CN101983637B - Radix trichosanthis saponin and application of radix trichosanthis saponin in preparing medicine for treating ischemic cerebrovascular diseases - Google Patents
Radix trichosanthis saponin and application of radix trichosanthis saponin in preparing medicine for treating ischemic cerebrovascular diseases Download PDFInfo
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Abstract
The invention discloses a radix trichosanthis saponin and application of radix trichosanthis saponin in preparing a medicine for treating ischemic cerebrovascular diseases. The extraction method of the radix trichosanthis saponin is as follows: a raw material radix trichosanthis is smashed and then water is added in the radix trichosanthis for ultrasonic extraction; the obtained extracting solution is extracted after extracting; the supernatant is separated and purified by macroporous absorption resins after extracting; further purification is carried out on the supernatant by adopting a silicagel column chromatography; elution is carried out by adopting water after purification to be in a transparent state; the elution is carried out by utilizing ethanol; and the collected ethanol eluent is successively subject to rotary evaporation drying vacuum drying in order to obtain the radix trichosanthis saponin. The radix trichosanthis saponin has the action of obviously prolonging the breath time and the breath times of a mouse after decollation, reducing cerebral edema, enhancing the survival rate and prolonging the survival time of an animal with cerebral ischemia, reducing the cerebral infarction area, restraining the formation of thrombosis in vivo and the like, and the toxin or side effect is less. Therefore, the obtained radix trichosanthis saponin of the invention has good treatment action on the ischemic cerebrovascular diseases.
Description
Technical field
[0001] the present invention relates to a kind of method for distilling and relevant purposes thereof of Radix Trichosanthis, particularly relate to a kind of Radix Trichosanthis saponin and the purposes in preparation treatment ischemic cerebrovascular medicine thereof.
Background technology
[0002] cerebrovascular is to cause one of human three dead big diseases, in the world, annual dead about 4,600,000 people, there are the high and high characteristics of disability rate of sickness rate again in it simultaneously.Cerebrovascular disease is divided into ischemic and hemorrhagic apoplexy.The former is clinical morely to see, accounts for whole cerebrovascular patients' 70～80%.Owing to reasons such as cerebral arteriosclerosis, make the cerebral arteries luminal stenosis, blood flow reduces or total blockage, brain blood circulation obstacle, the impaired and a series of symptoms that take place of cerebral tissue.The latter is many by due to the factors such as long-term hypertension, cerebrovascular malformation, intracranial aneurysm, accounts for about 20～30% of cerebrovascular.Because normal pressure is compeled cerebral tissue behind the intracranial hemorrhage, blood circulation is obstructed, and patient shows symptoms such as intracranial hypertension, disturbance of consciousness.Be primarily aimed at during ischemic cerebrovascular in treatment at present: anticoagulant therapy, thromboembolism treatment, cerebral protective agent, fall fine treatment, calcium ion antagonist treatment, antiplatelet aggregation treatment, remove methods such as free radical treatment.But the treatment target spot of these medicines is single, although certain curative effect is arranged, does not also have a kind of specific drug up to now.Because the incidence and development of cerebrovascular disease mechanism is complicated, relate to the unusual of multisystem too many levels.And Chinese medicine contains plurality of active ingredients, therefore when treatment, can play a role at multi-level, many target spots, too many levels simultaneously, and toxic and side effects is little.At present, the conventional Chinese medicine that is used to treat cerebrovascular disease clinically has Folium Ginkgo extract, Radix Salviae Miltiorrhizae extract, arasaponin etc.
Radix Trichosanthis is the dry root of cucurbitaceous plant Fructus Trichosanthis, sweet in the mouth, little hardship, and flavor is slightly cold, and has the effect of clearing away heat-fire, promoting the production of body fluid to quench thirst, detumescence and apocenosis.Mainly from Fructus Trichosanthis (Trichosanthes kirilowiiMax) and trichosanthes rosthornii Harms Trichosanthes rosthornii Harm), and Fructus Trichosanthis and trichosanthes rosthornii Harms are all 2010 editions, and " Chinese pharmacopoeia is recorded.But be not limited to southern Fructus Trichosanthis (T.da miaoshanensis C.Y Cheng et Yueh), big sub-Fructus Trichosanthis (T.truncata C.B.Clarke), Fructus Trichosanthis Cucumeroidis (T.cucumeroides (Ser.) Maxim), Fructus Trichosanthis anguinae (T.anguinaL.), trichosanthes japonica Regel (T.kirilowii Maxim Var japonica (Miq.) kitam), trichosanthes bracteataVoigy (T.tricuspidata Lour.), different strain Fructus Trichosanthis (T.dioica Roxb.), phoenix melon (T.integrifolia (Roxb.) kur) etc.Modern study shows: Fructus Trichosanthis contains chemical compounds such as multiple flavonoid, triterpenes and sterol; Have effects such as coronary artery dilator, raising hypoxia-bearing capability; And saponin has multiple effects such as antioxidation, antiplatelet aggregation, thereby performance valuable pharmacological effect in the control cardiovascular and cerebrovascular disease.Systematic study through to Radix Trichosanthis finds that the Radix Trichosanthis saponin is the effective site of Radix Trichosanthis, but does not see that so far the Radix Trichosanthis saponin is used for treating the relevant report that cerebrovascular disease medicaments such as cerebral ischemia, Cerebral Ischemia, cerebral infarction are used in preparation.
Summary of the invention
[0003] the present invention's technical problem that will solve is: a kind of Radix Trichosanthis saponin and the purposes in preparation treatment ischemic cerebrovascular medicine thereof are provided.The present invention passes through to extract to the raw material Radix Trichosanthis; Obtain Radix Trichosanthis saponin live part; The result shows through zoopery, the Radix Trichosanthis saponin extract that obtains through the present invention have significant prolongation mice broken end back breathing time with frequency of respiration, reduce cerebral edema, increase the survival rate of cerebral ischemia animal and prolong life span, reduce cerebral infarct size, the effects such as formation of inhibition thrombus in vivo.Thereby the Radix Trichosanthis saponin extract that the present invention obtains has therapeutical effect preferably to ischemic cerebrovascular.
Technical scheme of the present invention is:
The present invention provides a kind of Radix Trichosanthis saponin, and said Radix Trichosanthis saponin extracts through following method and obtains:
A, the raw material Radix Trichosanthis is pulverized; Pulverize the back and cross 20～60 mesh sieves, add entry in the Radix Trichosanthis after pulverizing, the addition of water is 10～30 times of pulverizing back Radix Trichosanthis; Adopt the ultrasonic extraction device to carry out supersound extraction after adding entry; Extraction time is 5～30min, and extraction time is 2～4 times, and extracting solution is merged;
B, the extracting solution that step a merging is obtained adopt abstraction instrument to extract; Obtain supernatant after the extraction; The gained supernatant is carried out separation and purification through macroporous adsorbent resin earlier, then adopt silica gel column chromatography to be further purified, the water elution that behind the purification its sample is adopted 5～10 times of column volumes earlier is to clarification; The ethanol elution of 4～8 times of column volumes of reuse is collected ethanol elution;
C, with the ethanol elution that step b collects, adopt Rotary Evaporators to carry out drying, obtain paste Radix Trichosanthis saponin after the drying, at last paste Radix Trichosanthis saponin is carried out vacuum drying, pulverizing, obtain powdery Radix Trichosanthis saponin.
According to Radix Trichosanthis saponin recited above, the frequency of the device of ultrasonic extraction described in the step a is 20～40kHz, and power is 300～800W.
According to Radix Trichosanthis saponin recited above, when adopting abstraction instrument to extract described in the step b, the extractant of employing is any in petroleum ether, n-butyl alcohol, chloroform and the ethyl acetate; When adopting abstraction instrument to extract described in the step b, the extracting solution that the extractant of employing and merging obtain between the two the volume ratio of addition be 1～50: 1.
According to Radix Trichosanthis saponin recited above, abstraction instrument described in the step b adopts Ninson ultrasonic extraction appearance.
According to Radix Trichosanthis saponin recited above, the model of macroporous adsorbent resin described in the step b is D101, D101B, D201, D301, D3520; AB-8, DM130, X-5, XDA-1, XDA-1B, XDA-7; H-20, H-30, H-40, H-60, HPD-100; HPD-300, HPD-600, NK-2, NKA-2, any among NK-9 and the WLD; Said macroporous adsorbent resin and the supernatant volume ratio between the two is 4～10: 1;
When said employing silica gel column chromatography was further purified, its silica gel order number was 60～300 orders; Adopt " chloroform-methanol-water " three's mixed liquor to carry out eluting as solvent when said employing silica gel column chromatography is further purified, elution flow rate is 8～12ml/min; Mixed volume ratio between said " chloroform-methanol-water " three is 50～85: 14～40: 1～10.
According to Radix Trichosanthis saponin recited above, when adopting the water elution of 5～10 times of column volumes described in the step b, its elution flow rate is 0.3～5ml/min; During with the ethanol elution of 4～8 times of column volumes, its elution flow rate is 0.3～5ml/min described in the step b, and alcoholic acid volumetric concentration is 20～95%.
According to Radix Trichosanthis saponin recited above, when adopting Rotary Evaporators to carry out drying described in the step c, pressure is 0.01～1Mpa, and baking temperature is 40～80 ℃, and the rotary speed of Rotary Evaporators is 1～120rmp, and rotary evaporation drying time is 2～5 hours;
When described in the step c paste Radix Trichosanthis saponin being carried out vacuum drying, vacuum is 0.07～0.09Mpa, and baking temperature is 50～80 ℃, and be 2～4 hours drying time.
The purposes of a kind of Radix Trichosanthis saponin recited above in preparation treatment ischemic cerebrovascular medicine.
According to the purposes of Radix Trichosanthis saponin recited above in preparation treatment ischemic cerebrovascular medicine, said Radix Trichosanthis saponin is Radix Trichosanthis saponin or Radix Trichosanthis saponin monomer.
According to the purposes of Radix Trichosanthis saponin recited above in preparation treatment ischemic cerebrovascular medicine, said Radix Trichosanthis saponin is ligroin extraction, n-butanol extract, chloroform extract or ethyl acetate extract.
Positive beneficial effect of the present invention:
1, can from Radix Trichosanthis, extract the Radix Trichosanthis saponin through technical scheme of the present invention, and the Radix Trichosanthis saponin that is extracted, its purity is greater than 90%.
2, the present invention is through a large amount of zooperies, and its experimental result shows: the Radix Trichosanthis saponin that the present invention extracts from Radix Trichosanthis has significant prolongation mice broken end back breathing time and increase frequency of respiration, reduces brain water content, increases the survival rate of cerebral ischemia animal and prolongs life span, minimizing cerebral infarct size, change hemostatic system, protection brain neuron, suppresses the effects such as formation of thrombus in vivo.Thereby the Radix Trichosanthis saponin that the present invention extracts from Radix Trichosanthis has good therapeutical effect to cerebral ischemia, cerebral thrombosis, the cerebral infarction ischemic cerebrovascular of waiting indefinitely, and the neuron after ischemic cerebrovascular is taken place has good protective effect; And Radix Trichosanthis saponin toxicity is low, side effect is little, (seeing zoopery embodiment in the specific embodiment for details) safe in utilization, quality controllable.
Four, the specific embodiment:
With embodiment the present invention is described further below, but the present invention is not limited to these embodiment.
Radix Trichosanthis saponin embodiment:
Embodiment 1:
Radix Trichosanthis saponin of the present invention obtains through following method extraction, and its detailed step is:
A, the raw material Radix Trichosanthis is pulverized, pulverized the back and cross 20～60 mesh sieves, add entry in the Radix Trichosanthis after pulverizing; The addition of water is 20 times of Radix Trichosanthis after pulverizing; Add and adopt the ultrasonic extraction device after the entry (frequency of said ultrasonic extraction device is 30kHz, and power is 400～450W) to carry out supersound extraction, and extraction time is 15～20min; Extraction time is 3 times, and extracting solution is merged;
B, the extracting solution that step a merging is obtained adopt abstraction instrument (adopting Ninson ultrasonic extraction appearance) to extract; The extractant that adopts is a petroleum ether; The extracting solution that petroleum ether extraction solvent and merge obtains between the two the volume ratio of addition be 20: 1; Obtain supernatant after the extraction; The gained supernatant is earlier carried out separation and purification through macroporous adsorbent resin (model of macroporous adsorbent resin is D101, and macroporous adsorbent resin and the supernatant volume ratio between the two is 6: 1), then adopt silica gel column chromatography to be further purified that (silica gel order number was 60～300 orders when the employing silica gel column chromatography carried out purification; Adopt " chloroform-methanol-water " three's mixed liquor to carry out eluting as solvent when adopting silica gel column chromatography to carry out purification, elution flow rate is 10ml/min; Mixed volume ratio between said " chloroform-methanol-water " three is 83: 16: 1); Water elution to the clarification of behind the purification its sample being adopted 8 times of column volumes earlier is (when adopting the water elution of 8 times of column volumes; Its elution flow rate is 1～2ml/min), and (during with the ethanol elution of 6 times of column volumes, its elution flow rate is 2～3ml/min to the ethanol elution of 6 times of column volumes of reuse; Alcoholic acid volumetric concentration is 50～60%), collect ethanol elution;
C, with the ethanol elution that step b collects, (pressure is 0.6Mpa, and baking temperature is 50 ℃ to adopt Rotary Evaporators to carry out drying; The rotary speed of Rotary Evaporators is 60rmp, and rotary evaporation drying time is 3 hours), obtain paste Radix Trichosanthis saponin after the drying; At last paste Radix Trichosanthis saponin is carried out vacuum drying (vacuum is 0.085Mpa, and baking temperature is 55 ℃, and be 3 hours drying time), pulverizes; Obtain powdery Radix Trichosanthis saponin, its purity is greater than 90%.
Embodiment 2: basic identical with embodiment 1, difference is:
Among the step a: the addition of water is 10 times of Radix Trichosanthis after pulverizing; Add and adopt the ultrasonic extraction device after the entry (frequency of said ultrasonic extraction device is 20kHz, and power is 300～350W) to carry out supersound extraction, and extraction time is 5～10min; Extraction time is 2 times, and extracting solution is merged;
Among the step b: the extractant of employing is a n-butyl alcohol; The extracting solution that n-butanol extraction solvent and merge obtains between the two the volume ratio of addition be 30: 1; Obtain supernatant after the extraction; (model of macroporous adsorbent resin is D301 through macroporous adsorbent resin with the gained supernatant; Macroporous adsorbent resin and the supernatant volume ratio between the two is 8: 1) carry out separation and purification, then adopt silica gel column chromatography to be further purified that (silica gel order number is 60～300 orders when adopting silica gel column chromatography to carry out purification; Adopt " chloroform-methanol-water " three's mixed liquor to carry out eluting as solvent when adopting silica gel column chromatography to carry out purification, elution flow rate is 8ml/min; Mixed volume ratio between said " chloroform-methanol-water " three is 76: 22: 2); Water elution to the clarification of behind the purification its sample being adopted 5 times of column volumes earlier is (when adopting the water elution of 5 times of column volumes; Its elution flow rate is 0.5～1ml/min), and (during with the ethanol elution of 4 times of column volumes, its elution flow rate is 0.5～1ml/min to the ethanol elution of 4 times of column volumes of reuse; Alcoholic acid volumetric concentration is 20～30%), collect ethanol elution;
Among the step c: (pressure is 0.5Mpa, and baking temperature is 60 ℃, and the rotary speed of Rotary Evaporators is 65rmp to adopt Rotary Evaporators to carry out drying; Rotary evaporation drying time is 3 hours); At last paste Radix Trichosanthis saponin is carried out vacuum drying (vacuum is 0.07Mpa, and baking temperature is 80 ℃, and be 3 hours drying time), pulverizes; Obtain powdery Radix Trichosanthis saponin, its purity is greater than 90%.
Embodiment 3: basic identical with embodiment 1, difference is:
Among the step a: the addition of water is 30 times of Radix Trichosanthis after pulverizing; Add and adopt the ultrasonic extraction device after the entry (frequency of said ultrasonic extraction device is 40kHz, and power is 750～800W) to carry out supersound extraction, and extraction time is 25～30min; Extraction time is 4 times, and extracting solution is merged;
Among the step b: the extractant of employing is a chloroform; The extracting solution that chloroform extraction solvent and merge obtains between the two the volume ratio of addition be 40: 1; Obtain supernatant after the extraction; The gained supernatant is carried out separation and purification through macroporous adsorbent resin (model of macroporous adsorbent resin is AB-8, and macroporous adsorbent resin and the supernatant volume ratio between the two is 4: 1), then adopt silica gel column chromatography to be further purified that (silica gel order number is 60～300 orders when adopting silica gel column chromatography to carry out purification; Adopt " chloroform-methanol-water " three's mixed liquor to carry out eluting as solvent when adopting silica gel column chromatography to carry out purification, elution flow rate is 12ml/min; Mixed volume ratio between said " chloroform-methanol-water " three is 68.5: 27.5: 4); Water elution to the clarification of behind the purification its sample being adopted 10 times of column volumes earlier is (when adopting the water elution of 10 times of column volumes; Its elution flow rate is 4～5ml/min), and (during with the ethanol elution of 8 times of column volumes, its elution flow rate is 4～5ml/min to the ethanol elution of 8 times of column volumes of reuse; Alcoholic acid volumetric concentration is 30～40%), collect ethanol elution;
Among the step c: (pressure is 1Mpa, and baking temperature is 40 ℃, and the rotary speed of Rotary Evaporators is 40rmp to adopt Rotary Evaporators to carry out drying; Rotary evaporation drying time is 5 hours); At last paste Radix Trichosanthis saponin is carried out vacuum drying (vacuum is 0.08Mpa, and baking temperature is 60 ℃, and be 4 hours drying time), pulverizes; Obtain powdery Radix Trichosanthis saponin, its purity is greater than 90%.
Embodiment 4: basic identical with embodiment 1, difference is:
Among the step a: the addition of water is 15 times of Radix Trichosanthis after pulverizing; Add and adopt the ultrasonic extraction device after the entry (frequency of said ultrasonic extraction device is 25kHz, and power is 400～450W) to carry out supersound extraction, and extraction time is 10～15min; Extraction time is 3 times, and extracting solution is merged;
Among the step b: the extractant of employing is an ethyl acetate; The extracting solution that ethyl acetate extraction solvent and merge obtains between the two the volume ratio of addition be 10: 1; Obtain supernatant after the extraction; (model of macroporous adsorbent resin is XDA-1 through macroporous adsorbent resin with the gained supernatant; Macroporous adsorbent resin and the supernatant volume ratio between the two is 5: 1) carry out separation and purification, then adopt silica gel column chromatography to be further purified that (silica gel order number is 60～300 orders when adopting silica gel column chromatography to carry out purification; Adopt " chloroform-methanol-water " three's mixed liquor to carry out eluting as solvent when adopting silica gel column chromatography to carry out purification, elution flow rate is 11ml/min; Mixed volume ratio between said " chloroform-methanol-water " three is 65: 30: 5); Water elution to the clarification of behind the purification its sample being adopted 6 times of column volumes earlier is (when adopting the water elution of 6 times of column volumes; Its elution flow rate is 1～2ml/min), and (during with the ethanol elution of 5 times of column volumes, its elution flow rate is 1～2ml/min to the ethanol elution of 5 times of column volumes of reuse; Alcoholic acid volumetric concentration is 70～95%), collect ethanol elution;
Among the step c: (pressure is 0.1Mpa, and baking temperature is 80 ℃, and the rotary speed of Rotary Evaporators is 100rmp to adopt Rotary Evaporators to carry out drying; Rotary evaporation drying time is 2 hours); At last paste Radix Trichosanthis saponin is carried out vacuum drying (vacuum is 0.09Mpa, and baking temperature is 50 ℃, and be 2 hours drying time), pulverizes; Obtain powdery Radix Trichosanthis saponin, its purity is greater than 90%.
Embodiment 5: basic identical with embodiment 1, difference is:
Among the step a: the addition of water is 25 times of Radix Trichosanthis after pulverizing; Add and adopt the ultrasonic extraction device after the entry (frequency of said ultrasonic extraction device is 35kHz, and power is 700～750W) to carry out supersound extraction, and extraction time is 20～25min; Extraction time is 4 times, and extracting solution is merged;
Among the step b: the extractant of employing is a n-butyl alcohol; The extracting solution that n-butanol extraction solvent and merge obtains between the two the volume ratio of addition be 5: 1; Obtain supernatant after the extraction; (model of macroporous adsorbent resin is H-30 through macroporous adsorbent resin with the gained supernatant; Macroporous adsorbent resin and the supernatant volume ratio between the two is 6: 1) carry out separation and purification, then adopt silica gel column chromatography to be further purified that (silica gel order number is 60～300 orders when adopting silica gel column chromatography to carry out purification; Adopt " chloroform-methanol-water " three's mixed liquor to carry out eluting as solvent when adopting silica gel column chromatography to carry out purification, elution flow rate is 9ml/min; Mixed volume ratio between said " chloroform-methanol-water " three is 61: 33: 6); Water elution to the clarification of behind the purification its sample being adopted 7 times of column volumes earlier is (when adopting the water elution of 7 times of column volumes; Its elution flow rate 2～3ml/min), (during with the ethanol elution of 6 times of column volumes, its elution flow rate is 2～3ml/min to the ethanol elution of 6 times of column volumes of reuse; Alcoholic acid volumetric concentration is 60～70%), collect ethanol elution;
Among the step c: (pressure is 0.8Mpa, and baking temperature is 50 ℃, and the rotary speed of Rotary Evaporators is 120rmp to adopt Rotary Evaporators to carry out drying; Rotary evaporation drying time is 3 hours); At last paste Radix Trichosanthis saponin is carried out vacuum drying (vacuum is 0.09Mpa, and baking temperature is 70 ℃, and be 3 hours drying time), pulverizes; Obtain powdery Radix Trichosanthis saponin, its purity is greater than 90%.
Embodiment 6: basic identical with embodiment 1, difference is:
Among the step a: the addition of water is 26 times of Radix Trichosanthis after pulverizing; Add and adopt the ultrasonic extraction device after the entry (frequency of said ultrasonic extraction device is 38kHz, and power is 600～650W) to carry out supersound extraction, and extraction time is 25～30min; Extraction time is 4 times, and extracting solution is merged;
Among the step b: the extractant of employing is a chloroform; The extracting solution that chloroform extraction solvent and merge obtains between the two the volume ratio of addition be 50: 1; Obtain supernatant after the extraction; (model of macroporous adsorbent resin is HPD-300 through macroporous adsorbent resin with the gained supernatant; Macroporous adsorbent resin and the supernatant volume ratio between the two is 7: 1) carry out separation and purification, then adopt silica gel column chromatography to be further purified that (silica gel order number is 60～300 orders when adopting silica gel column chromatography to carry out purification; Adopt " chloroform-methanol-water " three's mixed liquor to carry out eluting as solvent when adopting silica gel column chromatography to carry out purification, elution flow rate is 10ml/min; Mixed volume ratio between said " chloroform-methanol-water " three is 54: 38.5: 7.5); Water elution to the clarification of behind the purification its sample being adopted 9 times of column volumes earlier is (when adopting the water elution of 9 times of column volumes; Its elution flow rate 3～4ml/min), (during with the ethanol elution of 7 times of column volumes, its elution flow rate is 3～4ml/min to the ethanol elution of 7 times of column volumes of reuse; Alcoholic acid volumetric concentration is 70～80%), collect ethanol elution;
Among the step c: (pressure is 0.05Mpa, and baking temperature is 80 ℃, and the rotary speed of Rotary Evaporators is 120rmp to adopt Rotary Evaporators to carry out drying; Rotary evaporation drying time is 5 hours); At last paste Radix Trichosanthis saponin is carried out vacuum drying (vacuum is 0.08Mpa, and baking temperature is 70 ℃, and be 3 hours drying time), pulverizes; Obtain powdery Radix Trichosanthis saponin, its purity is greater than 90%.
Embodiment 7: basic identical with embodiment 1, difference is:
Among the step a: the addition of water is 18 times of Radix Trichosanthis after pulverizing; Add and adopt the ultrasonic extraction device after the entry (frequency of said ultrasonic extraction device is 28kHz, and power is 500～550W) to carry out supersound extraction, and extraction time is 20～25min; Extraction time is 3 times, and extracting solution is merged;
Among the step b: the extractant of employing is a chloroform; The extracting solution that chloroform extraction solvent and merge obtains between the two the volume ratio of addition be 25: 1, obtain supernatant after the extraction, (model of macroporous adsorbent resin is NK-9 through macroporous adsorbent resin with the gained supernatant; Macroporous adsorbent resin and the supernatant volume ratio between the two is 6: 1) carry out separation and purification; Then adopt silica gel column chromatography to be further purified, behind the purification water elution of 8 times of column volumes of its sample elder generation employing is extremely clarified (when adopting the water elution of 8 times of column volumes, its elution flow rate 2～3ml/min); The ethanol elution of 6 times of column volumes of reuse is (during with the ethanol elution of 6 times of column volumes; Its elution flow rate is 3～4ml/min, and alcoholic acid volumetric concentration is 80～95%), collect ethanol elution;
Among the step c: (pressure is 0.3Mpa, and baking temperature is 70 ℃, and the rotary speed of Rotary Evaporators is 80rmp to adopt Rotary Evaporators to carry out drying; Rotary evaporation drying time is 4 hours); At last paste Radix Trichosanthis saponin is carried out vacuum drying (vacuum is 0.08Mpa, and baking temperature is 70 ℃, and be 3 hours drying time), pulverizes; Obtain powdery Radix Trichosanthis saponin, its purity is greater than 90%.
Zoopery embodiment:
Embodiment 1: the Radix Trichosanthis saponin is to the influence of mice broken end back breathing time and frequency of respiration
(1) experimental technique: select 45 of the male mices of body weight 20 ± 2g, be divided into 3 groups (n=15) at random.Drug group is irritated the Radix Trichosanthis saponin that clothes the present invention extracts, and dosage is 40mg/kg, and positive controls is irritated clothes nimodipine 22.5mg/kg, and the blank group is irritated the isopyknic distilled water of clothes, administration every day 1 time, successive administration 3 days.Broken end (2mm place behind the ear) observation mice begins the time that stops to panting and the number of times of opening one's mouth Zi broken end behind last administration 1h.
(2) experimental result: as shown in table 1
The influence of breathing time and frequency of respiration after table 1 Radix Trichosanthis saponin breaks end to mice (X ± S)
*Expression P＜0.01;
*Expression P＜0.05.
(3) conclusion: visible by above-mentioned table 1 experimental result, the prolongation mice broken end back breathing time and the frequency of respiration of the Radix Trichosanthis saponin ability highly significant that the present invention extracts.Experimental result proof Radix Trichosanthis saponin has protective effect to the cerebral ischemia animal.
Embodiment 2: the Radix Trichosanthis saponin is to the influence of mice broken end back brain water content and cerebral index
(1) experimental technique: select 60 of the male mices of body weight 20 ± 2g, be divided into 4 groups (n=15) at random.Drug group is irritated the Radix Trichosanthis saponin that clothes the present invention extracts, and dosage is 40mg/kg, and positive controls is irritated clothes nimodipine 22.5mg/kg, and sham operated rats, cerebral ischemia matched group are irritated the isopyknic distilled water of clothes, administration every day 1 time, successive administration 3 days.Broken end (2mm place behind the ear) behind last administration 1h is got brain, is weighed, is dried to constant weight and weigh once more after weighing, both differences are brain water content.Significance with difference between each group of t value check and the blank.
(2) experimental result: as shown in table 2
Table 2 Radix Trichosanthis saponin is to the influence of mice broken end back brain water content
*Expression P＜0.01;
*Expression P＜0.05.
(3) conclusion: the experimental result by table 2 is visible; Cerebral ischemic model group brain water content and cerebral index and sham operated rats relatively have significant difference; Brain water content increases; And Radix Trichosanthis saponin treatment group brain water content obviously reduces, and with matched group significant difference is arranged, and proves that the Radix Trichosanthis saponin can alleviate the cerebral edema of cerebral ischemia animal.
Embodiment 3: the Radix Trichosanthis saponin is to the influence of cerebral ischemia mice survival rate
(1) experimental technique: male 40 of kunming mice, body weight 20 ± 2g is divided into normal saline matched group, sham operated rats, Radix Trichosanthis saponin group and nimodipine group (positive drug group) at random; Drug group is irritated the Radix Trichosanthis saponin that clothes the present invention extracts, and dosage is 40mg/kg, and positive controls is irritated clothes nimodipine 22.5mg/kg; Matched group, sham operated rats are irritated the isopyknic distilled water of clothes, administration every day 1 time, successive administration 3 days; After 3.5% chloral hydrate anesthesia behind the last administration 1h, the cervical region median incision, passivity is separated bilateral carotid; And on two on bilateral carotid underlying line, it is subsequent use to beat untwisting, wait animal clear-headed after; The ligation bilateral carotid is cut off between two lines.Observe reaction of animals and death condition in the ligation 6h.
(2) experimental result: as shown in table 3
Table 3 Radix Trichosanthis saponin is to the influence of cerebral ischemia mice survival rate
*Expression P＜0.01;
*Expression P＜0.05.
(3) conclusion: the experimental result by table 3 is visible, and the Radix Trichosanthis saponin that the present invention extracts can increase the survival rate and prolongation life span of cerebral ischemia animal.
Embodiment 4: the Radix Trichosanthis saponin is to the effect of focal cerebral ischemia
(1) experimental technique: 20 of healthy adult male wistar rats, body weight 250 ± 20g, animal is divided into two groups at random, 10 every group.The ischemic control group: normal saline is irritated stomach; Radix Trichosanthis saponin group is except that the medication difference, and other measures are identical.The foundation of animal model: adopt two vascular occlusions to merge depletion method, cause acute cerebral ischemia.Laboratory animal is earlier with being fixed on the operating-table after the anesthetics anesthesia.Through the neck median incision, passivity is separated bilateral carotid, silk thread ligation suspension 30min, and the blood-letting 0.8mL that docks simultaneously unclamps silk ligature 10min then, unclamps behind the ligation bilateral common carotid arteries 30min once more.The postoperative water inlet of taking food.
Observation index:
1. blood coagulation system thrombin time (TT), prothrombin time (PT), activated partial thrombin time (APTT), fibrinogen assay (FBG).
2. the mensuration of brain infarction area: put to death postoperative certain hour blood sampling back, gets some rat brains, removes XIAONAO and brain stem, and the crown brain sheet that is cut to 0.3cm is observed and had or not ischemic focus.The brain sheet is immersed among the TTC, and formaldehyde fixed behind the constant temperature is utilized image pathological analysis system measurement infarct size.
3. pathology detect: after the brain sheet is dyed, under light microscopic, observe the pathological change of cerebral tissue.
(2) experimental result:
1., the testing result of hemostatic system: as shown in table 4.
The testing result of table 4 hemostatic system
*Expression P＜0.01;
*Expression P＜0.05
Conclusion: visible by table 4 experimental result; The treatment group is irritated and can be found out that TT, APTT, FBG content are high than matched group behind the stomach; After bilateral ligation causes the rat brain ischemia injury, TT, APTT are prolonged, and the reduction of FBG is compared significant difference is all arranged with matched group.
2. behind the focal cerebral ischemia in rats certain hour infarct size and medicine influence the result: as shown in table 5.
Table 5 cerebral infarction scope
*Expression P＜0.01;
*Expression P＜0.05
Conclusion: visible by table 5 experimental result, the brain infarction area of Radix Trichosanthis saponin treatment group is starkly lower than the ischemic control group, proves that the Radix Trichosanthis saponin can reduce the brain infarction area of cerebral ischemia animal.
3., pathological change: the matched group light microscopic is observed down and is found interstice's broadening, and edema has cavity to form, and neuronal cell nuclear dwindles, even dissolving disappears.And Radix Trichosanthis saponin treatment group can see that edema is light than matched group between tissue, and intercellular substance changes not obvious, and neuronal cell nuclear size is normal basically, and nuclear is than engrain, and the focus center has a little cavity to form.
Embodiment 5: the influence that the Radix Trichosanthis saponin forms platelet thrombus in the body
(1) experimental technique:
Healthy adult wistar rat, body weight 250 ± 20g, animal is divided into two groups at random, and 10 every group, experimental group gives Radix Trichosanthis saponin (40mg/kg), and matched group gives the equivalent normal saline.Anaesthetize behind last 1 administration 1h, fixing, separate trachea and insert endotracheal tube, separate RCCA and left external jugular vein.In polyethylene tube, put into a silk thread of having weighed.Normal saline with containing heparin is full of polyethylene tube.One end of pipe is inserted RCCA, and the other end inserts left external jugular vein, interrupts behind the open blood flow 15min, takes out silk thread and weighs, and calculates thrombus weight.
(2) experimental result:
The matched group thrombosis weighs 32.67 ± 8.74mg as a result, and Radix Trichosanthis saponin treatment group thrombosis weighs 20.99 ± 6.82mg.
(3) conclusion: the Radix Trichosanthis saponin can suppress thrombus in vivo and form.
Embodiment 6: acute toxicity testing
(1) experimental technique:
1., the acute toxicity testing of oral administration: get 40 of body weight 20 ± 2g mices, male and female half and half, the Radix Trichosanthis saponin that extracts with the present invention is by 4.8g/kg (being equivalent to crude drug 120g/kg), administration every day 3 times was observed 7 days then continuously.
2., the acute toxicity test of intraperitoneal injection: get 40 of body weight 20 ± 2g mices, male and female half and half, the Radix Trichosanthis saponin that extracts with the present invention is by 4.8g/kg (being equivalent to crude drug 120g/kg), administration every day 3 times was observed 7 days then continuously.
(2) experimental result: being equivalent to does not have dead mouse under the situation of consumption of 100 times of human body medications, does not record LD
50
(3) conclusion: the toxicity of Radix Trichosanthis saponin of the present invention is lower.
In sum, more than experiment and experimental result thereof can draw as drawing a conclusion:
The Radix Trichosanthis saponin has significant prolongation mice broken end back breathing time and increase frequency of respiration, reduces brain water content, increases the survival rate of cerebral ischemia animal and prolongs life span, minimizing cerebral infarct size, change hemostatic system, protection brain neuron, suppresses the effects such as formation of thrombus in vivo; So cerebral ischemia, cerebral thrombosis, the cerebral infarction ischemic cerebrovascular of waiting indefinitely is had good therapeutical effect; Neuron to after the ischemic cerebrovascular generation has good protective effect; And toxicity is low; Side effect is little, and is safe in utilization, quality controllable.
Claims (1)
1. the method for distilling of a Radix Trichosanthis saponin is characterized in that, said Radix Trichosanthis saponin extracts through following method and obtains:
A, the raw material Radix Trichosanthis is pulverized; Pulverize the back and cross 20～60 mesh sieves, add entry in the Radix Trichosanthis after pulverizing, the addition of water is 10～30 times of pulverizing back Radix Trichosanthis; Adopt the ultrasonic extraction device to carry out supersound extraction after adding entry; Extraction time is 5～30min, and extraction time is 2～4 times, and extracting solution is merged;
B, the extracting solution that step a merging is obtained adopt abstraction instrument to extract; Obtain supernatant after the extraction; The gained supernatant is carried out separation and purification through macroporous adsorbent resin earlier, then adopt silica gel column chromatography to be further purified, the water elution that behind the purification its sample is adopted 5～10 times of column volumes earlier is to clarification; The ethanol elution of 4～8 times of column volumes of reuse is collected ethanol elution;
C, with the ethanol elution that step b collects, adopt Rotary Evaporators to carry out drying, obtain paste Radix Trichosanthis saponin after the drying, at last paste Radix Trichosanthis saponin is carried out vacuum drying, pulverizing, obtain powdery Radix Trichosanthis saponin.
2. the method for distilling of Radix Trichosanthis saponin according to claim 1 is characterized in that: the frequency of the device of ultrasonic extraction described in the step a is 20～40 kHz, and power is 300～800W.
3. the method for distilling of Radix Trichosanthis saponin according to claim 1 is characterized in that: when adopting abstraction instrument to extract described in the step b, the extractant of employing is any in petroleum ether, n-butyl alcohol, chloroform and the ethyl acetate; When adopting abstraction instrument to extract described in the step b, the extracting solution that the extractant of employing and merging obtain between the two the volume ratio of addition be 1～50:1.
4. the method for distilling of Radix Trichosanthis saponin according to claim 1 is characterized in that: abstraction instrument described in the step b adopts Ninson ultrasonic extraction appearance.
5. the method for distilling of Radix Trichosanthis saponin according to claim 1 is characterized in that: the model of macroporous adsorbent resin described in the step b is D101, D101B, D201, D301, D3520; AB-8, DM130, X-5, XDA-1, XDA-1B, XDA-7; H-20, H-30, H-40, H-60, HPD-100; HPD-300, HPD-600, NK-2, NKA-2, any among NK-9 and the WLD; Said macroporous adsorbent resin and the supernatant volume ratio between the two is 4～10:1;
When said employing silica gel column chromatography was further purified, its silica gel order number was 60～300 orders; Adopt " chloroform-methanol-water " three's mixed liquor to carry out eluting as solvent when said employing silica gel column chromatography is further purified, elution flow rate is 8～12 ml/min; Mixed volume ratio between said " chloroform-methanol-water " three is 50～85 ︰, 14～40 ︰ 1～10.
6. the method for distilling of Radix Trichosanthis saponin according to claim 1 is characterized in that: when adopting the water elution of 5～10 times of column volumes described in the step b, its elution flow rate is 0.3～5ml/min; During with the ethanol elution of 4～8 times of column volumes, its elution flow rate is 0.3～5ml/min described in the step b, and alcoholic acid volumetric concentration is 20～95%.
7, the method for distilling of Radix Trichosanthis saponin according to claim 1; It is characterized in that: when adopting Rotary Evaporators to carry out drying described in the step c; Pressure is 0.01～1MPa; Baking temperature is 40～80 ℃, and the rotary speed of Rotary Evaporators is 1～120rmp, and rotary evaporation drying time is 2～5 hours;
When described in the step c paste Radix Trichosanthis saponin being carried out vacuum drying, vacuum is 0.07～0.09MPa, and baking temperature is 50～80 ℃, and be 2～4 hours drying time.
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| CN1840034A (en) * | 2006-01-12 | 2006-10-04 | 李其全 | Medicine for treating acute hematogenous osteomyelitis and preparation method thereof |
| CN101007147A (en) * | 2006-01-27 | 2007-08-01 | 广州王老吉药业股份有限公司 | A dispersible tablet of traditional Chinese medicine composition for treating gastrointestinal diseases and its preparation method |
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| CN1840034A (en) * | 2006-01-12 | 2006-10-04 | 李其全 | Medicine for treating acute hematogenous osteomyelitis and preparation method thereof |
| CN101007147A (en) * | 2006-01-27 | 2007-08-01 | 广州王老吉药业股份有限公司 | A dispersible tablet of traditional Chinese medicine composition for treating gastrointestinal diseases and its preparation method |
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| 陈颖等.天花粉总皂苷的提取及其清除DPPH自由基作用的研究.《药物生物技术》.2010,第17卷(第5期),第397-399页. * |
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