CN102614243B - 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 the 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, Loganic acid etc. at present.Wherein morroniside, loganin, sweroside content are the highest, and the content of three in Fructus Corni reaches respectively 2.0%, 0.6% and 0.1%, has accounted for more than 85% of Fructus Corni total iridoid glycoside.
Fructus Corni total glycosides has widely biological activity: comprise neuroprotective, affect cytokine profiles 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 platelet aggregation that the antagonism several different methods is induced 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, the damage of heart, kidney and vascular tissue's cell 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 the characteristics such as specificity, high efficiency, mild condition, and the biological enzymolysis extraction is hydrolysis or the degradation of cell wall construction by enzyme, 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 adopts the auxiliary Fructus Corni total glycosides of the method to extract is arranged not yet.
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 by reducing body oxygen deprivation stress intensity, weaken anoxia to the damage of body, make body in anaerobic environment, keep the physiological function of compared with normal.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, the deficient syndromes such as the seminal emission enuresis have significant curative effect.Up to now, there is not yet the report that Fructus Corni total glycosides is used for control heart and brain anoxia.
Summary of the invention
A technical problem to be solved by this invention is to provide the preparation method of Fructus Corni total glycosides.
Another technical problem to be solved by this invention is to provide a kind of new purposes for Fructus Corni total glycosides.
Solving the problems of the technologies described above the technical scheme that adopts is comprised of following step:
1, boiling hot boiling
The Fructus Corni fresh fruit is dropped in the boiling water to submergence, and boiling water boiling 10～15 minutes is pulled rear cooling a moment out, squeezes out pit with hands, and 40 ℃ of oven dry of sarcocarp are pulverized, and mistake 20 mesh sieves get the Fructus Corni powder.
2, enzymolysis and extraction
Get the Fructus Corni powder, add 2～4 times of water-soluble expanding, add emulsin, the mass ratio of Fructus Corni powder and emulsin is 1: 0.005～0.015 again, regulates pH value to 4～4.5 with glacial acetic acid, 40～50 ℃ of enzymolysis of constant temperature 3～5 hours, add the ethanol with the quality such as water, stop enzymolysis, adding water to the ethanol mass fraction is 25%～27% again, be that 25kHz, power are the ultrasonic extraction 30～40 minutes of 300W with frequency, inclining supernatant; Ethanol water as 25%～27% extracts once Fructus Corni powder slag take liquid ratio as 12～16 times, mass fraction again, and decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, gets the Fructus Corni ethanol extraction.
3, purification
The Fructus Corni ethanol extraction with etc. water dilution and the mixing of quality, carry out purification by macroporous resin column, use the deionized water eluting, colourless to eluent, again take mass fraction as 48%～55% ethanol water eluting, 8～9 times of column volumes of elution volume, applied sample amount 40～50mg/g merges eluent, is evaporated to without the alcohol flavor, 50 ℃ of dryings in baking oven, pulverize, 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 powder, add 2～4 times of water-soluble expanding, add again emulsin, the mass ratio of Fructus Corni 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 the quality such as water, stop enzymolysis, adding water to the ethanol mass fraction is 25%～27% again, is that 25kHz, power are the ultrasonic extraction 30～40 minutes of 300W with frequency, and inclining supernatant; Ethanol water as 25%～27% extracts once Fructus Corni powder slag take liquid ratio as 8～10 times, mass fraction again, and decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, gets 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 such as 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, such as lactose, Pulvis Talci, cellulose, polyvinylpyrrolidone, starch, pectin, tween 80, polyvinyl alcohol etc.
Above-mentioned preparation can be made according to the preparation technology of various preparation routines.
Active component Fructus Corni total glycosides of the present invention is made 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 the anoxia symptom that anaerobic environment or cerebral ischemia cause, and is unable such as dizziness, headache, tinnitus, dim eyesight, tetraparesis; Perhaps feel sick, vomit, have palpitation, breathe hard, rapid breathing, heart beating be unable fast, the diseases such as anoxia.
The medicinal preparation for oral administration that said extracted thing Fructus Corni total glycosides is made according to a conventional method is the above tablet or granule or capsule or 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 the Fructus Corni 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, respectively extraction and the purifying process of Fructus Corni total glycosides are optimized, determined the extraction purification optimal procedure parameters of Fructus Corni total glycosides, 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
The present invention is described in more detail below in conjunction with embodiment, but the invention is not restricted to these embodiment.
Embodiment 1
Take the dry medicinal powder 100g of the extract Fructus Corni of Fructus Corni fresh fruit as example, used raw material and extracting method thereof are as follows:
1, boiling hot boiling
The Fructus Corni fresh fruit is dropped in the boiling water to submergence, and boiling water boiling 10～15 minutes is pulled rear cooling a moment out, squeezes out pit with hands, and 40 ℃ of oven dry of sarcocarp are pulverized, and mistake 20 mesh sieves get the Fructus Corni powder.
2, enzymolysis and extraction
Get Fructus Corni powder 100g, add 2～4 times of water-soluble expanding, add again emulsin 1g, 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 the quality such as water, stop enzymolysis, adding water to the ethanol mass fraction is 25%～27% again, be that 25kHz, power are the ultrasonic extraction 30～40 minutes of 300W with frequency, inclining supernatant; Ethanol water as 25%～27% extracts once Fructus Corni powder slag take liquid ratio as 12～16 times, mass fraction again, and decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, gets the Fructus Corni ethanol extraction.
3, purification
The Fructus Corni ethanol extraction with etc. the water dilution of quality, carry out purification by macroporous resin column, use the deionized water eluting, colourless to eluent, again take mass fraction as 48%～55% ethanol water eluting, 8～9 times of column volumes of elution volume, applied sample amount 40～50mg/g merges eluent, is evaporated to without the alcohol flavor, 50 ℃ of dryings in baking oven, pulverize, 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
Take the dry medicinal powder 100g of the extract Fructus Corni of Fructus Corni fresh fruit as example, used raw material and extracting method thereof are as follows:
In enzymolysis and extraction step 2, get Fructus Corni powder 100g, add 2～4 times of water-soluble expanding, add again emulsin 0.5g, regulate pH value to 4 with glacial acetic acid, 45 ℃ of enzymolysis of constant temperature 6 hours, add the ethanol with the quality such as water, stop enzymolysis, adding water to the ethanol mass fraction is 25%～27% again, be that 25kHz, power are the ultrasonic extraction 30～40 minutes of 300W with frequency, inclining supernatant; Ethanol water as 25%～27% extracts once Fructus Corni powder slag take liquid ratio as 12～16 times, mass fraction again, and decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, gets the Fructus Corni ethanol extraction.
Other steps are identical with embodiment 1.Extract Fructus Corni total glycosides.
Embodiment 3
Take the dry medicinal powder 100g of the extract Fructus Corni of Fructus Corni fresh fruit as example, used raw material and extracting method thereof are as follows:
In enzymolysis and extraction step 2, get Fructus Corni powder 100g, add 2～4 times of water-soluble expanding, add again emulsin 1.5g, regulate pH value to 4.5 with glacial acetic acid, constant temperature 50C enzymolysis 4 hours, add the ethanol with the quality such as water, stop enzymolysis, adding water to the ethanol mass fraction is 25%～27% again, be that 25kHz, power are the ultrasonic extraction 30～40 minutes of 300W with frequency, inclining supernatant; Ethanol water as 25%～27% extracts once Fructus Corni powder slag take liquid ratio as 12～16 times, mass fraction again, and decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, gets 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, and is raw materials used as follows with adjuvant and quality proportioning thereof for example 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, and day takes each 3 three times.
Embodiment 5
The pharmaceutical formulation Fructus Corni total glycosides is prepared into capsule, and raw materials used and adjuvant and quality proportioning thereof are as follows as example 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, and day takes each 3 three times.
Embodiment 6
Medicinal Fructus Corni total glycosides is prepared into granule, and is raw materials used as follows with adjuvant and quality proportioning thereof as an example of production granule product 1000g example:
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, and day takes twice, each 1 bag.
Embodiment 7
Medicinal Fructus Corni total glycosides is prepared into oral liquid, and is raw materials used as follows with adjuvant and quality proportioning thereof as an example of production oral liquid product 1000mL example:
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, and day takes each 1 bottle three times.
In order to determine optimum material proportion of the present invention and optimised process step, the inventor has carried out a large amount of laboratory study tests, and various test situation are as follows:
1, morroniside in the Fructus Corni, 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%; The relative standard deviation RSD of stability＜2.2%; Average recovery between 95%～105%, relative standard deviation RSD＜2.0%.
2, Different front processing method is on the impact of iridoid glycoside 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, pull rear cooling out, squeeze out pit with hands, again with 40 ℃ of oven dry of sarcocarp, get dry sarcocarp 100mg, the ethanol water 25mL as 25% carries out supersound extraction take mass fraction, and extracting solution carries out assay by high performance liquid chromatography.
Water at atmospheric pressure is steamed method: get 20g Fructus Corni fresh fruit and put into food steamer, steamed behind upper vapour 1,5,10,20 minute respectively, cooling after taking out, 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 by high performance liquid chromatography.
Microwave method: get 20g Fructus Corni fresh fruit, respectively microwave heating 20,30,40,50,60 seconds, cooling after taking out, 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 by high performance liquid chromatography.
Undressed Fructus Corni fresh fruit is directly removed nuclear with hands, and again with 40 ℃ of oven dry of sarcocarp, the ethanol water 25mL as 25% carries out supersound extraction take mass fraction, and extracting solution carries out assay by high performance liquid chromatography.As negative control.
Result of the test sees Table 1.
Table 1 Different front processing method is on the impact 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 |
By as seen from Table 1, best Pretreatment 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 on the impact of iridoid glycoside content in the Fructus Corni extract
Get totally 5 parts of Fructus Corni 100g, it is water-soluble swollen to add respectively 300ml, and wherein 4 parts respectively add Snailase, pectase, cellulase, each 1g of emulsin, and another part is as negative control.Be 4.5 with the glacial acetic acid adjust pH, 50 ℃ of constant temperature, enzymolysis 4 hours adds ethanol 300mL and stops enzymolysis.Adding water to the ethanol mass fraction is 25%, is that 25kHz, power are the ultrasonic extraction 40 minutes of 300W with frequency.Extracting solution carries out assay with high performance liquid chromatography.
Result of the test sees Table 2.
The kind of table 2 enzyme is on the impact 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 |
By as seen from 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 on the impact of iridoid glycoside content in the Fructus Corni extract
Get totally 5 parts of Fructus Corni 100g, it is water-soluble swollen to add respectively 300ml, adds 0.2g again, 0.5g, and 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%, is that 25kHz, power are the ultrasonic extraction 40 minutes of 300W with frequency.Extracting solution carries out assay with high performance liquid chromatography.
Result of the test sees Table 3.
The consumption of table 3 enzyme is on the impact 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 and emulsin is 1: 0.005～0.015, and the mass ratio of Fructus Corni 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 on the impact of iridoid glycoside content in the Fructus Corni extract
Get totally 5 parts of Fructus Corni 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 respectively 3.0,4.0,4.5,5.0,6.0,50 ℃ of constant temperature, enzymolysis 4 hours.Add 300mL ethanol and stop enzymolysis.Adding water to the ethanol mass fraction is 25%, is that 25kHz, power are the ultrasonic extraction 40 minutes of 300W with frequency.Extracting solution carries out assay with high performance liquid chromatography.
Result of the test sees Table 4.
Table 4pH value is on the impact of iridoid glycoside 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 on the impact of iridoid glycoside content in the Fructus Corni extract
Get totally 5 parts of Fructus Corni 100g, each adds, and 300ml is water-soluble to expand, and adds emulsin 1g again, is 4.5 with the glacial acetic acid adjust pH, and temperature remains on respectively 30,40,50,60,70 ℃, enzymolysis 4 hours.Add 300mL ethanol and stop enzymolysis.Adding water to the ethanol mass fraction is 25%, is that 25kHz, power are the ultrasonic extraction 40 minutes of 300W with frequency.Extracting solution carries out assay with high performance liquid chromatography.
Result of the test sees Table 5.
Table 5 hydrolysis temperature is on the impact of iridoid glycoside 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 on the impact of iridoid glycoside content in the Fructus Corni extract
Get totally 5 parts of Fructus Corni 100g, it is water-soluble swollen that each adds 300ml, adds emulsin 1g again, is 4.5 with the glacial acetic acid adjust pH, and 50 ℃ of constant temperature are distinguished enzymolysis 1,2,4,8,12 hours.Add 300mL ethanol and stop enzymolysis.Adding water to the ethanol mass fraction is 25%, is that 25kHz, power are the ultrasonic extraction 40 minutes of 300W with frequency.Extracting solution carries out assay with high performance liquid chromatography.
Result of the test sees Table 6.
Table 6 enzymolysis time is on the impact of iridoid glycoside 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 affects ultrasonic extraction process has concentration of alcohol (X
1), liquid ratio (X
2), extraction time (X
3) and extraction time.Because 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, represents (α of three factor punctate opacity of the corneas design=1.732) with code value-α ,-1,0,1, α.Factor level sees Table 7.
Table 7 Fructus Corni total glycosides extraction process factor level (n=3)
According to the factor level table, get Fructus Corni 100g, add the ethanol ultrasonic extraction of variable concentrations, extract 2 times, filter, get filtrate, measure respectively yield and the total extract yield of 3 kinds of iridoid glycosides, the results are shown in 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, get overall desirability.OD=(d
1, d
2..., d
n)
1/n, n is the index number, the effect that the smaller the better effect of value and value are the bigger the better adopts the Hassan method to carry out respectively the mathematics conversion 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 the smaller the better, 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 overall desirability independent variable is carried out multiple linear regression and binomial fitting, as remarkable standard, selecting has larger r value (multiple correlation coefficient) that equation is optimized with the P of equation＜0.05.
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 fitting dependence among equations 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%, and 10.96 times of liquid ratios, 17.08 minutes extraction times, these three theoretical values and practical operation approach and within rational scope very much.In conjunction with response surface and contour map notional result is planned in the more accurate scope more simultaneously, fix one of 3 independent variables and be intermediate value, take the object function of match as mathematical model, draw response surface and the contour map of dependent variable (OD), on the basis of theoretical optimum condition again according to response surface and contour map, make up each response surface and isocontour zone overlapping, obtain best region, choose again the extraction optimised process scope of Fructus Corni total glycosides in conjunction with the test data of practical operation.
The result: the Fructus Corni powder take frequency as 25kHz, power extracts as the ultrasound wave of 300W, liquid ratio is 12～16 times, mass fraction is 25%～27%, 30～40 minutes extraction times.Merge extractive liquid, filters, and is evaporated to without 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 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 arranges five levels, represents (α of three factor punctate opacity of the corneas design=1.732) with code value-α ,-1,0,1, α.Factor level sees 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 described optimum extraction process gained of joint, add the dilution of 100mL water, carry out enriching and purifying by macroporous resin, get eluent, measure respectively the yield of 3 kinds of iridoid glycosides.The eluent concentrating under reduced pressure gets system's thing extractum and calculates yield, the results are shown in 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 of the quality of the eluate extractum of surveying/extract extractum * 100%, the Y of eluate
4) numerical standard turns to 0～1 normalizing value, and the normalizing value is asked the calculation geometric mean, get overall desirability.OD=(d
1, d
2..., d
n) 1/n, n is the index number, the effect that the smaller the better effect of value and value are the bigger the better adopts the Hassan method to carry out respectively the mathematics conversion 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 the smaller the better, 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 technique punctate opacity of the cornea EXPERIMENTAL DESIGN and result
Response surface method date processing: adopt Matlab R2007a software, with each overall desirability independent variable (X) is carried out multiple linear regression and binomial fitting, as remarkable standard, selecting has larger r value (multiple correlation coefficient) that equation is optimized with the P of equation＜0.05.
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 fitting dependence among equations 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 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 consider in response surface and the contour map best region determines.Fix one of 3 independent variables and be intermediate value, take the object function of match as mathematical model, draw response surface and the contour map of dependent variable, according to response surface and contour map, carry out each response surface and isocontour zone overlapping, must more excellent zone, choose Fructus Corni total glycosides purification by macroporous resin enrichment optimised process scope in conjunction with the actual tests data again.
The result: the Fructus Corni ethanol extraction carries out purification with the dilution of equivalent pure water and mixing by macroporous resin column, first with the deionized water eluting, colourless to eluent, again take mass fraction as 48%～55% ethanol water eluting, elution volume 8～9BV, applied sample amount 40～50mg/g.Merge eluent, be evaporated to without the alcohol flavor, 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 as follows:
Test objective: this test is investigated the resisting oxygen lack of Fructus Corni total glycosides take mice respiratory arrest under the normobaric hypoxia condition as index.
Test drug: Fructus Corni total glycosides, nimodipine tablet, Folium Ginkgo.
Experimental animal: 60 of healthy ICR Strains of Mouses, 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 the laboratory in SPF, animal occupancy permit: SYXK (Shan) 2010-004.23 ± 2 ℃ of raising temperatures, relative humidity 40 ± 2%, artificial lighting, light and shade each 12 hours.SPF level feedstuff is purchased from Xi'an Jiaotong University Medical College experimental animal center, drinking public water supply, the drinking-water of freely ingesting, well-ventilated.Sweep indoor sanitation 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, 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), take last breathing as index, observe 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 all 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 dosage group of Fructus Corni total glycosides has certain dose-dependence to the prolongation of normal pressure anoxia enduring mouse survival time.
Table 11 Fructus Corni total glycosides is on the impact (x ± s, n=10) of mice hypoxia-bearing capability
| 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) |
| Dosage group 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 made 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 (2)
1. the extracting method of a Fructus Corni total glycosides is characterized in that it is comprised of following step:
(1) boiling hot boiling
The Fructus Corni fresh fruit is dropped in the boiling water to submergence, and boiling water boiling 10～15 minutes is pulled rear cooling a moment out, squeezes out pit with hands, and 40 ℃ of oven dry of sarcocarp are pulverized, and mistake 20 mesh sieves get the Fructus Corni powder;
(2) enzymolysis and extraction
Get the Fructus Corni powder, add 2～4 times of water-soluble expanding, add emulsin, the mass ratio of Fructus Corni powder and emulsin is 1:0.005～0.015 again, regulates pH value to 4～4.5 with glacial acetic acid, 40～50 ℃ of enzymolysis of constant temperature 3～5 hours, add the ethanol with the quality such as water, stop enzymolysis, adding water to the ethanol mass fraction is 25%～27% again, be that 25kHz, power are the ultrasonic extraction 30～40 minutes of 300W with frequency, inclining supernatant; Ethanol water as 25%～27% extracts once Fructus Corni powder slag take liquid ratio as 12～16 times, mass fraction again, and decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, gets the Fructus Corni ethanol extraction;
(3) purification
The Fructus Corni ethanol extraction with etc. water dilution and the mixing of quality, carry out purification by macroporous resin column, use the deionized water eluting, colourless to eluent, again take mass fraction as 48%～55% ethanol water eluting, 8～9 times of column volumes of elution volume, applied sample amount 40～50mg/g merges eluent, is evaporated to without the alcohol flavor, 50 ℃ of dryings in baking oven, pulverize, 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 extracting method of Fructus Corni total glycosides according to claim 1, it is characterized in that: in enzymolysis and extraction step (2), get the Fructus Corni powder, add 2～4 times of water-soluble expanding, add again emulsin, the mass ratio of Fructus Corni 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 the quality such as water, stop enzymolysis, adding water to the ethanol mass fraction is 25%～27% again, be that 25kHz, power are the ultrasonic extraction 30～40 minutes of 300W with frequency, inclining supernatant; Ethanol water as 25%～27% extracts once Fructus Corni powder slag take liquid ratio as 8～10 times, mass fraction again, and decompress filter merges extracted twice liquid, is evaporated to relative density and is 1.20 extractum, gets the Fructus Corni ethanol extraction.
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