CN101766664A - Extraction method of total saponin of Radix Ilicis Asprellae and quality detection method thereof - Google Patents
Extraction method of total saponin of Radix Ilicis Asprellae and quality detection method thereof Download PDFInfo
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- CN101766664A CN101766664A CN201010120930.2A CN201010120930A CN101766664A CN 101766664 A CN101766664 A CN 101766664A CN 201010120930 A CN201010120930 A CN 201010120930A CN 101766664 A CN101766664 A CN 101766664A
- Authority
- CN
- China
- Prior art keywords
- ilicis asprellae
- radix ilicis
- total saponin
- ethanol
- saponin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
The invention discloses an extraction method of total saponin of Radix Ilicis Asprellae and a quality detection method thereof, and the extraction method comprises the following steps: taking a medicinal material of Radix Ilicis Asprellae root or stem, adding 4-12 times volume of 60% ethanol, extracting for 1.5-2h, adding 2-10 times volume of 60% ethanol, extracting for 1.0-1.5h, filtering and merging filtrate; concentrating under reduced pressure until no alcohol taste exists, adding water, diluting crude drug with the loading concentration of 0.1-0.5g/ml, and loading a pre-treated AB-8 macroporous resin column; washing the resin column to be cleared by water, and eluting by 60-80% of ethanol; and collecting eluent of 60-80% ethanol and concentrating under reduced pressure, drying to obtain the total saponin of Radix Ilicis Asprellae. The quality detection method comprises the steps of TLC chromatograph identification and content determination of ilexoside X XIX in the total saponin of Radix Ilicis Asprellae. By using the refined technology of the invention, the retention rates of the total saponin of Radix Ilicis Asprellae and f ilexoside X XIX are improved greatly, and the obtained extract has higher purity; the obtained total saponin of Radix Ilicis Asprellae has the functions of inflammation resistance, analgesis, koortswerend, antivirus and the like; by using the characteristic saponin ilexoside X XIX as a reference sample, the accuracy is higher and the result has high reliability; and the quality detection method of the total saponin of Radix Ilicis Asprellae extract can provide a reference for quality control of the total saponin of Radix Ilicis Asprellae extract.
Description
Technical field
The present invention relates to a kind of method and purposes of from the Flos Ilicis Asprellae medical material, extracting total saponin of Radix Ilicis Asprellae, the invention still further relates to a kind of quality determining method of total saponin of Radix Ilicis Asprellae.
Background technology
Flos Ilicis Asprellae is dry root and the stem of holly plant Folium Mume Ilicis Purpureae, records under the D/WS-058-2003 item of " Guangdong Province's Chinese crude drug standard " (first) Guangdong, and main product is in Guangdong, Guangxi and other places, for commonly using Chinese medicine in the south of the Five Ridges.Function cures mainly and is heat-clearing and toxic substances removing, promoting the production of body fluid to quench thirst, relieving sore throat and diminishing swelling, eliminating stasis to stop pain; Be used for cold, fever, the cough due to lung-heat, calentura Tianjin wound is thirsty, laryngopharynx swelling and pain, traumatic injury stasis of blood pain.Mainly contain triterpene saponin, lactone (coumarin) and a small amount of alkaloid.
Macroporous adsorbent resin is a kind of organic high poly-adsorbent, ability with selective absorption organic compound, the enriching and purifying that is applied to saponin component has effect preferably, but its absorption property and desorbing thereof, purification condition parameter are different because of the difference of the physicochemical property of chemical compound.(the research of total saponins in the macroporous resin enrichment purification Radix Ilicis Asprellae of Feng Da magnitude, ACAD J GCP in June, 2004) adopt macroporous adsorbent resin LSA-20 that the total saponins in the Radix Ilicis Asprellae is extracted, the Radix Ilicis Asprellae total saponin content is 5.3% in the preceding total solid of purification, behind the purification in the total solid Radix Ilicis Asprellae total saponin content be 62.98%, refining degree reaches 11.86 times.But the LSA-20 resin is not very high to the ratio adsorbance and the resolution factor of total saponin of Radix Ilicis Asprellae, behind the purification in the total solid Radix Ilicis Asprellae total saponin content also not very high.
The extraction and purification process of total saponin of Radix Ilicis Asprellae is to investigate as index with the content of total saponin of Radix Ilicis Asprellae in the prior art, and it is reference substance that the ginsenoside Re is all adopted in the demarcation of total saponin of Radix Ilicis Asprellae.Ultraviolet-visible absorption curve basically identical after the colour developing of ginsenoside Re and total saponin of Radix Ilicis Asprellae in contrast under the prerequisite of product, is reference substance demarcation total saponin of Radix Ilicis Asprellae at no Flos Ilicis Asprellae characteristic saponin ilexosideX X IX with ginsenoside Re usually.Yet, on the one hand, be that accuracy and the degree of accuracy that reference substance is demarcated total saponin of Radix Ilicis Asprellae is not very high with ginsenoside Re; On the other hand, only adopt this a kind of index of content of total saponin of Radix Ilicis Asprellae to investigate the total saponin of Radix Ilicis Asprellae extraction process, have ambiguity, the checking of extraction process is had certain influence.Therefore, be badly in need of to adopt more convictive index, the extracting method of existing total saponin of Radix Ilicis Asprellae is made improvements.
In addition, prior art still belongs to blank to the quality determining method of the total saponin of Radix Ilicis Asprellae of extraction.
Summary of the invention
One of purpose of the present invention promptly is the defective at prior art, a kind of new extracting method of total saponin of Radix Ilicis Asprellae is provided, this extracting method adopts new technological parameter, and the content and the retention rate of the total saponin of Radix Ilicis Asprellae of demarcating with characteristic monomer saponin ilexosideX X IX in the total saponin of Radix Ilicis Asprellae with based on ilexoside X X IX are that index is investigated.
For achieving the above object, the present invention has taked following technical scheme:
A kind of extracting method of total saponin of Radix Ilicis Asprellae may further comprise the steps:
(1) gets Radix Ilicis Asprellae or stem medical material, add 4～12 times of amount 60% ethanol extractions for the first time, add 2～10 times of amount 60% ethanol extractions more for the second time, filter merging filtrate;
(2) filtrate decompression that step (1) is obtained is concentrated into does not have the alcohol flavor, and the supreme sample concentration of thin up is 0.1-0.5g crude drug/ml, on pretreated AB-8 macroporous resin column;
(3) the washed resin post discarded post liquid and water lotion to clarification, used the 60%-80% ethanol elution then;
(4) collect 60%-80% ethanol elution concentrating under reduced pressure, decompression or spray drying get total saponin of Radix Ilicis Asprellae.
Preferably, add 8 times of amount 60% ethanol extractions in the described step (1) for the first time, add 6 times of amount 60% ethanol extractions more for the second time; Going up sample concentration in the described step (2) is 0.2g crude drug/ml; Maximum applied sample amount is counted 119.43mg total saponin of Radix Ilicis Asprellae/g dried resin in total saponin of Radix Ilicis Asprellae; Described step (3) is middle with 3 times of column volume 80% ethanol elutions.
The total saponin of Radix Ilicis Asprellae that adopts the said extracted method to extract has effects such as antiinflammatory, analgesia, analgesic and antiviral, can be prepared as and be used for dosage forms such as acute pharyngitis and antiviral tablet, capsule, granule, drop pill, oral liquid, mixture by adding conventional pharmaceutic adjuvant, also can be used as additive and add in beverage, toothpaste, collutory, the chewing gum, be used for oral cavity health.
Another object of the present invention is to provide a kind of quality determining method that adopts the total saponin of Radix Ilicis Asprellae that the said extracted method obtains.
For achieving the above object, the present invention has taked following technical scheme:
A kind of quality determining method of total saponin of Radix Ilicis Asprellae extract may further comprise the steps:
(1) discriminating of total saponin of Radix Ilicis Asprellae (discriminating of TLC chromatograph)
Get total saponin of Radix Ilicis Asprellae extract 0.1-0.2g, add water 5-15ml and make dissolving, in order to water saturated n-butanol extraction 2-3 time, each 5-15ml merges n-butyl alcohol liquid, add 0.8%-1.2% sodium hydroxide solution washing 2-3 time, each 15-25ml, get n-butyl alcohol liquid in order to n-butyl alcohol saturated be washed to neutrality, get n-butyl alcohol liquid evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution; Make Flos Ilicis Asprellae control medicinal material solution with method; According to " appendix a VIB of Chinese pharmacopoeia version in 2005 thin layer chromatography test, draw each 5 μ l point of above-mentioned solution respectively on same silica gel g thin-layer plate, with volume ratio is 1: 6: 1.5: 0.7: 0.1 chloroform: ethyl acetate: methanol: water: formic acid is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to speckle colour developing at 105 ℃; In the need testing solution chromatograph, with control medicinal material solution chromatograph relevant position on, show the same color speckle;
(2) assay (HPLC-ELSD) of ilexoside X X IX in the total saponin of Radix Ilicis Asprellae
1. chromatographic condition
With C
18Post is an immobile phase, and acetonitrile-0.2% formic acid is mobile phase, carries out gradient elution; Drift tube temperature: 40 ℃, nebulizer gas pressure: 2.5bar, flow velocity: 1ml/min, column temperature: room temperature; Greater than 4000, symmetrical factor is 0.95～1.05 to theoretical cam curve in ilexosideX X IX, with the separating degree of adjacent peak greater than 1.5;
2. the preparation of reference substance solution
Get ilexosideX X IX reference substance 5mg, accurate claim surely, put in the 5ml measuring bottle, with dissolve with methanol and be diluted to scale, shake up, promptly;
3. the preparation of need testing solution
Get total saponin of Radix Ilicis Asprellae 2g, the accurate title, decide, and is settled to 100ml with 70% ethanol, and microporous filter membrane filters, promptly.
Draw each 10 μ l of reference substance solution and need testing solution respectively, inject chromatograph of liquid, press 1. chromatographic condition mensuration, calculate the content of ilexoside X X IX.
Preferably, the weight percentage of contained ilexoside X X IX is 5%-15% in the described total saponin of Radix Ilicis Asprellae extract.
Compared with prior art, the present invention has following beneficial effect:
1, adopts macroporous resin AB-8 process for refining of the present invention, through purification with macroreticular resin, the retention rate of total saponin of Radix Ilicis Asprellae and ilexosideX X IX is greatly improved, the retention rate of total saponins reaches 83.71% in the alcohol washing liquid, the retention rate of ilexosideX X IX reaches 89.46%, the leakage rate of total saponin of Radix Ilicis Asprellae and ilexoside X X IX is very low, and the content of extract obtained middle total saponins can reach 93%, and the content of ilexoside X X IX can reach more than 8%;
2, adopting characteristic saponin ilexoside XX IX in the process for refining of the extraction process of total saponin of Radix Ilicis Asprellae of the present invention and macroporous resin is reference substance, ilexoside X X IX and the consistent (see figure 1) of the ultraviolet absorption curve of total saponin of Radix Ilicis Asprellae, be that index is carried out quantitative expedition with the content of total saponin of Radix Ilicis Asprellae and the content of ilexoside X X IX simultaneously, with only adopt the ginsenoside Re to demarcate total saponins in the prior art to compare, accuracy and degree of accuracy are higher, and the result has more credibility.
3, the invention provides a kind of quality determining method of total saponin of Radix Ilicis Asprellae extract, HPLC-ELSD is to the good separating effect of ilexoside X X IX in the total saponin of Radix Ilicis Asprellae, highly sensitive, can be used for the assay of ilexosideX X IX in Flos Ilicis Asprellae medical material and the total saponin of Radix Ilicis Asprellae, the quality control that can be the total saponin of Radix Ilicis Asprellae extract provides reference.
Description of drawings
Fig. 1 is Flos Ilicis Asprellae ilexoside X X IX and the back ultraviolet absorption curve at the 400-700nm wavelength of total saponin of Radix Ilicis Asprellae colour developing, and wherein going up surface curve among the figure is ilexoside X X IX colour developing post-absorption curve; Following surface curve is a total saponin of Radix Ilicis Asprellae colour developing post-absorption curve;
Fig. 2 is the influence of not the same sample concentration to the total saponin of Radix Ilicis Asprellae adsorbance;
Fig. 3 is the dynamic adsorption curve of total saponin of Radix Ilicis Asprellae;
Fig. 4 is the eluting rates of different eluting solvents to total saponin of Radix Ilicis Asprellae;
Fig. 5 is the eluting rates of different eluting solvents to ilexoside X X IX;
Fig. 6 is the eluting rate of the eluting solvent of different volumes to total saponin of Radix Ilicis Asprellae;
Fig. 7 is the eluting rate of the eluting solvent of different volumes to ilexoside X X IX;
Fig. 8 crosses HPLC collection of illustrative plates before and after the post for the total saponin of Radix Ilicis Asprellae extracting solution, and wherein A is the HPLC collection of illustrative plates of upper prop liquid, and B is the HPLC collection of illustrative plates of eluent;
Fig. 9 is the total saponin of Radix Ilicis Asprellae thin-layer chromatogram, and wherein A is the Flos Ilicis Asprellae control medicinal material, and B-F is the total saponin of Radix Ilicis Asprellae extract.
The specific embodiment
Describe the present invention in detail below in conjunction with the drawings and specific embodiments.
The extraction process of embodiment 1 total saponin of Radix Ilicis Asprellae
1, Orthogonal Experiment and Design
Retention rate with total saponins and ilexoside X X IX is an index respectively, adopts the Orthogonal Experiment and Design of four factors, three levels.Take by weighing Flos Ilicis Asprellae medical material 20g, carry out experiment according to the orthogonal design of table 1, merging filtrate is measured volume.
Table 1 Flos Ilicis Asprellae medicinal material extract technology orthogonal experiment factor level table
2, the preparation of need testing solution
2.1 the preparation of total saponins need testing solution
The medicinal liquid that precision is measured under each orthogonal test item is an amount of, be evaporated to and do not have the alcohol flavor, the supreme sample concentration of thin up is 0.2mg crude drug/ml, precision is measured the concentrated solution that is equivalent to the 4.00g crude drug, on pretreated AB-8 macroporous resin column (13 * 150mm), be washed till clarification with the 40ml distilled water, reuse 95% ethanol elution, collect pure washing liquid to 100ml measuring bottle scale, shake up, get the quadrature test liquid.Accurate absorption test liquid is an amount of, put in the test tube, water bath method adds 5% vanillin glacial acetic acid solution 0.2ml and perchloric acid 0.8ml, in 60 ℃ of water-baths, heat 20min, take out, cold water cooling 10 minutes adds glacial acetic acid to 5ml, shake up, make blank with corresponding reagent, the 541nm place measures absorbance, is the content of total saponins.
2.2 the preparation of ilexoside X X IX need testing solution
The medicinal liquid of getting under each orthogonal test item is an amount of, and microporous filter membrane filters, and injects chromatograph of liquid, measures peak area, calculates the content of ilexoside X X IX.
Chromatographic condition: SHIMADZU high performance liquid chromatograph, SEDEX 75 evaporative light scattering detector.N2000 chromatographic work station (Zhejiang University) determines that (250 * 4.6mm) is immobile phase, is mobile phase with acetonitrile-0.2% formic acid, presses table 2 gradient elution with Phenomenex BDS C18 post.Drift tube temperature: 40 ℃, carrier gas (nitrogen) pressure: 2.5bar, flow velocity: 1ml/min, column temperature: 28 ℃ of room temperatures.Under above chromatographic condition, measure, the record spectrogram, greater than 4000, symmetrical factor is 0.95～1.05 to theoretical cam curve in ilexoside X X IX, with the separating degree of adjacent peak greater than 1.5.
Table 2 gradient elution program
3, Orthogonal experiment results and analysis
3.1 orthogonal test sample total saponin content measurement result and analysis
Table 3 orthogonal experiment sample total saponin content measurement result
3.2 orthogonal test Flos Ilicis Asprellae ilexoside X X IX assay result and analysis
Table 4 orthogonal experiment sample ilexoside X X IX assay result
With the retention rate of total saponin of Radix Ilicis Asprellae and ilexoside X X IX, the comprehensive retention rate of weighted calculation.
Comprehensive retention rate=(total saponins retention rate+ilexoside X X IX retention rate)/2
The comprehensive retention rate result of table 5 Flos Ilicis Asprellae medicinal material extract technology orthogonal experiment
The comprehensive retention rate result statistics of table 6 Flos Ilicis Asprellae medicinal material extract technology orthogonal experiment (intuitive analysis)
The comprehensive retention rate result statistics of table 7 Flos Ilicis Asprellae medicinal material extract technology orthogonal experiment (variance analysis)
From the comprehensive retention rate result of table 5-table 7 as can be known, concentration of alcohol and extraction time are comparatively remarkable to the retention rate influence, and level 2 retention rates are the highest in the concentration of alcohol, so selection level 2 (60% ethanol).Level 1 has significant difference with level 2,3 in the extraction time, and so level 2 and level 3 there was no significant differences are selection level 2 (twice).The influence of solvent consumption is less, and is bigger with level 2, and extraction time is selected less 2 times in addition, so the moderate level 2 (8 times of amounts, 6 times of amounts) of solvent consumption selection.Slightly variant between three levels of extraction time, take all factors into consideration selection level 2.So optimum extraction process is: 8 times of amount 60% ethanol extraction 1.5h, 6 times of amounts of reuse, 60% ethanol extraction 1h.
The AB-8 macroporous adsorbent resin process for refining of embodiment 2 total saponin of Radix Ilicis Asprellae extracting solution
1, goes up the investigation of sample concentration
1.1 the mensuration of total saponin of Radix Ilicis Asprellae in the preparation of Flos Ilicis Asprellae extracting solution and the extracting solution
Get Flos Ilicis Asprellae medicinal material coarse powder (Chinese medicine beverage factory of Guangzhou medical material company lot number 20080401) 905g, 60% ethanol that adds 8 times of amounts, reflux 1.5 hours filters, and filtering residue adds 60% ethanol of 6 times of amounts again, reflux 1 hour, filter, merging filtrate is measured volume, get extracting solution 11020ml, standby.Measure wherein total saponin content.
1.2 the total saponins retention rate of not the same sample concentration is investigated (centrifugal, static adsorption)
Measure Flos Ilicis Asprellae extracting solution 608.8ml (being equivalent to contain the 50g crude drug), decompression recycling ethanol is concentrated into 100ml (0.5g/ml), and centrifugal (3000r/min 10min), gets supernatant 80ml, and thin up supplies reagent liquid to 100ml as 0.5g crude drug/ml.
Get above-mentioned 0.5g crude drug/ml confession reagent liquid 20ml (being equivalent to contain the 10g crude drug), water-bath is steamed to the thick paste shape, and thin up is to 10ml, as the confession reagent liquid of 1.0g crude drug/ml.
Get above-mentioned 0.5g crude drug/ml for reagent liquid 20ml, add water be diluted to respectively 200ml, 100ml, 50ml as 0.05g crude drug/ml, 0.1g crude drug/ml, 0.2g crude drug/ml for reagent liquid.
Get pretreated AB-8 macroporous adsorbent resin (20ml column volume) (13*150mm), each 5 parts, put in the tool plug conical flask, add the confession reagent liquid of each variable concentrations respectively,, continue 2h every 10min concussion 10s, resin is fallen on the Ding Shi funnel together with test liquid, add the washing of 100ml distilled water, reuse 95% ethanol elution is collected 95% ethanol elution to 100ml measuring bottle scale.Precision is measured above each 5ml of pure washing liquid, adds 70% ethanol dilution to 10ml, measures test liquid, parallel two parts as total saponins.Measure wherein total saponin content, calculate the ratio adsorbance of AB-8 macroporous adsorbent resin total saponins in the not the same sample concentration liquid.The results are shown in Table 8.
The total saponin content of not the same sample concentration of table 8 is measured (centrifugal, static adsorption)
Annotate: saponin content is in total saponins percentage ratio in the Flos Ilicis Asprellae crude drug in the table.
The adsorbance of the total saponins of not the same sample concentration of table 9 is investigated
Shown in Fig. 2 and table 8,9, the Flos Ilicis Asprellae of different crude drug concentration clarification medicinal liquid is through AB-8 resin static adsorption, and the ratio adsorbance of total saponins is 54.48-64.90mg/g.Centrifuging process is bigger to the influence of the retention rate of total saponins in the Flos Ilicis Asprellae medicinal liquid, and stock solution is after centrifugal, and total saponin content reduces to 2.713%, loss 26.7% from 3.701% in the medicinal liquid.The prompting centrifuging process is bigger to the loss of total saponins in the Flos Ilicis Asprellae medicinal liquid, should not adopt in process for refining.
Process efficiency is higher when the upper prop liquor strength is higher, but may block up post during upper prop, influences the technology seriality; Concentration is too low, and sample solution is too many, influences efficient.In conjunction with the ratio adsorbance of different crude drug concentration Flos Ilicis Asprellae clarification medicinal liquids in AB-8 resin static adsorption, take all factors into consideration the operation possibility and the production efficiency of plant produced, will go up column liquid concentration and be defined as 0.2g crude drug/ml.
2, the maximum applied sample amount of Flos Ilicis Asprellae medicinal liquid macroporous adsorbent resin process for refining is investigated
Measure Flos Ilicis Asprellae extracting solution 243.5ml (being equivalent to the 20g crude drug), reclaim under reduced pressure is not to there being the alcohol flavor, and thin up promptly gets upper prop liquid to 100ml, (13mm * 150mm), collected post liquid, every 2.5ml are a to cross pretreated AB-8 macroporous resin column, collect in order, collect 34 pipes altogether.
To cross post liquid 1
#To 34
#Go up respectively pretreated AB-8 macroporous adsorptive resins (13 * 80mm), the washing 50ml, reuse 95% ethanol elution is collected pure washing liquid 60ml.Measure the wherein content of total saponins and ilexoside X X IX, with content in the upper prop liquid relatively, calculate not adsorption rate, the result is as follows.
2.1 with the total saponins is the maximum applied sample amount mensuration of index
The maximum applied sample amount of table 10 is investigated and is respectively managed in the upper prop liquid saponin concentration and adsorption rate not in the experiment
Annotate: the volume of every pipe is 2.5ml in the table, and saponin concentration is in 7.72mg/ml in the stock solution.
As shown in Figure 3, the 26th pipe total saponins concentration is 0.45mg/ml, surpasses 5% (7.72mg/ml * 5%=0.39mg/ml) of upper prop liquid.The resin dry weight is 4.04g, promptly gets leakage point in 5%, and its maximum applied sample amount is 25 * 0.5=12.5g crude drug, is equivalent to 3.09g crude drug/g dried resin; In total saponins, its maximum applied sample amount is 2.5ml * 25 pipe * 7.72=482.5mg total saponins, is equivalent to 119.43mg total saponins/g dried resin.
2.2 with ilexoside X X IX is the maximum applied sample amount mensuration of index
Get 10
#, 16
#, 25
#, 27
#, 34
#Ilexoside X X IX test liquid is an amount of, and microporous filter membrane filters, and measures peak area, calculates ilexoside X X IX content.
During investigating, the maximum applied sample amount of table 11 respectively manages in the upper prop liquid ilexoside X X IX concentration and adsorption rate not
The result shows, in 5% as leakage point, during the 34th pipe ilexoside X X IX not adsorption rate only be 3.91%, surpass 5% of upper prop liquid yet.Explanation is investigated the resin leakage point with ilexoside X X IX as index certain defective, is not enough to reflect the information of total saponins comprehensively, and total saponins is more responsive to the reaction of maximum applied sample amount, so maximum applied sample amount is in the total saponins result.
3, eluting solvent kind is investigated
According to the maximum applied sample amount of the definite Flos Ilicis Asprellae medicinal liquid of previous experiments, get Flos Ilicis Asprellae extracting solution 152.2ml (being equivalent to contain the 12.5g crude drug), being evaporated to does not have the alcohol flavor, gets the about 30ml of Flos Ilicis Asprellae concentrated solution, and thin up is to 62.5ml, on pretreated AB-8 macroporous adsorptive resins (13 * 150mm), be washed till clarification with the 150ml distilled water, more successively with 20%, 40%, 60%, 80%, 95% ethanol difference eluting 4 times of column volumes, i.e. 80ml, collect pure washing liquid, standby.Measure total saponins and ilexoside X X IX content in the different eluting solvents respectively, calculate total saponins and the distribution of ilexoside X X IX at different eluting solvent kind apoplexy due to endogenous wind, the result is as follows:
Table 12 eluting solvent kind is investigated the assay of total saponin of Radix Ilicis Asprellae in the experiment
Annotate: saponin content is in total saponins percentage ratio in the Flos Ilicis Asprellae crude drug in the table.
Table 13 total saponin of Radix Ilicis Asprellae is in the distribution of different eluting solvent kind apoplexy due to endogenous wind
Table 14 eluting solvent kind is investigated the assay of ilexoside X X IX in the experiment
Table 15 ilexoside X X IX is in the distribution of different eluting solvent kind apoplexy due to endogenous wind
From table 12-15, Fig. 4-Fig. 5 can draw, in the eluting solvent kind of the macroporous resin process for refining of Flos Ilicis Asprellae is investigated, with regard to total saponins, 60% ethanol can be accumulated under the eluting about 89% saponin, and 80% ethanol can be accumulated under the eluting about 98.5% saponin, and is complete near eluting; With regard to ilexoside X X IX, 60% ethanol can be accumulated under the eluting almost 100% ilexosideX X IX, and is complete near eluting.Take all factors into consideration technology cost and elution efficiency, selecting 80% ethanol is the eluting solvent of the macroporous resin process for refining of Flos Ilicis Asprellae.
4, eluting solvent consumption is investigated
According to the definite Flos Ilicis Asprellae medicinal liquid applied sample amount of above maximum applied sample amount experiment, precision is measured Flos Ilicis Asprellae extracting solution 152.2ml (being equivalent to the 12.5g crude drug), be evaporated to and do not have the alcohol flavor, thin up is to 62.5ml, on pretreated AB-8 macroporous resin column (13 * 150mm), be washed to clarification with the distilled water of 150ml, use 80% ethanol elution, each times column volume (20ml) is collected respectively, collects 6 times of column volumes altogether, and is standby.Total saponins and ilexoside X X IX content in measuring wherein respectively calculate total saponins and the distribution of ilexoside X X IX in different amounts eluting solvent, and the result is as follows:
Table 16 eluting solvent consumption is investigated the assay of total saponin of Radix Ilicis Asprellae in the experiment
Annotate: saponin content is in total saponins percentage ratio in the Flos Ilicis Asprellae crude drug in the table.
The distribution of table 17 total saponin of Radix Ilicis Asprellae in different eluting solvent consumptions
Table 18 eluting solvent consumption is investigated the assay of ilexoside X X IX in the experiment
Annotate: saponin content is in ilexoside X X IX percentage ratio in the Flos Ilicis Asprellae crude drug in the table.
The distribution of table 19 ilexoside X X IX in different eluting solvent consumptions
As show 16-table 19, and Fig. 6-shown in Figure 7, with regard to total saponins, the total saponins of 80% ethanol of three times of column volumes energy eluting down most (97%), and more eluting solvents only can increase a spot of total saponins; With regard to ilexoside X X IX, the ilexoside X X IX of the 80% ethanol energy eluting down most (99%) of three times of column volumes, and more eluting solvents only can increase a spot of ilexoside X X IX, take all factors into consideration total saponins retention rate and production cost, determine that eluting solvent consumption is three times of column volumes.
The checking of embodiment 3 total saponin of Radix Ilicis Asprellae extracting solution AB-8 macroporous resin process for refining
1, the mensuration of total saponin of Radix Ilicis Asprellae and ilexoside X X IX in the preparation of Flos Ilicis Asprellae extracting solution and the extracting solution
By drafting condition, get Flos Ilicis Asprellae medicinal material coarse powder (Chinese medicine beverage factory of Guangzhou medical material company, lot number: 20080401) 250g, 60% ethanol that adds 8 times of amounts, reflux 1.5 hours filters, and filtering residue adds 60% ethanol of 6 times of amounts again, reflux 1 hour, filter, merging filtrate is measured volume, get extracting solution 3016ml, standby.Measure the wherein content of total saponin of Radix Ilicis Asprellae and ilexosideX X IX.
2, Flos Ilicis Asprellae AB-8 macroporous adsorbent resin process for refining checking
The maximum applied sample amount of determining according to embodiment 2 of Flos Ilicis Asprellae medicinal liquid macroporous resin, get Flos Ilicis Asprellae extracting solution 1508ml, be equivalent to contain the 125g crude drug, reclaim under reduced pressure is to there not being the alcohol flavor, the about 250ml of Flos Ilicis Asprellae concentrated solution, thin up is to 625ml, after pretreated AB-8 macroporous adsorbent resin, flow velocity is 1ml/min, collects post liquid; Continue with the distilled water washing, be washed till clarification, collect water lotion 1026ml; Reuse 80% ethanol elution is collected three times of column volumes, and promptly 600ml is standby.Measure upper prop liquid respectively, cross total saponins and ilexoside X X IX content in post liquid, water lotion and the pure washing liquid, the result is as follows:
The assay and the retention rate of total saponin of Radix Ilicis Asprellae in the table 20 resin purification process certification experiment
Annotate: saponin content is in total saponins percentage ratio in the Flos Ilicis Asprellae crude drug in the table.
Assay and the retention rate of ilexoside X X IX in the table 21 resin purification process certification experiment
*The expression with the upper prop liquor ratio, saponin content is in total saponins percentage ratio in the Flos Ilicis Asprellae crude drug in the table.
The HPLC collection of illustrative plates of upper prop liquid and eluent is seen Fig. 8, and Fig. 8 shows the basic composition that keeps characteristic peak representative in the upper prop liquid HPLC collection of illustrative plates in the pure washing liquid.
3, cross the variation checking of post front and back the rate of extract
Precision is measured two parts of 60% ethanol extract 120.6ml (being equivalent to 10g crude drug amount), precision is measured two parts of 48ml (being equivalent to 10g crude drug amount) from alcohol eluen, decompression recycling ethanol is to fluid extract, be transferred to respectively in the evaporating dish of constant weight, water bath method, put baking oven and dry to constant weight for 105 ℃, weigh, calculate the rate of extract.The results are shown in Table 22.
Solid yield was measured before and after table 22 was crossed post
Take the fixed parameter of process for refining, through purification with macroreticular resin, the retention rate of total saponins reaches 83.71% in the alcohol washing liquid, the retention rate of ilexoside X X IX reaches 89.46%, cross that total saponins has leaked 5.62% in the post liquid, total saponins has leaked 1.31% in the water lotion, crosses the leakage that does not have ilexoside X X IX in the post liquid, and 0.57% leakage is only arranged in the water lotion.Result of calculation showed that the solid yield of the preceding Flos Ilicis Asprellae extracting solution of post was 7.76%, the total saponins theoretical content is 1 * 3.826%/(1 * 7.76%)=49.31% in the extracting solution solid content, and ilexoside X X IX theoretical content is 1 * 0.455%/(1 * 7.76%)=5.87%; Cross that the solid yield of Flos Ilicis Asprellae alcohol eluen is 3.68% behind the post, the total saponins theoretical content is 1 * 3.203%/(1 * 3.68%)=87.03% in the alcohol eluen solid content, and ilexoside X X IX theoretical content is 1 * 0.398%/(1 * 3.68%)=10.83% in the solid content.
The methodology checking of the quality determining method of embodiment 4 total saponin of Radix Ilicis Asprellae extracts
Differentiate (discriminating of TLC chromatograph)
Get total saponin of Radix Ilicis Asprellae extract 0.1g, add water 10ml and make dissolving, in order to water saturated n-butanol extraction 2 times, each 10ml merges n-butyl alcohol liquid, add 1% sodium hydroxide solution washing 2 times, each 20ml, get n-butyl alcohol liquid in order to n-butyl alcohol saturated be washed to neutrality, get n-butyl alcohol liquid evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution.Make Flos Ilicis Asprellae control medicinal material solution with method.According to thin layer chromatography (" appendix VIB of Chinese pharmacopoeia version in 2005) test, draw respectively above-mentioned solution each 51 on same silica gel g thin-layer plate, with chloroform-ethyl acetate-methanol-water-formic acid (1: 6: 1.5: 0.7: 0.1) is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to speckle colour developing at 105 ℃.In the test sample chromatograph, with control medicinal material chromatograph relevant position on, show the same color speckle, see Fig. 9.
Assay (HPLC-ELSD)
1, the preparation of reference substance solution
Get ilexoside X X IX reference substance (ilexoside XXIX, purity is 98.9%) 5mg, accurate title fixed (4.92mg), put in the 5ml measuring bottle, with dissolve with methanol and be diluted to scale, shake up, every 1ml contains the reference substance solution of ilexosideX X IX0.984mg.
2, the preparation of need testing solution
Get the about 2g of total saponin of Radix Ilicis Asprellae extract, precision is weighed, and is settled to 100mL with 70% ethanol, promptly gets total saponin of Radix Ilicis Asprellae extract need testing solution.
3, the drafting of standard curve
The above-mentioned reference substance solution 1 μ l of accurate respectively absorption, 2 μ l, 4 μ l, 10 μ l, 25 μ l, 30 μ l inject chromatograph of liquid, measure peak area.Natural logrithm with sample size is an abscissa, the natural logrithm of peak area is a vertical coordinate, carry out linear regression, getting regression equation is y=1.3535x+4.7495, r=0.9998 shows that the natural logrithm value of ilexoside X X IX sample size in 0.984 μ g～29.52 μ g scopes becomes the good linear relation with the natural logrithm value of peak area.
4, precision test
The accurate reference substance solution 10 μ l that draw under above-mentioned chromatographic condition, repeat to inject chromatograph of liquid 6 times, measure peak area, calculate the content of ilexoside XXIX, and RSD is 1.58%.
5, replica test
Each 6 parts of preparation total saponin of Radix Ilicis Asprellae extract need testing solutions by " a 3 " operation down, inject high performance liquid chromatograph, measure peak area, calculate the content of ilexoside XXIX, and RSD is 1.06%.
6, stability test
Accurate each 10 μ l of need testing solution that draw the total saponin of Radix Ilicis Asprellae extract, respectively 0,2,4,8,12,24, the 48h sample introduction, calculate ilexoside XXIX content, RSD is 1.37%.The result shows that need testing solution keeps stable in 48h.
7, average recovery test
Get the about 0.1g of total saponin of Radix Ilicis Asprellae extract of known content, each six parts, the accurate title, decide, the accurate respectively ilexosideX XIX 8mg that adds, and by the need testing solution preparation method preparation down of " 2 " item, HPLC measures ilexoside XXIX content, calculates average recovery.Its average recovery rate is 100.4%, and RSD is 1.43%.
8, the mensuration of sample
Get the about 2g of total saponin of Radix Ilicis Asprellae extract, the accurate title, decide, and is settled to 100ml with 70% ethanol, and microporous filter membrane filters, and injects chromatograph of liquid, by drafting chromatographic condition, measures peak area, calculates the content of ilexoside XXIX.The results are shown in Table 23.
Ilexoside XXIX assay result (%) in the table 23 total saponin of Radix Ilicis Asprellae extract
The HPLC-ELSD detection method is to ilexoside XXIX good separating effect in the Flos Ilicis Asprellae, and is highly sensitive, can be used for the assay of ilexoside XXIX in the total saponin of Radix Ilicis Asprellae extract.
The pharmacological action of embodiment 5 total saponin of Radix Ilicis Asprellae and ilexoside X X IX
One, pharmacological action (the ilexoside mistake of ilexoside X X IX! Do not find Reference source.Dichlorodiphenyl Acetate causes the abdominal cavity capillary permeability increases test)
1.1 be subjected to the reagent thing
A Flos Ilicis Asprellae ilexoside X X IXilexoside mistake! Do not find Reference source., purity is 98.9%.
1.2 modeling and contrast medicine
Aspirin, outstanding pharmaceutcal corporation, Ltd produces in Xinxiang, and it is standby to be made into 0.5% suspension.
1.3 test apparatus
Mettler toledo (AB204-N) ten thousand/electronic balance; Mettler toledo (CP225D) 100,000/electronic balance; Thermo (He λ ios-γ) uv-spectrophotometric instrument;
1.4 laboratory animal
Mice is the Kunming kind, SPF level animal, and mice, male and female half and half (are provided by Traditional Chinese Medicine University Of Guangzhou's Experimental Animal Center.)
1.5 experimental situation
Traditional Chinese Medicine University Of Guangzhou's zoopery environmental facility cleaning level Animal House.
2. experimental technique
2.1 animal for research and grouping
Mice, male and female half and half are divided into 3 groups, 15 every group, are respectively the blank group, positive drug aspirin matched group, ilexoside mistake! Do not find Reference source.The medicine group.
2.2 administration time and dosage
Every day, gastric infusion was 1 time, successive administration 7 days, and the blank group gives the distilled water of equal volume.Positive controls is given aspirin, and each administration group dosage is respectively 20ml/kg.
2.3 test method
0.5h after the last administration, tail vein injection 1% azovan blue normal saline solution 0.1ml/10g, and lumbar injection 0.6% acetic acid 0.2ml/ at once, behind the 20min, put to death mice (choking to death), cut off the abdominal cavity, collect cleaning mixture for several times, the centrifugal 5min of 1000r/min with 5ml normal saline flushing abdominal cavity, survey trap in 590nm wavelength place with ultraviolet spectrophotometer, the comparable group differences carries out the t check analysis, the results are shown in Table 24.
3. experimental result
Table 24
Annotate: administration group, positive controls compare with the blank group respectively.
A conclusion: compare the ilexoside mistake with the blank group! Do not find Reference source.Group has the effect that suppresses capillary permeability, and certain antiphlogistic effects is arranged.
Two, the pharmacological action of total saponin of Radix Ilicis Asprellae
I. antivirus action
1 experiment material
1.1 medicine name
Total saponin of Radix Ilicis Asprellae provides lot number by Traditional Chinese Medicine University Of Guangzhou new drug development research center: 20091101.
Content: every gram medicated powder (not adding adjuvant) contains crude drug 27g
1.2 animal
The NIH mice is provided by medical animal experiment center, Guangdong Province, the prescription that is rich in multiple nutritional components that mouse feed provides for Traditional Chinese Medicine University Of Guangzhou's Experimental Animal Center.Feeding environment: room temperature is 23 ± 2 ℃, and relative humidity is 75 ± 10%.
1.3 seed culture of viruses
First I type influenza virus FMI strain is provided by Chinese medicine bioassay, after mice strengthens virulence, goes down to posterity 2 times in chick embryo allantoic cavity, and measures its median lethal dose(LD 50).
1.4 equipment
The medical clean work station of YJ-875, Suzhou cleaning equipment company; Pressure steam sterilizer, Nanhua, Shanghai medical apparatus and instruments factory; Superpure water machine, Millipore; Ultra cold storage freezer, Sanyo Japan; Ten thousand/electronic analytical balance, FA/JA series, the flat instrument and meter company limited of last current chart; Ether, Tianjin chemical reagent company limited;
0.9% normal saline is opened and is helped Biology Pharmacy Co., Ltd; Table balance, Foochow balance equipment factory;
Plate, dissecting scissors, ophthalmology tweezer, disposable syringe etc.
2.1 virus dilution
Before the experiment, get seed culture of viruses flowing water, be diluted to every 0.05ml with physiological saline solution and contain 15 LD50, put in the frozen water and preserve towards soaking thawing.
2.2 infecting mouse
The mice random packet, divide positive drug group (virazole), virus control group and trial drug group, male and female half and half.Under the slight anesthesia of ether, with titration influenza virus drop nose infection, 4 of every Mus, about 0.05ml.Establish the normal mouse matched group simultaneously.
2.3 administration
According to the dosage of body surface area method calculating total saponin of Radix Ilicis Asprellae and positive drug, medicine is divided into the administration of high, medium and low dosage group, is respectively 20g/kg, 10g/kg, 5g/kg crude drug.Positive drug matched group dosage is 0.07g/kg.Above medicine all begins administration the previous day in virus attack.Administration is 5 days altogether, once a day, irritates stomach 0.3ml administration.
2.4 measure the lung index
Infected back four days, and cutd open mice extremely, fasting was prohibited water more than 4 hours before cuing open extremely.Claim the Mus body weight, the oxter arterial blood letting is put to death the back and is taken out the Mus lung, the record lesion degree, and criterion is the same as a result, lung is placed in the plate that fills 0.9% normal saline washs secondary, blots surface moisture with absorbent paper, and it is heavy to weigh up lung.Calculate lung index and lung index suppression ratio as follows:
Lung index=(mouse lung weight/mice body weight) * 100
Lung index suppression ratio=[(the average lung index of the average lung index-experimental group of model control group)/average lung index of model control group] * 100
3 statistical procedures
The gained experimental data is carried out the t check with SPSS12.0, ask statistical significance.
4 experimental results
Table 25 total saponin of Radix Ilicis Asprellae causes the inhibitory action result of mice pneumonia to influenza virus
Annotate: compare with the virus control group
*P＜0.01,
The result shows that the total saponin of Radix Ilicis Asprellae extract can improve the lung index of mouse infection first I type influenza virus, and lung index suppression ratio has dose-effect relationship, and prompting total saponin of Radix Ilicis Asprellae extract has resisiting influenza virus effect in the body.
II. antiinflammatory test
1, experiment material
1.1 be subjected to the reagent thing
Title: the total saponin of Radix Ilicis Asprellae extract provides lot number by Traditional Chinese Medicine University Of Guangzhou new drug development research center: 20091101.
Content: every gram medicated powder (not adding adjuvant) contains crude drug 27g
Compound method: with distilled water diluting to desired concn
Route of administration: gavage administration
Clinical dosage: 30g crude drug/60Kg/ days, promptly 0.5g crude drug/Kg/ days.
1.2 modeling and contrast medicine
Prednisone acetate tablets is produced by Guangdong Huanan Pharmaceutical Co., Ltd, 5mg/ sheet total saponin of Radix Ilicis Asprellae extract 20,10,5g crude drug/kg, and positive controls gavages prednisolone acetate (prednisone) 10mg/kg, and be administered once every day, continuous 5 days; The blank group gavages the equal-volume distilled water.30min after the last administration, at every Mus sufficient plantar subcutaneous injection 1% carrageenin suspension 0.08ml in a left side,, calculate swelling rate and suppression ratio as follows with 30min, 1h, 2h, 4h, 6h pedal swelling degree after the self-control survey volume glass container measurement administration, the t check the results are shown in Table 3.
3, experimental result
The influence of rat paw edema due to the table 26 total saponin of Radix Ilicis Asprellae extract on Carrageenan (x ± s)
Annotate: administration group, positive controls and blank group compare,
*P＜0.05,
*P＜0.01.
4. conclusion
Table 26 shows: with the blank group relatively, total saponin of Radix Ilicis Asprellae extract 20,10,5g crude drug/kg all can suppress rat paw edema (p＜0.01～0.05) due to the carrageenin significantly.
Prompting total saponin of Radix Ilicis Asprellae extract has antiinflammatory action.
III, analgesic test (hot plate method, hot water contract tail method)
1. experiment material
1.1 be subjected to the reagent thing
Test with antiinflammatory.
1.2 modeling and contrast medicine
Indometacin, for Sancai Medicine Group Co., Ltd., Zhongshan City produces, the 25mg/ sheet
1.3 test apparatus
Test with antiinflammatory.
1.2 modeling and contrast medicine
Indometacin, for Sancai Medicine Group Co., Ltd., Zhongshan City produces, the 25mg/ sheet
1.3 test apparatus
Electric-heated thermostatic water bath is produced by Shanghai medical apparatus and instruments two factories.
Stopwatch, self-control medicine box (dress mice, a side are cut into fence for grabbing usefulness, leave a blank and use for whipping in the bottom).
2. experimental technique
2.1 body weight placed at the female mice of 20 ± 2g on 55 ℃ ± 0.5 ℃ the metallic plate, licking metapedes with mice is the pain reaction signal, with pain reaction incubation period be threshold of pain index, twice of administration before measurement (5min at interval), with its average as the basic threshold of pain, select pain threshold 50 of 5～30 seconds mices, be divided into 5 groups (n=10) at random.Drug component does not gavage total saponin of Radix Ilicis Asprellae extract 20,10,5g crude drug/kg, and positive controls gavages indometacin 8.33mg/kg, and be administered once every day, for three days on end; The blank group gavages the equal-volume distilled water.Surveyed pain threshold twice (5min at interval) in 1 hour after the last administration, average, pain threshold is poor before and after calculating, i.e. threshold of pain improvement value, and the t check sees Table 27.
2.2 body weight is immersed 50 ℃ of water bath with thermostatic control 3cm at the kunming mice tail of 20 ± 2g, record contract tail incubation period is a threshold of pain index, twice of administration before measurement (5min at interval), with its average as the basic threshold of pain, select pain threshold 50 of 5～30 seconds mices, male and female half and half are divided into 5 groups (n=10) at random.It is the same that each organizes the administration situation.After the last administration, survey pain threshold in 1 hour twice (5min at interval), average.Pain threshold is poor before and after calculating, and promptly threshold of pain improvement value is carried out the t check, sees Table 28.
3. experimental result
Table 27 total saponin of Radix Ilicis Asprellae extract is to the influence of hot plate method induced mice pain (x ± s)
Annotate: administration group, positive controls and blank group are relatively.
Table 28 total saponin of Radix Ilicis Asprellae extract is to the contract influence (n=10 of x ± s) of tail of hot-water process's induced mice
Annotate: administration group, positive controls and blank group are relatively.
4. conclusion
Table 27 shows: compare with the blank group, total saponin of Radix Ilicis Asprellae extract 20,10g crude drug/kg can prolong the incubation period (p＜0.05) that the hot plate induced mice is licked the metapedes reaction, 5g crude drug/kg also has certain analgesic activity, but difference does not have significance meaning (P＞0.05).
Table 28 shows: with the blank group relatively, total saponin of Radix Ilicis Asprellae extract 20,10g crude drug/kg can prolong hot water induced mice tail incubation period (p＜0.05) that contracts, 5g crude drug/kg also has certain analgesic activity, but difference does not have significance meaning (P＞0.05).
Experiment prompting total saponin of Radix Ilicis Asprellae extract has and prolongs hot water and cause contract tail incubation period and hot plate of mice and cause effect incubation period that mice licks the metapedes reaction, shows that total saponin of Radix Ilicis Asprellae has analgesic activity.
IV, separate heat test (the total saponin of Radix Ilicis Asprellae extract is to 2, the influence of rat fever due to the 2, 4-dinitrophenol)
1. experiment material
1.1 be subjected to the reagent thing
Test with antiinflammatory.
1.2 modeling and contrast medicine
2,2, 4-dinitrophenol (C.P) is produced by Shanghai reagent three factories
Aspirin is produced by Shijiazhuang martial prowess Pharmaceutical joint-stock company
1.3 test apparatus
Omron electronic clinical thermometer (precision is 0.05 ℃) is produced by Japanese Omron Corp.
2. experimental technique
Select for use body weight 50 of 200 ± 10g rats, male and female half and half are selected the rat of body temperature in normal range for use, are divided into 5 groups (n=10) at random.Drug component does not gavage total saponin of Radix Ilicis Asprellae extract 20,10,5g crude drug/kg, and positive controls gavages aspirin 0.1g/kg, and the blank group gavages the equal-volume distilled water.Behind the single administration immediately to rat back subcutaneous injection 2%2,2, 4-dinitrophenol 15mg/kg, after administration 30,60,120,180,240min surveys its anus temperature changing value, the t check the results are shown in Table 29.
3. experimental result
Table 29 total saponin of Radix Ilicis Asprellae extract is to 2, and the influence of rat fever due to the 2, 4-dinitrophenol (x ± s)
4. conclusion
Table 29 shows: with the blank group relatively, total saponin of Radix Ilicis Asprellae extract 20,10,5g crude drug/kg all can suppress 2 significantly, rat fever (p＜0.01～0.05) due to the 2, 4-dinitrophenol.
Experiment prompting total saponin of Radix Ilicis Asprellae extract has inhibition 2, and 2, 4-dinitrophenol causes the rat fever effect.
The above is a preferred implementation of the present invention, should be pointed out that for those skilled in the art, under the premise of not departing from the present invention, can also make some improvement and modification, and these improvement and modification also are considered as protection scope of the present invention.
Claims (8)
1. the extracting method of a total saponin of Radix Ilicis Asprellae is characterized in that: may further comprise the steps:
(1) gets Radix Ilicis Asprellae or stem medical material, added 4～12 times of amounts 60% ethanol extraction 1.5-2 hour, add 2～10 times of amounts 60% ethanol extraction 1.0-1.5 hour again, filter merging filtrate;
(2) filtrate decompression that step (1) is obtained is concentrated into does not have the alcohol flavor, and the supreme sample concentration of thin up is 0.1-0.5g crude drug/ml, on pretreated AB-8 macroporous resin column;
(3) the washed resin post is used the 60%-80% ethanol elution then to clarification;
(4) collect 60%-80% ethanol elution concentrating under reduced pressure, the dry total saponin of Radix Ilicis Asprellae that gets.
2. the extracting method of total saponin of Radix Ilicis Asprellae according to claim 1 is characterized in that: added 8 times of amount 60% ethanol extractions in the described step (1) 1.5 hours, and added 6 times of amount 60% ethanol extractions 1 hour again.
3. the extracting method of total saponin of Radix Ilicis Asprellae according to claim 1 is characterized in that: going up sample concentration in the described step (2) is 0.2g crude drug/ml.
4. the extracting method of total saponin of Radix Ilicis Asprellae according to claim 1, it is characterized in that: in total saponin of Radix Ilicis Asprellae, maximum applied sample amount is 119.43mg total saponin of Radix Ilicis Asprellae/g dried resin in the described step (2).
5. the extracting method of total saponin of Radix Ilicis Asprellae according to claim 1 is characterized in that: in the described step (3) with 3 times of column volume 80% ethanol elutions.
6. the total saponin of Radix Ilicis Asprellae according to each described extracting method gained of claim 1-5 is preparing antiinflammatory, analgesia, analgesic or anti-influenza virus medicament, or the application in preparation beverage, collutory, toothpaste or chewing gum daily use chemicals health-oriented products.
7. the quality determining method of a total saponin of Radix Ilicis Asprellae extract is characterized in that: may further comprise the steps:
(1) discriminating of total saponin of Radix Ilicis Asprellae
Get total saponin of Radix Ilicis Asprellae extract 0.1-0.2g, add water 5-15ml and make dissolving, in order to water saturated n-butanol extraction 2-3 time, each 5-15ml merges n-butyl alcohol liquid, add 0.8%-1.2% sodium hydroxide solution washing 2-3 time, each 15-25ml, get n-butyl alcohol liquid in order to n-butyl alcohol saturated be washed to neutrality, get n-butyl alcohol liquid evaporate to dryness, residue adds methanol 1ml makes dissolving, as need testing solution; Make Flos Ilicis Asprellae control medicinal material solution with method; According to " appendix a VIB of Chinese pharmacopoeia version in 2005 thin layer chromatography test, draw each 5 μ l point of above-mentioned solution respectively on same silica gel g thin-layer plate, with volume ratio is 1: 6: 1.5: 0.7: 0.1 chloroform: ethyl acetate: methanol: water: formic acid is developing solvent, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to speckle colour developing at 105 ℃; In the need testing solution chromatograph, with control medicinal material solution chromatograph relevant position on, show the same color speckle;
(2) assay of ilexosideX X IX in the total saponin of Radix Ilicis Asprellae
1. chromatographic condition
With C
18Post is an immobile phase, and acetonitrile-0.2% formic acid is mobile phase, carries out gradient elution; Drift tube temperature: 40 ℃, nebulizer gas pressure: 2.5bar, flow velocity: 1ml/min, column temperature: room temperature; Greater than 4000, symmetrical factor is 0.95～1.05 to theoretical cam curve in ilexosideX X IX, with the separating degree of adjacent peak greater than 1.5;
2. the preparation of reference substance solution
Get ilexosideX X IX reference substance 5mg, accurate claim surely, put in the 5ml measuring bottle, with dissolve with methanol and be diluted to scale, shake up, promptly;
3. the preparation of need testing solution
Get total saponin of Radix Ilicis Asprellae 2g, the accurate title, decide, and is settled to 100ml with 70% ethanol, and microporous filter membrane filters, promptly;
Draw each 10 μ l of reference substance solution and need testing solution respectively, inject chromatograph of liquid, press 1. chromatographic condition mensuration, calculate the content of ilexosideX X IX.
8. the quality determining method of total saponin of Radix Ilicis Asprellae according to claim 7, it is characterized in that: the weight percentage of ilexosideX X IX is 5%-15% in the described total saponin of Radix Ilicis Asprellae extract.
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| WO2012100612A1 (en) * | 2011-01-24 | 2012-08-02 | 北京大学 | Extraction method, total saponin and use of ilex kudingcha c.j.tseng leaves |
| CN102830199A (en) * | 2012-08-23 | 2012-12-19 | 涂瑶生 | Thin-layer chromatography method for identifying Ilex asprella formula particles |
| CN110346463A (en) * | 2018-04-08 | 2019-10-18 | 暨南大学 | A kind of method for building up of Flos Ilicis Asprellae root herb HPLC-ELSD finger-print |
| CN111366549A (en) * | 2020-03-24 | 2020-07-03 | 广西壮族自治区人民医院 | Method for measuring content of total saponins in red kiwi fruits |
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| CN1695504A (en) * | 2005-06-20 | 2005-11-16 | 张玉生 | Beverage of increasing the body fluid |
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| CN1695504A (en) * | 2005-06-20 | 2005-11-16 | 张玉生 | Beverage of increasing the body fluid |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012100612A1 (en) * | 2011-01-24 | 2012-08-02 | 北京大学 | Extraction method, total saponin and use of ilex kudingcha c.j.tseng leaves |
| CN102830199A (en) * | 2012-08-23 | 2012-12-19 | 涂瑶生 | Thin-layer chromatography method for identifying Ilex asprella formula particles |
| CN110346463A (en) * | 2018-04-08 | 2019-10-18 | 暨南大学 | A kind of method for building up of Flos Ilicis Asprellae root herb HPLC-ELSD finger-print |
| CN111366549A (en) * | 2020-03-24 | 2020-07-03 | 广西壮族自治区人民医院 | Method for measuring content of total saponins in red kiwi fruits |
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