CN102614243A - Method for extracting common macrocarpium fruit total glycoside and application of common macrocarpium fruit total glycoside to preparation of hypoxia tolerant medicines - Google Patents
Method for extracting common macrocarpium fruit total glycoside and application of common macrocarpium fruit total glycoside to preparation of hypoxia tolerant medicines Download PDFInfo
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Abstract
The invention relates to a method for extracting a common macrocarpium fruit total glycoside and application of the common macrocarpium fruit total glycoside serving as active ingredients to the preparation of hypoxia tolerant medicines. The method comprises the following steps of: boiling, performing enzymolysis for extracting and purifying. The pharmacodynamic test proves that the common macrocarpium fruit total glycoside extracted by the method can improve the cardio-cerebral hypoxia capacity of mice, enhance physical strength, improve antifatigue capacity of organisms, and promote the establishment of hypoxia tolerant acclimatization.
Description
Technical field
The invention belongs to the medicinal preparation technical field of the product that contains raw material or itself and not clear structure, be specifically related to derive from the material of plant.
Background technology
Fructus Corni is the drying and ripening sarcocarp of Cornaceae, Macrocarpium defoliation small arbor Fructus Corni.Have another name called Fructus Corni, Fructus Corni etc., sour-puckery flavor, slightly warm in nature has the temperature compensation Liver and kidney, and the merit of astringent or styptic treatment for spontaneous sweating vital essence cures mainly diseases such as lumbocrural pain, dizzy, tinnitus, frequent micturition, persistent vaginal bloody discharge, is one of clinical conventional Chinese medicine.
The Fructus Corni main chemical compositions is mainly polysaccharide, organic acid and esters, iridoid glycoside and aglycon, tannin and volatile ingredient.Wherein iridoid glycoside is the main active site in the Fructus Corni.Clear and definite iridoid glycoside constituents comprises morroniside, loganin, sweroside, cornin, 7-O-ethyl morroniside, 7-oxygen-butyl morroniside, 7-dehydrogenation loganin, Semen Strychni thuja acid etc. at present.Wherein morroniside, loganin, sweroside content are the highest, and the content of three in Fructus Corni reaches 2.0%, 0.6% and 0.1% respectively, has accounted for more than 85% of Fructus Corni total iridoid glycoside.
Fructus Corni total glycosides has wide biological activity: comprise neuroprotective, influence the various kinds of cell factor in the brain, suppress neuronal apoptosis and neuronal death that various models cause, thereby performance is to the therapeutical effect of cerebral infarction and Alzheimer's disease.Act on cardio-cerebrovascular, suppress inside and outside thrombosis, activate anticoagulant factor, the inductive platelet aggregation of antagonism several different methods increases, and the myocardial ischemia behind the coronary ligation is played a protective role.The treatment diabetes all have mitigation to diabetic vascular complications, cardiac complication and renal complication, heart, kidney and vascular tissue's cells injury that the protection diabetes cause.
Enzymatic isolation method uses increasingly extensive in the extraction of active ingredient of natural product.The catalytic reaction of enzyme has characteristics such as specificity, high efficiency, mild condition, and it is hydrolysis or the degradation of cell wall construction through enzyme that biological enzymolysis extracts, and intracellular composition fully is dissolved in the solvent, thereby increases extraction ratio.Many examples of many successful in recent years show the Enzymatic Extraction mild condition, and extract color stability and repeatability are all better, and extraction ratio is higher than traditional handicraft.But the report that Shang Weiyou adopts the auxiliary Fructus Corni total glycosides of the method to extract.
Anoxia is the most general stressors of special environment such as plateau, aviation, diving, and acute exposure will produce the oxygen deprivation stress reaction in the human or animal of anaerobic environment.Improve the hypoxia-bearing capability of body, will promote the foundation that anoxia enduring adapts to, weaken the damage of anoxia, make body in anaerobic environment, keep normal physiological function body through reducing body oxygen deprivation stress intensity.The Fructus Corni liver and kidney tonifying, essence astringing and desertion stemming.In clinical to vertigo and tinnitus, soreness of waist and knee joint, profuse sweating collapse, deficient syndromes such as the seminal emission enuresis have significant curative effect.Up to now, do not see that as yet Fructus Corni total glycosides is used to prevent and treat the anoxybiotic report of heart and brain.
Summary of the invention
A technical problem to be solved by this invention is to provide the method for preparing of Fructus Corni total glycosides.
Another technical problem to be solved by this invention is for Fructus Corni total glycosides a kind of new purposes to be provided.
Solving the problems of the technologies described above the technical scheme that is adopted is made up of following step:
1, boiling hot boiling
The Fructus Corni fresh fruit is dropped in the boiling water to submergence, and boiling water boiled 10～15 minutes, pulled postcooling a moment out, squeezed out pit with hands, and 40 ℃ of oven dry of sarcocarp are pulverized, and cross 20 mesh sieves, the Fructus Corni medicinal powder.
2, enzymolysis and extraction
Get the Fructus Corni medicinal powder, add 2～4 times of water-soluble expanding, add emulsin again; The mass ratio of Fructus Corni medicinal powder and emulsin is 1: 0.005～0.015, regulates pH value to 4～4.5,40～50 ℃ of enzymolysis of constant temperature 3～5 hours with glacial acetic acid; Add the ethanol with quality such as water, stop enzymolysis, adding water to the ethanol mass fraction again is 25%～27%; Use frequency to be the ultrasonic extraction of 300W 30～40 minutes as 25kHz, power, inclining supernatant; Fructus Corni medicinal powder slag again with the liquid material than being that 12～16 times, mass fraction are that 25%～27% ethanol water extracts once, decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, the Fructus Corni ethanol extraction.
3, purification
The Fructus Corni ethanol extraction with etc. the water dilution and the mixing of quality, carry out purification through macroporous resin column, use the deionized water eluting, colourless to eluent; Be 48%～55% ethanol water eluting with mass fraction again, 8～9 times of column volumes of elution volume, applied sample amount 40～50mg/g merges eluent; Being evaporated to does not have the alcohol flavor, and 50 ℃ of dryings in baking oven are pulverized, and cross 20 mesh sieves; Get Fructus Corni total glycosides, in Fructus Corni total glycosides, morroniside, loganin, sweroside total content are not less than 70%.
In enzymolysis and extraction step 2 of the present invention, get the Fructus Corni medicinal powder, add 2～4 times of water-soluble expanding; Add emulsin again, the mass ratio of Fructus Corni medicinal powder and emulsin is 1: 0.01, regulates pH value to 4.5 with glacial acetic acid; 50 ℃ of enzymolysis of constant temperature 4 hours add the ethanol with quality such as water, stop enzymolysis; Adding water to the ethanol mass fraction again is 25%～27%, uses frequency to be the ultrasonic extraction of 300W 30～40 minutes as 25kHz, power, and inclining supernatant; Fructus Corni medicinal powder slag again with the liquid material than being that 8～10 times, mass fraction are that 25%～27% ethanol water extracts once, decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, the Fructus Corni ethanol extraction.
The purposes of effective ingredient Fructus Corni total glycosides in preparation anoxia enduring medicine.
The effective ingredient Fructus Corni total glycosides prepares the anoxia enduring medicine to be used with the form of conventional pharmaceutical formulation.Described conventional pharmaceutical formulation contains the Fructus Corni total glycosides as active component, and this active component mixes like solid or the liquid excipient that is suitable for the organic or inorganic of administration in the gastrointestinal with pharmaceutically acceptable carrier in preparation.This pharmaceutical formulation can be solid form such as tablet, granule, capsule; Also can be liquid form such as suspending agent, syrup, Emulsion etc.
Can contain auxiliary substance, stabilizing agent, wetting agent, solubilizing agent and other additive commonly used in the above-mentioned preparation, like lactose, Pulvis Talci, cellulose, polyvinylpyrrolidone, starch, pectin, tween 80, polyvinyl alcohol etc.
Above-mentioned preparation can be processed according to the conventional preparation technology of various preparations.
Active component Fructus Corni total glycosides of the present invention is processed the oral drugs of various dosage forms, becomes human oral, common dose 180mg every day, and one after each meal, children is taken the circumstances into consideration decrement.
The indication of Fructus Corni total glycosides: treatment is in anaerobic environment or anoxia symptom that cerebral ischemia caused, and is unable like dizziness, headache, tinnitus, dim eyesight, tetraparesis; Perhaps feel sick, vomit, have palpitation, breathe hard, rapid breathing, heart beating be unable fast, diseases such as anoxia.
The medicinal preparation for oral administration that said extracted thing Fructus Corni total glycosides is processed by conventional method is the above tablet or granule or capsule or an oral liquid of galenic pharmacy.
The present invention is applied to the leaching process of Fructus Corni total glycosides with enzymatic isolation method, and emulsin carries out enzymolysis to Fructus Corni medical material powder, and the effective ingredient stripping quantity has increased by 20% in the Fructus Corni alcohol extraction thing; Adopt punctate opacity of the cornea design-response surface method; Extraction and purifying process to Fructus Corni total glycosides is optimized respectively, confirmed the extraction purification optimum process parameter of Fructus Corni total glycosides, and effective ingredient in the prepared Fructus Corni total glycosides (morroniside, loganin and sweroside) content reaches 70%; The response rate reaches 80%; Preparation efficiency is high, and raw material availability is high, is fit to suitability for industrialized production.
The Fructus Corni total glycosides that adopts the inventive method to extract proves that through the test of pesticide effectiveness it can improve the patient cerebral anoxia ability of mice, and physical strength reinforcing improves body fatigue resistance, promotes the foundation that anoxia enduring adapts to.
The specific embodiment
To further explain of the present invention, but the invention is not restricted to these embodiment below in conjunction with embodiment.
Embodiment 1
Dry medicinal powder 100g is an example with the extract Fructus Corni of Fructus Corni fresh fruit, and used raw material and method for distilling thereof are following:
1, boiling hot boiling
The Fructus Corni fresh fruit is dropped in the boiling water to submergence, and boiling water boiled 10～15 minutes, pulled postcooling a moment out, squeezed out pit with hands, and 40 ℃ of oven dry of sarcocarp are pulverized, and cross 20 mesh sieves, the Fructus Corni medicinal powder.
2, enzymolysis and extraction
Get Fructus Corni medicinal powder 100g, add 2～4 times of water-soluble expanding, add emulsin 1g again, the model of emulsin is M0034-1; Emulsin is the commodity of market sale, is sold by the emerging commerce and trade company limited of Shanghai section, regulates pH value to 4.5 with glacial acetic acid; 50 ℃ of enzymolysis of constant temperature 4 hours add the ethanol with quality such as water, stop enzymolysis; Adding water to the ethanol mass fraction again is 25%～27%, uses frequency to be the ultrasonic extraction of 300W 30～40 minutes as 25kHz, power, and inclining supernatant; Fructus Corni medicinal powder slag again with the liquid material than being that 12～16 times, mass fraction are that 25%～27% ethanol water extracts once, decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, the Fructus Corni ethanol extraction.
3, purification
The Fructus Corni ethanol extraction with etc. the water dilution of quality, carry out purification through macroporous resin column, use the deionized water eluting, colourless to eluent; Be 48%～55% ethanol water eluting with mass fraction again, 8～9 times of column volumes of elution volume, applied sample amount 40～50mg/g merges eluent; Being evaporated to does not have the alcohol flavor, and 50 ℃ of dryings in baking oven are pulverized, and cross 20 mesh sieves; Get Fructus Corni total glycosides, in Fructus Corni total glycosides, morroniside, loganin, sweroside total content are not less than 70%.
Embodiment 2
Dry medicinal powder 100g is an example with the extract Fructus Corni of Fructus Corni fresh fruit, and used raw material and method for distilling thereof are following:
In enzymolysis and extraction step 2, get Fructus Corni medicinal powder 100g, add 2～4 times of water-soluble expanding; Add emulsin 0.5g again, regulate pH value to 4,45 ℃ of enzymolysis of constant temperature 6 hours with glacial acetic acid; Add the ethanol with quality such as water, stop enzymolysis, adding water to the ethanol mass fraction again is 25%～27%; Use frequency to be the ultrasonic extraction of 300W 30～40 minutes as 25kHz, power, inclining supernatant; Fructus Corni medicinal powder slag again with the liquid material than being that 12～16 times, mass fraction are that 25%～27% ethanol water extracts once, decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, the Fructus Corni ethanol extraction.
Other steps are identical with embodiment 1.Extract Fructus Corni total glycosides.
Embodiment 3
Dry medicinal powder 100g is an example with the extract Fructus Corni of Fructus Corni fresh fruit, and used raw material and method for distilling thereof are following:
In enzymolysis and extraction step 2, get Fructus Corni medicinal powder 100g, add 2～4 times of water-soluble expanding; Add emulsin 1.5g again, regulate pH value to 4.5, constant temperature 50C enzymolysis 4 hours with glacial acetic acid; Add the ethanol with quality such as water, stop enzymolysis, adding water to the ethanol mass fraction again is 25%～27%; Use frequency to be the ultrasonic extraction of 300W 30～40 minutes as 25kHz, power, inclining supernatant; Fructus Corni medicinal powder slag again with the liquid material than being that 12～16 times, mass fraction are that 25%～27% ethanol water extracts once, decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, the Fructus Corni ethanol extraction.
Other steps are identical with embodiment 1.Extract Fructus Corni total glycosides.
Embodiment 4
The pharmaceutical formulation Fructus Corni total glycosides is prepared into tablet, is that example is raw materials used following with adjuvant and quality proportioning thereof to produce 1000 of tablet products:
Its preparation technology is undertaken by the common process of galenic pharmacy tablet, every heavy 0.3g, and every contains Fructus Corni total glycosides 20mg, day clothes three times, each 3.
Embodiment 5
The pharmaceutical formulation Fructus Corni total glycosides is prepared into capsule, is that example is raw materials used following with adjuvant and quality proportioning thereof to produce 1000 of capsule products:
Fructus Corni total glycosides 20g
Magnesium stearate 1g
Starch adds to 300g
Its preparation method is undertaken by the conventional method of galenic pharmacy capsule, every heavy 0.3g, and every contains Fructus Corni total glycosides 20mg, day clothes three times, each 3.
Embodiment 6
Medicinal Fructus Corni total glycosides is prepared into granule, is that example is raw materials used following with adjuvant and quality proportioning thereof to produce granule product 1000g:
Its preparation method is undertaken by the conventional method of galenic pharmacy granule, every bag heavy 5g, and every gram contains Fructus Corni total glycosides 18mg, day clothes twice, each 1 bag.
Embodiment 7
Medicinal Fructus Corni total glycosides is prepared into oral liquid, is that example is raw materials used following with adjuvant and quality proportioning thereof to produce oral liquid product 1000mL:
Fructus Corni total glycosides 6g
Cyclamate 10g
Distilled water adds to 1000mL
Its preparation method is undertaken by the conventional method of galenic pharmacy oral liquid, every bottle heavy 10mL, and 1mL contains Fructus Corni total glycosides 6mg, day clothes three times, each 1 bottle.
In order to confirm optimum material proportion of the present invention and optimised process step, the inventor has carried out the development test of lot of test chamber, and various test situation are following:
1, morroniside in the Fructus Corni medical material, the quantitative analysis of loganin and sweroside
Content assaying method: chromatographic column Welchrom-C18 post (250mm * 4.6mm, 5 μ m), 35 ℃ of column temperatures; Mobile phase: methanol (A)-0.1% phosphoric acid water (B), gradient elution (0～8 minute, 28%A; 8～15 minutes, 28%A → 35%A; 15～20 minutes, 28%A), flow velocity 1mL/min; Detect wavelength 240nm; Sample size 10 μ L.Under above-mentioned chromatographic condition, morroniside, loganin and sweroside can reach baseline separation with other compositions.
Methodological study: the range of linearity 10.0～600.0 of this method morroniside, detectability 0.21 μ g/ml, quantitative limit 0.67 μ g/ml, correlation coefficient 0.9996; The range of linearity 7.2～432.0 of loganin, detectability 0.18 μ g/ml, quantitative limit 0.58 μ g/ml, correlation coefficient 0.9998; The range of linearity 3.1～186.0 of sweroside, detectability 0.16 μ g/ml, quantitative limit 0.50 μ g/ml, correlation coefficient 0.9999; This assay method in a few days reach day to day precision, reproducible relative standard deviation RSD＜1.2%; Relative standard deviation RSD＜2.2% of stability; Average recovery between 95%～105%, relative standard deviation RSD＜2.0%.
2, different pre-treating methods are to the influence of iridoid glycosides content in the Fructus Corni extract
Water-boiling method: get 20g Fructus Corni fresh fruit and drop in the boiling water; Boiled respectively 1,5,10,20 minute, and pulled postcooling out, squeeze out pit with hands; Again with 40 ℃ of oven dry of sarcocarp; Getting dry sarcocarp 100mg, is that 25% ethanol water 25mL carries out supersound extraction with mass fraction, and extracting solution carries out assay through HPLC.
Water at atmospheric pressure is steamed method: get 20g Fructus Corni fresh fruit and put into food steamer, treat respectively to steam 1,5,10,20 minute after the Shanghai Automobile Factory, take out postcooling; Squeeze out pit with hands; With 40 ℃ of oven dry of sarcocarp, carry out supersound extraction with 25% ethanol 25mL again, extracting solution carries out assay through HPLC.
Microwave method: get 20g Fructus Corni fresh fruit, microwave heating is 20,30,40,50,60 seconds respectively, takes out postcooling; Squeeze out pit with hands; With 40 ℃ of oven dry of sarcocarp, carry out supersound extraction with 25% ethanol 25mL again, extracting solution carries out assay through HPLC.
Undressed Fructus Corni fresh fruit is directly removed nuclear with hands, again with 40 ℃ of oven dry of sarcocarp, is that 25% ethanol water 25mL carries out supersound extraction with mass fraction, and extracting solution carries out assay through HPLC.As negative control.
Result of the test is seen table 1.
The different pre-treating methods of table 1 are to the influence of 3 kinds of iridoid glycoside content in the Fructus Corni extract
| Change active constituent content | Negative control | Water-boiling method | Water at atmospheric pressure is steamed method | Microwave method |
| Morroniside (mg/g) | 16.04 | 24.40 | 21.15 | 18.75 |
| Loganin (mg/g) | 8.12 | 10.13 | 9.85 | 9.27 |
| Sweroside (mg/g) | 0.85 | 1.30 | 1.17 | 0.98 |
Visible by table 1, best pre-treatment condition is that Fructus Corni fresh fruit 20g is dropped in the boiling water, scalds to boil after 10 minutes and pulls out, and a moment is removed pit in cooling, dries to constant weight for 40 ℃.
3, the kind of enzyme is to the influence of iridoid glycosides content in the Fructus Corni extract
Get totally 5 parts of Fructus Corni medical material 100g, add respectively that 300ml is water-soluble to expand, wherein 4 parts respectively add Snailase, pectase, cellulase, each 1g of emulsin, and another part is as negative control.Using the glacial acetic acid adjust pH is 4.5,50 ℃ of constant temperature, and enzymolysis 4 hours adds ethanol 300mL and stops enzymolysis.Adding water to the ethanol mass fraction is 25%, uses frequency to be the ultrasonic extraction of 300W 40 minutes as 25kHz, power.Extracting solution carries out assay with HPLC.
Result of the test is seen table 2.
The kind of table 2 enzyme is to the influence of 3 kinds of iridoid glycoside content in the Fructus Corni extract
| Active constituent content | Negative control | Snailase | Pectase | Cellulase | Emulsin |
| Morroniside (mg/g) | 28.5 | 24.48 | 24.47 | 29.95 | 38.78 |
| Loganin (mg/g) | 10.28 | 8.12 | 8.53 | 11.67 | 14.34 |
| Sweroside (mg/g) | 0.92 | 0.657 | 0.532 | 0.94 | 1.258 |
Visible by table 2, the hydrolysis result of emulsin is best, and other enzymes do not have remarkable result, so the extraction of using emulsin can improve the effective ingredient in the Fructus Corni total glycosides.
4, the consumption of enzyme is to the influence of iridoid glycosides content in the Fructus Corni extract
Get totally 5 parts of Fructus Corni medical material 100g, add respectively that 300ml is water-soluble to expand, add 0.2g again, 0.5g, 1g, 1.5g, the 2g emulsin is transferred pH value 4.5 with glacial acetic acid, 50 ℃ of constant temperature, enzymolysis 4 hours adds 300mL ethanol and stops enzymolysis.Adding water to the ethanol mass fraction is 25%, uses frequency to be the ultrasonic extraction of 300W 40 minutes as 25kHz, power.Extracting solution carries out assay with HPLC.
Result of the test is seen table 3.
The consumption of table 3 enzyme is to the influence of 3 kinds of iridoid glycoside content in the Fructus Corni extract
As shown in table 3, extraction effect is better when the mass ratio of Fructus Corni medical material and emulsin is 1: 0.005～0.015, and the mass ratio of Fructus Corni medical material and emulsin is 1: 0.01 o'clock, and the extraction effect of effective ingredient reaches maximum in the Fructus Corni extract.
5, pH value is to the influence of iridoid glycosides content in the Fructus Corni extract
Get totally 5 parts of Fructus Corni medical material 100g, each adds, and 300ml is water-soluble to expand, and adds emulsin 1g again, with glacial acetic acid each part pH value is transferred to 3.0,4.0,4.5,5.0,6.0 respectively, 50 ℃ of constant temperature, enzymolysis 4 hours.Add 300mL ethanol and stop enzymolysis.Adding water to the ethanol mass fraction is 25%, uses frequency to be the ultrasonic extraction of 300W 40 minutes as 25kHz, power.Extracting solution carries out assay with HPLC.
Result of the test is seen table 4.
Table 4pH value is to the influence of iridoid glycosides content in the Fructus Corni extract
As shown in table 4, extraction effect is better when enzymolysis pH is 4.0～4.5, and the extraction of effective ingredient reaches maximum in the Fructus Corni extract when enzymolysis pH is 4.5.
6, hydrolysis temperature is to the influence of iridoid glycosides content in the Fructus Corni extract
Get totally 5 parts of Fructus Corni medical material 100g, each adds, and 300ml is water-soluble to expand, and adds emulsin 1g again, and using the glacial acetic acid adjust pH is 4.5, and temperature remains on 30,40,50,60,70 ℃ respectively, enzymolysis 4 hours.Add 300mL ethanol and stop enzymolysis.Adding water to the ethanol mass fraction is 25%, uses frequency to be the ultrasonic extraction of 300W 40 minutes as 25kHz, power.Extracting solution carries out assay with HPLC.
Result of the test is seen table 5.
Table 5 hydrolysis temperature is to the influence of iridoid glycosides content in the Fructus Corni extract
As shown in table 5, when temperature was 40～50 ℃, the enzymolysis and extraction effect was better, and when temperature was 50 ℃, the enzymolysis and extraction effect reached maximum.
7, enzymolysis time is to the influence of iridoid glycosides content in the Fructus Corni extract
Get totally 5 parts of Fructus Corni medical material 100g, each adds, and 300ml is water-soluble to expand, and adds emulsin 1g again, and using the glacial acetic acid adjust pH is 4.5,50 ℃ of constant temperature, and enzymolysis is 1,2,4,8,12 hours respectively.Add 300mL ethanol and stop enzymolysis.Adding water to the ethanol mass fraction is 25%, uses frequency to be the ultrasonic extraction of 300W 40 minutes as 25kHz, power.Extracting solution carries out assay with HPLC.
Result of the test is seen table 6.
Table 6 enzymolysis time is to the influence of iridoid glycosides content in the Fructus Corni extract
By shown in the table 6, during enzymolysis time 3～5h, hydrolysis result is better, and enzymolysis time can reach best hydrolysis result when being 4h.
8, punctate opacity of the cornea design-response surface method is optimized the test of Fructus Corni total glycosides extraction process
The punctate opacity of the cornea EXPERIMENTAL DESIGN: the principal element that influences ultrasonic extraction process has concentration of alcohol (X
1), the liquid material is than (X
2), extraction time (X
3) and extraction time.Because of extraction time is discontinuous variable, regression treatment is difficulty, in conjunction with trial test result and industrial reality, is fixed as temporarily 2 times.Above 3 factors are made independent variable, set the factor level table, according to the punctate opacity of the cornea design principle, every factor is established five levels, representes (α=1.732 of three factor punctate opacity of the corneas design) with code value-α ,-1,0,1, α.Factor level is seen table 7.
Table 7 Fructus Corni total glycosides extraction process factor level (n=3)
According to the factor level table, get Fructus Corni medical material 100g, add the Different concentrations of alcohol supersound extraction, extract 2 times, filter, get filtrating, measure the yield and the total extract yield of 3 kinds of iridoid glycosides respectively, the result sees table 8.
In addition, with extract morroniside (Y in the table 8
1), loganin (Y
2) and sweroside (Y
3) yield (quality/quality of medicinal material of each iridoid glycoside * 100% in the extract), extract yield (quality/quality of medicinal material of total extract * 100%, Y
4) numerical standard turn to 0～1 normalizing value, and the normalizing value is asked the calculation geometric mean, general comment normalizing value.OD=(d
1, d
2..., d
n)
1/n, n is the index number, the effect that the more little good more effect of value and value are the bigger the better adopts the Hassan method to carry out the mathematics conversion respectively and asks normalizing value d
MinAnd d
Max, d
Min=(Y
Max-Y
i)/(Y
Max-Y
Min), d
Max=(Y
i-Y
Min)/(Y
Max-Y
Min).In addition, the yield of the morroniside that this test requirements document is extracted, loganin and sweroside is the bigger the better, and the amount of total extract is more little good more, respectively extracts the component content height and impurity content is few thereby make.
Table 8 Fructus Corni total glycosides extraction process punctate opacity of the cornea EXPERIMENTAL DESIGN and result (n=3)
Response surface method date processing: adopt Matlab R2007a software; With each general comment normalizing value independent variable is carried out multiple linear regression and binomial match; As remarkable standard, select to have big r value (multiple correlation coefficient) that equation is optimized with P＜0.05 of equation.
Multiple linear equation: Y=0.3119-3.4 * 10
-3X
1+ 8.6 * 10
-3X
2+ 3.1 * 10
-3X
3R=0.0815, F=0.3252, root-mean-square error is 0.0561, P＞0.05.
Binomial equation: Y=2.4528-0.1046X
1+ 3.81 * 10
-2X
2+ 1.3 * 10
-3X
3+ 1 * 10
-4X
1X
2-1 * 10
-4X
1X
3+ 1 * 10
-4X
2X
3+ 1 * 10
-3X
1 2-1.3 * 10
-3X
2 2+ 1 * 10
-4X
3 2R=0.9177, F=6.1987, root-mean-square error is 0.005, P＜0.05.
Can learn that from the correlation coefficient r of these two kinds of fit equation the correlation coefficient of multiple linear regression equations is low than binomial equation; Linear dependence is poor between expression independent variable X and the dependent variable Y; So should not use linear regression model (LRM); And polynary binomial fit equation correlation coefficient is higher, and fitting effect is better.Again according to binomial equation respectively to X
1, X
2, X
3Ask partial derivative, and make it equal 0, the theoretical process conditions that draw are: concentration of alcohol 52%, liquid material be than 10.96 times, 17.08 minutes extraction times, these three theoretical values and practical operation very near and within reasonable range.Combine simultaneously response surface and contour map that notional result is planned in the more accurate scope again; One of fixing 3 independent variables are intermediate value; Object function with match is a mathematical model; Draw the response surface and the contour map of dependent variable (OD), on the basis of theoretical optimum condition,, make up each response surface and isocontour zone overlapping again according to response surface and contour map; Obtain best region, combine the test data of practical operation to choose the extraction optimised process scope of Fructus Corni total glycosides again.
The result: the Fructus Corni medicinal powder is that 25kHz, power are that the ultrasound wave of 300W extracts with the frequency, and liquid material ratio is 12～16 times, and mass fraction is 25%～27%, 30～40 minutes extraction times.Merge extractive liquid, filters, and being evaporated to does not have the alcohol flavor, gets the Fructus Corni ethanol extraction.
9, punctate opacity of the cornea design-response surface method is optimized the test of Fructus Corni total glycosides purifying process
The punctate opacity of the cornea EXPERIMENTAL DESIGN: the examination factor of Fructus Corni medicinal substances extract purification by macroporous resin technological parameter is concentration of alcohol, elution volume, applied sample amount.Above 3 factors as independent variable, are set the factor level table, and according to the principle of punctate opacity of the cornea design, every factor is provided with five levels, representes (α=1.732 of three factor punctate opacity of the corneas design) with code value-α ,-1,0,1, α.Factor level is seen table 9.
Table 9 Fructus Corni total glycosides purification by macroporous resin examination factor level
According to this factor level table, get the extract 100g of the said optimum extraction process gained of joint, add the dilution of 100mL water, carry out enriching and purifying through macroporous resin, get eluent, measure the yield of 3 kinds of iridoid glycosides respectively.The eluent concentrating under reduced pressure gets system's thing extractum and calculates yield, and the result sees table 10.
With morroniside (Y in the eluate in the table 10
1), loganin (Y
2), sweroside (Y
3) yield (quality of the content/extract of each iridoid glycoside * 100% in the eluate), yield (quality * 100% of the quality/extract extractum of the eluate extractum of surveying, the Y of eluate
4) numerical standard turns to 0～1 normalizing value, and the normalizing value asked the calculation geometric mean, general comment normalizing value.OD=(d
1, d
2..., d
n) 1/n, n is the index number, the effect that the more little good more effect of value and value are the bigger the better adopts the Hassan method to carry out the mathematics conversion respectively and asks normalizing value d
MinAnd d
Max, d
Min=(Y
Max-Y
i)/(Y
Max-Y
Min), d
Max=(Y
i-Y
Min)/(Y
Max-Y
Min).In addition, the yield of the morroniside that this test requirements document is extracted, loganin and sweroside is the bigger the better, and the amount of eluate is more little good more, thereby makes the component content height of each eluting purification and impurity content is few.
Table 10 Fructus Corni total glycosides purification by macroporous resin technology punctate opacity of the cornea EXPERIMENTAL DESIGN and result
Response surface method date processing: adopt Matlab R2007a software; With each general comment normalizing value independent variable (X) is carried out multiple linear regression and binomial match; As remarkable standard, select to have big r value (multiple correlation coefficient) that equation is optimized with P＜0.05 of equation.
Multiple linear equation: Y=-0.9798+2.59 * 10
-2X
1+ 0.118X
2-4.4 * 10
-3X
3R=0.8616, root-mean-square error is 0.0141, F=24.911, P＜0.01.
Binomial equation: Y=-2.4361+3.21 * 10
-2X
1+ 0.8012X
2-3.18 * 10
-2X
3+ 9 * 10
-4X
1X
2+ 2 * 10
-4X
1X
3+ 2.5 * 10
-3X
2X
3-3 * 10
-4X
1 2-6.99 * 10
-2X
2 2+ 1 * 10
-4X
3 2R=0.939, root-mean-square error is 0.0062, F=10.26, P＜0.05.
Can learn that from the correlation coefficient r of these two kinds of fit equation the correlation coefficient of multiple linear regression equations is low than binomial equation; Linear dependence is poor between expression independent variable X and the dependent variable Y; So should not use linear regression model (LRM); And polynary binomial fit equation correlation coefficient is higher, and fitting effect is better.Again according to binomial equation respectively to X
1, X
2, X
3Ask partial derivative, and make it equal 0, the theoretical process conditions that draw are: concentration of alcohol 67.12%; Elution volume 6.38BV, applied sample amount 12.135mg/g, but this is a notional result; With practical operation the place of coming in and going out is mutually arranged still, can not be as unique criterion of optimum condition.In addition, also will take all factors into consideration in response surface and the contour map best region confirms.One of fixing 3 independent variables are intermediate value; Object function with match is a mathematical model; Draw the response surface and the contour map of dependent variable,, carry out each response surface and isocontour zone overlapping according to response surface and contour map; Must more excellent zone, combine the actual tests data to choose Fructus Corni total glycosides purification by macroporous resin enrichment optimised process scope again.
The result: the Fructus Corni ethanol extraction carries out purification with dilution of equivalent pure water and mixing through macroporous resin column, earlier with the deionized water eluting; Colourless to eluent; Be 48%～55% ethanol water eluting with mass fraction again, elution volume 8～9BV, applied sample amount 40～50mg/g.The merging eluent, being evaporated to does not have the alcohol flavor, and 50 ℃ of dryings in baking oven are pulverized, and sieve, and get Fructus Corni total glycosides, and in Fructus Corni total glycosides, morroniside, loganin, sweroside total content are not less than 70%.
In order to verify beneficial effect of the present invention, the inventor adopts the embodiment of the invention 1 preparation Fructus Corni total glycosides to carry out the test of pesticide effectiveness, and test situation is following:
Test objective: this test is an index with mice respiratory arrest under the normobaric hypoxia condition, investigates the resisting oxygen lack of Fructus Corni total glycosides.
Test drug: Fructus Corni total glycosides, nimodipine tablet, Folium Ginkgo.
Experimental animal: 60 of healthy ICR strain mices, body weight 20 ± 2, male and female half and half, Xi'an Jiaotong University Medical College experimental animal center provides, the animal quality certification: the moving card of doctor word 08-004 number.Raise laboratory, animal occupancy permit: SYXK (Shan) 2010-004 in SPF.23 ± 2 ℃ of raising temperatures, relative humidity 40 ± 2%, artificial lighting, light and shade each 12 hours.SPF level feedstuff is purchased in Xi'an Jiaotong University Medical College experimental animal center, drinking public water supply, the drinking-water of freely ingesting, well-ventilated.Sweep indoor health every day 2 times.All zooperies all strictly observe Northwest University's zoopery codes of ethics.
Test method: 60 mices are divided into 6 groups at random; Dosage 0.1mL/10g/day is respectively: dosed administration group, the heavy dose of administration group of Fructus Corni total glycosides 20mg/mL, the positive group of nimodipine (5mg/mL), the positive group of Folium Ginkgo (5mg/mL), water matched group among the low dose of administration group of Fructus Corni total glycosides 5mg/mL, the Fructus Corni total glycosides 10mg/mL.Once a day, administration is 7 days.
Mice after the administration 7 times is put into the ground wide mouthed bottle of 250mL, and sealing (is put sodica calx 30g in the bottle, in order to absorb titanium dioxide and water; Be lined with filter paper on the sodica calx, put one of mice for every bottle, bottleneck is coated with vaseline with anti-gas-leak); With last breathing is index, observes the time-to-live of mice.
Result of the test: as shown in table 11, positive drug group (nimodipine group, Folium Ginkgo group), Fructus Corni total glycosides be little, in, heavy dose of group and water matched group relatively, but equal time-to-live of significant prolongation normobaric hypoxia mice, and difference highly significant (P＜0.01).In the Fructus Corni total glycosides, heavy dose of compare with the positive drug group, highly significant difference (P＜0.01) is also arranged.Each dose groups of Fructus Corni total glycosides has certain dose-dependence to the prolongation of normal pressure anoxia enduring mice time-to-live.
Table 11 Fructus Corni total glycosides to the influence of mice hypoxia-bearing capability (x ± s, n=10)
| Group | Dosage (mg/kg) | Time-to-live (min) |
| The water matched group | - | 17.44±2.30 |
| Nimodipine tablet positive drug group | 60 | 21.14±2.921) |
| Folium Ginkgo positive drug group | 25 | 21.11±1.901) |
| The Fructus Corni total glycosides small dose group | 25 | 23.32±3.921) |
| Dose groups in the Fructus Corni total glycosides | 50 | 30.78±7.871，2，3) |
| The heavy dose of group of Fructus Corni total glycosides | 100 | 31.46±7.651，2，3) |
Annotate: compare 1 with the water matched group) P＜0.01; Compare 2 with the nimodipine tablet group) P＜0.01; Compare 3 with the Folium Ginkgo group) P＜0.01.
Points for attention: preferably only put 1 mice in each wide mouthed bottle, in case the mensuration of mutual disturbing influence hypoxia-bearing capability; Lid for jar must airtightly be obturaged, otherwise result of the test is inaccurate; The wide mouthed bottle volume should be suitable, can the activity of having by oneself be advisable with mice.
Conclusion: Fructus Corni total glycosides can improve the hypoxia-bearing capability of mice, and physical strength reinforcing improves body fatigue resistance, promotes the foundation that anoxia enduring adapts to.Fructus Corni total glycosides has the highly significant effect to improving the mice hypoxia-bearing capability, is better than positive drug nimodipine and Folium Ginkgo far away.Fructus Corni total glycosides is processed the oral drugs of various dosage forms, becomes human oral, common dose 180mg on the one, and one after each meal, children is taken the circumstances into consideration decrement.
Claims (3)
1. the method for distilling of a Fructus Corni total glycosides is characterized in that it is made up of following step:
(1) boiling hot boiling
The Fructus Corni fresh fruit is dropped in the boiling water to submergence, and boiling water boiled 10～15 minutes, pulled postcooling a moment out, squeezed out pit with hands, and 40 ℃ of oven dry of sarcocarp are pulverized, and cross 20 mesh sieves, the Fructus Corni medicinal powder;
(2) enzymolysis and extraction
Get the Fructus Corni medicinal powder, add 2～4 times of water-soluble expanding, add emulsin again; The mass ratio of Fructus Corni medicinal powder and emulsin is 1: 0.005～0.015, regulates pH value to 4～4.5,40～50 ℃ of enzymolysis of constant temperature 3～5 hours with glacial acetic acid; Add the ethanol with quality such as water, stop enzymolysis.Adding water to the ethanol mass fraction again is 25%～27%, uses frequency to be the ultrasonic extraction of 300W 30～40 minutes as 25kHz, power, and inclining supernatant; Fructus Corni medicinal powder slag again with the liquid material than being that 12～16 times, mass fraction are that 25%～27% ethanol water extracts once, decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, the Fructus Corni ethanol extraction;
(3) purification
The Fructus Corni ethanol extraction with etc. the water dilution and the mixing of quality, carry out purification through macroporous resin column, use the deionized water eluting, colourless to eluent; Be 48%～55% ethanol water eluting with mass fraction again, 8～9 times of column volumes of elution volume, applied sample amount 40～50mg/g merges eluent; Being evaporated to does not have the alcohol flavor, and 50 ℃ of dryings in baking oven are pulverized, and cross 20 mesh sieves; Get Fructus Corni total glycosides, in Fructus Corni total glycosides, morroniside, loganin, sweroside total content are not less than 70%.
2. the method for distilling of Fructus Corni total glycosides according to claim 1 is characterized in that: in enzymolysis and extraction step (2), get the Fructus Corni medicinal powder; Add 2～4 times of water-soluble expanding, add emulsin again, the mass ratio of Fructus Corni medicinal powder and emulsin is 1: 0.01; Regulate pH value to 4.5 with glacial acetic acid, 50 ℃ of enzymolysis of constant temperature 4 hours add the ethanol with quality such as water; Stop enzymolysis; Adding water to the ethanol mass fraction again is 25%～27%, uses frequency to be the ultrasonic extraction of 300W 30～40 minutes as 25kHz, power, and inclining supernatant; Fructus Corni medicinal powder slag again with the liquid material than being that 8～10 times, mass fraction are that 25%～27% ethanol water extracts once, decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, the Fructus Corni ethanol extraction.
3. the purposes of effective ingredient Fructus Corni total glycosides in preparation anoxia enduring medicine.
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| CN107929347A (en) * | 2017-12-25 | 2018-04-20 | 浙江工业大学 | Application of the Fructus Corni total glycosides in the medicine for preparing prevention remodeling ventricle |
| CN109053849A (en) * | 2018-06-21 | 2018-12-21 | 长沙湘资生物科技有限公司 | The method of Fructus Corni comprehensive utilization |
| CN112047984A (en) * | 2019-06-06 | 2020-12-08 | 南京宸翔医药研究有限责任公司 | Extraction, separation and purification method of various components of dogwood |
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| CN102389162A (en) * | 2011-08-04 | 2012-03-28 | 深圳烟草工业有限责任公司 | Method for preparing cornus officinalis extract, application in cigarettes and cigarette |
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| CN1510037A (en) * | 2002-12-20 | 2004-07-07 | 首都医科大学宣武医院 | Cornel extractive, extracting method and use for preparing medicine and health food |
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| CN102127140A (en) * | 2010-11-25 | 2011-07-20 | 南京泽朗医药科技有限公司 | Method for combinedly extracting Morroniside, dogwood polysaccharide and ursolic acid from dogwood |
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| CN107929347A (en) * | 2017-12-25 | 2018-04-20 | 浙江工业大学 | Application of the Fructus Corni total glycosides in the medicine for preparing prevention remodeling ventricle |
| CN109053849A (en) * | 2018-06-21 | 2018-12-21 | 长沙湘资生物科技有限公司 | The method of Fructus Corni comprehensive utilization |
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