CN105232891A - Preparation method of wake robin extracts and saponins of wake robin extracts and application of wake robin extracts and saponins to preparation of medicine for resisting ischemic heart disease - Google Patents
Preparation method of wake robin extracts and saponins of wake robin extracts and application of wake robin extracts and saponins to preparation of medicine for resisting ischemic heart disease Download PDFInfo
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Abstract
The invention relates to a preparation method of wake robin extracts and saponins of wake robin extracts and application of wake robin extracts and saponins in preparation of medicine for resisting ischemic heart diseases. Prepared wake robin extracts are 85% ethyl alcohol eluting parts of large-hole adsorption resin of wake robin total extracts, and the total saponin content of wake robin extracts is larger than 90%, and wake robin extracts are mainly prepared from 66-75% of pennogenin, 3-7% of wake robin saponin and 5-8% of dioscin. Prepared wake robin saponin compounds include one or more of pennogenin-3-O-alpha-L-pyran rhamanopyranosyl(1-2)-beta-D-glucoside, pennogenin-3-O-alpha-L-pyran rhamanopyranosyl(1-4)-[alpha-L-pyran rhamanopyranosyl(1-2)]-beta-D-glucoside and pennogenin-3-O-alpha-L-pyran rhamanopyranosyl(1-4)-alpha-L-pyran rhamanopyranosyl(1-4)-[alpha-L-pyran rhamanopyranosyl(1-2)]-beta-D-glucoside and have similar treatment functions in human bodies due to the sharing of structural units of pennogenin-3-O-alpha-L-pyran rhamanopyranosyl(1-2)-beta-D-glucoside and the like.
Description
Technical field
The present invention relates to Trillium tschonoskii Maxim extract and wherein saponins compound preparation method and preparing the application in ischemia resisting cardiotropic formulation, belong to technical field of traditional Chinese medicine preparation.
Background technology
Trillium tschonoskii Maxim is Liliaceae Trillium plant Trillium tschonoskii Maxim TrilliumtschonoskiiMaxim., and popular name Rhizoma Trillii Tschonoskii, Rhizoma Trillii Tschonoskii, lion ERQI etc., be traditional rare Chinese medicine, have effect of life lengthening.Cure mainly have a dizzy spell, have a sleepless night, traumatic injury, traumatic hemorrhage, neurasthenia, hypertension, the disease such as cerebral concussion sequela, be one of famous Folk medicine of Tujia Nationality in Enshi.The domestic and international pharmacology activity research to Trillium plant shows that Trillium tschonoskii Maxim has stronger antiinflammatory, immunomodulating, improves learning and memory function and the anti-ageing effect of waiting for a long time at present.Inventor adopts coronary ligation to cause myocardial ischemia in rats experimental model, confirms that Trillium tschonoskii Maxim 85% ethanol elution thing has significant preventive and therapeutic effect to myocardial ischemia from aspect researchs such as the vigor of electrocardiogram, myocardial infarct size, serum creatine kinase (CK) and serum lactate dehydrogenase (SLD) (LDH).
Chemical research shows, effective site 85% alcohol elution is based on steroidal saponin constituents, steroidal saponin total content is greater than 90%, wherein be mainly pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides, pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides, above-mentioned three kinds of saponin are index and the characteristic chemical constituent of Trillium tschonoskii Maxim medical material, again index and the characteristic chemical constituent of effective site 85% alcohol elution.Further pharmacological research confirms that in effective site, pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides, pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides is the effective ingredient of Trillium tschonoskii Maxim ischemia resisting cardiotropic formulation.Above-mentioned research is not also reported, we are studied by the experiment in vivo and vitro of system, first finds that its antagonism ischemic heart desease has good therapeutical effect.
Summary of the invention
The object of the invention be to provide a kind of Trillium tschonoskii Maxim extract and wherein saponins compound preparation method and preparing the application in ischemia resisting cardiotropic formulation.
Present invention employs following technical proposals.
The preparation method of Trillium tschonoskii Maxim extract: Trillium tschonoskii Maxim pulverizing medicinal materials is become coarse powder, by 0-100% soak with ethanol 0.5-2 hour, 4-12 times amount solvothermal reflux, extract, 2-4 time, each 1-3 hour, filters, merging filtrate.Above-mentioned filtrate and 45-80 DEG C are evaporated to 0.5-1.5g raw medicinal herbs/ml, 0-4 DEG C of refrigerated overnight, analyse glue, filter, filtrate is reclaimed the upper macroporous adsorbent resin of extractum dilution or filtrate directly go up macroporous adsorbent resin AB-8 type or D-101 type or HPD-400 or various model macroporous adsorbing resin for purification with n-butanol extraction again, use ethanol water by 0-50% eluting successively, each eluting position is obtained by 50-95% eluting again with ethanol water, the eluting position of main collection ethanol water 60-85%, each eluting position at 45-80 DEG C of decompression and solvent recovery to appropriate amount of ethanol concentration, drying is carried out with freeze-drying or spraying dry, collect lyophilized powder or spray drying powder, obtain.
Pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides (pennogenin-3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside) in Trillin compounds, the preparation method of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides (pennogenin-3-O-α-L-rhamnopyranosyl-(1 → 4)-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-glucopyranoside) and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides (pennogenin-3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-glucopyranoside): by silicagel column on claim 6 gained Trillium tschonoskii Maxim extract, chloroform is used successively: methanol or dichloromethane: methanol or chloroform: methanol: water or dichloromethane: methanol: water gradient elution after loading, with pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides in above-mentioned Trillin compounds, 3 kinds of saponin of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides in contrast product TLC or HPLC detect, merge respective streams part.Above-mentioned stream part is in 45-80 DEG C of concentrating under reduced pressure evaporate to dryness, with appropriate distilled water suspendible or dissolving, upper reverse phase silica gel OD5-C18 column chromatography or SephadexLH-20 column chromatography or preparative high-performance liquid chromatographic technology separation purification, use ethanol water by 0-50% eluting successively, use ethanol water 50-95% eluting again, eluent freeze-drying or spraying dry carry out drying, collect lyophilized powder or spray drying powder, with 10-95% alcohol crystal and recrystallization, to obtain final product.
Trillium tschonoskii Maxim extract prepared by preceding method and wherein saponins compound, its effective ingredient is pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β D-Glucose glycosides, one or more in pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides, or in its this material the 21st of sapogenin and the hydroxyl of the conversion of hydroxymethyl methyl of the 27th or the alcoholic extract hydroxyl group of 17 hydroxyls or all glycosyls are by acetyl group, aliphatic chain alkyl, aromatic substituents, any one in amide groups replaces, or the material of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides after hydrolysis, can be produced, can be used for preparing ischemia resisting cardiotropic formulation, described medicine comprises oral liquid, capsule, tablet, effervescent tablet, injectable powder, aqueous injection or injection or various dosage formulation, described medicine also comprises various folk prescription and compound preparation.
Involved ischemic heart desease is caused by myocardial ischemia and any one of the ischemic heart desease such as the coronary heart diseases and angina pectoris occurred and myocardial infarction.
Advantage of the present invention is: provide a kind of Trillium tschonoskii Maxim extract and the wherein extraction of saponins compound and preparation method, in gained material, effective ingredient is mainly pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides, one or more in pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides, gained material can be used for the oral liquid preparing ischemia resisting cardiotropic formulation, capsule, tablet, effervescent tablet, injectable powder, the crude drug of aqueous injection or injection or various dosage formulation.
In a kind of Trillium tschonoskii Maxim extract and wherein saponins compound, pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides, pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides is in vivo and in vitro to be caused by myocardial ischemia and the ischemic heart desease such as the coronary heart diseases and angina pectoris occurred and myocardial infarction has good therapeutical effect and stronger biological activity.
Pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides in a kind of Trillium tschonoskii Maxim extract and wherein saponins compound, the preparation method of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides mainly through extracting from natural drug, for pure natural component and monomer, there is good biocompatibility.
Accompanying drawing explanation
Fig. 1 is the molecular structural formula of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides;
Fig. 2 is the molecular structural formula of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides;
Fig. 3 is the molecular structural formula of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described:
Embodiment 1
A kind of Trillium tschonoskii Maxim extract and wherein pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides, the preparation method of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides: Trillium tschonoskii Maxim pulverizing medicinal materials is become coarse powder, by 70% soak with ethanol 1 hour, 10 times amount solvothermal reflux, extract, 3 times, each 2 hours, filter, merging filtrate.Above-mentioned filtrate and 65 DEG C are evaporated to 1.0g raw medicinal herbs/ml, 0-4 DEG C refrigerated overnight, analyse glue, filter, filtrate, again with organic solvent extractions such as n-butyl alcohol, reclaims organic solvent, obtains extractum and to add water suspendible, upper AB-8 model macroporous adsorbing resin for purification, use water, 30% ethanol, 60% ethanol and 85% ethanol elution successively, obtain each eluting position, carry out drying with freeze-drying, collect lyophilized powder, to obtain final product.Wherein the eluting position of 85% is based on steroidal saponin constituents, and steroidal saponin total content is greater than 95%, obtains Trillium tschonoskii Maxim extract.The 100-200 order silica gel extractum at the eluting position of above-mentioned Trillium tschonoskii Maxim extract both 85% ethanol being added equivalent is mixed thoroughly, 50 DEG C of decompressions volatilize solvent, grind well, dry method loading, upper normal pressure silica gel column (200-300 order, silica gel amount is 30 times of extractum), chloroform is used successively: methanol: water gradient elution after loading, main collection chloroform: methanol: water is stream part of 65: 35: 1, with pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides in above-mentioned Trillin class material, 3 kinds of saponin of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides in contrast product TLC or HPLC detect, merge respective streams part.Above-mentioned stream part is in 65 DEG C of concentrating under reduced pressure evaporates to dryness, with appropriate distilled water suspendible, upper reverse phase silica gel ODS-C18 column chromatography, use ethanol water by 0-50% eluting successively, use ethanol water 50-95% eluting again, main 60%-80% eluent freeze-drying of collecting carries out drying, collects lyophilized powder, with 10-95% alcohol crystal and recrystallization, to obtain final product.The yield adopting said method to prepare pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β D-Glucose glycosides in Trillin class material is 1.5-2.6g/kg, and detecting purity through HPLC is 99.0%; The yield of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides is 0.8-1.9g/kg, and detecting purity through HPLC is 98.6%; The yield that pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-yield of [α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides respectively is is 0.3-0.9g/kg, and detecting purity through HPLC is 98.2%; Chromatographic condition UItimateRXB-C18 chromatographic column (4.6mm × 150mm, 3 μm), mobile phase is acetonitrile-water (10%-100% gradient elution), and determined wavelength is 203nm, and flow velocity is 1.0ml/min, column temperature 30 DEG C, sample size 20 μ l.
Embodiment 2
A kind of Trillium tschonoskii Maxim extract and wherein pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β D-Glucose glycosides, the preparation method of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides: Trillium tschonoskii Maxim pulverizing medicinal materials is become coarse powder, by 60% soak with ethanol 2.0 hours, 12 times amount solvothermal reflux, extract, 3 times, each 2 hours, filter, merging filtrate.Above-mentioned filtrate and 70 DEG C are evaporated to 1g raw medicinal herbs/ml, 0-4 DEG C of refrigerated overnight, analyse glue, filter, D101 model macroporous adsorbing resin for purification on filtrate, uses water, 30% ethanol, 60% ethanol and 85% ethanol elution successively, obtain each eluting position, carry out drying with spray drying method, collect spray dried powder, to obtain final product.Wherein the eluting position of 85% is based on steroidal saponin constituents, and steroidal saponin total content is greater than 92%, obtains Trillium tschonoskii Maxim extract.The 100-200 order silica gel extractum at the eluting position of above-mentioned Trillium tschonoskii Maxim extract both 85% ethanol being added equivalent is mixed thoroughly, 50 DEG C of decompressions volatilize solvent, grind well, dry method loading, upper normal pressure silica gel column (200-300 order, silica gel amount is 25 times of extractum), chloroform is used successively: methanol: water gradient elution after loading, main collection chloroform: methanol: water is eluting stream part of 60: 30: 1, with pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β D-Glucose glycosides in above-mentioned Trillin class material, 3 kinds of saponin of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides in contrast product TLC or HPLC detect, merge respective streams part.Above-mentioned stream part is in 65 DEG C of concentrating under reduced pressure evaporates to dryness, with appropriate distilled water suspendible, upper SephadexLH-20 column chromatography, use ethanol water by 0-50% eluting successively, use ethanol water 50-95% eluting again, eluent spraying dry carries out drying, collects spray drying powder, with 10-95% alcohol crystal and recrystallization, to obtain final product.The yield adopting said method to prepare pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides in Trillin class material is 1.5-2.6g/kg, and detecting purity through HPLC is 98.8%; The yield of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides is 0.8-1.9g/kg, and detecting purity through HPLC is 98.5%; The yield that pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-yield of [α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides respectively is is 0.3-0.9g/kg, and detecting purity through HPLC is 97.8%; Chromatographic condition UItimateRXB-C18 chromatographic column (4.6mm × 150mm, 3 μm), mobile phase is acetonitrile-water (10%-100% gradient elution), and determined wavelength is 203nm, and flow velocity is 1.0ml/min, column temperature 30 DEG C, sample size 20 μ l.
Embodiment 3
A kind of Trillium tschonoskii Maxim extract and wherein pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides, the preparation method of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides: Trillium tschonoskii Maxim pulverizing medicinal materials is become coarse powder, by 50% soak with ethanol 1.0 hours, 8 times amount solvothermal reflux, extract, 3 times, each 2 hours, filter, merging filtrate.Above-mentioned filtrate and 70 DEG C are evaporated to 1g raw medicinal herbs/ml, 0-4 DEG C of refrigerated overnight, analyse glue, filter, HPD400 model macroporous adsorbing resin for purification on filtrate, uses water, 30% ethanol, 50% ethanol and 85% ethanol elution successively, obtain each eluting position, carry out drying with spray drying method, collect spray dried powder, to obtain final product.Wherein the eluting position of 85% is based on steroidal saponin constituents, and steroidal saponin total content is greater than 90%, obtains Trillium tschonoskii Maxim extract.The 100-200 order silica gel extractum at the eluting position of above-mentioned Trillium tschonoskii Maxim extract both 85% ethanol being added equivalent is mixed thoroughly, 50 DEG C of decompressions volatilize solvent, grind well, dry method loading, upper normal pressure silica gel column (200-300 order, silica gel amount is 20 times of extractum), chloroform is used successively: methanol: water gradient elution after loading, main collection chloroform: methanol: water is eluting stream part of 65: 35: 1, with pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides in above-mentioned Trillin class material, 3 kinds of saponin of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides in contrast product TLC or HPLC detect, merge respective streams part.Above-mentioned stream part, in 65 DEG C of concentrating under reduced pressure evaporates to dryness, purifies with preparative high-performance liquid chromatographic technology separation, with ethanol water 50-95% eluting, eluent carries out drying through suitable concentrated spraying dry, collect spray drying powder, with 10-95% alcohol crystal and recrystallization, to obtain final product.The yield adopting said method to prepare pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β D-Glucose glycosides in Trillin class material is 1.5-2.6g/kg, and detecting purity through HPLC is 99.3%; The yield of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides is 0.8-1.9g/kg, and detecting purity through HPLC is 99.1%; The yield that pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-yield of [α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides respectively is is 0.3-0.9g/kg, and detecting purity through HPLC is 98.8%; Chromatographic condition UItimateRXB-C18 chromatographic column (4.6mm × 150mm, 3 μm), mobile phase is acetonitrile-water (10%-100% gradient elution), and determined wavelength is 203nm, and flow velocity is 1.0ml/min, column temperature 30 DEG C, sample size 20 μ l.
Embodiment 4
Inventor adopts coronary ligation to cause myocardial ischemia in rats experimental model, confirms that Trillium tschonoskii Maxim 85% ethanol elution thing has significant preventive and therapeutic effect and stronger biological activity to myocardial ischemia from aspect researchs such as the vigor of electrocardiogram, myocardial infarct size, serum creatine kinase (CK) and serum lactate dehydrogenase (SLD) (LDH).
1 medicinal material extract: by the Trillium tschonoskii Maxim root and rhizome 30kg of drying, be ground into coarse powder, with 70% alcohol reflux 3 times, each 2.0 hours, filter, merge extractive liquid, reclaim under reduced pressure, got dry extract (total extract).Be separated after dry cream water suspendible through AB-8 type macroporous adsorbent resin, use water, 30% ethanol, 60% ethanol, 85% ethanol elution successively, obtain each eluting position.By Trillium tschonoskii Maxim 70% ethanol total extract (1g is equivalent to crude drug 6.7g), water elution thing (1g is equivalent to crude drug 12.2g), 30% ethanol elution thing (1g is equivalent to crude drug 83.3g), 60% ethanol elution thing (1g is equivalent to crude drug 55.6g), 85% ethanol elution thing (1g is equivalent to crude drug 26.7g), prepare test liquid with 2% Polysorbate aqueous solution respectively.
2 animal grouping and administrations: rat 60, be divided into sham operated rats, model group at random by body weight, positive group (DIAOXINXUE KANG JIAONANG is 60mg/kg), 85% ethanol elution thing high dose group be 88mg/kg, dosage group is 44mg/kg in 85% ethanol elution thing, dosage is 20ml/kg, sham operated rats, model group are filled with and are given equivalent distilled water, every day 1 time, continuous 14 days.
3 model preparations: after last administration 1h, lumbar injection 3% pentobarbital sodium 45mg/kg anesthetized animal, is fixed on supine position on Mus platform, cut off throat's skin, blunt separation subcutaneous fascia, muscle, find trachea, row endotracheal intubation, and connect animal respirator assisted respiartion.In chest unhairing, sterilization, be about 2cm along left mid-clavicular line longitudinal incision skin, in the 4th, 5 intercostal blunt separation muscle layer, open thoracic cavity, draw out heart with ring of coring from round.Left coronary artery anterior descending branch initial part can be seen, apart from left auricle lower edge 1 ~ 2mm place, with 6/0 stitching thread ligation at pulmonary conus and left auricle lower edge.Depth of needle controls at about 2mm, and width is about 2mm, after ligation, heart is put back to thoracic cavity, and after getting rid of the air in thoracic cavity, rapid mosquito forceps closes thoracic cavity.Ligation refused by a sham operated rats threading.
4 observation index:
4.1II lead electrocardiogram Animal Anesthesia dorsal position is fixed on Mus platform, extremity subcutaneous insertion electrocardiogram needle electrode, to record after normal II lead electrocardiogram and coronary ligation half an hour electrocardiogram (after ligation, the ECG ST section back of a bow is obviously raised, and ligation cardiac muscle color and luster shoal can represent ligation success), observe the change of electrocardiogram, 30minST section and T ripple after ligation.
After 4.2 myocardial infarct size abdominal aortic bloods, rapid taking-up heart, with blood remaining in the clean heart of PBS wash buffer, moisture is sucked with filter paper, in-20 DEG C of refrigerators, take out heart after freezing 8min, rapidly by the parallel lamellar being cut into thickness and being about 2mm of left ventricle ligature lower part cardiac muscle, lamellar cardiac muscle recovers lucifuge dyeing in the rearmounted 1%TTC buffer of now joining of room temperature, and 37 DEG C of water bath with thermostatic control 15min, period vibrates 4 times fully to dye.After right dyeing, cardiac muscular tissue is placed in 10% formalin and soaks 24h, normal myocardium is contaminated for redness, and infarct cardiac muscle is not painted.Cardiac muscular tissue's sequence is taken pictures, ImageProPlus6.0 medical image analysis system is used to analyze the myocardial infarction district area of taking pictures, countandmeasureabjects instrument is used to calculate Infarct area and whole myocardial area, the infarction size=Infarct area/heart gross area × 100%.
Abdominal aortic blood 6ml after the detection myocardial infarction 3h of 4.3CK, LDH vigor, after leaving standstill 2h, the centrifugal 20min of 4000r/min gets supernatant.Test kit detects the vigor of CK, LDH.
5 statistical processing methods:
Result is all used
represent, electrocardiogram represents with the difference of basic value before different time point measured value and ligation.Utilize between SPSS13.0 statistical software one factor analysis of variance method group and compare.
6 result of the tests:
The impact that 6.1 Trillium tschonoskii Maxim 85% ethanol elution things change rats with myocardial ischemia II lead electrocardiogram ST section and T ripple
The results are shown in Table 1, table 2.Sham operated rats is compared, model group rats after coronary ligation 5,10,15,30min ECG ST section, T ripple all significantly raise.Compared with model group, in positive drug group, Trillium tschonoskii Maxim 85% ethanol elution thing high dose group and Trillium tschonoskii Maxim 85% ethanol elution thing, dosage group all significantly can resist the rising (p < 0.05 or p < 0.01) that ligation arteria coronaria causes rat ST section, T ripple.
6.2 Trillium tschonoskii Maxim 85% ethanol elution things are on the impact of rats with myocardial ischemia myocardial infarct size
The results are shown in Table 3.Compared with sham operated rats, model group rats myocardial infarct size significantly raises; Compared with model group, in positive drug group, Trillium tschonoskii Maxim 85% ethanol elution thing high dose group, Trillium tschonoskii Maxim 85% ethanol elution thing, dosage group all significantly can reduce myocardial infarct size (p < 0.01), illustrate that in positive drug group, Trillium tschonoskii Maxim 85% ethanol elution thing high dose group, Trillium tschonoskii Maxim 85% ethanol elution thing, dosage group all can increase ischemic region blood supply, reduce myocardial ischemia infringement.
Table 3 Trillium tschonoskii Maxim 85% ethanol elution thing on the impact of rats with myocardial ischemia myocardial infarct size (
n=11)
Note: compare with sham operated rats, ##P < 0.01; Compare with model group, * * P < 0.01.
6.3 Trillium tschonoskii Maxim 85% ethanol elution things are on the impact of rats with myocardial ischemia sero-enzyme CK, LDH
The results are shown in Table 4.Compared with sham operated rats, model group rats after ligation arteria coronaria in serum CK, LDH vigor significantly raise; Compared with model group; in positive drug group, Trillium tschonoskii Maxim 85% ethanol elution thing high dose group, Trillium tschonoskii Maxim 85% ethanol elution thing dosage group serum in CK, LDH activity significantly reduce (p < 0.01), in prompting positive drug group, Trillium tschonoskii Maxim 85% ethanol elution thing high dose group, Trillium tschonoskii Maxim 85% ethanol elution thing, the ischemic injuries of dosage group to cardiac muscle has protective effect.
Table 4 Trillium tschonoskii Maxim 85% ethanol elution thing on the impact of rats with myocardial ischemia sero-enzyme CK, LDH vigor (
n=11)
Note: compare with sham operated rats, ##P < 0.01, compares with model group, * * P < 0.01.
7 experiment brief summaries:
Myocardial ischemia is caused by the supply and demand disequilibrium due to cardiac blood and oxygen, is the main pathophysiological mechanism that the ischemic heart desease such as coronary heart disease occur.In the research of myocardial ischemia regarding assay, the ECG change of laboratory animal is one of important observation index, the change of ST section and T wave height is that myocardial ischemia earliest period occurs, the most responsive and the most definite index one, is usually used in judging the drug effect that resists myocardial ischemia copying whether success and evaluation medicine of myocardial infarction and ischemia model.It is that it is due to subendocardiac muscle ischemia that T ripple raises caused by point transient current of injury formed between the unpolarized myocardial cell of generation and non-ischemic region myocardial cell of inside, visceral pericardium ischemic region that the ST section that during myocardial ischemia, electrocardiogram shows is raised.Therefore, usually using the changing value of ST section and T ripple as the leading indicator evaluating myocardial ischemia in rats damage.
CK is that one is transported with intracellular energy, muscle contraction, ATP regenerate the important kinases having direct relation, and the phosphoryl that turns between the catalysis creatine that it is reversible and ATP reacts.Mainly be distributed in skeletal muscle, cardiac muscle and brain cell.Its major function is: under adenosine triphosphate (ATP) effect, catalysis creatine phosphate, form phosphagen and adenosine diphosphate (ADP) (ADP), and the energy-rich phosphate bond of catalytic phosphatase creatine is transferred to adenosine diphosphate (ADP) (ADP) and generates adenosine triphosphate (ATP), and form creatine.After myocardial ischemia, cell membrane is subject to damage in various degree, and creatine kinase can be released into blood from damaged cell, and creatine kinase level in serum is improved rapidly, and creatine kinase is the important blood biochemistry index of myocardial infarction.
LDH is a kind of oligomerization enzyme, can the reversible transition of catalysis lactic acid and acetone acid.This enzyme is extensively distributed in cardiac muscle, erythrocyte, skeletal muscle and liver, and its content in Cell sap is very abundant, and thus the reaction of institute's catalysis is very fast, is the important biomolecule enzyme on Cellular respiration chain.During histanoxia, cell glycolysis is vigorous, and the process that acetone acid is transformed into lactic acid under LDH effect is strengthened, and the rising of LDH activity means that anerobic glycolysis process is strengthened, and aerobic oxidation process is suppressed, energy supply obstacle.So the activity of LDH can the scope of reflecting myocardium ischemic injuries and the order of severity in serum, its activity is higher, and reflecting myocardium damage is heavier.
Originally experimental studies have found that, after the ligation of rat left coronary artery anterior descending branch, ECG ST section, T ripple all significantly raise, and myocardial infarct size significantly increases, and in serum, CK, LDH vigor obviously raises, prompting myocardial ischemia modeling success.The exception that in Trillium tschonoskii Maxim 85% ethanol elution thing high dose group, Trillium tschonoskii Maxim 85% ethanol elution thing, dosage group all significantly can reduce rat model ST section and T ripple is raised, reduce the myocardial infarct size of rats with myocardial ischemia, suppress the vigor of CK, LDH in serum, prompting Trillium tschonoskii Maxim 85% ethanol elution thing has certain preventive and therapeutic effect to coronary ligation Myocardial Ischemia.
Embodiment 5
Trillium tschonoskii Maxim component analysis and quality standard research, seminar, in the research to Trillium tschonoskii Maxim composition, finds Trillium tschonoskii Maxim pennogenin B wherein
3(pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β D-Glucose glycosides), B
1(pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides), B
2(pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides) content is larger, it is the specificity composition and characteristic composition in Trillium tschonoskii Maxim, and have uv absorption at 203nm place, direct UV-detector can carry out assay, method is easy.HPLC method is adopted to measure Trillin B in Trillium tschonoskii Maxim herein
2, B
1, B
3content, for research and the quality standard of formulating Trillium tschonoskii Maxim medical material provide foundation.
B
3: pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β D-Glucose glycosides (pennogenin-3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside), its molecular structural formula is shown in Fig. 1.
B
1: pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides (pennogenin-3-O-α-L-rhamnopyranosyl-(1 → 4)-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-glucopyranoside), its molecular structural formula is shown in Fig. 2.
B
2: pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides (pennogenin-3-O-α-L-rhamnopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-[α-L-rhamnopyranosyl-(1 → 2)]-β-D-glucopyranoside), its molecular structural formula is shown in Fig. 3.
Reference substance Trillin B
2(pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides), B
1(pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides), B
3(pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β D-Glucose glycosides) is made by oneself by this laboratory, through efficient liquid phase areas of peak normalization method quality measurement mark 99%.Trillium tschonoskii Maxim medical material picks up from Shennongjia, Hubei Province, is accredited as Liliaceae Trillium plant Trillium tschonoskii Maxim TrilliumtschonoskiiMaxim. through Jiangxi University of Traditional Chinese Medicine associate professor Zuo Yueming.Acetonitrile is chromatographically pure (α Cygni friend biomedical technology company limited), and water is redistilled water (self-control).
Sample determination takes Trillium tschonoskii Maxim medicinal powder and (crosses No. 4 sieves, oven drying at low temperature is to constant weight) about 1.0g, prepares according to the preparation method of need testing solution, accurate pipette samples solution 20 μ l, injection high performance liquid chromatograph measures, according to the Trillin B in external standard two-point method calculation sample
2, B
1, B
3content be respectively 0.13%, 0.19% and 0.28% (n=3).
HPLC method measures the preliminary study of Trillium tschonoskii Maxim constituents absorbed into blood
This seminar has also carried out the Contained Serum chromatographic fingerprinting research of Trillium tschonoskii Maxim, but only tentatively establishes preparation method and the detection method of Contained Serum, and tentatively determining that Trillium tschonoskii Maxim enters blood component is B
1(pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides) and B
3(pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β D-Glucose glycosides), thus make the quality evaluating method of Trillium tschonoskii Maxim more perfect, for its effective substance clear and definite and the mechanism of action provide scientific basis widely.
Embodiment 6
Inventor adopts coronary ligation to cause myocardial ischemia in rats experimental model, from electrocardiogram, myocardial infarct size, the aspect researchs such as the vigor of serum creatine kinase (CK) and serum lactate dehydrogenase (SLD) (LDH) confirm pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides in Trillium tschonoskii Maxim 85% ethanol elution thing effective site, pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides have significant preventive and therapeutic effect and stronger biological activity to myocardial ischemia.
1 medicine and reagent: Trillin 1 (pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β D-Glucose glycosides), Trillin 2 (pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides), Trillin 3 (pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides) is white powder, be separated voluntarily by the author's laboratory and obtain, mass fraction is greater than 98%.
2 experimental techniques:
2.1 animal grouping and administrations: rat 90, to be divided in sham operated rats, model group, positive group (DIAOXINXUE KANG JIAONANG concentration is 6mg/ml), glycosides 1 high dose group (29.14mg/kg), glycosides 1 in dosage group (14.57mg/kg), glycosides 2 high dose group (17.14mg/kg), glycosides 2 dosage group (14.28mg/kg) in dosage group (8.86mg/kg), glycosides 3 high dose group (26.86mg/kg), glycosides 3 at random by body weight, often to organize 10 mouse.Dosage is 10ml/kg, and sham operated rats, model group are filled with and given equivalent distilled water, every day 1 time, continuous 14 days.
2.2 model preparations: after last administration 1h, lumbar injection 3% pentobarbital sodium 45mg/kg anesthetized animal, is fixed on supine position on Mus platform, cut off throat's skin, blunt separation subcutaneous fascia, muscle, find trachea, row endotracheal intubation, and connect animal respirator assisted respiartion.In chest unhairing, sterilization, be about 2cm along left mid-clavicular line longitudinal incision skin, in the 4th, 5 intercostal blunt separation muscle layer, open thoracic cavity, draw out heart with ring of coring from round.Left coronary artery anterior descending branch initial part can be seen, apart from left auricle lower edge 1 ~ 2mm place, with 6/0 stitching thread ligation at pulmonary conus and left auricle lower edge.Depth of needle controls at about 2mm, and width is about 2mm, after ligation, heart is put back to thoracic cavity, and after getting rid of the air in thoracic cavity, rapid mosquito forceps closes thoracic cavity.Ligation refused by a sham operated rats threading.
2.3 observation index:
2.3.1II lead electrocardiogram Animal Anesthesia dorsal position is fixed on Mus platform, extremity subcutaneous insertion electrocardiogram needle electrode, to record after normal II lead electrocardiogram and coronary ligation half an hour electrocardiogram (after ligation, the ECG ST section back of a bow is obviously raised, and ligation cardiac muscle color and luster shoal can represent ligation success), observe the change of electrocardiogram, 30minST section and T ripple after ligation.
2.3.2 after myocardial infarct size abdominal aortic blood, rapid taking-up heart, with blood remaining in the clean heart of PBS wash buffer, moisture is sucked with filter paper, in-20 DEG C of refrigerators, take out heart after freezing 8min, rapidly by the parallel lamellar being cut into thickness and being about 2mm of left ventricle ligature lower part cardiac muscle, lamellar cardiac muscle recovers lucifuge dyeing in the rearmounted 1%TTC buffer of now joining of room temperature, and 37 DEG C of water bath with thermostatic control 15min, period vibrates 4 times fully to dye.After right dyeing, cardiac muscular tissue is placed in 10% formalin and soaks 24h, normal myocardium is contaminated for redness, and infarct cardiac muscle is not painted.Cardiac muscular tissue's sequence is taken pictures, ImageProPlus6.0 medical image analysis system is used to analyze the myocardial infarction district area of taking pictures, countandmeasureabjects instrument is used to calculate Infarct area and whole myocardial area, the infarction size=Infarct area/whole myocardium gross area × 100%.
2.3.3CK, abdominal aortic blood 6mi after the detection myocardial infarction 3h of LDH vigor, after leaving standstill 2h, the centrifugal 20min of 4000r/min gets supernatant.Test kit detects the vigor of CK, LDH.
2.4 statistical processing methods
Result is all used
represent, electrocardiogram represents with the difference of basic value before different time point measured value and ligation.Utilize between SPSS13.0 statistical software one factor analysis of variance method group and compare.
3 experimental results:
The impact that 3.1 Trillins change rats with myocardial ischemia II lead electrocardiogram ST section and T ripple
The results are shown in Table 1, table 2.Compared with sham operated rats, model group rats after coronary ligation 5,10,15,30min ECG ST section, T ripple all significantly raise.Compared with model group, positive drug group, Trillin 1 (Trillin 2,3) are high, middle dosage group all significantly can resist the rising (p < 0.05 or p < 0.01) that ligation arteria coronaria causes rat ST section, T ripple.
3.2 Trillins are on the impact of rats with myocardial ischemia myocardial infarct size
The results are shown in Table 3.Compared with sham operated rats, model group rats myocardial infarct size significantly raises; Compared with model group, positive drug group, Trillin 1 (Trillin 2,3) are high, middle dosage group all significantly can reduce myocardial infarct size (p < 0.01 or p < 0.05), illustrate that positive drug group, Trillin 1 (Trillin 2,3) are high, middle dosage group all can increase ischemic region blood supply, reduce myocardial ischemia infringement.
Table 3 Trillin on the impact of rats with myocardial ischemia myocardial infarct size (
n=8)
Note: compare with sham operated rats,
##p < 0.01; Compare with model group, * P < 0.05, * * P < 0.01.
3.3 Trillins are on the impact of rats with myocardial ischemia sero-enzyme CK, LDH
The results are shown in Table 4.Compared with sham operated rats, model group rats after ligation arteria coronaria in serum CK, LDH vigor significantly raise; Compared with model group; in positive drug group, Trillin 1 (Trillin 2,3) height, middle dosage group serum, CK, LDH activity significantly reduce (p < 0.05 or p < 0.01), and prompting positive drug group, Trillin 1 (Trillin 2,3) are high, the ischemic injuries of middle dosage group to cardiac muscle has protective effect.
Table 4 Trillin on the impact of rats with myocardial ischemia sero-enzyme CK, LDH vigor (
n=8)
Note: compare with sham operated rats,
##p < 0.01; Compare with model group, * P < 0.05, * * P < 0.01.
4 experiment brief summaries:
Myocardial ischemia is caused by the supply and demand disequilibrium due to cardiac blood and oxygen, is the main pathophysiological mechanism that the ischemic heart desease such as coronary heart disease occur.In the research of myocardial ischemia regarding assay, the ECG change of laboratory animal is one of important observation index, the change of ST section and T wave height is that myocardial ischemia earliest period occurs, the most responsive and the most definite index one, is usually used in judging the drug effect that resists myocardial ischemia copying whether success and evaluation medicine of myocardial infarction and ischemia model.It is that it is due to subendocardiac muscle ischemia that T ripple raises caused by point transient current of injury formed between the unpolarized myocardial cell of generation and non-ischemic region myocardial cell of inside, visceral pericardium ischemic region that the ST section that during myocardial ischemia, electrocardiogram shows is raised.Therefore, usually using the changing value of ST section and T ripple as the leading indicator evaluating myocardial ischemia in rats damage.
CK is that one is transported with intracellular energy, muscle contraction, ATP regenerate the important kinases having direct relation, and the phosphoryl that turns between the catalysis creatine that it is reversible and ATP reacts.Mainly be distributed in skeletal muscle, cardiac muscle and brain cell.Its major function is: under adenosine triphosphate (ATP) effect, catalysis creatine phosphate, form phosphagen and adenosine diphosphate (ADP) (ADP), and the energy-rich phosphate bond of catalytic phosphatase creatine is transferred to adenosine diphosphate (ADP) (ADP) and generates adenosine triphosphate (ATP), and form creatine.After myocardial ischemia, cell membrane is subject to damage in various degree, and creatine kinase can be released into blood from damaged cell, and creatine kinase level in serum is improved rapidly, and creatine kinase is the important blood biochemistry index of myocardial infarction.
LDH is a kind of oligomerization enzyme, can the reversible transition of catalysis lactic acid and acetone acid.This enzyme is extensively distributed in cardiac muscle, erythrocyte, skeletal muscle and liver, and its content in Cell sap is very abundant, and thus the reaction of institute's catalysis is very fast, is the important biomolecule enzyme on Cellular respiration chain.During histanoxia, cell glycolysis is vigorous, and the process that acetone acid is transformed into lactic acid under LDH effect is strengthened, and the rising of LDH activity means that anerobic glycolysis process is strengthened, and aerobic oxidation process is suppressed, energy supply obstacle.So the activity of LDH can the scope of reflecting myocardium ischemic injuries and the order of severity in serum, its activity is higher, and reflecting myocardium damage is heavier.
Originally experimental studies have found that, after the ligation of rat left coronary artery anterior descending branch, ECG ST section, T ripple all significantly raise, and myocardial infarct size significantly increases, and in serum, CK, LDH vigor obviously raises, prompting myocardial ischemia modeling success.Trillin 1 (pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β D-Glucose glycosides), the height of Trillin 2 (pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides) and Trillin 3 (pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides), the exception that middle dosage group all significantly can reduce rat model ST section and T ripple is raised, the aobvious myocardial infarct size reducing rats with myocardial ischemia, suppress CK in serum, the vigor of LDH, prompting Trillin 1 (pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β D-Glucose glycosides), the height of Trillin 2 (pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides) and Trillin 3 (pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides), middle dosage group has certain preventive and therapeutic effect to coronary ligation Myocardial Ischemia.
Embodiment 7
The spectral data information of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β D-Glucose glycosides, pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β D-Glucose glycosides and parsing in Trillin class material.
1. pennogenin-3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-Glucose glycosides (pennogenin-3-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside), colorless needles (methanol), molecular formula is C
39h
62o
13.
1H-NMR(CDCl
3，400MHz)：δ0.76(3H，d，J＝6.0Hz，CH
3-27)，0.83(3H，s，CH
3-18)，0.87(3H，s，CH
3-19)，1.04(3H，brs，CH
3-21)，1.19(3H，m，RhaCH
3-6″)；5.36(1H，s，H-6)，3.62(2H，m，H-26)，4.85(1H，d，GlcH-1′)，5.92(1H，s，RhaH-1″)。
13C-NMR(CDCl
3，100MHz)：δ36.8(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′)，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″)。Molecular structural formula is shown in Fig. 1.
2. pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides (pennogenin-3-O-α-L-rhamnopyranosyl-(1 → 4)-[α-L-rhamnopyranosyl-(1 → 2)] β-D-glucopyranoside), white needles (methanol), molecular formula is C
45h
71o
17.
1H-NMR(CDCl
3，400MHz)：δ0.76(3H，d，J＝6.0Hz，CH
3-27)，0.83(3H，s，CH
3-18)，0.87(3H，s，CH
3-19)，1.04(3H，brs，CH
3-21)，1.20(3H，d，J＝6.0Hz，Rha′CH
3-6)，1.23(3H，d，J＝6.8HzRha″CH
3-6)，5.36(1H，brs，H-6)，4.88(1H，d，GlcH-1)，5.49(1H，s，Rha′H-1)，6.17(1H，s，Rha″H-1)。
13C-NMR(CDCl
3，100MHz)：δ36.8(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(GlcC-1′)，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″′)。Molecular structural formula is shown in Fig. 2.
Claims (7)
1. the cardiopathic Trillium tschonoskii Maxim extract of ischemia resisting, it is characterized in that, be the 60-85% alcohol elution of macroporous adsorbent resin on Trillium tschonoskii Maxim total extract and its total saponin content is greater than 90%, wherein pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides of bisnosaponin, the content 66-75% of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides, the trillenosideA of Trillin class, trillenosideB, trillenosideC, Epitrillenogenin-24-O-acetate-1-O-[2, 3, 4,-tri-O-acetyl-α-L-rhamn-opyranosyl-(1 → 2)-α-L-arabinopyranoside], Epitrillenogenin-1-O-[2, 3, 4,-tri-O-acetyl-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranoside], Epitrillenogenin-24-O-acetate-1-O-[2, 4,-di-O-acetyl-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranoside], Epitrille-nogenin-1-O-[2, 4,-di-O-acetyl-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranoside], the content 3-7% of Epitrillenogenin-1-O-[4-O-acetyl-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosi-de], diosgenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides of dioscin class, the content 5-8% of diosgenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides.
2. the cardiopathic Trillin compounds of ischemia resisting, it is characterized in that, itself be one or more in pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides, pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides.
3. the cardiopathic Trillin compounds of ischemia resisting, it is characterized in that, described Trillin class material is pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides, the 21st of sapogenin in one or more these materials in pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and the hydroxyl of the conversion of hydroxymethyl methyl of the 27th or the alcoholic extract hydroxyl group of 17 hydroxyls or all glycosyls are by acetyl group, aliphatic chain alkyl, aromatic substituents, any one in amide groups replaces, or the material of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides after hydrolysis, can be produced.
4. Trillium tschonoskii Maxim extract and wherein saponins compound preparing the application in ischemia resisting cardiotropic formulation, wherein: involved ischemic heart desease is caused by myocardial ischemia and any one of the ischemic heart desease that formed of the coronary heart diseases and angina pectoris occurred and myocardial infarction.
5. the Trillium tschonoskii Maxim extract as described in claim 1-4 and wherein saponins compound are preparing the application in ischemia resisting cardiotropic formulation, it is characterized in that, described medicine comprises oral liquid, capsule, tablet, effervescent tablet, injectable powder, aqueous injection or injection or various dosage formulation, and described medicine also comprises various folk prescription and compound preparation.
6. the preparation method of Trillium tschonoskii Maxim extract described in claim 1: Trillium tschonoskii Maxim pulverizing medicinal materials is become a coarse powder, by 0-100% soak with ethanol 0.5-2 hour, 4-12 times amount solvothermal reflux, extract, 2-4 time, each 1-3 hour, filters, merging filtrate, above-mentioned filtrate and 45-80 DEG C are evaporated to 0.5-1.5g raw medicinal herbs/ml, 0-4 DEG C of refrigerated overnight, analyse glue, filter, filtrate is reclaimed the upper macroporous adsorbent resin of extractum dilution or filtrate directly go up macroporous adsorbent resin AB-8 type or D-101 type or HPD-400 or various model macroporous adsorbing resin for purification with n-butanol extraction again, use ethanol water by 0-50% eluting successively, each eluting position is obtained by 50-95% eluting again with ethanol water, collect the eluting position of ethanol water 60-85%, each eluting position at 45-80 DEG C of decompression and solvent recovery to appropriate amount of ethanol concentration, drying is carried out with freeze-drying or spraying dry, collect lyophilized powder or spray drying powder, obtain.
7. the preparation method of Trillin compounds described in a claim 2: by silicagel column on claim 6 gained Trillium tschonoskii Maxim extract, chloroform is used successively: methanol or dichloromethane: methanol or chloroform: methanol: water or dichloromethane: methanol: water gradient elution after loading, with pennogenin-3-O-α-L-rhamnopyranosyl (1 → 2)-β-D-Glucose glycosides in above-mentioned Trillin compounds, 3 kinds of saponin of pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides and pennogenin-3-O-α-L-rhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 4)-[α-L-rhamnopyranosyl (1 → 2)]-β-D-Glucose glycosides in contrast product TLC or HPLC detect, merge respective streams part, above-mentioned stream part is in 45-80 DEG C of concentrating under reduced pressure evaporate to dryness, with appropriate distilled water suspendible or dissolving, upper reverse phase silica gel ODS-C18 column chromatography or SephadexLH-20 column chromatography or preparative high-performance liquid chromatographic technology separation purification, use ethanol water by 0-50% eluting successively, use ethanol water 50-95% eluting again, eluent freeze-drying or spraying dry carry out drying, collect lyophilized powder or spray drying powder, with 10-95% alcohol crystal and recrystallization, to obtain final product.
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