CN105168235B - Application of pennogenin compound in preparing medicament for treating senile dementia and Alzheimer disease - Google Patents

Application of pennogenin compound in preparing medicament for treating senile dementia and Alzheimer disease Download PDF

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CN105168235B
CN105168235B CN201510689816.4A CN201510689816A CN105168235B CN 105168235 B CN105168235 B CN 105168235B CN 201510689816 A CN201510689816 A CN 201510689816A CN 105168235 B CN105168235 B CN 105168235B
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rhamnopyranosyl
alpha
pennogenin
beta
glucoside
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CN105168235A (en
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张忠立
左月明
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Jiangxi University of Traditional Chinese Medicine
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Abstract

The invention relates to the application of pennogenin compounds in preparing medicaments for treating senile dementia and Alzheimer's disease, wherein the pennogenin compounds are pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside. The pennogenin compounds have the functions and the efficacies of treating cerebral ischemia in a cerebral hypoperfusion state caused by aging and cardiovascular and cerebrovascular diseases, senile dementia caused by the pathogenesis of amyloid A beta deposition and Alzheimer disease.

Description

Application of pennogenin compound in preparing medicament for treating senile dementia and Alzheimer disease
Technical Field
The invention relates to a preparation method of trillium saponins, a preparation method of a preparation of the trillium saponins, and application of the trillium saponins in preparation of medicines for treating senile dementia and Alzheimer's disease, belonging to the technical field of traditional Chinese medicine preparation.
Background
The Trillium tschosokii maxim, commonly named as parietal barberry, rhizoma podocarpi, leonurus potamonum and the like, is a traditional rare traditional Chinese medicine and has the effect of prolonging life. Mainly treats diseases such as dizziness, insomnia, traumatic injury, traumatic hemorrhage, neurasthenia, hypertension, cerebral concussion sequelae and the like, and is one of famous folk medicines of the Enshi indigenous family. At present, the pharmacological activity research of the Trillium plants at home and abroad shows that the Trillium has stronger effects of resisting inflammation, regulating immunity, improving the learning and memory functions of rats, resisting aging and the like. The inventor uses an H/MAD model to carry out preliminary research on the pharmacological activity of the Trillium vulgare, and proves that the 70% ethanol extract of the root and the rhizome of the Trillium vulgare has the effect of obviously improving the dementiaThe rat has learning and memory function, and the drug-containing cerebrospinal fluid of the Trillium japonicum has anti-Abeta25-35The biological activity of causing PC12 cell apoptosis is deeply researched and determined that the 85% ethanol eluate of the trillium fortunei is the effective part of the trillium fortunei for treating heterogeneous and multifactorial senile dementia and Alzheimer disease model rats.
Chemical research shows that 85% of ethanol elution parts of the effective part are mainly composed of steroid saponin components, the total content of the steroid saponin is more than 90%, wherein the total content is greater than 90%, and the main components are pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucopyranoside, pennogenin-3-O-alpha-L-rhamnopyranosyl- (1 → 2) -beta-D-glucopyranoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside (pennogenin-3-O-alpha-L-rhamnopyranosyl- (1 → 4) - [ alpha-L-rhamnopyranosyl- (1 → 2) ] -beta-D-glucopyranoside) and pennogenin- 3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside (pennogenin-3-O-alpha-L-rhamnopyranosyl- (1 → 4) - [ alpha-L-rhamnopyranosyl- (1 → 2) ] -beta-D-glucopyranoside), which are both an index and characteristic component of a Trillium tenuifolium medicinal material and an index and characteristic component of an 85% ethanol elution part of an effective part. Further pharmacological research proves that the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside in the effective part, the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside are effective components of tril grass for treating heterogeneous and multifactorial senile dementia and Alzheimer's disease. The research has not been reported, and through systematic in vitro and in vivo experimental research, the medicine firstly finds that the medicine has good therapeutic effect on cerebral hypoperfusion state caused by aging, cardiovascular and cerebrovascular diseases (heart disease, hypertension, atherosclerosis, transient ischemic attack and the like), senile dementia caused by amyloid A beta deposition and Alzheimer disease.
Disclosure of Invention
The invention aims to provide a trillium saponin substance, a preparation method of a preparation of the trillium saponin substance and application of the trillium saponin substance in preparation of medicines for treating senile dementia and Alzheimer disease.
The invention adopts the following technical scheme.
In the trillium saponin substance, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside (pennogenin-3-O-alpha-L-rhamnopyranosyl- (1 → 2) -beta-D-glucopyranoside), pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside (pennogenin-3-O-alpha-L-rhamnopyranosyl- (1 → 4) - [ alpha-L-rhamnopyranosyl- (1 → 2) ] -beta-D-glucopyranoside) and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) -alpha-L-rhamnopyranosyl (1 → 2) — -rhamnopyranosyl (1 → 4) - [ α -L-rhamnopyranosyl (1 → 2) ] - β -D-glucoside (pennogenin-3-O- α -L-rhamnopyranosyl- (1 → 4) - [ α -L-rhamnopyranosyl- (1 → 2) ] - β -D-glucopyranoside), characterized by the following steps:
(1) pulverizing Trillium into coarse powder, soaking in 0-100% ethanol, adding 4-12 times of solvent, reflux-extracting under heating for several times, filtering, and mixing filtrates;
(2) concentrating the filtrate under reduced pressure to 0.5-1.5g crude drug/ml, refrigerating overnight, separating gel, filtering, extracting the filtrate with organic solvent or subjecting the filtrate to gradient elution with macroporous adsorbent resin, recovering organic solvent to obtain extract, and loading to silica gel column;
(3) after application to a silica gel column, sequentially applying chloroform: methanol or methylene chloride: methanol or chloroform: methanol: water or dichloromethane: methanol: performing gradient elution with water, eluting with pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside in the trilobate saponins, detecting 3 saponins of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside as reference substances by TLC or HPLC, and combining the corresponding fractions;
(4) concentrating corresponding fractions of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside in the trillium bengal saponins by reduced pressure, evaporating to dryness, dissolving with appropriate amount of ethanol, filtering, recovering ethanol under reduced pressure until the ethanol content is 10-40%, separating gel, filtering, adding appropriate amount of polyamide into the filtrate, stirring for 0.5-3 hr, standing for 4-24 hr, filtering, adding appropriate amount of activated carbon into the filtrate, stirring for 0.5-3 hr, standing for 4-24 hr, filtering, drying the filtrate by spray drying or freeze drying, collecting spray dried powder or lyophilized powder, crystallizing with 10-95% ethanol, and recrystallizing to obtain the final product.
More specifically, in the step (1), 4-12 times of solvent is added, heated and refluxed for extraction for 2-4 times, and each time lasts for 1-3 hours.
More specifically, in step (2), the mixture is refrigerated overnight at 0-4 ℃.
More specifically, in the step (2), the concentration temperature under reduced pressure is 45 to 80 ℃.
More specifically, in the step (2), the filtrate is enriched by macroporous adsorption resin, is sequentially eluted by 0-50% of ethanol water, is eluted by 50-95% of ethanol water to obtain various eluted parts, the eluted parts with 60-85% of ethanol water are mainly collected, and the eluent is recovered to obtain the extract which is applied to a silica gel column.
Further preferably, chloroform is mainly collected in step (3): methanol: the water content is 65: 35: 1 or 60: 30: fraction eluted of 1.
The active ingredients of the trillium saponins substance prepared by the method are one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside, or the hydroxyl group at the 21 st position and the hydroxyl group at the 27 th position of the conversion hydroxymethyl group or the hydroxyl group at the 17 th position of the sapogenin in the substance or all the hydroxyl groups at the 21 st position and the 27 th position of the sapogenin in the substance The alcoholic hydroxyl group of glycosyl is substituted by any one of acetyl, aliphatic alkane group, aromatic substituent and amido, or the substance which can generate pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside after hydrolysis can be used for preparing the medicaments for treating senile dementia and Alzheimer disease, the medicaments comprise various formulations such as oral liquid, capsules, tablets, effervescent tablets, injections and the like, and the medicaments also comprise various single and compound preparations.
The related senile dementia and Alzheimer disease are any of senile dementia and Alzheimer disease caused by aging, cerebral hypoperfusion state caused by cardiovascular and cerebrovascular diseases (heart disease, hypertension, atherosclerosis, transient ischemic attack, etc.), amyloid A beta deposition.
The invention discloses a preparation method of a tablet containing one or more components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside, the paint is characterized by comprising the following components: one or more components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside are 0.25-15g, lactose is 3-8g, dextrin is 3-12g, 3-8g of microcrystalline cellulose and 0.2-0.5g of magnesium stearate, uniformly mixing according to a proper proportion, granulating by a wet method, finally adding magnesium stearate, uniformly mixing and tabletting to prepare tablets, wherein each tablet contains one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside 5-300mg of effective component.
The invention discloses a method for preparing capsules containing one or more components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside, the paint is characterized by comprising the following components: one or more components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside are 0.25-15g, lactose is 3-8g, dextrin is 3-12g, 3-8g of microcrystalline cellulose, evenly mixing according to a proper proportion, granulating by a wet method, filling into capsules, each capsule contains 5-300mg of one or more effective components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside.
The invention discloses a method for preparing a water injection containing one or more components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside, the paint is characterized by comprising the following components: one or more components of the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside are 100-6000mg dissolved in a proper amount of water for injection, and are added with metered water-soluble auxiliary materials for injection, adding 0.2-1% of active carbon to remove heat source, adjusting pH to 6.0-7.5, ultrafiltering with microporous membrane, adding water for injection to constant volume, filling into ampoule or infusion bottle, sealing/capping, checking, and packaging to obtain the final product containing pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-pyriproxyfen (1 → 4) ], and the final product in each ampoule or infusion bottle 5-300mg of one or more effective components in rhamnopyranosyl (1 → 2) ] -beta-D-glucoside.
The invention discloses a method for preparing a powder injection containing one or more components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside, the paint is characterized by comprising the following components: 100-6000mg of one or more components of the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside are dissolved in a proper amount of water for injection, a proper amount of mannitol is added, the volume is kept to 200-, adjusting pH to 5.5-7.5, sterilizing, filtering, lyophilizing, making into ampoule bottle containing one or more effective components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside 5-300 mg.
The invention has the advantages that: provides an extraction and preparation method of trillium saponins substances, the obtained substances are one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside, the obtained substance can be used for preparing tablet, capsule, powder for injection, and water injection for treating heterogeneous and multi-cause senile dementia and Alzheimer disease or preparing various application medicines.
In the trillium saponin substance, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside cause cerebral hypoperfusion state, cerebral and cerebral vascular diseases (heart disease, hypertension, atherosclerosis and transient ischemic attack, etc.) in vitro and in vivo, Senile dementia and Alzheimer disease caused by amyloid A beta deposition have good treatment effect and strong biological activity.
In the trillium saponin substance, the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside are mainly used for the symptomatic treatment of etiology and pathogenesis senile dementia and Alzheimer disease, can treat both principal and secondary aspect of disease.
The preparation method of the benomygenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, the benomygenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and the benomygenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside in the trillium bengal saponin substances mainly comprises the steps of extracting the benomygenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside from natural medicines to obtain pure natural components and monomers, has good biocompatibility.
Drawings
FIG. 1 is a molecular structural formula of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside;
FIG. 2 is a molecular structural formula of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside;
FIG. 3 is a molecular structural formula of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside.
FIG. 4 is a graph of the spatial trajectory of Trillium repens extract versus rats in example 5.
FIG. 5 shows β -APP, A β in hippocampal neurons of AD rats in example 51-40 Caspase 3 immunohistochemistry (400 ×).
FIG. 6 is a plot of the spatial exploration trace of trillium saponin in example 7 against rats.
Detailed Description
The invention is further described below with reference to the following examples:
example 1
A method for preparing pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside in the trillium bengal saponin substances, wherein: pulverizing Trillium into coarse powder, soaking in 60% ethanol for 1 hr, reflux-extracting with 8 times of solvent under heating for 3 times (2 hr each time), filtering, and mixing filtrates. Concentrating the filtrate with 65 deg.C under reduced pressure to 1.0g crude drug/ml, refrigerating at 0-4 deg.C overnight, separating gel, filtering, extracting the filtrate with organic solvent such as n-butanol, recovering organic solvent to obtain extract, adding 100-mesh silica gel of 200 meshes, stirring, volatilizing solvent under reduced pressure at 50 deg.C, grinding, loading with dry method, loading onto normal pressure silica gel column (200-mesh 300 meshes, silica gel amount 20 times of the extract), sequentially adding chloroform: methanol: water gradient elution, collecting mainly chloroform: methanol: the water content is 65: 35: 1, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside in the trillium bengal saponin substances, the 3 kinds of saponins of the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside are used as reference substances to be detected by TLC or HPLC, and the corresponding flow parts are combined. Concentrating the above fractions under reduced pressure at 65 deg.C, evaporating to dryness, dissolving with appropriate amount of ethanol, filtering, recovering ethanol under reduced pressure until the ethanol content is 30%, refrigerating at 0-4 deg.C overnight, separating gel, filtering, adding 8% crude drug polyamide into the filtrate, stirring for 2 hr, standing for 12 hr, filtering, adding 4% crude drug active carbon into the filtrate, stirring for 2 hr, standing for 12 hr, filtering, drying the filtrate with freeze-drying method, collecting lyophilized powder, crystallizing with 95% ethanol, and recrystallizing to obtain the final product. The yield of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside in the trilobate saponins prepared by the method is 1.5-2.6g/kg, and the purity is 98.8% by HPLC detection; the yield of the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside is 0.8-1.9g/kg, and the purity is 97.5% by HPLC detection; the yields of the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside are respectively 0.3-0.9g/kg and 96.2% by HPLC detection; chromatographic conditions UItimeateR O XB-C18 chromatographic column (4.6mm x 150mm, 3 μm), mobile phase acetonitrile-water (10% -100% gradient elution), detection wavelength of 203nm, flow rate of 1.0ml/min, column temperature of 30 ℃, sample volume of 20 μ l.
Example 2
The preparation method of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside in the trillium bengal saponin substances is as follows: pulverizing Trillium into coarse powder, soaking in 70% ethanol for 1.5 hr, reflux-extracting with 10 times of solvent under heating for 3 times (each for 3 hr), filtering, and mixing filtrates. Concentrating the filtrate with 70 deg.C under reduced pressure to 1g crude drug/ml, refrigerating at 0-4 deg.C overnight, separating gel, filtering, enriching the filtrate with AB-8 type macroporous adsorbent resin, sequentially eluting with water, 30% ethanol, 60% ethanol and 85% ethanol to obtain each eluate part, wherein 85% of the eluate parts mainly contain steroid saponins, the total content of steroid saponins is greater than 90%, recovering eluate to obtain extract, loading to silica gel column, sequentially loading with chloroform: methanol: water gradient elution, collecting mainly chloroform: methanol: the water content is 60: 30: 1, detecting 3 saponins of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 4) ] -beta-D-glucoside in the trillium bengal saponin substances as reference substances by TLC or HPLC, the corresponding fractions are combined. Concentrating the above fractions at 65 deg.C under reduced pressure, evaporating to dryness, dissolving in 95% ethanol, filtering, crystallizing, and recrystallizing to obtain the final product. The yield of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside in the trilobate saponins prepared by the method is 1.5-2.6g/kg, and the purity is 99.1% by HPLC detection; the yield of the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside is 0.8-1.9g/kg, and the purity is 98.3% by HPLC detection; the yields of the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside are respectively 0.3-0.9g/kg and 98.1% by HPLC detection; chromatographic conditions UItimate O RXB-C18 chromatographic column (4.6mm X150 mm, 3 μm), mobile phase acetonitrile-water (10% -100% gradient elution), detection wavelength of 203nm, flow rate of 1.0ml/min, column temperature of 30 ℃, sample volume of 20 μ l.
Example 3
The preparation method of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside in the trillium bengal saponin substances is as follows: pulverizing Trillium into coarse powder, soaking in 50% ethanol for 1.0 hr, reflux-extracting with 10 times of solvent under heating for 3 times (2 hr each time), filtering, and mixing filtrates. Concentrating the filtrate with 70 deg.C under reduced pressure to 1g crude drug/ml, refrigerating at 0-4 deg.C overnight, separating gel, filtering, enriching the filtrate with D-101 type macroporous adsorbent resin, sequentially eluting with water, 30% ethanol, 50% ethanol and 85% ethanol to obtain each eluate part, wherein 85% of eluate parts mainly contains steroid saponins, recovering eluate to obtain extract, loading to silica gel column, sequentially loading with chloroform: methanol: water gradient elution, collecting mainly chloroform: methanol: the water content is 65: 35: 1, detecting 3 saponins of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 4) ] -beta-D-glucoside in the trillium bengal saponin substances as reference substances by TLC or HPLC, the corresponding fractions are combined. Concentrating the above fractions at 65 deg.C under reduced pressure, evaporating to dryness, dissolving in 95% ethanol, filtering, crystallizing, and recrystallizing to obtain the final product. The yield of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside in the trilobate saponins prepared by the method is 1.5-2.6g/kg, and the purity is 97.6% by HPLC detection; the yield of the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside is 0.8-1.9g/kg, and the purity is 96.2% by HPLC detection; the yields of the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside are respectively 0.3-0.9g/kg and 95.8% by HPLC detection; chromatographic conditions UItimeateR O XB-C18 chromatographic column (4.6mm x 150mm, 3 μm), mobile phase acetonitrile-water (10% -100% gradient elution), detection wavelength of 203nm, flow rate of 1.0ml/min, column temperature of 30 ℃, sample volume of 20 μ l.
Example 4
Preparing raw materials and preparations:
examples one, two or three of the trillium saponin substance prepared by the method of example one, two or three of the examples include pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside as raw materials, or pennogenin-3- O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside are used as starting materials for the preparation of the following formulations.
1. Tablet preparation
Prescription 1(50 tablets) containing pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside 5g, lactose 5g, dextrin 8g, microcrystalline cellulose 5g, magnesium stearate is 0.2 g. According to the operation method of the conventional tablet, the components are uniformly mixed, granulated by a wet method, finally added with magnesium stearate, uniformly mixed and tabletted, each tablet is prepared to contain 100mg of one or more effective components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside.
Prescription 2(50 tablets) containing one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside 250mg, lactose 3g, dextrin 12g, microcrystalline cellulose 8g, magnesium stearate is 0.2 g. According to the operation method of the conventional tablet, the components are uniformly mixed, granulated by a wet method, finally added with magnesium stearate, uniformly mixed and tabletted, each tablet is prepared to contain 5mg of one or more effective components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside.
Prescription 3(50 tablets) containing 15g of one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside, 8g of lactose, 3g of dextrin and 3g of microcrystalline cellulose, magnesium stearate is 0.5 g. According to the operation method of the conventional tablet, the components are uniformly mixed, granulated by a wet method, finally added with magnesium stearate, uniformly mixed and tabletted, each tablet is prepared to contain 300mg of one or more effective components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside.
2. Capsule preparation
Prescription 1(50 granules), the component contains pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside is 5g, lactose is 5g, dextrin is 8g, microcrystalline cellulose is 5 g. Mixing the above materials, granulating by wet method, and making into capsule, each capsule contains 100mg of one or more effective components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside.
Prescription 2(50 granules), the component contains pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside is 250mg, lactose is 3g, dextrin is 12g, microcrystalline cellulose is 8 g. Mixing the above materials, granulating by wet method, and making into capsule, each capsule contains 5mg of one or more effective components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside.
Prescription 3(50 granules), the component contains pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside is 15g, lactose is 8g, dextrin is 3g, and microcrystalline cellulose is 3 g. Mixing the above materials, granulating by wet method, and making into capsule, each capsule contains 300mg of effective component (one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside).
3 preparation of powder injection
Prescription 1(20 bottles), the ingredients of which contain one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside is 200mg dissolved in a proper amount of water for injection, and is added with a proper amount of mannitol, fixing volume to 200ml, adjusting pH to 7-7.2, sterilizing, filtering, freeze drying, checking, packaging, and making into ampoule bottle containing 10mg of one or more effective components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside. Prescription 2(20 bottles), the ingredients of which contain 100mg of one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside, dissolved in a proper amount of water for injection, added with a proper amount of mannitol, fixing volume to 200ml, adjusting pH to 7-7.2, sterilizing, filtering, freeze drying, checking, packaging, and making into ampoule bottle containing 5mg of one or more effective components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside. Prescription 3(20 bottles), the ingredients of which contain one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside is 6000mg dissolved in a proper amount of water for injection, and is added with a proper amount of mannitol, diluting to 2000ml, adjusting pH to 7-7.2, sterilizing, filtering, freeze drying, checking, packaging, and making into ampoule bottle containing 300mg of one or more effective components of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside.
4 preparation of injection
Prescription 1(20 bottles), the ingredients of which contain one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside is 500mg dissolved in a proper amount of water for injection, and is added with a metered amount of water-soluble auxiliary materials for injection, adding 0.2-1% of activated carbon to remove heat source, adjusting pH to 6.0-7.5, ultrafiltering with microporous membrane, adding water for injection to constant volume, filling into ampoule or infusion bottle, sealing/capping, checking, and packaging to obtain the final product containing pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 4) ] 25mg of one or more effective components in rhamnosyl (1 → 2) ] -beta-D-glucoside.
Prescription 2(20 bottles), the ingredients of which contain 100mg of one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside, are dissolved in a proper amount of water for injection, and are added with metered water-soluble auxiliary materials for injection, adding 0.2-1% of active carbon to remove heat source, adjusting pH to 6.0-7.5, ultrafiltering with microporous membrane, adding water for injection to constant volume, filling into ampoule or infusion bottle, sealing/capping, checking, and packaging to obtain the final product containing pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 4) ] One or more effective components of the plum sugar group (1 → 2) ] -beta-D-glucoside are 5 mg.
Prescription 3(20 bottles), the ingredients of which contain one or more of pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside is 6000mg dissolved in a proper amount of water for injection, and is added with metered water-soluble auxiliary materials for injection, adding 0.2-1% of activated carbon to remove heat source, adjusting pH to 6.0-7.5, ultrafiltering with microporous membrane, adding water for injection to constant volume, filling into ampoule or infusion bottle, sealing/capping, checking, and packaging to obtain the final product containing pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 4) ] 300mg of one or more effective components in rhamnosyl (1 → 2) ] -beta-D-glucoside.
Example 5
The medicinal effect of the trillium fortunei on the cerebral hypoperfusion caused by aging, cardiovascular and cerebrovascular diseases (heart disease, hypertension, atherosclerosis, transient ischemic attack, etc.), senile dementia caused by amyloid A beta deposition and Alzheimer disease is studied in vitro and in vivo:
1 extracting dried root and rhizome of Trillium wilfordii 30kg, pulverizing into coarse powder, extracting with 70% ethanol under reflux for 3 times, each for 2.0 hr, filtering, mixing extractive solutions, and recovering under reduced pressure to obtain dry extract. Suspending the dry extract with water, separating with AB-8 type macroporous adsorbent resin, and sequentially eluting with water, 30% ethanol, 60% ethanol, and 85% ethanol to obtain each eluted part. A test solution was prepared from 70% ethanol total extract (1g corresponding to 6.7g crude drug), water eluate (1g corresponding to 12.2g crude drug), 30% ethanol eluate (1g corresponding to 83.3 g crude drug), 60% ethanol eluate (1g corresponding to 55.6g crude drug), and 85% ethanol eluate (1g corresponding to 26.7g crude drug) of Trillium japonicum by using 2% polysorbate aqueous solution.
Grouping and model replication of 2 animals 182 healthy SD male rats were randomly divided into blank group, sham-injured group, H/MAD model group, total-extract high-dose group of trillium, total-extract medium-dose group of trillium, water-wash high-dose group, water-wash medium-dose group, 30% ethanol-elute high-dose group, 30% ethanol-elute medium-dose group, 60% ethanol-elute high-dose group, 60% ethanol-elute medium-dose group, 85% ethanol-elute high-dose group, 85% ethanol-elute medium-dose group, donepezil hydrochloride group. Each group was tested for 13 rats.
Heterogeneous and multifactorial animal models of senile dementia: each group of experimental rats was injected with D-galactose (50 mg. kg) intraperitoneally daily-1) The subacute aging is caused, after 4W, the bilateral common carotid artery is repeatedly ligated for blocking blood flow in a time-phased manner, cerebral ischemic focus is caused (chloral hydrate abdominal cavity injection anesthesia, incision in the middle of the neck, bilateral common carotid artery is separated, and number 4 silk thread buckle is sleeved. Tightening the screw thread, blocking blood flow for 10min, loosening the screw thread to perfuse blood for 10min, blocking blood flow for 10min, perfusing for 2 nd time, observing for 5min, and suturing skin. ) After the operation, the rats were fed for 3 days, and the non-dead rats in good condition were injected intracerebrally with condensed Abeta1-40(Chloral hydrate anesthesia, fixed on brain stereotaxic apparatus, the head is preserved, the operation is carried out under aseptic condition, an incision with the length of 2-3 cm is made along the midline of the skull, periosteum is separated, a human-shaped suture and a cross-shaped suture are exposed, 3.0mm behind bregma and 2.0mm beside the right side of the midline are opened, the skull is drilled by a No. 7 needle, dura mater is exposed, a micro-injector is vertically inserted 4.0mm from the surface of the brain, and then 5 mu 1 Abeta is slowly injected into the brain tissue1-40Injecting for 5min, keeping the needle for 5min, and sealing the skull with dental mud after injection. After diminishing inflammation, the skin is sutured.
Blank groups are not processed; the model group and the administration group are respectively processed according to the molding method; the abdominal cavity of the sham-injured group is injected with 4W of normal saline, after 4W, the common carotid artery is separated, only threading is carried out but ligation is not carried out, and then the same amount of normal saline is injected intracerebrally. The sham-injured group and the model group were gavaged with a 2% polysorbate aqueous solution for 6 days 1 time a day on the 3 rd day after the intracerebral injection surgery. Each group started continuous gavage for 6 days 1 time per day on day 3 after surgery.
In the 3Morris water maze experiment, clear water is injected into a water tank in advance to enable the water surface to be 1-2 cm higher than a platform, and the water temperature is controlled to be about 24 seconds and 2 ℃. The inner wall of the water tank is coated with black paint, a proper amount of black ink is added into the water tank before each test, and the water tank is divided into 4 quadrants. PlatformThe rats were placed in the middle of the first quadrant, optionally in three other quadrants with a point facing the pool wall, and placed in water for 5 days, each quadrant being tested once a day in the morning for 1min each time. If the rat does not find the platform within 1min, the rat is taken to the platform and allowed to stand on the platform for 15s, if the rat finds the platform within l min, the rat is allowed to stand on the platform for 15s, and the training is finished once, and the training is carried out for 4 days. On day 5, the platform was removed and each rat traveled l min, the time to first pass the original platform (latency) was recorded for each rat, the number of times each rat passed the platform in l min, the total distance traveled and the time to stay in the quadrant of the original platform and the other three quadrants, and the time to swim in the fourth quadrant (t) was calculated4) Occupying the whole swimming time (t)General assembly) Percentage of (d) and distance to swim in the fourth quadrant (I)4) Occupying the whole swimming distance (I)General assembly) Percentage of (c).
4 spectrophotometry for measuring T-AOC, TChE, SOD, MDA and GSH-PXContent of (B) 2.3 rats after behavioral testing, rats in each group were treated with chloral hydrate (300 mg. kg)-1) After anesthesia, the chest is opened rapidly, the heart is sampled, the blood serum is taken out after 3500r/min centrifugation for 10min, and the blood serum is stored at the low temperature of 70 ℃ below zero. Detecting T-AOC, TChE, SOD, MDA and GSH-P by ultraviolet spectrophotometry according to the kit specificationXThe content of (a).
5, adopting SAS software analysis, expressing the experimental data by x +/-s, and analyzing the experimental data by single-factor variance. P <0.05 is statistically significant.
6 results
6.1 comparing the Morris water maze experiment with the blank group and the false injury group, the model group rats have prolonged latency, reduced number of passing stages, short stay time in the first quadrant and obvious difference (P is less than 0.01); compared with the model group, the donepezil hydrochloride group and the trillium fortunei extract group can shorten the latent period and increase the number of times of passing through the table, the residence time is prolonged in the first quadrant (P is less than 0.05 or P is less than 0.01), which indicates that the treatment effect of each administration group is effective, and suggests that the trillium fortunei extract (especially the dosage group in 85% ethanol eluate) has obvious effect. See table 1. The trace diagram is shown in fig. 4.
TABLE 1 Trillium PolygalumResults of spatial exploration test of H/MAD model rat by extract (
Figure BDF0000006866170000161
n＝10)
Figure BDF0000006866170000162
Figure BDF0000006866170000171
Compared with the blank group and the false lesion group,1)p < 0.01; in comparison with the set of models,2)p﹤0.05，3)influence of p < 0.016.2 Trillium arvense extract on serum T-AOC and TChE content in H/MAD model rats
Compared with a blank group and a false injury group, the content of T-AOC in the serum of the rat in the model group is reduced, the activity of TChE is enhanced, and the difference is obvious (P is less than 0.01); the administration of each group can improve the content of T-AOC and reduce the activity of TChE, has obvious difference (P is less than 0.05 or P is less than 0.01) compared with a model group, shows that the Trillium fortunei extract (especially the dosage group in 85 percent ethanol eluate) has obvious treatment effect, can recover the activity of T-AOC in the brain of an H/MAD model rat, reduce the content of TChE, enhance the capacity of scavenging free radicals, antagonize oxidation reaction, prevent the reduction of neurotransmitter and play a role in preventing and treating AD. See table 2.
TABLE 2 influence of Trillium arvense extract on the T-AOC and TChE content in serum of H/MAD model rats ((for T-AOC and TChE))
Figure BDF0000006866170000172
n＝10)
Figure BDF0000006866170000173
Figure BDF0000006866170000181
And emptyThe white group and the sham-injured group were compared,1)p < 0.01; in comparison with the set of models,2)p﹤0.05，3)influence of p < 0.016.3 Trillium ramorum extract on contents of SOD, MDA and GSH-PX in serum of H/MAD model rat
Model group rat SOD, GSH-P compared with blank group and false injury groupXSignificantly decreased (P <0.01), while MDA levels were significantly increased (P < 0.01); compared with the model group, the donepezil hydrochloride group has unobvious and insignificant difference in MDA level reduction, but can improve SOD and GSH-PXActivity of (P < 0.05); compared with model group, MDA level of Trillium arvense extract in each administration group is significantly reduced (P is less than 0.05 or P is less than 0.01), and SOD and GSH-P are significantly reducedXThe obvious rise (P is less than 0.05 or P is less than 0.01) indicates that the treatment of the administration group is effective and superior to that of the donepezil hydrochloride group. The results are shown in Table 3.
TABLE 3 Trillium arvense extract on SOD, MDA, GSH-P in serum of H/MAD model ratXInfluence of content (
Figure BDF0000006866170000182
n＝10)
Figure BDF0000006866170000183
Figure BDF0000006866170000191
Compared with the blank group and the false lesion group,1)p < 0.01; in comparison with the set of models,2)p﹤0.05，3)influence of p < 0.016.4 Trillium arvense total extract on expression of hippocampal neurons beta-APP, Abeta 1-40 and Caspase-3 in H/MAD model rats
β -APP immunoreaction: the normal control group and the sham operation group have small positive expression of the hippocampal neuron cytoplasm; the model group hippocampal neuron cytoplasm has a large amount of positive expression and is in a tan finger ring shape, fusiform shape or pyramidal shape; the positive expression of hippocampal cytoplasmic beta-APP in each group was significantly reduced (P <0.01 or P < 0.05). A beta 1-40 immune response: the cells of the visible part of the hippocampal neurons of the normal control group and the false operation group have light positive reaction of anti-Abeta antibody; the model group hippocampal neurons widely distributed Abeta positive cells; the positive expression of hippocampal neuron cytoplasm Abeta 1-40 of each administration group is obviously weakened (P <0.01 or P < 0.05). Caspase 3 immune response: the normal control group and the false operation group have a small amount of weak positive expression cells in hippocampal neurons; the model group hippocampal neurons showed strong positive expression and cell nucleus contraction phenomenon. Hippocampal neurons in the Trillium arvense total extract group were positively expressed (P <0.01), and the results are shown in Table 4 and FIG. 5.
TABLE 4 influence of Trillium arvense extract on the expression of A D rat brain hippocampal β -APP, A β 1-40 and Caspase 3 (for)
Figure BDF0000006866170000201
n＝10)
Figure BDF0000006866170000202
Note: compared with a normal control group and a false operation group,△△p<0.01; comparison with model group﹡p<0.05，﹡﹡p<0.016.5 study on protective effect of drug-containing cerebrospinal fluid on PC12 cell injury caused by Abeta 25-35
Preparing medicated cerebrospinal fluid: 5 healthy white rabbits were selected, 2 were used in the blank group, and 3 were used in the administration group. Perfusing the blank group with distilled water for 1 time every day, perfusing the stomach for 3 days, and collecting cerebrospinal fluid as blank cerebrospinal fluid after anesthesia on the 3 rd day; the administration group drenches Trillium wilfordii total extract 1 time per day, the administration dosage is 10 times of the therapeutic dosage, and after 3 days of drenching administration and 45 minutes of the last administration, the cerebrospinal fluid is taken as the drug-containing cerebrospinal fluid.
② the protective action determination of drug-containing cerebrospinal fluid to PC12 cell damage caused by Abeta 25-35: (operating as a method for screening for Alzheimer's disease activity at the cellular level). The absorbance (A492nm) was measured at 492nm with a microplate reader, and the results are shown in Table 3.
The drug-containing cerebrospinal fluid group with different concentrations of the total extracts of the Trillium fortunei has a protective effect on PC12 cell damage caused by A beta 25-35, so that the cell viability of the Trillium fortunei is enhanced, and the results show that the A492nm value is increased, and the differences are obvious when compared with the A beta model group respectively (p is less than 0.05 or p is less than 0.01). The fact shows that after the total extract of the Trillium fortunei is administrated by gastric lavage, the components moving in blood can enter into cerebrospinal fluid through a blood-brain barrier to play a certain role in protecting nerve cells.
Table 5 different concentrations of drug-containing cerebrospinal fluid protect damaged PC12 cells (n ═ 10)
Figure BDF0000006866170000203
Figure BDF0000006866170000211
Note:*comparison with Abeta model group p<0.05；**Comparison with Abeta model group p<0.01
7 conclusion and discussion
Senile dementia is a nervous system degenerative disease with unknown reasons and mainly clinically manifested by cognitive hypofunction, and the incidence of senile dementia is high. The clinical manifestations of senile dementia mainly include hypomnesis, disorientation, impaired judgment and reasoning ability, etc. The currently comparatively well-recognized classifications of senile Dementia (senile Dementia) include senile Dementia (AD), Vascular Dementia (VD), and mixed Dementia. Because the pathogenesis and pathological process of the senile dementia are complex, it is extremely difficult to find an animal model which can completely simulate all clinical symptoms and pathological features of the senile dementia, the senile dementia caused by cardiovascular and cerebrovascular diseases has a high prevalence trend, so people combine AD and VD, the existing model preparation can strictly distinguish the senile dementia and the senile dementia, most of the senile dementia is only aimed at one aspect of pathological features of diseases, and the heterogeneity and multiformity of the senile dementia are ignored. It is found that aging and cerebral hypoperfusion are two main factors indispensable for the pathogenesis of AD and VD, and amyloid (amyloid beta-protein, Abeta) is one of the main components and pathological features of senile plaque. Based on the heterogeneity and multifactorial nature of AD and VD, cerebral hypoperfusion and brain pathological changes caused by cerebral aging and vascular risk factors (heart disease, hypertension, atherosclerosis, transient ischemic attack and the like) are combined, and an H/MAD model is prepared by utilizing intraperitoneal injection of galactose to promote aging, repeated ligation of cerebral hypoperfusion caused by bilateral common carotid arteries and one-time injection of beta amyloid in rat ventricles, so that the real brain pathological damage state of senile dementia patients can be reflected. Relevant researches prove that the H/MAD model has the characteristics of overall regulation action related to multiple layers, multiple ways and multiple targets, is an effective screening model which is in line with the characteristics of the traditional Chinese medicine and has relatively definite pathogenesis and is suitable for anti-H/MAD medicines (comprising traditional Chinese medicines, natural medicines, effective parts and monomer compounds), and provides a new method for researching medicines for treating the degenerative diseases of the central nervous system.
The experiment is carried out by measuring T-AOC, TChE, SOD, MDA and GSH-P in the serum of the H/MAD model ratXThe content of (b) can be used for understanding the damage of the organism caused by oxygen free radicals and the strength of the organism in the capacity of eliminating the free radicals, thereby providing a basis for the research of the action mechanism of the organism. The results show that after modeling, the generation of free radicals in the brain tissues of rats is increased, obvious lipid peroxidation reaction occurs, the application group (especially the middle dose group) of the Trillium arvense extract can recover the ethological activity of the H/MAD model rats, can recover the activity of T-AOC in serum, and can obviously improve SOD and GSH-PXThe activity level (P is less than 0.05 or P is less than 0.01), and the capability of the organism for eliminating free radicals is enhanced; can reduce TChE content, prevent neurotransmitter decrease, and simultaneously can reduce MDA content (P <0.05 or P <0.01) to reduce organism free radical injury degree, and show good antioxidant injury effect. The pathological manifestations of senile dementia include that a large amount of Senile Plaques (SP), neurofibrillary tangles (NFT) and a large amount of neurons are lost in cerebral cortex and hippocampus, wherein A beta aggregation is a central link and also is a key factor and a key therapeutic target for AD onset, interference on A beta generation and prevention of A beta aggregation are effective ways for preventing and treating AD, and development of a new drug for inhibiting A beta fibril formation and aggregation has important significance. Trillium has pharmacological effects of interfering with the production of A.beta.and preventing its aggregation. Synthesize the above mentioned researchThe 85% ethanol eluate of the trillium fortunei is proved to be the effective part of the trillium fortunei for treating heterogeneous and multi-cause senile dementia model rats.
Example 6
The component analysis and quality standard research of Trillium arvense, and the subject group found Trillium arvense pennogenin B in the research of Trillium arvense component3(pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside), B1(pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2)]-beta-D-glucoside), B2(pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2))]beta-D-glucoside) is a special component and a characteristic component in the Trillium wilfordii, has ultraviolet absorption at 203nm, can be directly used for content determination by an ultraviolet detector, and has simple and convenient method. The HPLC method is adopted to determine the trillin B in the trillin2、 B1、B3The content of (b) provides a basis for researching and formulating the quality standard of the Trillium wilfordii medicinal material.
B3: the molecular structural formula of the pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside (pennogenin-3-O-alpha-L-rhamnopyranosyl- (1 → 2) -beta-D-glucopyranoside) is shown in figure 1.
B1: pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2)]-beta-D-glucoside (pennogenin-3-O-alpha-L-rhamnopyranosyl- (1 → 4) - [ alpha-L-rhamnopyranosyl- (1 → 2)]- β -D-glucopyranoside), the molecular structural formula of which is shown in figure 2.
B2: pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2)]-beta-D-glucoside (pennogenin-3-O-alpha-L-rhamnopyranosyl- (1 → 4) - [ alpha-L-rhamnopyranosyl- (1 → 2)]- β -D-glucopyranoside), the molecular structural formula of which is shown in fig. 3.
The control product of trillium saponin B2 (pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside), B1 (pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside), b3 (pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside) is self-made by the laboratory, and the mass fraction is determined to be 99% by a high performance liquid phase peak area normalization method. The Trillium repens is collected from Shennongjia area of Hubei province and identified as Trillium tschonoski Maxim of Trillium repens of Trillium of Liliaceae by the assistant professor of Leimemingming of the Hodgia-western traditional Chinese medicine institute. Acetonitrile is chromatographically pure (Tianjin Si you biomedicine technology Co., Ltd.), and water is redistilled water (self-made).
Sample determination about 1.0g of trillium fortunei medicinal powder (passing through a No. 4 sieve and drying at low temperature to constant weight) is weighed, the preparation method is adopted to prepare a test sample solution, 20 mu l of the sample solution is precisely absorbed and injected into a high performance liquid chromatograph for determination, and the trillium fortunei saponin B2, B1 and B3 contents in the sample are respectively calculated to be 0.13%, 0.19% and 0.28% (n is 3) according to an external standard two-point method.
Preliminary study on determination of transitional components in Trillium wilfordii blood by HPLC method
The subject group also develops the research of the chromatographic fingerprint of the medicament-containing serum of the trillium, but only establishes a preparation method and a detection method of the medicament-containing serum preliminarily, and preliminarily determines that the blood-entering components of the trillium are B1 (pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside) and B3 (pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside), so that the quality evaluation method of the trillium is more complete, and a wider scientific basis is provided for determining the basis and action mechanism of the medicament-acting substances of the trillium.
Example 7
The method in the second embodiment of the invention is used for preparing the raw materials of the metanosapogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, the metanosapogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and the metanosapogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl from the trill 85% ethanol eluate of the trillium bengal saponin substances, wherein the raw materials of the metanosapogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl are subjected to the treatment of aging, cardiovascular and cerebrovascular diseases (heart diseases), heart diseases, blood pressure and the like in vitro, Hypertension, atherosclerosis, transient ischemic attack, etc.) resulting in cerebral hypoperfusion, senile dementia with the etiology of amyloid a β deposition, and alzheimer's disease.
1. Medicines and reagents trillium saponin 1 (pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside), trillium saponin 2 (pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2)]-beta-D-glucoside), trillin 3 (pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2)]-beta-D-glucoside) are white powders which are obtained by self-separation in a pencil laboratory and have the mass fraction of 98 percent. Donepezil hydrochloride (wei-defense pharmaceutical co., ltd., national drug standard H20050978, lot No. 131246 a); d-galactose (Sigma, Lot #060M 00631V); abeta (beta)25-35(Sigma Co., Lot #053M 4804V); T-AOC, TChE, CAT, SOD, MDA and GSH-PXDetection kit (Nanjing institute of bioengineering, batch No. 20150512).
2. Grouping, modeling and dosing of animals rats were randomly divided into 9 groups of 12 animals: blank group, H/MAD model group, donepezil hydrochloride group, trillium saponin 1 high dose group, trillium saponin 1 middle dose group, trillium saponin 2 high dose group, trillium saponin 2 middle dose group, trillium saponin 3 high dose group, trillium saponin 3 middle dose group. Blank groups are not processed; the rats in the model group and the administration group were injected with D-galactose (50 mg. kg) intraperitoneally every day-1) The subacute aging is caused, and the bilateral common carotid arteries are repeatedly ligated after 4W to block blood flow in a time-interval manner, so that cerebral ischemic lesions are caused. After the operation, the rats were fed for 3 days, and the non-dead rats in good condition were injected intracerebrally with condensed Abeta25-35. The model group started intracerebrally gavage 2% polysorbate aqueous solution 1 time daily on day 3 after the surgery; the donepezil hydrochloride group is 1mg kg-1Infusing polysorbate solution of donepezil hydrochloride for 1 time every day; the high (middle) dose group of trillium saponin 1, the high (middle) dose group of trillium saponin 2 and the high (middle) dose group of trillium saponin 3 are respectively 34.38 mg.kg-1、17.19mg·kg-1、20.51 mg·kg-1、10.25mg·kg-1、15.19mg·kg-1、7.59mg·kg-1Adding polysorbate water solution of Trillium wilfordii saponin 1, 2, 3, 1 times daily, and administering for 6 days.
Comparing the Morris water maze experiment with the blank group, the model group rats have prolonged latency, reduced number of passing stages, short stay time in the fourth quadrant and obvious difference (P is less than 0.01); compared with the model group, the administration groups of donepezil hydrochloride and trillin 1 and 2 can shorten the latent period and increase the number of times of passing through the platform, and the stay time in the fourth quadrant is prolonged (P is less than 0.05 or P is less than 0.01), which shows that the treatment effect of donepezil hydrochloride and trillin 1 and 2 is obvious, but the treatment effect of trillin 3 is not obvious. The results are shown in Table 6. The track diagram is shown in FIG. 6
TABLE 6 Trilligenin pair H/MAD model rat space exploration test results: (
Figure BDF0000006866170000241
n＝10)
Figure BDF0000006866170000242
Figure BDF0000006866170000251
In comparison with the blank set, the results,#p < 0.01; in comparison with the set of models,*p﹤0.05，**p﹤0.01
4. influence of Trillium fortunei saponin on T-AOC, TChE and CAT contents in serum of H/MAD model rat
Compared with the blank group, the content of T-AOC and CAT in the serum of the rat in the model group is obviously reduced, the activity of TChE is enhanced, and the difference is obvious (P is less than 0.01); compared with a model group, the trillium saponin 3 enables the activity of T-AOC to be recovered unobviously, has insignificant difference, can improve CAT content and reduce TChE content (P is less than 0.05); the administration of donepezil hydrochloride group and trillin 1 and 2 groups can improve the T-AOC and CAT content and reduce the activity of TChE, and have obvious difference (P is less than 0.05 or P is less than 0.01) compared with a model group, which shows that the administration groups of donepezil hydrochloride and trillin 1 and 2 have obvious treatment effect, can recover the activity of T-AOC in the brain of an H/MAD model rat, increase the CAT content of antioxidase, reduce the TChE content, enhance the capacity of removing free radicals, antagonize oxidation reaction, prevent the reduction of neurotransmitter, and play a role in preventing and treating AD. The results are shown in Table 7.
TABLE 7 influence of Trilligenin on T-AOC, TChE and CAT content in H/MAD model rat serum ((for T-AOC), TChE and CAT)
Figure BDF0000006866170000252
n＝10)
Figure BDF0000006866170000253
Figure BDF0000006866170000261
In comparison with the blank set, the results,#p < 0.01; in comparison with the set of models,*p﹤0.05，**p﹤0.01
5. influence of Trillium fortunei saponin on contents of SOD, MDA and GSH-PX in serum of H/MAD model rat
Compared with blank group, model group rat SOD and GSH-PXSignificantly decreased (P <0.01), while MDA levels were significantly increased (P < 0.01); compared with the model group, the donepezil hydrochloride group and the trillin 1 and 2 groups can obviously reduce the MDA level (P is less than 0.01), and the SOD and the GSH-PXThe obvious increase (P is less than 0.01) indicates that the treatment effect of the administration of donepezil hydrochloride and trilobenin 1 and 2 is effective; compared with the model group, the trillium saponin 3 has the advantages of unobvious and insignificant reduction of MDA level, but can improve SOD and GSH-PXActivity (P < 0.05). The results are shown in Table 8.
TABLE 8 Trillium fortunei Saponin vs. H/MAD model rat serum SOD, MDA, GSH-PXInfluence of content (
Figure BDF0000006866170000262
n＝10)
Figure BDF0000006866170000263
Figure BDF0000006866170000271
In comparison with the blank set, the results,#p < 0.01; in comparison with the set of models,*p﹤0.05，**p﹤0.01
the experiment is carried out by measuring T-AOC, TChE, CAT, SOD, MDA and GSH-P in the serum of the H/MAD model ratXThe content of (b) can be used for understanding the damage of the organism caused by oxygen free radicals and the strength of the organism in the capacity of eliminating the free radicals, thereby providing a basis for the research of the action mechanism of the organism. The results show that after modeling, the free radical production in the brain tissue of rats is increased, obvious lipid peroxidation reaction occurs, and the trilobatin 1 (pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside), the trilobatin 2 (pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2))]beta-D-glucoside) (especially in each medium dosage group) can recover the behavioral activity of H/MAD model rat, recover the activity of T-AOC in serum, increase the content of antioxidase CAT, and obviously improve SOD and GSH-PXThe activity level (P is less than 0.05 or P is less than 0.01), and the capability of the organism for eliminating free radicals is enhanced; can reduce TChE content, prevent neurotransmitter decrease, reduce MDA content (P <0.05 or P <0.01) to reduce organism damage caused by free radicals, and exhibit good antioxidant injury effect, and trillium grass saponin 3 (pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2))]- β -D-glucoside) is not significant. However, the pathogenesis of AD is very complex, and the pharmacodynamic material basis and mechanism of action remain to be further studied.
Example 8
The information and the analysis of the spectrum data of the metanosapogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside, the metanosapogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside and the metanosapogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2) ] -beta-D-glucoside in the trillium grass saponin substances.
1. pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 2) -beta-D-glucoside (pennogenin-3-O-alpha-L-rhamnopyranosyl- (1 → 2) -beta-D-glucopyranoside), colorless needle crystal (methanol), and molecular formula C39H62O13。1H-NMR(CDCl3，400MHz)：δ0.76(3H,d,J＝6.0Hz,CH3-27)，0.83(3H, s,CH3-18)，0.87(3H,s,CH3-19)，1.04(3H,brs,CH3-21)，1.19(3H,m,Rha CH3-6″)；5.36(1H,s,H-6)，3.62(2H,m,H-26)，4.85(1H,d,Glc H-1′)，5.92(1H,s,Rha H-1″)。13C-NMR(CDCl3100 MHz): Δ 36.8(C-1), 31.4(C-2), 80.5(C-3), 37.1(C-4), 141.4(C-5), 121.9(C-6), 33.6(C-7), 31.3(C-8), 54.1(C-9), 36.1(C-10), 22.4(C-11), 31.7(C-12), 43.8(C-13), 51.3(C-14), 30.6(C-15), 90.5(C-16), 90.3(C-17), 16.8(C-18), 19.1(C-19), 46.9(C-20), 18.6(C-21), 110.0(C-22), 33.9(C-23), 26.9(C-24), 30.3(C-25), 66.9(C-26), 16.7(C-27), 99.8 (C-1'),99,1,27,99,1, 79.2(C-2 '), 77.9(C-3 '), 73.7(C-4 '), 77.1(C-5 '), 62.7(C-6 '), 100.8(C-1 '), 72.3(C-2 '), 71.8(C-3 '), 71.7(C-4 '), 68.5(C-5 '), 19.2(C-6 '). The molecular structural formula is shown in figure 1.
2. pennogenin-3-O-alpha-L-rhamnopyranosyl (1 → 4) - [ alpha-L-rhamnopyranosyl (1 → 2)]-beta-D-glucoside (pennogenin-3-O-alpha-L-rhamnopyranosyl- (1 → 4) - [ alpha-L-rhamnopyranosyl- (1 → 2)]-beta-D-glucopyranoside), white needle crystals (methanol), molecular formula C45H71O17。1H-NMR(CDCl3， 400MHz)：δ0.76(3H,d,J＝6.0Hz,CH3-27)，0.83(3H,s,CH3-18)，0.87(3H,s, CH3-19)，1.04(3H,brs,CH3-21)，1.20(3H,d,J＝6.0Hz,Rha′CH3-6)，1.23(3H,d, J＝6.8Hz Rha″CH3-6)，5.36(1H,brs,H-6)，4.88(1H,d,Glc H-1)，5.49(1H,s,Rha′H-1)， 6.17(1H,s,Rha″H-1)。13C-NMR(CDCl3100 MHz): Δ 36.8(C-1), 31.3(C-2), 80.5(C-3), 37.2(C-4), 141.4(C-5), 121.9(C-6), 33.6(C-7), 31.4(C-8), 54.1(C-9), 36.1(C-10), 22.4(C-11), 31.7(C-12), 43.8(C-13), 51.3(C-14), 30.8(C-15), 90.5(C-16), 90.3(C-17), 16.8(C-18), 19.1(C-19), 46.9(C-20), 18.6(C-21), 110.2(C-22), 33.9(C-23), 26.9(C-24), 30.3(C-25), 66.9(C-26), 16.8(C-27), 100.2 (Glc-1'),100, 79.2(C-2 '), 77.9 (C-3'), 76.7(C-4 '), 77.1 (C-5'), 62.7(C-6 '), 101.8 (Rha' C-1 '), 72.3 (C-2'), 71.8(C-3 '), 71.0 (C-4'), 69.7(C-5 '), 19.2 (C-6'), 102.5(Rha "C-1 '), 72.7 (C-2'), 72.4(C-3 '), 77.1 (C-4'), 70.6(C-5 '), 19.2 (C-6'). The molecular structural formula is shown in figure 2.

Claims (5)

1. The application of the trillium saponins compound as the only active component in the preparation of the medicine for treating the Alzheimer disease is characterized in that: the trillium saponin compound is pennogenin-3-O-α-LRhamnopyranosyl (1 → 2) - β -DGlucoside (i.e. Paris Saponin VI), pennogenin-3-O-α-LRhamnopyranosyl (1 → 4) - [ alpha-LRhamnopyranosyl (1 → 2)]-β-D-at least one of the glucosides, the alzheimer's disease involved is any one of the alzheimer's diseases caused by aging, cardio-cerebrovascular diseases resulting in cerebral hypoperfusion states, amyloid a β deposits.
2. The use of trillium saponin compounds as claimed in claim 1 as the sole active ingredient in the manufacture of a medicament for the treatment of alzheimer's disease, characterized in that: the cardiovascular and cerebrovascular diseases can be any one of heart disease, hypertension, atherosclerosis and transient ischemic attack.
3. The use of trillium saponin compounds as claimed in claim 1 as the sole active ingredient in the manufacture of a medicament for the treatment of alzheimer's disease, characterized in that: the preparation formulation of the medicine is oral liquid, capsules, tablets and injection.
4. The use of trillium saponin compounds as claimed in claim 1 as the sole active ingredient in the manufacture of a medicament for the treatment of alzheimer's disease, characterized in that: the medicine comprises a single preparation and a compound preparation.
5. The use of trillium saponin compounds as claimed in claim 1 as the sole active ingredient in the manufacture of a medicament for the treatment of alzheimer's disease, characterized in that: the Trillium age saponin compound is derived from Trillium age of Trillium of LiliaceaeTrillium tschonoskii Maxim.。
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