYANG HUANWU TANG group difference.6 when week experiment medicines 1) group and experiment medicine 2) group do not have significant difference with matched group; And experiment medicine 1) group and experiment medicine 2) group has the significance meaning than difference, and experiment medicine 2) group is better than testing medicine 1) group.

(2) nerve conduction velocity (NCV): 2 when week experiment medicines 1) group, experiment medicine 2) group, BUYANG HUANWU TANG group and matched group be than all being better than matched group, but the difference not statistically significant.4 weeks the time were wherein tested medicine 1) group, experiment medicine 2) group, BUYANG HUANWU TANG group and matched group be than all being better than matched group, and difference has remarkable meaning, and difference nonsignificance between three groups of medication groups.

(3) histological observation: each group of medication is all significantly better than matched group during 2 weeks, but each medication group difference is meaningless, medication is respectively organized all significantly better than matched group during 4 weeks, and experiment medicine 2) group is significantly better than full side's group and BUYANG HUANWU TANG group, and square full group is meaningless with the BUYANG HUANWU TANG group difference.In 6 whens week,, Chinese medicine compound group of the present invention was better than matched group and statistics has notable difference.Experiment medicine 2) group obviously is better than matched group and tests medicine 1) group and experiment medicine 2) organize between there was no significant difference.

Hedysamn polysaccharide assay in embodiment 12 traditional Chinese medicine compound extract of the present invention

Can effectively promote on the regenerated basis of rat sciatic nerve injury at the extract recipe that contains 70 parts of Radix Hedysari, 25 parts of Herba Epimedii, 5 parts of Pheretimas, adopt phenol-concentrated sulphuric acid method to make assay main matter composition Hedysamn polysaccharide among this side.

Material and method

1.1 material: phenol (analytical pure) is made into the 50g/l aqueous solution after heavily steaming; Concentrated sulphuric acid (analytical pure, d=1.84g/cm ^-3); Absorbance uses optical path 10mm quartz colorimetric utensil with 721 type spectrophotometric determinations, inhales with the Optifix quantitative charger to add 50g/l phenol and concentrated sulphuric acid.The pure product of glucose.The Chinese medicine compound of the present invention of having purified the side's of subtracting extracting solution (concentration 86mg/ml)

1.2 method: the preparation of standard solution: accurately take by weighing the pure product 10mg of exsiccant glucose, be dissolved in the 100ml water, dilution is 2,4,8,10 for concentration respectively then, 20ug/ml, and extracting solution dilution in the Chinese medicine compound of the present invention side of subtracting is 4.3ug/ml..Get earlier and diluted each each 0.4ml of standard glucose solution, place the 10ml test tube, after adding 50g/l phenol solution 0.8ml mixes, add the 4ml concentrated sulphuric acid rapidly, behind the mix homogeneously, room temperature is placed 30min, measure absorbance at wavelength 490nm, blank replaces sugar juice with distilled water.After waiting to measure standard curve, get the Chinese medicine compound of the present invention side of the subtracting extracting solution 0.4ml that has diluted, place the 10ml test tube, after adding 50g/l phenol solution 0.8ml mixing, add the 4ml concentrated sulphuric acid rapidly, behind the mix homogeneously, room temperature is placed 30min, measure absorbance at wavelength 490nm, this absorbance substitution standard curve equation is obtained the Hedysamn polysaccharide amount that contains, thereby draw Hedysamn polysaccharide content in the prescription.

2. result and discussion

2.1 obtain standard curve by the sample light absorption value shown in the following table, trying to achieve linear equation is Y=14.73X-0.0343Chi-Square:0.00091

Wherein Y is institute's sugar content, and X is a 490nm place absorbance

It is 0.076 that the Chinese medicine compound of the present invention side of the subtracting extracting solution of dilution records absorbance, and it is 1.0908ug that the substitution equation is tried to achieve institute's sugar content, and diluent contains prescription 1.72ug, and contained Hedysamn polysaccharide accounts for 63.41% of total amount in the prescription thereby try to achieve.

Sample number	Contained standard glucose amount (ug)	The 490nm light absorption value
			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 side's of subtracting prescription,, promptly be Hedysamn polysaccharide so the sugar that records can be thought because Herba Epimedii and Pheretima contain saccharide hardly in the side.Record the result and be 63.41% of prescription total amount

The pharmacodynamic experiment of embodiment 13 single medicine Radix Hedysari

1. experiment purpose: observe Radix Hedysari list medicine to promoting the regeneration behind the peripheral nerve injury

2. experimental technique

Radix Hedysari list medicament extracting solution and NGF were by dilution in 1: 16000, and as positive reference, normal culture fluid is made negative control.Put into the fresh SD rat sciatic nerve that cuts respectively.37 ℃ of cultivations.3 in parallel sample.Take out sciatic nerve respectively at cultivating back 48h, 72h and 96h ,-20 ℃ of refrigerators are preserved.Tyrosine protein kinase test macro: U.S. Gibco company product (NO:13154-018).Contain substrate solution and do not contain the contrast liquid of RR-SRC.With preceding by 100uCi/ml add [γ- ³²P] ATP (＞5000Ci/mmol) (Beijing inferior brightness biomedical engineering company), press the reagent system recommended formula and prepare extract.Sample preparation reference reagent box explanation before detecting, every nerve adds extract 200ul, and 20min is placed in the homogenate of glass homogenate product on ice, the centrifugal 2min of 12000g, the Coomassie brilliant blue method is surveyed protein concentration ^[7]Press the protein concentration sampling, be diluted to 0.5mg/ml with extract.Add substrate solution or contrast liquid 10ul respectively, hatch the centrifugal 10min of 12000g (4 ℃) behind the 10min on ice.Draw reactant liquor supernatant 20ul, drip on the cellulose phosphate paper of labelling, 1% (v/v) acetic acid and tap water respectively wash twice, at every turn 5min.Add liquid and dodge detection in the cup.Pattern detection adopts Wallac 1410 liquid scintillation counters (Finland) counting, every sample counting 1min.

3. interpretation of result:

The cpm of 1. every pmol phosphorus presses formula and calculates: 10ul contains ³²Cpm value/1200 of P substrate (a) and contrast (b); 2. the peptide pmol number that participates in phosphorus is pressed formula calculating: sample cpm value * 2/ 1. _a-contrast cpm value * 2/ 1. _b3. kinase activity is pressed formula calculating: 2./30.The sample result of variable concentrations and different time is carried out statistical procedures with the rank test of multisample comparison.The difference of average between variance analysis 48h Radix Hedysari list medicine, NGF and contrast.

4. conclusion: Radix Hedysari list medicine has the effect that promotes regulation of Schwann cell proliferation and differentiation, and effect is similar to NGF.Regenerated effect after the single Radix Hedysari also has the promotion peripheral nerve injury is described.

The preparation of embodiment 14 injections

Preparation injection: the water decoction of prescription is steamed the instrument purification with revolving earlier, the moisture that will contain in the vacuum machine then proposes, be dried by water-bath again and be powdery, by the accurate weighing of institute's expense, the rat local injection is used during use with water for injection dilution back.

The preparation of embodiment 15 oral agents

The water decoction of prescription is steamed the instrument purification with revolving earlier, and simmer down to institute expense gavages experiment to rat.

Claims (9)

1. a medicine that promotes that peripheral nerve is repaired is made raw materials of effective components and is consisted of: Radix Hedysari 6-16 weight portion, Herba Epimedii 4-10 weight portion, Pheretima 2-6 weight portion.

2. the medicine repaired of the described promotion peripheral nerve of claim 1, described raw material consists of: Radix Hedysari 8-14 weight portion, Herba Epimedii 5-9 weight portion, Pheretima 3-5 weight portion.

3. the medicine repaired of the described promotion peripheral nerve of claim 1, described raw material consists of: Radix Hedysari 9-13 weight portion, Herba Epimedii 6-8 weight portion, Pheretima 3-4 weight portion.

4. the medicine repaired of the described promotion peripheral nerve of claim 1, described raw material also has Radix Angelicae Sinensis 1-6 weight portion.

5. the medicine repaired of the described promotion peripheral nerve of claim 4, described raw material also has Rhizoma Chuanxiong 1-6 weight portion and/or Radix Paeoniae Rubra 1-6 weight portion.

6. the medicine repaired of the described promotion peripheral nerve of claim 4, described raw material also has Flos Carthami 1-4 weight portion and/or Semen Persicae 1-4 weight portion.

7. the single Radix Hedysari is preparing the purposes that promotes in the peripheral nerve repair medicine.

8. the medicine that the described promotion peripheral nerve of arbitrary claim is repaired among the claim 1-7, dosage form is an oral agents.

9. the medicine that the described promotion peripheral nerve of arbitrary claim is repaired among the claim 1-7, dosage form is an injection.