CN101983637A - 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
The present invention relates to a kind of extracting method 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
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 generation development mechanism complexity of cerebrovascular disease relates to the unusual of multisystem too many levels.And Chinese medicine contains plurality of active ingredients, therefore can play a role at multi-level, many target spots, too many levels simultaneously when treatment, and toxic and side effects is little.At present, the conventional Chinese medicine that is used for the treatment of 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 rosthorniiHarm), and Fructus Trichosanthis and trichosanthes rosthornii Harms be all 2010 editions " Chinese pharmacopoeia recorded.But be not limited to southern Fructus Trichosanthis (
T.da miaoshanensisC. Y. Cheng et Yueh), big sub-Fructus Trichosanthis (
T.truncataC. B. Clarke), Fructus Trichosanthis Cucumeroidis (
T. cucumeroides(Ser.) Maxim), Fructus Trichosanthis anguinae (T. anguinaL.), trichosanthes japonica Regel (
T. kirilowiiMaxim Var japonica (Miq.) kitam), trichosanthes bracteataVoigy (
T. tricuspidataLour.), different strain Fructus Trichosanthis (
T. dioicaRoxb.), the 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.Find that through systematic study 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 the treatment of the relevant report of using in the cerebrovascular disease medicaments such as cerebral ischemia, Cerebral Ischemia, cerebral infarction in preparation to Radix Trichosanthis.
Summary of the invention
The technical problem to be solved in the present invention 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, and the Radix Trichosanthis saponin extract that obtains by the present invention has significant prolongation mice broken end back breathing time and frequency of respiration, reduction cerebral edema, increases the survival rate of cerebral ischemia animal and prolong life span, minimizing cerebral infarct size, suppresses the effects such as formation of 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 invention provides a kind of Radix Trichosanthis saponin, described Radix Trichosanthis saponin extracts by the 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 Radix Trichosanthis after pulverizing, 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 by macroporous adsorbent resin earlier, then adopt silica gel column chromatography to be further purified, adopt the water elution of 5～10 times of column volumes to clarification earlier in its sample behind the purification, 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 20～40 kHz, 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 instrument.
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; Described macroporous adsorbent resin and the supernatant volume ratio between the two is 4～10:1;
When described 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 described employing silica gel column chromatography is further purified, elution flow rate is 8～12 ml/min; Mixed volume ratio between described " 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, described 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, described 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 by 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 zoopery, 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 increases frequency of respiration, reduces brain water content, increases the survival rate of cerebral ischemia animal and prolong life span, reduces cerebral infarct size, changes hemostatic system, the 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:
The invention will be further described with embodiment below, but the present invention is not limited to these embodiment.
Radix Trichosanthis saponin embodiment:
Embodiment 1:
Radix Trichosanthis saponin of the present invention extracts by the following method and obtains, and its detailed step is:
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 20 times of Radix Trichosanthis after pulverizing, add and adopt the ultrasonic extraction device after the entry (frequency of described ultrasonic extraction device is 30 kHz, 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, step a is merged the extracting solution that obtains adopts abstraction instrument (adopting Ninson ultrasonic extraction instrument) 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, (model of macroporous adsorbent resin is D101 by macroporous adsorbent resin earlier 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 10ml/min; Mixed volume ratio between described " chloroform-methanol-water " three is 83:16:1), the water elution that behind the purification its sample is adopted 8 times of column volumes earlier is to clarifying (when adopting the water elution of 8 times of column volumes, its elution flow rate is 1～2ml/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 2～3ml/min, alcoholic acid volumetric concentration is 50～60%), collect ethanol elution;
C, the ethanol elution that step b is collected, (pressure is 0.6Mpa to adopt Rotary Evaporators to carry out drying, baking temperature is 50 ℃, and 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 carried out vacuum drying (vacuum is 0.085Mpa, and baking temperature is 55 ℃, and be 3 hours drying time), pulverize, obtain powdery Radix Trichosanthis saponin, its purity is greater than 90%.
Embodiment 2: substantially the same manner as Example 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 described ultrasonic extraction device is 20 kHz, 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 by 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 described " chloroform-methanol-water " three is 76:22:2), the water elution that behind the purification its sample is adopted 5 times of column volumes earlier is to clarifying (when adopting the water elution of 5 times of column volumes, its elution flow rate is 0.5～1ml/min), the ethanol elution of 4 times of column volumes of reuse is (during with the ethanol elution of 4 times of column volumes, its elution flow rate is 0.5～1ml/min, alcoholic acid volumetric concentration is 20～30%), collect ethanol elution;
Among the step c: (pressure is 0.5Mpa to adopt Rotary Evaporators to carry out drying, baking temperature is 60 ℃, the rotary speed of Rotary Evaporators is 65rmp, rotary evaporation drying time is 3 hours), at last paste Radix Trichosanthis saponin carried out vacuum drying (vacuum is 0.07Mpa, and baking temperature is 80 ℃, and be 3 hours drying time), pulverize, obtain powdery Radix Trichosanthis saponin, its purity is greater than 90%.
Embodiment 3: substantially the same manner as Example 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 described ultrasonic extraction device is 40 kHz, 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, (model of macroporous adsorbent resin is AB-8 by macroporous adsorbent resin with the gained supernatant, macroporous adsorbent resin and the supernatant volume ratio between the two is 4:1) 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 12ml/min; Mixed volume ratio between described " chloroform-methanol-water " three is 68.5:27.5:4), the water elution that behind the purification its sample is adopted 10 times of column volumes earlier is to clarifying (when adopting the water elution of 10 times of column volumes, its elution flow rate is 4～5ml/min), the ethanol elution of 8 times of column volumes of reuse is (during with the ethanol elution of 8 times of column volumes, its elution flow rate is 4～5ml/min, alcoholic acid volumetric concentration is 30～40%), collect ethanol elution;
Among the step c: (pressure is 1Mpa to adopt Rotary Evaporators to carry out drying, baking temperature is 40 ℃, the rotary speed of Rotary Evaporators is 40rmp, rotary evaporation drying time is 5 hours), at last paste Radix Trichosanthis saponin carried out vacuum drying (vacuum is 0.08Mpa, and baking temperature is 60 ℃, and be 4 hours drying time), pulverize, obtain powdery Radix Trichosanthis saponin, its purity is greater than 90%.
Embodiment 4: substantially the same manner as Example 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 described ultrasonic extraction device is 25 kHz, 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 by 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 described " chloroform-methanol-water " three is 65:30:5), the water elution that behind the purification its sample is adopted 6 times of column volumes earlier is to clarifying (when adopting the water elution of 6 times of column volumes, its elution flow rate is 1～2ml/min), the ethanol elution of 5 times of column volumes of reuse is (during with the ethanol elution of 5 times of column volumes, its elution flow rate is 1～2ml/min, alcoholic acid volumetric concentration is 70～95%), collect ethanol elution;
Among the step c: (pressure is 0.1Mpa to adopt Rotary Evaporators to carry out drying, baking temperature is 80 ℃, the rotary speed of Rotary Evaporators is 100rmp, rotary evaporation drying time is 2 hours), at last paste Radix Trichosanthis saponin carried out vacuum drying (vacuum is 0.09Mpa, and baking temperature is 50 ℃, and be 2 hours drying time), pulverize, obtain powdery Radix Trichosanthis saponin, its purity is greater than 90%.
Embodiment 5: substantially the same manner as Example 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 described ultrasonic extraction device is 35 kHz, 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 by 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 described " chloroform-methanol-water " three is 61:33:6), the water elution that behind the purification its sample is adopted 7 times of column volumes earlier is to clarifying (when adopting the water elution of 7 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 2～3ml/min, alcoholic acid volumetric concentration is 60～70%), collect ethanol elution;
Among the step c: (pressure is 0.8Mpa to adopt Rotary Evaporators to carry out drying, baking temperature is 50 ℃, the rotary speed of Rotary Evaporators is 120rmp, rotary evaporation drying time is 3 hours), at last paste Radix Trichosanthis saponin carried out vacuum drying (vacuum is 0.09Mpa, and baking temperature is 70 ℃, and be 3 hours drying time), pulverize, obtain powdery Radix Trichosanthis saponin, its purity is greater than 90%.
Embodiment 6: substantially the same manner as Example 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 described ultrasonic extraction device is 38 kHz, 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 by 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 described " chloroform-methanol-water " three is 54:38.5:7.5), the water elution that behind the purification its sample is adopted 9 times of column volumes earlier is to clarifying (when adopting the water elution of 9 times of column volumes, its elution flow rate 3～4ml/min), the ethanol elution of 7 times of column volumes of reuse is (during with the ethanol elution of 7 times of column volumes, its elution flow rate is 3～4ml/min, alcoholic acid volumetric concentration is 70～80%), collect ethanol elution;
Among the step c: (pressure is 0.05Mpa to adopt Rotary Evaporators to carry out drying, baking temperature is 80 ℃, the rotary speed of Rotary Evaporators is 120rmp, rotary evaporation drying time is 5 hours), at last paste Radix Trichosanthis saponin carried out vacuum drying (vacuum is 0.08Mpa, and baking temperature is 70 ℃, and be 3 hours drying time), pulverize, obtain powdery Radix Trichosanthis saponin, its purity is greater than 90%.
Embodiment 7: substantially the same manner as Example 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 described ultrasonic extraction device is 28 kHz, 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 by 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, the water elution that behind the purification its sample is adopted 8 times of column volumes earlier is to clarifying (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, alcoholic acid volumetric concentration is 80～95%), collect ethanol elution;
Among the step c: (pressure is 0.3Mpa to adopt Rotary Evaporators to carry out drying, baking temperature is 70 ℃, the rotary speed of Rotary Evaporators is 80rmp, rotary evaporation drying time is 4 hours), at last paste Radix Trichosanthis saponin carried out vacuum drying (vacuum is 0.08Mpa, and baking temperature is 70 ℃, and be 3 hours drying time), pulverize, 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.The medicine group is gavaged the Radix Trichosanthis saponin that the present invention extracts, and dosage is 40mg/kg, and positive controls gavages nimodipine 22.5 mg/kg, and the blank group gavages isopyknic distilled water, 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: by above-mentioned table 1 experimental result as seen, the prolongation mice broken end back breathing time and the frequency of respiration of the Radix Trichosanthis saponin energy 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.The medicine group gavages the Radix Trichosanthis saponin that the present invention extracts, and dosage is 40mg/kg, and positive controls gavages nimodipine 22.5 mg/kg, and sham operated rats, cerebral ischemia matched group gavage isopyknic distilled water, 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: as seen by the experimental result of table 2, 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, with matched group significant difference is arranged, prove 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, be divided into the normal saline matched group at random, sham operated rats, Radix Trichosanthis saponin group and nimodipine group (positive drug group), the medicine group gavages the Radix Trichosanthis saponin that the present invention extracts, dosage is 40mg/kg, positive controls gavages nimodipine 22.5mg/kg, matched group, sham operated rats gavages isopyknic distilled water, 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 standby 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: by the experimental result of table 3 as seen, the Radix Trichosanthis saponin that the present invention extracts can increase the survival rate of cerebral ischemia animal and prolong life span.
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, and 30min is hung in the silk thread ligation, 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 cerebellum and brain stem, and the crown brain sheet that is cut to 0.3 cm 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, observe the pathological change of cerebral tissue under light microscopic.
(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: as seen by table 4 experimental result, the treatment group is irritated behind the stomach as can be seen TT, APTT, FBG content than the matched group height, 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: by table 5 experimental result as seen, 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 substantially, 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 right common carotid artery and left external jugular vein.In polyethylene tube, put into a silk thread of having weighed.Be full of polyethylene tube with the normal saline that contains heparin.One end of pipe is inserted right common carotid artery, 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 equivalent to crude drug 120g/kg by 4.8g/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 equivalent to crude drug 120g/kg by 4.8g/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 increases frequency of respiration, reduces brain water content, increases the survival rate of cerebral ischemia animal and prolong life span, reduces cerebral infarct size, changes hemostatic system, the 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 after the ischemic cerebrovascular generation there is good protective effect; and toxicity is low; side effect is little, and is safe in utilization, quality controllable.
Claims (10)
1. a Radix Trichosanthis saponin is characterized in that, described Radix Trichosanthis saponin extracts by the 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 Radix Trichosanthis after pulverizing, 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 by macroporous adsorbent resin earlier, then adopt silica gel column chromatography to be further purified, adopt the water elution of 5～10 times of column volumes to clarification earlier in its sample behind the purification, 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. 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. 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. Radix Trichosanthis saponin according to claim 1 is characterized in that: abstraction instrument described in the step b adopts Ninson ultrasonic extraction instrument.
5. 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; Described macroporous adsorbent resin and the supernatant volume ratio between the two is 4～10:1;
When described 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 described employing silica gel column chromatography is further purified, elution flow rate is 8～12 ml/min; Mixed volume ratio between described " chloroform-methanol-water " three is 50～85:14～40:1～10.
6. 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. 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, and baking temperature is 40～80 ℃, 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.
8. the described Radix Trichosanthis saponin of claim 1 is preparing the purposes for the treatment of in the ischemic cerebrovascular medicine.
9. the purposes of Radix Trichosanthis saponin according to claim 8 in preparation treatment ischemic cerebrovascular medicine, it is characterized in that: described Radix Trichosanthis saponin is Radix Trichosanthis saponin or Radix Trichosanthis saponin monomer.
10. the purposes of Radix Trichosanthis saponin according to claim 8 in preparation treatment ischemic cerebrovascular medicine, it is characterized in that: described Radix Trichosanthis saponin is ligroin extraction, n-butanol extract, chloroform extract or ethyl acetate extract.
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| CN102670695A (en) * | 2012-05-31 | 2012-09-19 | 河南科技学院 | Application of saponins in radix trichosanthin in protecting nerve cells after subarachnoid hemorrhage |
| CN103893242A (en) * | 2014-03-20 | 2014-07-02 | 浙江工业大学 | Radix trichosanthes triterpene extract as well as preparation method and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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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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| Publication number | Priority date | Publication date | Assignee | Title |
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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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102670695A (en) * | 2012-05-31 | 2012-09-19 | 河南科技学院 | Application of saponins in radix trichosanthin in protecting nerve cells after subarachnoid hemorrhage |
| CN103893242A (en) * | 2014-03-20 | 2014-07-02 | 浙江工业大学 | Radix trichosanthes triterpene extract as well as preparation method and use thereof |
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