CN102233009A - Chinese medicinal composition for promoting nerve regeneration and preparation method and use thereof - Google Patents

Chinese medicinal composition for promoting nerve regeneration and preparation method and use thereof Download PDF

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CN102233009A
CN102233009A CN2011101673556A CN201110167355A CN102233009A CN 102233009 A CN102233009 A CN 102233009A CN 2011101673556 A CN2011101673556 A CN 2011101673556A CN 201110167355 A CN201110167355 A CN 201110167355A CN 102233009 A CN102233009 A CN 102233009A
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ethanol
ginsenoside
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贝伟剑
郭姣
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Guangdong Pharmaceutical University
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Abstract

The invention discloses a Chinese medicinal composition for promoting nerve regeneration and a preparation method and use thereof. The Chinese medicinal composition for promoting nerve regeneration consists of 2 to 10 parts of multiflower knotweed root, 1 to 10 parts of ginseng and 1 to 10 parts of ginkgo leaves; and the Chinese medicinal composition for promoting nerve regeneration also may consist of commercial multiflower knotweed root extract, ginseng extract and ginkgo leaf extract in a ratio. Besides the conventional preparation method, the invention also provides a preparation method of the Chinese medicinal composition, which comprises: subjecting the raw materials to C1 to C1 alcohol extraction respectively to obtain C1 to C3 alcohol extracts; concentrating, passing through a macroporous adsorption resin column, eluting with ethanol, collecting eluent, recovering ethanol, and drying; or recovering ethanol, concentrating to obtain thick paste, drying, grinding, obtaining the final extracts, and mixing to obtain the Chinese medicinal composition. The Chinese medicinal compostion has an obvious nerve regeneration promoting effect, and has the effects of resisting cerebral ischemia injury, protecting nerves, improving learning and memory and resisting senile dementia; the main active ingredients are definite, the quality is stable, and the dosage is small; and the Chinese medicinal composition is a high-quality natural medicine or health-care food.

Description

A kind of Chinese medicine composition that promotes neuranagenesis and its production and application
Technical field
The present invention relates to technical field of Chinese medicines, be specifically related to a kind of Chinese medicine composition that promotes neuranagenesis and its production and application.
Background technology
Diseased associated with cerebral ischemia injury comprises apoplexy, apoplexy sequela, vascular dementia.Cerebral infarction is the important killer who threatens human health, and is very high at the mid-aged population sickness rate.The whole nation estimates ischemic stroke patients is arranged approximately up to nearly 5 million peoples, and the whole world is especially up to more than 2,000 ten thousand people, and age of onset is to get over rejuvenation, many ischemic stroke patients disabilities, grievous injury social labor power and creativity.
Senile dementia is an old common disease, have vascular dementia (Vascular Dementia, VD) and alzheimer disease (Alzheimer disease, AD) etc. dissimilar.Vascular dementia is a kind of important kind of senile dementia, also is one of sequela of cerebral infarction, and quite general in the ischemic cerebrovascular patient, prevalence can reach more than 10% in crowd more than 60 years old, has a strong impact on the life of elderly person quality.Along with the aging gradually of China's population, cerebral infarction and VD patient are in continuous increase.
The medical expense that is used for cerebral infarction and vascular dementia domestic every year is up to last 20,000,000,000 yuan, and heavy burden up to tens billion of dollars, is brought to family social especially in the whole world.But up to now, cerebral infarction and VD still there are not specific treatment and prophylactic agent.Effectively prevention and treatment cardiovascular and cerebrovascular disease have become the serious and arduous problem of pendulum in face of us.
Chinese medical theory think cerebral ischemia apoplexy and senile dementia particularly the pathogenesis of vascular dementia be: one for empty, and promptly Kidney-Yin element loses, positive QI-insufficiency; Two is real, and the QI rising in reverse order blood stasis is hoodwinked brain god in last.Be old suffering from a deficiency of the kidney, blood stasis due to qi deficiency and cause the brain venation and be subjected to stagnation of blood stasis, thrombosis, brain inanition, brain decline forgetful.Control suitable QI invigorating kidney tonifying brain-strengthening, blood circulation promoting and blood stasis dispelling.
Chinese medicine has effect unique to treatment cerebral infarction and vascular dementia.But the Chinese patent medicine that has the treatment cerebral infarction disease of unique curative effect and vascular dementia at present is very few, and lacks especially through strict therapeutic evaluation, efficient, safe, quality controllable effective treatment cerebral infarction and vascular dementia modern traditional Chinese patent medicine.The big compound recipe of Chinese patent medicine ubiquity of existing control cerebral infarction disease and vascular dementia, form that flavour of a drug are many, effective ingredient is complicated, production technology is unstable, quality is restive, curative effect is difficult to assurance and waits until defective, be difficult to meet the modern Chinese medicine requirement.On the future market those complicated components not clear, can't or be difficult to carry out effective mass control, the mechanism of action is unclear, uncertain therapeutic efficacy is cut old Chinese patent medicine product will be eliminated; Single preparations of ephedrine and folk prescription effective parts formulation are though composition understands that effect is clear and definite, and is quality controllable, in the face of refractory diseases such as the cerebral ischemia diseases (apoplexy) of etiology and pathogenesis complexity and senile dementia obviously are difficult to obtain satisfactory effect.And Chinese medical theory instructs little compound drug down will be expected to solve the puzzlement that big compound Chinese patent medicine and simple prescription preparation and folk prescription effective parts formulation are faced, and the research and development prescription form simplify, effective ingredient relatively clearly, quality is easy to the direction that control and stable processing technique, safely and effectively little compound recipe modern Chinese medicine preparation or Chinese medicine active component new drug have become future development.
Open day on February 4th, 2009 of Chinese patent 200810198306.7() disclose the medicine and the health product of a kind of cerebral ischemia disease and vascular dementia, formed by plant active components such as 2 ~ 10 parts in 2 ~ 10 parts of ginsenoside Rb1s, 2 ~ 10 parts of ginsenoside Rg1s, 2 ~ 10 parts of ginsenoside Rds, 2 ~ 10 parts of ginsenoside Res, 2 ~ 10 parts of stilbene glucosides, 2 ~ 10 parts of bilobalides, nimbecetin and Quercetin mixture.It is through screening and the plant active component compatibility that gets, be used for cerebral ischemia disease and vascular dementia and obtain desirable curative effect, experimentation also shows, this side is to experimental control cerebral ischemia disease (cerebral arteriosclerosis, cerebral infarction, apoplexy sequela) and vascular dementia determined curative effect, have good anti-cerebral ischemia damnification, improve the learning and memory effect, this prescription patent is obtained the authorization.
Though this prescription has good anti-cerebral ischemia damnification, improves the effect of learning and memory, because this prescription is the pure chemistry component prescription, each composition raw material sources is subjected to certain restriction, and manufacturing cost is relatively costly, and extensive use is restricted.
Promote that neuranagenesis is a kind of new treatment trend and the development in future direction of treatment cerebral ischemia diseases, but about the also rare report of the Chinese medicine of this respect.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, provide a kind of raw material to be easy to get, preparation method is easy, the Chinese medicine composition of promotion neuranagenesis evident in efficacy, and called after crow ginseng refreshment side is called for short WSXN.
Another object of the present invention provides the preparation method of above-mentioned Chinese medicine composition.
Another purpose of the present invention provides the application of above-mentioned Chinese medicine composition.
The present invention is achieved through the following technical solutions above-mentioned purpose:
A kind of Chinese medicine composition that promotes neuranagenesis is made up of following component and parts by weight: the Radix Polygoni Multiflori 2 ~ 10, Radix Ginseng 1 ~ 10 and Folium Ginkgo 1 ~ 10.
More than promote the Chinese medicine composition of neuranagenesis also can form with commercial Radix Polygoni Multiflori extract 2 ~ 10 weight portions, Radix Ginseng extract 1 ~ 10 weight portion and Folium Ginkgo extract 1 ~ 10 weight portion.
As a kind of preferred version, this promotes the Chinese medicine composition of neuranagenesis to be made up of following component and parts by weight: the Radix Polygoni Multiflori 2 ~ 5, Radix Ginseng 1 ~ 5 and Folium Ginkgo 1 ~ 5.
Further, the Chinese medicine composition of above-mentioned promotion neuranagenesis can be through C with three kinds of material medicines 1-3Be prepared into the extract remix after the alcohol extraction and form, the concrete material medicine Radix Polygoni Multiflori 2 ~ 5 weight portions, Radix Ginseng 1 ~ 5 weight portion and Folium Ginkgo 1 ~ 5 weight portion.
The Chinese medicine composition of above-mentioned promotion neuranagenesis can also directly select for use the main effective ingredient of the extract that contains the Radix Polygoni Multiflori, Radix Ginseng and Folium Ginkgo to form, and effective ingredient and weight ratio are: stilbene glucoside: the ginsenoside Rb1: the ginsenoside Rg1: the ginsenoside Rd: the ginsenoside Re: bilobalide: nimbecetin: Quercetin equals 1 ~ 18:1 ~ 10:1 ~ 10:1 ~ 2:1 ~ 4:1 ~ 4:1 ~ 2:1 ~ 2.
The Chinese medicine composition of above-mentioned promotion neuranagenesis directly water mixing decocts to such an extent that decoction is taken, and also can use C 1-3Alcohol separately or mixed extraction, merging, concentrate, dry mixed becomes extractive composition.
Provide a kind of preferred extraction to prepare scheme below, step is as follows:
Pass through C respectively after the material medicine Radix Polygoni Multiflori, Radix Ginseng and Folium Ginkgo taken by weighing by proportioning 1-3After the alcohol extraction, obtain C 1-3Ethanol extract merges total extract, and total extract obtains concentrated solution after concentrating, and concentrated solution is added on the macroporous adsorbent resin of having handled well, uses the Different concentrations of alcohol eluting successively, collects corresponding eluent, reclaims ethanol, drying; Or recovery ethanol, be condensed into the thick paste medicine, vacuum drying, pulverize, promptly get impurity components such as having removed non-saponins in the concentrated solution, non-stilbene glycoside, non-bilobalide-like and non-flavonoid, obtain the final extract of material medicine,, promptly obtain the Chinese medicine composition of described promotion neuranagenesis the final extract mixing of three kinds of material medicines.
Described C 1-3Alcohol extraction is the C with 30 ~ 95 volume % 1-3 Alcohol extraction 1 ~ 5 time, each C that extracts 1-3The alcohol volume is 1 ~ 15 times of quality of medicinal material, and each extraction time is 5min ~ 5h; Described C 1-3Alcohol is methanol, ethanol or propanol.
The amount ratio of described macroporous adsorbent resin and concentrated solution is that the 1kg macroporous resin adds concentrated solution 1 ~ 5L.
Described ethanol elution is with 50 ~ 95 volume % ethanol, consumption be every 1kg macroporous resin with 5 ~ 20 L alcoholic solution, with 1 ~ 20 ml/min flow velocity eluting, be eluted to colourless; Described macroporous resin is polyamides amine type resin or polystyrene weak-base anion-exchange resin.
The above any one Chinese medicine composition promotes the medicine of neuranagenesis and/or the application in the health food in preparation.
The application of above-mentioned Chinese medicine composition in preparation control cerebral ischemia disease or senile dementia disease medicament and/or health food.Described cerebral ischemia disease comprises apoplexy, cerebral thrombosis, arteriosclerosis, apoplexy sequela and vascular dementia etc., and described senile dementia disease is constitutional alzheimer disease (Alzheimer disease) and/or vascular dementia.
Chinese medical theory thinks that Radix Ginseng is a rare Chinese medicine, and property is flat, sweet in the mouth, little hardship.Have that strongly invigorating primordial QI, multiple arteries and veins take off admittedly, the function of invigorating the spleen to benefit the lung, the Fructus Alpiniae Oxyphyllae of promoting the production of body fluid, calm the nerves.Be used for weak body and prostration, cold extremities faint pulse, prolonged illness weakness with emaciation, palpitation with fear insomnia, heart failure, cardiogenic shock etc.
Modern medicine study proves that the effective ingredient of Radix Ginseng is the ginsenoside, and medical value is high, effect such as have defying age, Fructus Alpiniae Oxyphyllae, calm the nerves, lengthen the life.Be generally used for treating cardiovascular disease, forgetful insomnia etc.Pharmacological research shows that Radix Ginseng, Radix Ginseng total saponins and each saponin monomer thereof can improve the cardio-cerebrovascular function; to the acute and chronic cerebral ischemia reperfusion injury of multiple animals such as rat, mice, pallasiomy have obvious protective effect, can also nervous system regulation function and immunologic function; improve brain cell hypoxia-bearing capability and antistress function; improve cerebral ischemia pallasiomy neural stem cell survival rate and ability of learning and memory; improve transient cerebral ischemia and cause the learning capacity of learning memory disorder model mice; in addition can also blood fat reducing, be widely used in the control of apoplexy and vascular dementia.
The Radix Polygoni Multiflori has another name called Radix Polygoni Multiflori ,Warm in nature, bitter in the mouth, sweet, puckery is gone into liver, kidney channel.Radix Polygoni Multiflori Preparata is a tonic medicine, has functions such as invigorating the liver and kidney, benefiting essence-blood, black beard and hair, bone and muscle strengthening, can be used for blood deficiency and yellow complexion, dizzy ear toot, early whitening of beard and hair, soreness of the waist and knees, numb limbs and tense tendons.Modern pharmacological research shows that the Radix Polygoni Multiflori has effects such as slow down aging, arteriosclerosis and cerebral ischemia.
Contain the active component stilbene glucoside in the Radix Polygoni Multiflori and have cerebral protection; Can improve the pallasiomy damage in learning and memory that ischemia-reperfusion causes, and the dull-witted cell model of multiple plan is had neuroprotective; Experimental atherosclerosis rat fat and inflammatory factor also there is regulating action.
The Radix Ginseng fleece-flower root is the conventional Chinese medicine of Chinese medicine cerebral ischemia apoplexy and senile dementia, and composition is relative with effect clear and definite, and this national standard Chinese patent medicine of ginseng and fleece-flower root capsule is arranged, and has invigorating the liver and kidney, the replenishing QI and blood function.Be used for deficient qi and blood, early whitening of beard and hair, neurasthenia, forgetful insomnia, inappetence, overfatigue etc.But further evaluation and raisings such as its curative effect of ginseng and fleece-flower root square preparation has mainly are that Radix Ginseng, Radix Polygoni Multiflori blood circulation-promoting functions are not strong, affect the treatment.We think that assistant with the class Chinese medicine of invigorating blood circulation, can significantly improve drug effect.
Folium Ginkgo, property is flat, sweet in the mouth, hardship, puckery, GUIXIN, lung meridian.Effect with blood circulation promoting and blood stasis dispelling, pain relieving is used for coronary heart disease, angina pectoris, hyperlipemia.Near find during the last ten years and prevent in the assault of fortified positions of cerebral ischemia that these mankind ginkgo agent is a dark horse, extremely World Focusing.The main effective ingredient of Semen Ginkgo is ginkgo flavone and lactone and flavone.Its preparation XINGLING KELI, Ginaton tablets etc. have the function of blood circulation promoting and blood stasis dispelling, TONGMAI SHULUO, be mainly used in apoplexy, thoracic obstruction diseases such as (coronary heart disease) that blood stasis causes, disease is seen: chest distress and palpitation symptoms, stiff tongue and retardation in speech, hemiplegia, hemianesthesia, crooked mouth and tongue, dementia etc.But single ginkgo leaf extract preparation is restricted its use because of the existence of toxic component ginkgoic acid, and its drug effect and curative effect still remain further to be improved.
Our discovery that studies for a long period of time, Radix Ginseng fleece-flower root, compatibility Folium Ginkgo, ischemia resisting brain tissue impairment and promote neuranagenesis and improve the learning and memory effect significantly to strengthen.The three share drug effect and significantly is better than Radix Ginseng fleece-flower root, Radix Ginseng and Folium Ginkgo or Folium Ginkgo, Radix Ginseng, the independent effect of the Radix Polygoni Multiflori.And Radix Ginseng, the Radix Polygoni Multiflori and Folium Ginkgo be when share, and ginsenoside and stilbene glucoside isoreactivity composition are accelerated to enter cerebral tissue, and increase their concentration and bioavailability in cerebral tissue.
In addition, the ginkgetin composition can promote ginseng and fleece-flower root active component stilbene glucoside, ginsenoside Rb1, Rg1, Rd, Re to enter cerebral tissue and play a role.The Radix Ginseng fleece-flower root, the compatibility Folium Ginkgo, drug action significantly improves; And can reduce the share of toxic component ginkgoic acid, and reduce toxic and side effects, strengthen the drug effect that promotes neuranagenesis, anti-cerebral ischemia damnification and improve learning and memory.Synergism is arranged on the pharmacology, can bring into play better anti-cerebral ischemia damnification and neuroprotective and promote the neuranagenesis effect.
Compared with prior art, the present invention has following beneficial effect:
Chinese medicine composition of the present invention has good short neuranagenesis effect; and have outstanding anti-cerebral ischemia damnification, neuroprotective, improve the learning and memory effect; and main active is clear and definite; steady quality; using dosage is few; can be made into various sustained release preparations and carry out large-scale production, is a kind of good natural drug and/or health food.
Medicine material kind of the present invention is few, preparation method is simple to operation, has extensive use and is worth.
Description of drawings
Fig. 1. the Chinese medicine composition extract (WSXN) of embodiment 1 is analyzed the finger printing that obtains through UHPLC-MS;
Fig. 2. the Chinese medicine composition extract (WSXN) of embodiment 1 is analysed the flavonoid component finger printing that obtains through HPLC;
Fig. 3. different pharmaceutical is to the body weight change rate impact analysis figure of global brain ischemia rat;
Fig. 4. rat hippocampal is organized HE stained microscope figure;
Fig. 5. rat hippocampal is organized CA1 district cone neurocyte microscope figure;
Fig. 6. each medicine is to the impact analysis figure of cerebral ischemia re-pouring learning and memory in rats (constant-bearing navigation experiment latent time);
Fig. 7. each medicine is to the impact analysis figure of cerebral ischemia re-pouring rat space exploration learning and memory (spanning platform number of times);
Fig. 8. the single mark of the capable BrdU of rat hippocampus tissue immunohistochemical reaction (showing neuranagenesis) confocal laser scanning microscope, CLSM photo effect after each drug treating.
The specific embodiment
Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.(the following stated ethanol all by volume percentage calculation)
The preparation (WSXN1) of embodiment 1 Chinese medicine compound total extract
The Radix Polygoni Multiflori 100 kg, Radix Ginseng 90 kg, Folium Ginkgo 60 kg.
Get the Radix Ginseng of recipe quantity, be ground into coarse powder, respectively extract 3 times with 10 times, 8 times of medical material amount and 6 times 60 % alcohol refluxs respectively, each 2 h, merge extractive liquid,, vacuum decompression reclaim ethanol and make alcohol extraction part concentration is that 1 ml is equivalent to 1 g crude drug.
The Folium Ginkgo processing method is identical with Radix Ginseng, and final alcohol extraction part concentration is that 1 ml is equivalent to 1 g crude drug.
The Radix Polygoni Multiflori is with 10 times, 8 times and 6 times water of medical material amount, decoct respectively and extract 3 times, each 2 h, merge 3 times the water extract, vacuum decompression is concentrated into the 1ml extracting solution and is equivalent to 1 g raw medicinal herbs, adds 95% ethanol and makes and contain alcohol amount and reach 70%, standing over night, filtration is got the alcoholic solution vacuum decompression and is reclaimed ethanol, concentrates to the concentrated alcohol extract of 1 g medical material/ml, partly merges with aforementioned Radix Ginseng, Semen Ginkgo alcohol extraction to obtain total alcohol extract concentrated solution.
Total ethanol extract concentrated solution is added on the macroporous adsorbent resin of having handled well (PD100 polystyrene weak-base anion-exchange resin), and the amount ratio of macroporous resin and concentrated solution is that the 1kg macroporous resin adds concentrated solution 2L.Use 50%, 70%, 80% and 95 % ethanol elutions successively, consumption is respectively 4,4,4,4 L, with 10 ml/min flow velocity eluting, collects corresponding eluent, reclaims ethanol, drying; Or recovery ethanol, being condensed into the thick paste medicine, vacuum drying is pulverized and is promptly got final extract (WSXN1) 10.1 kg.
Contain stilbene glucoside, ginsenoside Rb through this extract of UHPLC-MS determination and analysis (WSXN1) 1, the ginsenoside Rg 1, ginsenoside Rd, ginsenoside Re, bilobalide, nimbecetin and Quercetin etc.Their content is as shown in table 1.
The HPLC assay result of table 1 WSXN1
Test item Content (%) Test item Content (%)
Stilbene glucoside 8.8 The ginsenoside Rb1 4.8
The ginsenoside Rg1 4.2 The ginsenoside Re 3.1
The ginsenoside Rd 1.3 Bilobalide 1.0
Nimbecetin 0.89 Quercetin 0.86
Ginkgoic acid 2PPM ? ?
Embodiment 2 Chinese medicine compound total extracts preparations (WSXN1-2)
The Radix Polygoni Multiflori 100 kg, Radix Ginseng 90 kg, Folium Ginkgo 60 kg.
Above-mentioned medical material, be ground into coarse powder, respectively with 12 times of medical material amount, the water of 10 times and 6 times, boil respectively and boil 2 h, 1.5 h and 1 h, filter, merging filtrate, decompression is concentrated into 1 ml and is equivalent to contain the 1g crude drug below 80 ℃, adding 95% ethanol makes and contains alcohol amount and reach 70%, standing over night filters and gets alcoholic solution vacuum decompression recovery ethanol, concentrates to the concentrated alcohol extract of 1g medical material/ml, total ethanol extract concentrated solution is added on the macroporous adsorbent resin of having handled well (PD100 polystyrene weak-base anion-exchange resin), and the amount ratio of macroporous resin and concentrated solution is that the 1kg macroporous resin adds concentrated solution 2L.Use 50%, 70%, 80% and 95 % ethanol elutions successively, consumption is respectively 4,4,4,4 L, with 10 ml/min flow velocity eluting, collects corresponding eluent, reclaims ethanol, drying; Or recovery ethanol, being condensed into the thick paste medicine, vacuum drying is pulverized and is promptly got final extract (WSXN1-2) 9.61 kg.
Contain stilbene glucoside, ginsenoside Rb through this extract of UHPLC-MS determination and analysis (WSXN1-2) 1, the ginsenoside Rg 1, ginsenoside Rd, ginsenoside Re, bilobalide, nimbecetin and Quercetin etc.Their content is as shown in table 2.
The HPLC assay result of table 2 WSXN1-2
Test item Content (%) Test item Content (%)
Stilbene glucoside 8.3 The ginsenoside Rb1 4.1
The ginsenoside Rg1 4.0 The ginsenoside Re 2.6
The ginsenoside Rd 1.1 Bilobalide 0.9
Nimbecetin 0.85 Quercetin 0.836
Ginkgoic acid 2PPM ? ?
The preparation of embodiment 3 Radixs Polygoni Multiflori, Radix Ginseng, Folium Ginkgo extract
The preparation of 1 Radix Polygoni Multiflori extract 1
Get 500 kg radix polygoni multiflori powders and be broken into coarse powder, respectively with 70 % alcohol reflux of 10 times, 8 times of quality of medicinal material and 6 times heavy 3 times, each 2 h, merge extractive liquid,, vacuum decompression reclaim ethanol and make alcohol extraction part concentration is total alcohol extract that 1 ml is equivalent to 1 g crude drug; Total alcohol extract is added on the macroporous adsorbent resin of having handled well (PD100 polystyrene weak-base anion-exchange resin), and macroporous resin is that the 1kg macroporous resin adds up alcohol extract 2L with the amount ratio of total alcohol extract.Use 50%, 70%, 80% and 95% ethanol elution successively, consumption is 4 L, with 10 ml/min flow velocity eluting, collects corresponding eluent, reclaims ethanol, drying; Or recovery ethanol, be condensed into the thick paste medicine, vacuum drying, pulverizing promptly gets Radix Polygoni Multiflori extract 1(and abbreviates EHSW1 as).The yield of Radix Polygoni Multiflori extract 1 is 5.1%; Wherein stilbene glucoside content is 20.8%.
The preparation of 2 Radix Polygoni Multiflori extracts 2
The radix polygoni multiflori powder of getting 500kg is broken into coarse powder, respectively with the water of 10 times, 8 times and 6 times weights of quality of medicinal material, decoct respectively and extract 3 times, each 2 h merge 3 times the water extract, and vacuum decompression is concentrated into 1 g medical material/ml extracting solution, adding 95% ethanol makes and contains alcohol amount and reach 70%, standing over night filters and to get the alcoholic solution vacuum decompression and reclaim ethanol, concentrate concentrated solution.And to make alcohol extraction part concentration be the Radix Polygoni Multiflori crude extract that 1 ml is equivalent to 1 g crude drug; Concentrated solution is added on the macroporous adsorbent resin of having handled well (PD100 polystyrene weak-base anion-exchange resin), and the amount ratio of macroporous resin and concentrated solution is that the 1kg macroporous resin adds concentrated solution 2L.Use 50%, 70%, 80% and 95 % ethanol elutions successively, consumption is 4 L, with 10 ml/min flow velocity eluting, collects corresponding eluent, reclaims ethanol, drying; Or recovery ethanol, be condensed into the thick paste medicine, vacuum drying, pulverizing promptly gets Radix Polygoni Multiflori extract 2(and abbreviates EHSW2 as).The yield of Radix Polygoni Multiflori extract 2 is 10%; Wherein stilbene glucoside content is 10.2%.
The preparation of 3 Radix Ginseng extracts 1
The Radix Ginseng powder who gets 500 kg is broken into coarse powder, reflux respectively with 60 % ethanol of 10 times, 8 times of quality of medicinal material and 6 times heavy and respectively to extract 3 times, each 2 h, merge extractive liquid,, vacuum decompression reclaims ethanol and makes alcohol extraction part concentration is that 1 ml is equivalent to 1 g crude drug, obtains concentrated solution; Be added on the macroporous adsorbent resin of having handled well (PD100 polystyrene weak-base anion-exchange resin), the amount ratio of macroporous resin and concentrated solution is that the 1kg macroporous resin adds concentrated solution 2.5L.Use 60%, 70%, 80% and 95 % ethanol elutions successively, consumption is 5 L, with 12 ml/min flow velocity eluting, collects corresponding eluent, reclaims ethanol, drying; Or recovery ethanol, being condensed into the thick paste medicine, vacuum drying is pulverized and is promptly got Radix Ginseng extract 1 (ERS1).The yield of extract is 5%; Wherein Radix Ginseng total saponins content is 30.3%.
The preparation of 4 Radix Ginseng extracts 2
The Radix Ginseng powder who gets 500 kg is broken into coarse powder, and reflux respectively with 75 % propanol of 10 times, 8 times of quality of medicinal material and 6 times and respectively extract 3 times, each 2 h, merge extractive liquid,, vacuum decompression reclaim propanol and make alcohol extraction part concentration is that 1 ml is equivalent to 1 g crude drug; Be added on the macroporous adsorbent resin of having handled well (PD800 polystyrene weak-base anion-exchange resin), the amount ratio of macroporous resin and alcohol extract is that the 1kg macroporous resin adds alcohol extract 2.2L.Use 60%, 70%, 80% and 95 % ethanol elutions successively, consumption is respectively 4.5,4,3.5,4 L, with 10 ml/min flow velocity eluting, collects corresponding eluent, reclaims ethanol, drying; Or recovery ethanol, being condensed into the thick paste medicine, vacuum drying is pulverized and is promptly got Radix Ginseng extract 2 (ERS2).The yield of extract is 4.32%; Wherein Radix Ginseng total saponins content is 29.6%.
The preparation of 5 Folium Ginkgo extract 1
The ginkgo leaf powder of getting 500 kg is broken into coarse powder, distinguish reflux, extract, 3 times with 10 times, 8 times of quality of medicinal material and 6 times 70 % ethanol, each time be 2.0 h, 1.5 h and 1.5 h respectively, merge each extracting solution, vacuum decompression reclaims ethanol and makes alcohol extraction part concentration is that 1 ml is equivalent to 1 g crude drug, be added on the macroporous adsorbent resin of having handled well (PD800 polystyrene weak-base anion-exchange resin), the amount ratio of macroporous resin and alcohol extract is that the 1kg macroporous resin adds alcohol extract 2.2L.Use 60%, 70%, 80% and 95 % ethanol elutions successively, consumption is respectively 4.5,4,3.5,4 L, with 10 ml/min flow velocity eluting, collects corresponding eluent, reclaims ethanol, drying; Or recovery ethanol, be condensed into the thick paste medicine, vacuum drying is pulverized and is promptly got Folium Ginkgo extract 1(EGB1), yield is 2.5%; Bilobalide content is 6.2%, nimbecetin content is 5.6%, quercetin content is that 5.8% ginkgoic acid content is＜5 ppm.
The preparation of 6 Folium Ginkgo extract 2
The ginkgo leaf powder of getting 500 kg is broken into coarse powder, with 70 % ethanol of 10 times of quality of medicinal materials, 8 times and 6 times heavy reflux, extract, 3 times respectively, each time be 2.0 h respectively, 1.5 h, 1.5 h, merge extractive liquid,, vacuum decompression reclaims ethanol and makes alcohol extraction part concentration is that 1 ml is equivalent to 1 g crude drug, adjust pH is 6.0, is added on the macroporous adsorbent resin of having handled well (polyamides amine type resin), and the amount ratio of macroporous resin and concentrated solution is that the 1kg macroporous resin adds alcohol extract 2.0L.Use 60%, 70%, 80% and 95% ethanol elution successively, consumption is respectively 4.5 L, 5 L, and 3.5 L, 3 L with 10 ml/min flow velocity eluting, collect corresponding 70%, 80% ethanol elution, reclaim ethanol, drying; Or recovery ethanol, be condensed into the thick paste medicine, vacuum drying is pulverized and is promptly got Folium Ginkgo flavone extract 2-A(EGB2-A) 2.25 kg, yield is 0.45%.Wherein nimbecetin content is 15.1%, and quercetin content is 16.0%, and ginkgoic acid content is＜5 ppm.
Above-mentioned 60% ethanol and 95% ethanol elution are collected corresponding eluent, reclaiming ethanol and making alcohol extraction part concentration is that 1 ml is equivalent to 1 g crude drug, adjust pH is 8.0, be added on the macroporous adsorbent resin of having handled well (PD800 polystyrene weak-base anion-exchange resin), the amount ratio of macroporous resin and alcohol extract is that the 1kg macroporous resin adds alcohol extract 2.0L.Use 60%, 70%, 80% and 95% ethanol elution successively, consumption is respectively 4.5 L, 4 L, and 3.5 L, 5 L with 10 ml/min flow velocity eluting, collect corresponding eluent, reclaim ethanol, drying; Or recovery ethanol, be condensed into the thick paste medicine, vacuum drying is pulverized and is promptly got Folium Ginkgo lactone extract 2-B(EGB2-B) 2.35 kg, yield is 0.47%.Wherein bilobalide content is 40.8%, and nimbecetin content is 2.7%, and quercetin content is 2.8%, and ginkgoic acid content is＜5 ppm.
Embodiment 4 crow ginseng refreshment side 2(WSXN2) preparation
Select each extract of gained among the embodiment 3 for use:
Radix Polygoni Multiflori extract 1(stilbene glucoside content is 20.8%) 9 kg;
Radix Ginseng extract 1(Radix Ginseng total saponins content is 30.3%) 9 kg;
Folium Ginkgo extract 1(bilobalide content is 6.2%, nimbecetin content is 5.6%, quercetin content is that 5.8% ginkgoic acid content is＜5 ppm) 3.6 kg.
Take by weighing the above-mentioned three kinds of extracts of recipe quantity, by equivalent incremental method mix homogeneously.Promptly get a kind of herbal mixture extract (WSXN2) that promotes neuranagenesis, control cerebral ischemia disease and senile dementia, analyze finger printing such as the accompanying drawing 1, shown in Figure 2 that obtains through UHPLC-MS, this extract contains stilbene glucoside, ginsenoside Rb after measured 1, the ginsenoside Rg 1, composition such as ginsenoside Rd, ginsenoside Re, bilobalide, nimbecetin and Quercetin.Their content is as shown in table 3.
The HPLC assay result of table 3 WSXN2
Test item Content (%) Test item Content (%)
Stilbene glucoside 8.6 The ginsenoside Rb1 4.5
The ginsenoside Rg1 4.3 The ginsenoside Re 3.0
The ginsenoside Rd 1.1 Bilobalide 1.0
Nimbecetin 0.88 Quercetin 0.89
Ginkgoic acid 1PPM ? ?
Embodiment 5 crow ginseng refreshment side 3(WSXN3) preparation
Select each extract of gained among the embodiment 2 for use:
Radix Polygoni Multiflori extract 2(stilbene glucoside content is 10.2%) 15kg;
Radix Ginseng extract 2(Radix Ginseng total saponins content is 29.6%) 10kg;
Folium Ginkgo extract 2[bilobalide extract (EGB2-B, content are 40.8%) 1.0g+ gingko flavone extractive (EGB2-A, nimbecetin content are 15.1%, quercetin content be 16.0%)] 3 kg.
Take by weighing the above-mentioned three kinds of extracts of recipe quantity, by equivalent incremental method mix homogeneously.Promptly get a kind of herbal mixture extract (WSXN3) that promotes neuranagenesis, control cerebral ischemia disease and senile dementia, UHPLC-MS analyzes, and this extract contains stilbene glucoside, ginsenoside Rb after measured 1, the ginsenoside Rg 1, ginsenoside Rd, ginsenoside Re, bilobalide, nimbecetin and Quercetin etc.Their content is as shown in table 4.
The HPLC assay result of table 4 WSXN3
Test item Content (%) Test item Content (%)
Stilbene glucoside 5.3 The ginsenoside Rb1 3.0
The ginsenoside Rg1 2.9 The ginsenoside Re 2.5
The ginsenoside Rd 0.41 Bilobalide 1.4
Nimbecetin 1.58 Quercetin 1.66
Ginkgoic acid 1PPM ? ?
Embodiment 6 crow ginseng refreshment side 4(WSXN4) preparation
Radix Polygoni Multiflori extract 9 kg(stilbene glucoside content 20.5%, buy) by Ningbo of Zhejiang Holley plant development corporation, Ltd.;
Radix Ginseng extract 9 kg(total saponin contents 30.5% are bought from Jilin Hongjiu Biotech Co., Ltd.);
The total bilobalide content 6.1% of Folium Ginkgo extract 3.5kg[; Total flavones 24.3% (quercetin content 4.8% and nimbecetin content 4.6%), ginkgoic acid＜5ppm; Buy by Zhejiang Holley plant development corporation, Ltd.].
Get Radix Polygoni Multiflori extract, ginsenoside extract, Folium Ginkgo extract, make a kind of herbal mixture extractive composition (WSXN4) 21.5kg that promotes neuranagenesis, control cerebral ischemia disease and vascular dementia by equivalent incremental method mixing.
Carry out UHPLC-MS and analyze, said composition contains stilbene glucoside, ginsenoside Rb after measured 1, the ginsenoside Rg 1, ginsenoside Rd, ginsenoside Re, bilobalide, nimbecetin and Quercetin.Their content is as shown in table 5.
The HPLC assay result of table 5 WSXN4
Test item Content (%) Test item Content (%)
Stilbene glucoside 8.41 The ginsenoside Rb1 2.21
The ginsenoside Rg1 2.20 The ginsenoside Re 2.01
The ginsenoside Rd 0.46 Bilobalide 0.99
Nimbecetin 0.75 Quercetin 0.77
Ginkgoic acid <1PPM ? ?
The preparation of embodiment 7 crow ginseng XINGNAO DIWAN (WSXN5)
Crow ginseng refreshment side 2(WSXN2) 7 kg, PEG-6000 7 kg, PEG-4000 7kg makes every 72mg drop pill by the drop pill routine fashion, polishing, quality inspection packing.Make 29.16 ten thousand of crow ginseng XINGNAO DIWAN altogether, every contains WSXN2 24mg.
Measure each active constituent content in the drop pill (SWXN5) with the HPLC method, result such as following table:
The HPLC assay result of table 6 WSXN5
Test item Content (mg/ grain) Test item Content (mg/ grain)
The ginsenoside Rb1 1.03 Stilbene glucoside 1.95
The ginsenoside Rg1 1.01 Bilobalide 0.23
The ginsenoside Rd 0.26 Quercetin 0.21
The ginsenoside Re 0.71 Nimbecetin 0.20
Carry out clinic trial treatment vascular dementia with this drop pill capsule, every day 3 times, each 10, serve on 90 days, patient's memory has obvious reinforcement after the treatment of treatment group as a result, and total effective rate is 76.7%.
The preparation of embodiment 8 crow ginseng Awakes (WSXN6)
Get the extract WSXN1 8kg of embodiment 1, add starch 5kg, dextrin 7kg, mixing adds 75% ethanol and granulates, 60 ℃ of dryings, granulate, it is an amount of to add magnesium stearate, tabletting, bag Opadry or other film-coat make 1,000,000 altogether.Every heavy 0.20g contains WSXN1 80mg/ sheet, measures with the HPLC method, and each active constituent content result is as follows in every:
The HPLC assay result of table 7 crow ginseng Awake (WSXN6)
Test item Content (mg/ sheet) Test item Content (mg/ sheet)
The ginsenoside Rb1 9.62 Stilbene glucoside 17.12
The ginsenoside Rg1 8.81 Bilobalide 2.04
The ginsenoside Rd 2.56 Quercetin 1.76
The ginsenoside Re 6.02 Nimbecetin 1.75
Ginkgoic acid <1PPM ? ?
The preparation of embodiment 9 crow ginseng refreshment soft capsules (WSXN7)
Compositions WSXN1 7.2 kg, gelatin 7.2 kg, soybean oil 7.2 kg make every soft capsule that weighs 0.36g by the soft capsule routine fashion, the quality inspection packing.Make 120,000 of crow ginseng refreshment soft capsules altogether, every contains WSXN1120mg.Measure each active constituent content among the soft capsule WSXN7, result such as following table with the HPLC method.
The HPLC assay result of table 8 crow ginseng Awake (WSXN7)
Test item Content (mg/ grain) Test item Content (mg/ grain)
The ginsenoside Rb1 5.73 Stilbene glucoside 10.14
The ginsenoside Rg1 5.07 Bilobalide 1.26
The ginsenoside Rd 1.53 Quercetin 1.03
The ginsenoside Re 3.63 Nimbecetin 1.06
Carry out clinic trial treatment arteriosclerosis with this soft capsule, every day 3 times, each 2, serve on 90 days, patient's memory has obvious reinforcement after the treatment of treatment group as a result, and total effective rate is 76.7%.
The preparation of embodiment 10 crow ginseng refreshment capsules (WSXN8)
With WSXN3 compositions 15 kg, add starch 9 kg, dry granulation behind the dextrin 6 kg mix homogeneously, 60 ℃ of dryings, granulate is filled No. 2 capsule, polishing, quality inspection packing.Every heavy 0.25g makes 120,000 of capsules altogether, and every 0.25g contains WSXN3 0.125g/ grain.
Measure each active constituent content in the compound recipe crow ginseng refreshment capsule, result such as following table with the HPLC method.
The HPLC assay result of table 9 crow ginseng refreshment capsule (WSXN8)
Test item Content (mg/ grain) Test item Content (mg/ grain)
The ginsenoside Rb1 3.74 Stilbene glucoside 6.61
The ginsenoside Rg1 3.61 Bilobalide 1.74
The ginsenoside Rd 0.53 Quercetin 2.07
The ginsenoside Re 3.14 Nimbecetin 1.94
Carry out clinic trial treatment vascular dementia with this capsule, every day 3 times, each 1, serve on 90 days, patient's memory has obvious reinforcement after the treatment of treatment group as a result, and total effective rate is 76.7%.
Certainly, the invention is not restricted to above-mentioned dosage form, also can make injection, but need further do the tests of pesticide effectiveness such as toxicity.Add cosolvent as WSXN, make injectable powder by the injectable powder requirement.Every bottle contains WSXN240mg.
Embodiment 11 crows are joined refreshment side's anti-cerebral ischemia damnifications, improve the effect of learning and memory
1. experiment material
Experiment medicine: embodiment 1 crow ginseng refreshment side (WSXN1) and ginseng and fleece-flower root extract (being called for short RSSW) are provided by the Chinese medicine academy of Guangdong Pharmaceutical University, positive control drug Ginaton tablets (EGB761, Dr Willmar Schwabe produces); With 1% sodium carboxymethyl cellulose (CMC-Na) each medicine is mixed with suspension oral gavage during application.
Reagent: dehydrated alcohol, dimethylbenzene, paraffin, sodium hydrogen phosphate, sodium dihydrogen phosphate, formaldehyde, sodium chloride etc. are homemade analytical pure.
Instrument: electric heating constant temperature training incubator (Shanghai), Leica RM2135 cycle type histotome (Germany), optical microscope (Olympus BX51).
Laboratory animal: the Sprague-Dawley rat of male, body weight 220 ~ 280g, the cleaning grade standard provides the (quality certification number: Guangdong probatio inspectionem pecuoarem word 2008A053 number) by Nanfang Medical Univ's Experimental Animal Center.Experimental temperature is controlled at 25 ± 1 ℃, and light controlled for 12 little time, and 12 hours dark, freely drinks water and the edible animal feedstuff.Raise and begin experiment after 3 days.
2. experimental technique
(1) grouping and administration
Rat is sorted, numbers by the body weight size; Be divided into 6 groups at random with district's group method of dividision into groups at random, A: sham operated rats (Sham), B: model group (Model), C:WSXN 1 high dose group (80mg/kg), D:WSXN1 low dose group (40mg/kg), E:RSSW organizes (120mg/kg), H: positive drug EGB761(40.0mg/kg), and 10 every group.
(2) experimental establishment
Sham operated rats and model group rat are irritated stomach 1 time at preceding 5 days of modeling, postoperative CMC-Na 14 day every day 1%, and operation art on the same day was irritated stomach 1 time in preceding 2 hours.All the other drug components An be not separately dosage at preceding 5 days of modeling, postoperative gastric infusion 14 day every day, operation art on the same day was irritated stomach 1 time in preceding 2 hours.After weighing in 2 days after surgery, 5 days, 8 days and 12 days, carry out the evaluation of body weight change rate.(body weight change rate=(B-A)/A * 100%, A: first day body weight, B: the same day body weight).Postoperative is observed rat behavior every day, allows rat free swimming 2 minutes to adapt to surrounding on the 8th day, and the 9th day begins to carry out the test of Morris water maze, lasts 6 days, the test period administration that does as usual.Postoperative was put to death animal on the 15th day, selected 5 rats to get full brain at random for every group and was stored in 4% paraformaldehyde, was used to do the cerebral ischemia degree that brain section carries out each group of HE dyeing observation of full wafer; And carry out the degree of injury that the cerebral hippocampus Nissl's staining is observed the CA1 district pyramidal cell of each group.
(3) improvement Pulsinelli four blood vessel blocking manufactured VD rat models
After the rat abdominal cavity anesthesia, row dorsal part neck median incision, successively passivity is separated, expose bilateral the 1st transverse process of cervical vertebra wing aperture, coagulate vertebral artery in the pin calcination double side wings aperture, cause the permanent occlusion with the electricity of the about 0.15mm of diameter, it is fixing rat to be lain on the back again, row veutro neck median incision, passivity is separated bilateral common carotid arteries, and is standby with No. 4 silk thread threading.Behind the 24h, close bilateral common carotid arteries 15min with arteriole folder folder, before local wound is sewed up, protect from infection with the processing of sulfa powder localized pulverization, postoperative gave penicillin 2U/kg intramuscular injection infection in 3 days.The sham operated rats step is the same, does not do ischemia and handles.
Estimate the standard of modeling success: folder closes about bilateral common carotid arteries 10s, loss of consciousness, bilateral platycoria, corneal reflex and righting reflex loss appear in rat, and realize in ischemic stage and the equal recuperator not of above-mentioned reflection at 15min, expression modeling success can be implemented to pour into again.Dead or lethargy only occurs and the rat of having a convulsion all goes out of use in whole process in ischemic stage.
(4) Morris water maze laboratory
Constant-bearing navigation experiment (Place navigation test):
This experiment is used for the ability of learning and memory of test animal.Allowed rat free swimming 2 minutes to adapt to surrounding after operation on the 8th day, carried out the test of Morris water maze since the 9th day, every rat is trained 3 times every day, promptly trains 3 place of entry every day, and each 2 ~ 3 hours at interval, experiment lasted 5 days.Select place of entry during training at random, rat is put into water towards sidewall, treat that rat finds after the platform, be allowed to condition on the platform and stopped 10 seconds, observe and the record rat searching platform time (Latency of the rats to reach the platform, latent time).To seek the platform time is that incubation period is as the index that detects learning and memory.During training,, need it to be caused platform, at this moment be designated as 90 seconds incubation period, continuous 5 days with hands if rat was not found platform in 90 seconds.
Space exploration experiment (Spatial probe test):
The 14th day laggard row space in operation back explored experiment.Remove platform, from rat being put into water towards sidewall from a target quadrant place of entry farthest, 90 seconds inherent target quadrants of record rat over against quadrant in the exploration distance and the spanning platform number of times to observe memory ability.
(5) date processing
Experimental data is represented with mean ± SEM, carries out significance test of difference with the variance analysis of SPSS 13.0 software repeated measurement datas.
3. experimental result
(1) to the influence of rat body weight rate of change:
As shown in Figure 3, sham operated rats postoperative body weight shows a rising trend, and model group is because the damage that causes of global brain ischemia, causes body weight gain slow even the weight loss phenomenon arranged.(each time point and administration before relatively) body weight change rate, with sham operated rats with time point relatively, significant difference was all arranged after the model group modeling in 2 days, 5 days, 8 days and 12 days.Compare with model group with time point body weight change rate, the WSXN1 high dose group had significant difference in 5 days after surgery, 8 days and 12 days; Positive controls had significant difference in 8 days after surgery and 12 days, showed that giving these medicines can improve the weight loss that cerebral ischemia causes.
(2) to the influence of cerebral tissue ischemic injuries
Normal rat cerebral hippocampal organism optical microscopically tissue morphology: under the low power lens, sham operated rats rat hippocampal tissue shows " C " shape, is divided into CA1, CA2, CA3 district, and there are 3 ~ 4 floor pyramidal cell in the CA1 district, marshalling (Fig. 4, Fig. 5); Under the high power lens, pyramidal cell nuclear is justified greatly, and 1 ~ 2 kernel is arranged, and pyramidal cell density is 172.1 ± 17.4(table 10).
Rat disappearance, righting reflex loss and platycoria occur realizing immediately through the rat of Pulsinelli four blood vessel blockings, preceding cerebral ischemic model success.Preceding cerebral ischemia re-pouring 7 days, visible integral animal is in poor shape, and the activity of rat outward appearance obviously reduces, and fur is alarmmed, and is rough, loses weight; Rat cerebral tissue's cell obviously damages, extra large CA1 district pyramidal cell is most of dead, cell debris distribution at random (table 10).
Give crow ginseng refreshment side (WSXN1), ginseng and fleece-flower root extract (RSSW) and the Semen Ginkgo extrac sheet (EGB761) of various dose, the integral animal situation is improved, and the activity of rat outward appearance is normal, and fur is smooth, and it is not obvious to lose weight; The animals survived number is obvious more than model control group behind the ischemia-reperfusion; With model group relatively, the CA1 district pyramidal cell density of living significantly increases, many CA1 districts cellular morphology normally, rat hippocampal histiocyte form be improved significantly.Low, the black ginseng of high dose refreshment side group is lived pyramidal cell density than the obvious increase of model group, and apparent doses dependency ( P＜0.01, see Table 10 and Fig. 4, Fig. 5 WSXN1).Experiment shows that crow ginseng refreshment side and ginseng and fleece-flower root extract (RSSW) damage rat hippocampal CA1 district neuronal damage due to the cerebral ischemia re-pouring before acute and whole rat obvious protective effect is arranged.
The rat hippocampal CA1 district cone neurocyte density of positive control drug Semen Ginkgo extrac sheet 40.0mg/kg group also than the obvious increase of model group ( P＜0.05, table 10, Fig. 5 EGB761 group).Prompting Semen Ginkgo extrac sheet also has obvious protective effect to rat hippocampal histiocyte damage due to the ischemia-reperfusion.
From anti-cerebral ischemia damnification pharmacodynamic study result, the drug effect of crow ginseng refreshment side is better than the Folium Ginkgo extract sheet (Ginaton tablets EGB761) and the compound ginseng Radix Polygoni Multiflori of the international mark post medicine of like product Dr Willmar Schwabe import.
Table 10 crow ginseng refreshment sides etc. are to preceding cerebral ischemia reperfusion injury rat hippocampus CA1 cone protecting neuron from acute
Group Dosage (mg/kg) Number of animals The CA1 cone of living neurocyte density (it is individual/mm,
Figure 198971DEST_PATH_IMAGE001
±S）
Normal control 0 10 162.1±18.1
Model group (SHAM) 0 7 7.1±3.4**
RSSW 120 7 56.7±6.9 ★★
The WSXN1 low dosage 40 9 58.6±9.5 ★★
The WSXN1 high dose 80 8 81.5±10.8 ★★
EGB761 80 8 59.9±6.8 ★★
Annotate: compare * * with the normal control group P＜0.01; Compare with model group: ★ ★ P＜0.01.
(3) to the influence of learning and memory
By the constant-bearing navigation experiment, choosing the most important index searching platform time is latent time analysis, has detected the protective effect of medicine to the damage of global brain ischemia learning and memory in rats ability.The results are shown in Figure 6, model group shows that than the latent time significant prolongation of sham operated rats global brain ischemia causes the decline of ability of learning and memory.And WSXN1 high and low dose group, the incubation period of RSSW group and positive controls EGB, wherein high low dosage WSXN1 was the most remarkable all than the model group shortening, illustrated that high and low dose WSXN1 is to the tool protective effect of the inductive learning memory injury of cerebral ischemia.
Global brain ischemia rat space exploration experiment is chosen important index spanning platform number of times and is explored distance at the target quadrant and analyze.The result as shown in Figure 7.
The result shows and organizes respectively that spanning platform number of times (promptly passing through the original platform position) model group significantly descends than sham operated rats in the rat 90 seconds; and low, high dose WSXN1 increases rats with cerebral ischemia spanning platform number of times to a certain extent, shows that may there be protective effect in high and low dose WSXN1 to the spatial memory damage that global brain ischemia causes.Simultaneously; as shown in Figure 7; compare with sham operated rats; the exploration distance significance of 90 seconds inherent original platforms of model group rat place quadrant (being the purpose quadrant) shortens; illustrate that global brain ischemia causes the remarkable decline of the spatial memory power ability of rat; except positive control drug; each administration group all shows the trend of improving global brain ischemia rat spatial memory ability; WSXN1 height wherein; the low dose group rat shortens at the exploration distance significance of purpose quadrant; have significant difference, show height; low dosage WSXN1 has significant protective effect to the spatial memory damage that global brain ischemia causes.
Morris water maze result shows the swimming rate no difference of science of statistics of each group, and the difference that shows each observation index of Morris water maze is mainly by learning and memory and the decision of spatial memory ability.The constant-bearing navigation experiment shows; global brain ischemia causes the decline of ability of learning and memory; and WSXN 1 high and low dose group, the incubation period of RSSW group and positive controls EGB761 is all than the model group shortening, and wherein low dosage WSXN1 is to the tool protective effect of the inductive learning memory injury of cerebral ischemia.Simultaneously; the space exploration experiment shows; global brain ischemia causes the spatial memory power ability drop of rat, and WSXN 1 high and low dose group rat shortens at the exploration distance significance of purpose quadrant, and wherein high and low dose WSXN1 is to the tool protective effect of the inductive learning memory injury of cerebral ischemia.Simultaneously; the space exploration experiment shows; global brain ischemia causes the spatial memory power ability drop of rat, and WSXN 1 high and low dose group rat shortens at the exploration distance significance of purpose quadrant, shows that WSXN1 has significant protective effect to the spatial memory damage that global brain ischemia causes.
From anti-cerebral ischemia damnification, prevent and treat apoplexy and vascular dementia pharmacodynamic study result, the drug effect of crow ginseng refreshment side is better than the Folium Ginkgo extract sheet (Ginaton EGB761) and the compound ginseng Radix Polygoni Multiflori of the international mark post medicine of like product Dr Willmar Schwabe import.
Embodiment 12 crow ginseng refreshment sides are to the influence of cerebral ischemia neuranagenesis
(1) experiment material
10 the week age SD rat, male, 250 ~ 350g is provided by Guangdong Medical Lab Animal Center; Semen Ginkgo extrac sheet (Ginaton EGB761, Dr Willmar Schwabe produces); Ginseng and fleece-flower root extract (RSSW), crow ginseng refreshment formula extraction (WSXN2) provides by this laboratory.5-bromodeox yuridine (BrdU, sigma company); Mouse anti BrdU monoclonal antibody (CST company), horseradish peroxidase-labeled goat anti-mouse IgG (H+ L), goat anti-mouse Cy 5, anti-fluorescent quenching mounting liquid, DAB horseradish peroxidase colour reagent box (green skies biotechnology research institute, Haimen, Jiangsu).
Key instrument: CO 2Incubator (U.S. Heraeus HERAcell 150); Inverted fluorescence microscope (German ZEISS AXIO OBSERVER A1); Paraffin specimen embedding machine (German Leica-2000 type); Paraffin specimen microtome (German Leica-2135 type); Automatic photomicrography system and colored cytological image analyses instrument (German AxioVision Rel. 4.7); Microscope (Japanese Olympus BX-51 type); Confocal laser scanning microscope, CLSM shooting (Japanese Olympus LSM-GB200).
(2) experimental technique
Rat is divided into sham operated rats (SH), ischemia model group (Mo), EGB 761 Ginaton Semen Ginkgo extrac sheets (EGB 761,40 mg/ kg), RSSW group (120 mg/ kg), WSXN2 high and low dose group (WSXN2 80,40mg/kg) at random.The administration group gives each corresponding dosage medicine 5 d in advance before cerebral ischemia.
Rat with 10% chloral hydrate (300 mg/ kg, ip) anesthesia is poured into after setting up intraluminal middle cerebral artery occlusion in rats ischemia-reperfusion injury model (MCAO) 2 h again, infusion time continues 7 d again.Pouring into 24 h again, each is organized rat and carries out behavioristics's scoring.Adopt 5 fens system behavioristics standards of grading: behavior normally is 0 minute, and right front pawl can not full extension be 1 minute, and spontaneous to turn-take or advance to the right be 2 minutes, and only advancing when being upset is 3 minutes, is 4 minutes to stimulating reactionless.With the rat of scoring between 1 ~ 3 is experimental subject.
Administration group rat 5 d before cerebral ischemia re-pouring begin to give various medicines by grouping dosage; Behind last administration 24 h, rat i. p. BrdU (100 mg/kg, 3d, 24h at interval continuously).6h puts to death rat behind last 1 injection BrdU.Separate the single mark of Hippocampus row BrdU immunohistochemical reaction immediately.
The fluorescent labeling of neuranagenesis: tissue slice heats (85 ℃, 5 min) in the solution that contains BrdU (10 mo l/ L), hatches among the 2 mol/ L HCl that (RT, 30min), rinsing 10min in the 0.1 mol/ L boric acid (pH 8.5) contains 1% H 2O 2PBS in hatch 30 min, sealing 1 h (room temperature) is then hatched (4 ℃ are spent the night) with mouse anti BrdU monoclonal antibody (1:200) among the PBS (containing 3% normal goats serum, 0.3% (w/v) Triton X 100 and 0. 1%BSA).Hatch with goat anti-mouse Cy5 (1:1000), rinsing is dried, and with the embedding of fluorescence mountant, the matched group tissue slice is handled and saved an antiserum.Every animal is got 5 sections and carries out confocal laser scanning microscope, CLSM shooting and analysis, and experimental result sees that (SH is false ischemic control group to Fig. 8; Mo is an ischemia group; EGB761 is the Semen Ginkgo extrac group; RSSW is a ginseng and fleece-flower root extract group; WSXN is a crow ginseng refreshment formula extraction.Point shows experimental rat hippocampus regenerating nerve unit quantity among the figure).
Data analysis is the result show, model group rat 24 h behind cerebral ischemia re-pouring occurred tangible behavioristics unusual (with the matched group ratio, P＜0.01), the behavioristics of each administration group rat scoring all be lower than model group (with the model group ratio, P＜0.01), the WSXN2 high dose group significantly be lower than EGB 761 and RSSW group ( P＜0.01), sees Table 11.
Table 11 medicine to cerebral ischemia re-pouring 24 h rats after the influence (X ± SD, n=6) of Hippocampus neural behavior scoring
Group Dosage (mg/ kg) Behavior scoring
SH - 0
Mo - 2.91±0.61 # #
EGb761 40 1.98±0.33* *
RSSW 120 1.65±0.46* *
WSXN2 is low 40 1.45±0.35* *
The WSXN2 height 80 1.30±0.52* * §§※※
Annotate: compare with sham operated rats, # P＜0. 05, # # P＜0. 01; Compare * with model group P＜0. 05, * * P＜0. 01.
Compare with EGB761, § § P＜0. 01; Compare with the RSSW group, ※ ※ P＜0. 01.
Regeneration situation for Hippocampus nerve behind definite cerebral ischemia re-pouring adopts laser confocal microscope to analyze the Hippocampus nerve (Fig. 8) of respectively organizing rat.The result shows, model group rat hippocampus district BrdU +Cell number obviously increase (with the matched group ratio, P＜0. 01); EGb761 group, RSSW group and WSXN2 group rat hippocampus district BrdU +The cell number increase more remarkable (with the model group ratio, P＜0. 01).Wherein, the WSXN2 high dose group is to rat hippocampus district BrdU +The cell increase is the most remarkable, and value added significantly is better than other administration group.See Table 12, Fig. 8.
The regeneration of Hippocampus nerve after table 12 cerebral ischemic reperfusion in rats (X ± SD, n=6)
Group Dosage (mg/ kg) BrdU +(cell number/mm 2)
SH - 65±11
Mo - 398±46 # #
EGb761 40 998±123* *
RSSW 120 1110±146* *
WSXN2 is low 40 1050±185* *
The WSXN2 height 80 1310±160* *
Annotate: compare with sham operated rats, # P＜0. 05, # # P＜0. 01; Compare * with model group P＜0. 05, * * P＜0. 01.
 
Embodiment 13 crow ginseng refreshment sides are to amyloid-beta 25-35 (A β 25-35 ) protective effect of induced mice hippocampal neurons injury
(Alzheimer's disease AD) is the common central nervous system degenerative disease of old people to Alzheimer.Amyloid-beta plays an important role in the AD morbidity, the present invention amyloid-beta 25-35 (A β 25-35) the induced mice hippocampal neurons injury induce the AD model.Research crow ginseng refreshment side, ginseng and fleece-flower root extract and Folium Ginkgo extract are to the drug effect of preventing and treating of this model AD.
1. laboratory animal: Healthy female C57BL/ 6 mices are 40, and body constitution amount (20 ± 2) g is provided by Guangdong Province's Experimental Animal Center.Be placed under (25 ± 2.0) ℃ room temperature the illumination of 12 ~ 12h day-night cycle, ad lib, drinking-water.Animal adapts to 1 week of laboratory environment before the experiment.
2. medicine and reagent: WSXN1, RSSW, EGB761 are provided by the Chinese medicine academy of Guangdong Pharmaceutical University; A β 25-35Available from U.S. Sigma company; The reverse transcription test kit is available from U.S. Promeg a company.Tag DNA polymerase is available from Japanese T AKARA company.All the other reagent are homemade analytical pure.
3. condensed state A β 25-35Preparation: with 1 mg A β 25-35Be dissolved in the 1 mL sterile saline, concentration is 1 mmol/ L.Sealing is placed in 37 ℃ of cell culture incubators hatches 96 h, makes it become the A β of condensed state 25-35, be placed in 4 ℃ of refrigerators standby.
4. mice AD Preparation of model and administration
C57BL/ 6 mices through chloral hydrate (after the anesthesia of 400 mg/kg), the excision bilateral ovaries.Perform the operation behind 10 d, mice is divided into matched group, A β at random 25-35Group, A β 25-35+ WSXN1 group, A β 25-35+ RSSW group and A β 25-35+ EGB761 group, 10 every group.Control group mice intracerebroventricular injection normal saline.Other group mice intracerebroventricular injections 1 mmol/L A β 25-35Preparation AD mouse model.After model prepares 3d, matched group and A β 25-35Group mouse stomach normal saline 5 mL/kg, A β 25-35+ WSXN1 organizes mouse stomach WSXN1 (80 mg/kg); A β 25-35+ RSSW organizes mouse stomach RSSW (120 mg/kg); A β 25-35+ EGB761 organizes mouse stomach EGB761 (40mg/kg).Continuous use 14d.
5. training of Morris water maze and test
The training of self administration of medication beginning in the 8th day water maze, 7d altogether.Special water maze platform is put in the 4th quadrant, and every mice detects 2 times every day, gets rid of platform place quadrant, adherently in the centre of other 3 quadrants puts into mice.No longer descend water to think that once test finishes with the mice 10s that appears on the stage; The full 90s of mice record that can not appear on the stage, and the guiding mice makes it continue 30 s on platform, to enforce one's memory to platform.Photograph the swimming process of mice, write down for 1 week altogether, this is the learning process of mice.Learning process finishes 1 week of back, removes platform, the spatial memory ability (number of times with mice spanning platform position in 90 s is represented) of test mice.Then,, get the fresh hippocampal tissue of mice, be stored in-80 ℃ of refrigerators, be used for gene test through the chloral hydrate anesthesia mice.
6. statistical analysis: experimental result with
Figure 556266DEST_PATH_IMAGE001
± s represents, relatively carries out one factor analysis of variance (One-Way ANOVA) with SSPS13. 0 statistical software between group, and compares in twos with the Tukey method.
7. experimental result
Medicines such as table 13 WSXN1 are to A β 25-35The influence of inductive AD ability of learning and memory in mice and Bcl-2 gene expression
Group Water maze incubation period (t/s) The spanning platform number of times Bcl-2/GADPH
Matched group 21.3±6.8 3.78±1.96 0.81±0.16
Aβ 25-35 53.2±13.6** 0.35±0.61** 0.33±0.09**
Aβ 25-35+ WSXN1 26.5±17.8 ## 3.66±1.56 ## 1.09±0.24 ##
Aβ 25-35+ RSSW 31.6±14.5 ## 3.21±1.66 ## 0.98±0.28 ##
Aβ 25-35+ EGB761 32.1±13.9 ## 3.38±1.84 ## 0.96±0.23 ##
Compare with matched group, F=11.3 ~ 19.61, q=5. 848 ~ 9.186, * * P＜0. 01; With A β 25-35Group is compared, # q=5. 848 ~ 9.186, ## P＜0. 01.
(1) WSXN1 is to A β 25-35The influence of inductive mice space learning memory ability
Training to the is in the time of 7 days, A β 25-35The group incubation period than the obvious prolongation of matched group ( P＜0.01); And the incubation period of WSXN1, RSSW and EGB761 administration group is than A β 25-35Model group obviously shortens (P＜0.01); Training removes platform after finishing for 1 week, compares A β with control group mice 25-35The number of times of group mice spanning platform obviously reduces; And WSXN1, RSSW and EGB761 gastric infusion can obviously improve A β 25-35The memory ability of group mice.See table 13 for details.
(2) WSXN1 is to A β 25-35The influence of inductive mice Hippocampus bcl-2 gene expression
A β 25-35Intracerebroventricular injection can obviously reduce bcl-2 expression of gene in the mice hippocampal tissue, and WSXN1, RSSW and EGB761 gastric infusion can improve A β 25-35This damaging action, increase anti-apoptotic genes expression bcl-2 expression of gene, protection mouse brain neuronal damage.
The The above results prompting: WSXN1, RSSW and EGB761 prevent and treat drug effect to Alzheimer (AD).The drug effect of crow ginseng refreshment side and ginseng and fleece-flower root extract and Folium Ginkgo extract relatively have certain superiority.
Embodiment 14 Folium Ginkgo extract promote ginsenoside's composition to enter cerebral tissue
(1) experiment material
Reagent: crow ginseng refreshment side (WSXN) and ginseng and fleece-flower root extract (RSSX), ginsenoside extract (ERS), Radix Polygoni Multiflori extract (EHSW), positive control Folium Ginkgo extract (EGB761) are provided by the Chinese medicine academy of Guangdong Pharmaceutical University, and each reagent is used 1% sodium carboxymethyl cellulose (CMC-Na) and is mixed with suspension oral gavage.
Ginsenoside Rg1, Re, Rb1 reference substance: (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number is respectively 110703-200424,110754-200421,110704-200420).
Reagent: chromatographically pure methanol, acetonitrile, phosphoric acid, formic acid, dehydrated alcohol, etc. be German import.Water is ultra-pure water (distilled water passes through Millipore ultra-pure water systems produce again).
Instrument: three grades of quadrupole mass spectrometers of the AB I4000 Q TRAP of American AB company, be furnished with electric spray ion source (ESI); U.S. Agilent company-1100 liquid chromatographic system; U.S. Waters solid-phase extracting instrument, Waters Oasis HLB post.
Laboratory animal: the Sprague-Dawley rat of male and female half and half, body weight 220 ~ 250g, the cleaning grade standard provides the (quality certification number: Guangdong probatio inspectionem pecuoarem word 2008A053 number) by Nanfang Medical Univ's Experimental Animal Center.4 one cages.Temperature is controlled at 25 ℃ ± 1 ℃, and light controlled for 12 little time, and 12 hours dark, freely drinks water and the edible animal feedstuff.Raise and begin experiment after 3 days.
(2) test method
Administration: rat is divided into 6 groups of WSXN, RSSW, ERS, EHSW and Folium Ginkgo extract positive control (EGB761) and normal controls at random, every group 6, gastric infusion WSXN 120 mg/kg, RSSW 100mg/kg, ERS 50 mg/kg, EHSW 50 mg/kg, EGb761 20 mg/kg and normal saline are adjusted drug level by the capacity of 0.2mL/100g (body constitution amount) with solvent respectively.Wherein WSXN, RSSW and ERS three are contained the Radix Ginseng total saponins of same dose 15mg/kg; WSXN, RSSW and EHSW three are contained the stilbene glucoside of same dose 10mg/kg, and WSXN and EGB761 group contain equivalent EGB(Folium Ginkgo extract) (12mg/kg).Respectively at after the administration 0,30,60,120, the 240min femoral artery gets the about 5mL of blood, the femoral artery blood-letting in the test tube that heparin is handled, centrifuging and taking blood plasma.At once put to death animal after getting blood, get the homonymy cerebral cortex respectively ,-20 ℃ of refrigerators are saved to analysis.
Wherein, WSXN 120 mg/kg[are equivalent to (Radix Ginseng total saponins 15mg+ stilbene glucoside 10mg+EGB 20mg)/kg], RSSW 100mg/kg [being equivalent to (Radix Ginseng total saponins 15mg+stilbene glucoside 10mg)/kg], ERS 50 mg/kg (are equivalent to Radix Ginseng total saponins 15mg/kg), EHSW 50 mg/kg (being equivalent to stilbene glucoside 10mg/kg); EGB 20 mg/kg and normal saline.
(3) determination of drug concentration
1. sample treatment
Blood plasma: use the Waters solid-phase extracting instrument, Waters Oasis HLB post with each the 2 mL activation of first alcohol and water, is got blood plasma 0.5 mL decompression constant speed by the SPE post successively, and water 2 mL clean pillar then, drain; Use methanol 2 mL eluting at last, collect eluent, dry up under 35 ℃ of water-bath nitrogen current, after residue fully dissolves with mobile phase 500 μ L, cross 0.45 μ m filter membrane, get 5 μ L sample introductions, carry out HPLC/MS/MS and analyze.
Cerebral tissue: about rat respective organization 0.1g, add 1. 0mL ultra-pure waters, after organizing pulverizer to make homogenate at a high speed, 5 min that vibrate, centrifugal 5 min of 18 000 r/min get supernatant 0.5 mL decompression constant speed by the SPE post, water 2 mL clean pillar then, drain; Use methanol 2 mL eluting at last, collect eluent, dry up under 35 ℃ of water-bath nitrogen current, after residue fully dissolves with mobile phase 500 μ L, cross 0.45 μ m filter membrane, get 5 μ L sample introductions, carry out HPLC/MS/MS and analyze.
2. analysis condition
Chromatographic condition: chromatographic column is RESTEK Pinnacle II C18 post (50 mm * 211 mm, 5 μ m); Column temperature: 20 ℃; Flow velocity is 200 μ L/min; Mobile phase is A water (volume fraction 0.5 ‰ formic acid), B acetonitrile (volume fraction 0.5 ‰ formic acid), gradient elution (0～12 min, Mobile phase B volume fraction 20%～50%; 12～12.1min, Mobile phase B volume fraction 50%～20%; 12.1～21 min, Mobile phase B volume fraction 20%).
Mass spectrum condition: electron spray ESI ion source, gas curtain gas is 10 psi, atomization gas (GAS1) is 40 psi, the auxiliary gas (GAS2) of heating is 40 psi, collision gas CAD is Medium, and spray voltage IS is 5.5 Kv, and atomization temperature is 500 ℃, detection mode is a cation polyion reaction detection (MRM), and the ion that is used for quantitative analysis is M/zRg 1832.8 → 643.6; Re 969.8 → 789.7; Rb 11132.1 → 365.3.
3. the HPLC method is measured stilbene glucoside content in blood plasma and the brain tissue homogenate
Adopting C18 chromatographic column (250 mm * 4.6 mm, 5 μ m), is interior mark with polygonin, and acetonitrile: methanol: 0. 1% glacial acetic acid (12:10:78) is a mobile phase, detects wavelength 320 nm, and flow velocity is 1.0 mL/min.
(4) result and discussion
Rb1, Rg1 and the concentration of Re in rat cerebral tissue that each organizes different time points see Table 14 ~ 17.
Table 14 as seen, concentration ratio RSSW and the ERS group of the Rb1 of the different time points of WSXN group in rat cerebral tissue significantly raises, and do not detect Rb1 in the rat cerebral tissue of normal control and EGB group different time points.The existence that EGB is described can promote that significantly Rb1 enters cerebral tissue in the WSXN side, helps bringing into play in cerebral tissue drug action.
The concentration of the Rb1 that table 14 is respectively organized different time points in rat cerebral tissue (ng/g,
Figure 549629DEST_PATH_IMAGE001
± S)
Time point (min) 0 30 60 120 240
Normal group - - - - -
EGB - - - - -
ERS - 2.4±1.8 4.6±1.5 5.9±1.3 6.5±2.1
RSSW - 2.5±1.5 4.8±1.5 6.2±1.4 7.2±1.9
WSXN - 5.3±2.5 ★★## 8.2±1.3 ★★## 10.3±2.3 ★★## 11.6±1.8 ★★##
★ ★Compare with the ERS group, P＜0.01; ##Compare with the RSSW group, P＜0.01.
By table 15 as seen, concentration ratio RSSW and the ERS group of the Rg1 of WSXN group different time points in rat cerebral tissue significantly raises, and do not detect Rg1 in the rat cerebral tissue of normal control and EGB group different time points.The existence that EGB is described can promote that significantly Rg1 enters cerebral tissue in the WSXN side, helps bringing into play in cerebral tissue drug action.
The concentration of the Rg1 that table 15 is respectively organized different time points in rat cerebral tissue (ng/g,
Figure 241642DEST_PATH_IMAGE001
± S)
Time point (min) 0 30 60 120 240
Normal group - - - - -
EGB - - - - -
ERS - 2.2±1.4 4.3±1.4 5.3±1.4 6.1±1.6
RSSW - 2.3±1.3 4.5±1.5 5.6±1.6 6.4±1.5
WSXN - 5.1±2.1 ★★## 7.8±1.4 ★★## 9.3±2.1 ★★## 10.1±1.9 ★★##
★ ★Compare with the ERS group, P＜0.01; ##Compare with the RSSW group, P＜0.01.
By table 16 as seen, concentration ratio RSSW and the ERS group of the Re of WSXN group different time points in rat cerebral tissue significantly raises, and do not detect Re in the rat cerebral tissue of normal control and EGB group different time points.The existence that EGB is described can promote that significantly Re enters cerebral tissue in the WSXN side, helps bringing into play in cerebral tissue drug action.
The concentration of the Re that table 16 is respectively organized different time points in rat cerebral tissue (ng/g,
Figure 978654DEST_PATH_IMAGE001
± S)
Time point (min) 0 30 60 120 240
Normal group - - - - -
EGB - - - - -
ERS - 1.1±0.8 1.6±1.0 2.5±1.3 2.8±1.1
RSSW - 1.2±0.6 1.8±0.8 3.0±0.8 3.2±1.0
WSXN - 1.5±0.5 ★★## 3.2±1.0 ★★## 4.3±1.3 ★★## 4.8±1.1 ★★##
★ ★Compare with the ERS group, P＜0.01; ##Compare with the RSSW group, P＜0.01.
Table 17 as seen, concentration ratio RSSW group and the EHSW group of the stilbene glucoside of the different time points of WSXN group in rat cerebral tissue significantly raises, and normal control and EGB organize in the rat cerebral tissue of different time points and do not detect stilbene glucoside.The existence that EGB is described can promote that significantly stilbene glucoside enters cerebral tissue in the WSXN side, helps bringing into play in cerebral tissue drug action.
Table 17 different time points respectively organize stilbene glucoside in the rat cerebral tissue concentration (ng/g,
Figure 688990DEST_PATH_IMAGE001
± S)
Time (min) 0 30 60 120 240
Normal group - - - - -
EGB - - - - -
ERS - - - - -
EHSW - 50.2±6.8 133.6±12.5 208.9±19.1 265.3±32.1
RSSW - 65.5±12.5 158.3±26.5 250.6±38.1 317.5±35.5
WSXN - 105.3±15.2 ★★## 208.2±31.3 ★★## 373.3±42.3 ★★## 480.6±51.3 ★★##
★ ★Compare with EGB group and EHSW group, P＜0.01; ##Compare with the RSSW group, P＜0.01
Embodiment 15 crow ginseng refreshment sides are to the influence of vascular dementia
(1) clinical data and method
The case situation: all case all is the department of neurology inpatient, male's 70 examples, women's 20 examples; 58 ~ 76 years old age, average 63.9 years old; The course of disease is the shortest 1 year, and is the longest 10 years, average 3.5 years.Through clinical examination, the test of neural scale, and through head CT or MR prover.
Diagnostic criteria: adopt the diagnostic criteria of DSM-IV medium vessels dementia.
Diagnostic criteria: adopt the diagnostic criteria of DSM-IV medium vessels dementia.
Exclusion standard: primary disease and Hai Jinsiji ischemia index scales (HIS) such as serious nerve, blood, endocrine are arranged, total points 18 minutes, score＜7 are divided into alzheimer disease person.
Scale is selected: 1 U.S.'s simple intelligent scale, and total points 30 minutes is if score＜16 minute person is a disturbance of intelligence; 2 Japanese Chang Gu river Dementia scale, total points 30 minutes is if score＜16 minute person is dull-witted the establishment; 3 Hai Jinsiji ischemia index scales (HIS), if total points 18 minutes is score〉person was a vascular dementia 7 minutes, score＜7 minute person is an alzheimer disease.
Traditional Chinese medical science cardinal symptom: with reference to new Chinese medicine clinical research guideline; The clinical research guideline of new Chinese medicine treatment senile dementia.
In conjunction with clinical experience, with following symptom as observation index: dull expression, dysphonia, or speaking scarcely,taciturn or language perversion, forgetful, be insomnia dizziness, headache, body of the tongue petechia.
Criterion of therapeutical effect: adopt the Comprehensive Assessment method, comment content with the change of aspects such as the intellectual status before and after the patient treatment, traditional Chinese medical science cardinal symptom, sign as combining, and change into emphasis with intelligence.Cured person Chang Gu river Dementia scale test score is increased to normal value, and produce effects person's score increased more than 5 minutes, and responder's score increases less than 5 minutes, and nonresponder's score does not only have increase and descends on the contrary.
Administrated method: make drop pill (seeing embodiment 6) with WSXN prescription, morning, noon and afternoon every day are respectively obeyed 10, and 2 months was 1 course of treatment, and all 3 courses of treatment of medication, other cerebral vasodilators medicine of stopping using therebetween, brain cell metabolic drug, function of nervous system regulate medicine.Folium Ginkgo extract sheet (Ginaton tablets) 40mg*20 sheet/box, one day 2-3 time, each 1-2 sheet.
The ginseng and fleece-flower root capsule is produced by Guiyang Medical College pharmaceutical Co. Ltd, adorns 0.3 gram for every, and is oral, one time 2,3 times on the one, taking medicine before meal is used.
(2) curative effect and result
The scale integration changes before and after the treatment: (seeing Table 18,19,20,21)
Simple intelligent scale integration variation before and after table 18 treatment ( ± s)
The medicine group n Before the treatment After the treatment P
The WSXN drop pill 30 13.66±5.31 19.63±6.03 <0.05
Ginaton tablets 30 13.53±5.32 18.35±5.06 <0.05
Dementia scale integration variation in Chang Gu river before and after table 19 treatment (x ± s)
Medication name n Before the treatment After the treatment P
The WSXN drop pill 30 12.65±5.66 18.86±6.17 <0.05
Ginaton tablets 30 12.61±5.71 18.12±5.31 <0.05
Traditional Chinese medical science cardinal symptom changes: (seeing Table 20)
Cardinal symptom scoring variation before and after table 20 treatment (
Figure 298142DEST_PATH_IMAGE001
± s)
Cardinal symptom Medicine n Before the treatment After the treatment P
Dull expression The WSXN drop pill 30 3.33±0.52 2.35±0.82 <0.01
Dull expression Ginaton tablets 30 3.29±0.50 2.38±0.83 <0.01
Dysphonia The WSXN drop pill 29 3.36±0.66 2.26±0.91 <0.01
Dysphonia Ginaton tablets 29 3.31±0.65 2.35±0.93 <0.01
Forgetful being insomnia The WSXN drop pill 29 3.33±0.56 1.32±0.91 <0.01
Forgetful being insomnia Ginaton tablets 30 3.32±0.55 1.41±0.95 <0.01
Dizziness headache The WSXN drop pill 30 3.16±0.82? 1.33±0.87 <0.01
Dizziness headache Ginaton tablets 30 ?3.14±0.81 1.43±0.81 <0.01
The body of the tongue petechia The WSXN drop pill 30 2.16±0.43? 1.26±0.63 <0.01
The body of the tongue petechia Ginaton tablets 30 ?2.15±0.45 1.31±0.62 <0.01
Efficacy analysis: (seeing Table 21)
Two kinds of medicines of table 21 are added up the vascular dementia curative effect
Figure 573266DEST_PATH_IMAGE002
(3) conclusion
The WSXN drop pill is to the intelligence improvement effect of vascular dementia: as can be seen from Table 21,60 routine patients are through clothes WSXN drop pill and Ginaton tablets after 3 courses of treatment, the scale score of two medicines treatment all obviously increases (P＜0.01), illustrates all to regain the strength of memory, improve the intelligence effect.This active ingredient composition WSXN is 80.0% to the total effective rate of primary disease, although cured person is zero, produce effects 5 examples only, effective percentage accounts for more than half many (63.3%).This active ingredient composition curative effect is better slightly than Ginaton tablets.
(4) to the preventive and therapeutic effect of senile dementia
Crow ginseng XINGNAO DIWAN and the capsular efficacy analysis of ginseng and fleece-flower root: see Table 22
Two kinds of medicines of table 22 are added up the alzheimer disease curative effect
Figure 216737DEST_PATH_IMAGE003
Can know that from 22 tables crow ginseng XINGNAO DIWAN is to the total effective rate 77.1% of senile dementia, produce effects 17.1%, slightly higher than 70% total effective rate of medicine at present commonly used, and do not observe apparent side effect.
Conclusion: this product has the improvement effect to the hypophrenia of alzheimer disease, and as can be seen from Table 22,35 routine patients are through obeying crow ginseng refreshment side compositions after 2 courses of treatment, the scale score obviously increase ( P＜0.01), illustrated to regain the strength of memory, improve intelligence effect, and doing well,improvings such as dysphonia, insomnia, agitation, irritability, body of the tongue petechia are very obvious, to turbid phlegm blocking the clear orifices, the alzheimer disease of qi depression to blood stasis (AD disease) 32 routine effective percentage reach 77.1%.Curative effect is than the oral ginseng and fleece-flower root capsule height of taking, particularly but to symptom more remarkable treatment effect such as insomnia, agitation, irritabilities.
Clinical research is the result show, this product has significant anti-cerebral ischemia damnification, improves the learning and memory effect, and apoplexy sequela that the treatment cerebral ischemia is relevant and vascular dementia and senile dementia clinical efficacy are definite.

Claims (10)

1. a Chinese medicine composition that promotes neuranagenesis is characterized in that being made up of following component and parts by weight: the Radix Polygoni Multiflori 2 ~ 10, Radix Ginseng 1 ~ 10 and Folium Ginkgo 1 ~ 10.
2. according to the Chinese medicine composition of the described promotion neuranagenesis of claim 1, it is characterized in that forming: Radix Polygoni Multiflori extract 2 ~ 10, Radix Ginseng extract 1 ~ 10 and Folium Ginkgo extract 1 ~ 10 by following component and parts by weight.
3. according to the Chinese medicine composition of the described promotion neuranagenesis of claim 1, it is characterized in that forming: the Radix Polygoni Multiflori 2 ~ 5, Radix Ginseng 1 ~ 5 and Folium Ginkgo 1 ~ 5 by following component and parts by weight.
4. according to the Chinese medicine composition of the described promotion neuranagenesis of claim 3, it is characterized in that it being through C by the Radix Polygoni Multiflori 2 ~ 5 weight portions, Radix Ginseng 1 ~ 5 weight portion and Folium Ginkgo 1 ~ 5 bulk drugs thing 1 ~ 3The mixture of extract obtained composition after the alcohol extraction.
5. according to the Chinese medicine composition of the described promotion neuranagenesis of claim 4, it is characterized in that it being C by the Radix Polygoni Multiflori, Radix Ginseng and Folium Ginkgo 1 ~ 3Alcohol extract is formed, and the effective ingredient of compositions and weight ratio are: stilbene glucoside: the ginsenoside Rb1: the ginsenoside Rg1: the ginsenoside Rd: the ginsenoside Re: bilobalide: nimbecetin: Quercetin equals 1 ~ 18:1 ~ 10:1 ~ 10:1 ~ 2:1 ~ 4:1 ~ 4:1 ~ 2:1 ~ 2.
6. claim 1,3,4 or 5 described any one promote the preparation method of the Chinese medicine composition of neuranagenesis, it is characterized in that step is as follows:
Pass through C respectively after the material medicine Radix Polygoni Multiflori, Radix Ginseng and Folium Ginkgo taken by weighing by proportioning 1-3After the alcohol extraction, obtain C 1-3Ethanol extract merges total extract, and total extract obtains concentrated solution after concentrating, and concentrated solution is added on the macroporous adsorbent resin of having handled well, uses the Different concentrations of alcohol eluting successively, collects corresponding eluent, reclaims ethanol, drying; Or recovery ethanol, being condensed into the thick paste medicine, vacuum drying is pulverized, and obtains the final extract of three kinds of medical materials, and the final extract mixing with three kinds of medical materials promptly obtains described Chinese medicine composition.
7. according to the preparation method of the Chinese medicine composition of the described promotion neuranagenesis of claim 6, it is characterized in that described C 1-3Alcohol extraction is the C with 30 ~ 95 volume % 1-3Alcohol extraction 1 ~ 5 time, each C that extracts 1-3The alcoholic solution volume is 1 ~ 15 times of quality of medicinal material, and each extraction time is 5min ~ 5h;
Described C 1-3Alcohol is methanol, ethanol or propanol;
The amount ratio of described macroporous adsorbent resin and concentrated solution is that the 1kg macroporous resin adds concentrated solution 1 ~ 5L;
Described ethanol elution is with 50 ~ 95 volume % ethanol, consumption be every 1kg macroporous resin with 5 ~ 20 L alcoholic solution, with 1 ~ 20 ml/min flow velocity eluting, be eluted to colourless; Described macroporous resin is polyamides amine type resin or polystyrene weak-base anion-exchange resin.
8. described any one Chinese medicine composition of claim 1 ~ 5 promotes the medicine of neuranagenesis and/or the application in the health food in preparation.
9. described any one Chinese medicine composition of claim 1 ~ 5 is in preparation control cerebral ischemia disease and/or the medicine of senile dementia disease and/or the application in the health food.
10. application according to claim 9 is characterized in that described cerebral ischemia disease is apoplexy, cerebral thrombosis, arteriosclerosis, apoplexy sequela and/or vascular dementia;
Described senile dementia disease is constitutional alzheimer disease and/or vascular dementia.
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Publication number Priority date Publication date Assignee Title
WO2012175018A1 (en) * 2011-06-21 2012-12-27 广东药学院 Traditional chinese medicine composition for promoting nerve regeneration and preparation method and use thereof
CN103055014A (en) * 2012-12-31 2013-04-24 青岛华仁信息技术开发有限公司 Folium ginkgo health product for preventing senile dementia and preparation method thereof
CN105031212A (en) * 2015-07-07 2015-11-11 广东药学院 Drug for treating parkinson disease
CN106728015A (en) * 2016-12-07 2017-05-31 孟光 A kind of Chinese medicine preparation for nerve degenerative diseases and preparation method thereof
WO2018113027A1 (en) * 2016-12-22 2018-06-28 广东药科大学 Application of bilobalide as synergist in preparation of drugs for preventing cranial nerve injury diseases

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012175018A1 (en) * 2011-06-21 2012-12-27 广东药学院 Traditional chinese medicine composition for promoting nerve regeneration and preparation method and use thereof
CN103055014A (en) * 2012-12-31 2013-04-24 青岛华仁信息技术开发有限公司 Folium ginkgo health product for preventing senile dementia and preparation method thereof
CN103055014B (en) * 2012-12-31 2014-09-17 青岛华仁技术孵化器有限公司 Folium ginkgo health product for preventing senile dementia and preparation method thereof
CN105031212A (en) * 2015-07-07 2015-11-11 广东药学院 Drug for treating parkinson disease
CN106728015A (en) * 2016-12-07 2017-05-31 孟光 A kind of Chinese medicine preparation for nerve degenerative diseases and preparation method thereof
WO2018113027A1 (en) * 2016-12-22 2018-06-28 广东药科大学 Application of bilobalide as synergist in preparation of drugs for preventing cranial nerve injury diseases

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