CN109406708A - Join the thin layer measuring method that Rong is good for waist wine - Google Patents
Join the thin layer measuring method that Rong is good for waist wine Download PDFInfo
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Abstract
The invention discloses the thin layer measuring method that ginseng Rong is good for waist wine, this method comprises: step 1, ginsenoside mixed reference substance solution and Echinacea purpurea is sweet, preparation of acteoside and Hyperoside reference substance solution；Step 2, ginseng Rong are good for the preparation of waist wine test solution: step 3, the respectively accurate test solution and ginsenoside mixed reference substance solution point drawn are in silica gel g thin-layer plate, spray is with 10% ethanol solution of sulfuric acid, it is clear that it is heated to spot development at 105 DEG C, is placed under ultraviolet lamp 365nm wavelength and inspects；It is accurate respectively to draw test solution and Echinacea purpurea is sweet, acteoside and Hyperoside reference substance solution point are on polyam ide TLC plate, using methanol-acetic acid-water as solvent, expansion, it takes out, it dries, sets and inspected under 365nm ultraviolet light, show the fluorescence spot of same color.This method has many advantages, such as easy to operate, efficient, at low cost, accuracy and reproducible, and the quality for being good for waist wine for control ginseng Rong is of great significance.
Description
Technical field
The invention belongs to Chinese medicine detection technique fields, and in particular to a seed ginseng Rong is good for the thin layer measuring method of waist wine.
Background technique
Join Rong and be good for waist wine, the ginseng Rong produced such as Jiangsu Hai Sheng pharmaceutcal corporation, Ltd is good for waist wine (bringing up logical) by ginseng, Radix Astragali, Huang
Essence, radix polygonati officinalis, Rehmannia glutinosa, prepared fleece flower root, Cortex Eucommiae, fructus lycii, Herba Cistanches, Semen Cuscutae, dog kidney (system), radix aucklandiae, dried orange peel, fingered citron, chicken
Blood rattan is made, and has Yishen Jianpi, strong spinal column, relaxing tendons and activating collaterals.For ache of the spinal column caused by speen-kidney dificiency, two knees are powerless etc.
Disease.
In existing standards of pharmacopoeia only one ginseng Rong be good for waist wine in single ingredient as Index for examination, cannot be comprehensive, objective, quasi-
True evaluation ginseng Rong is good for the quality of waist wine, and the present invention filters out the strong waist of ginseng Rong that can detect multiple active constituents by many experiments
The thin layer measuring method of wine.This method has many advantages, such as easy to operate, efficient, at low cost, accuracy and reproducible, for standard
The quality that really control ginseng Rong is good for waist wine is of great significance.
Summary of the invention
Goal of the invention: the purpose of the present invention is overcome the deficiencies in the prior art, the ginseng Rong for developing a kind of multiple determination is strong
The thin layer measuring method of waist wine, can guarantee by means of the present invention benefit ginseng Rong be good for the quality stability of waist wine, consistency and
Controllability, so that it is guaranteed that benefit ginseng Rong is good for the safety and validity of waist wine.
Technical solution: in order to achieve the above object, the technical solution adopted by the present invention are as follows:
One seed ginseng Rong is good for the thin layer measuring method of waist wine, comprising the following steps:
The preparation of step 1, reference substance solution
(1.1) ginsenoside Rb1 is taken, ginsenoside Re and ginsenoside Rg1's reference substance are appropriate, and it is accurately weighed respectively, it sets
In volumetric flask, add methanol that ginsenoside mixed reference substance solution is made, it is spare；
(1.2) take the sweet reference substance of Echinacea purpurea appropriate, it is accurately weighed, it is placed in volumetric flask, adds methanol that Echinacea purpurea is made sweet right
It is spare according to product solution；
(1.3) take acteoside reference substance appropriate, it is accurately weighed, it is placed in volumetric flask, adds methanol that verbascose is made
Glycosides reference substance solution, it is spare；
(1.4) take Hyperoside reference substance appropriate, it is accurately weighed, it is placed in volumetric flask, adds methanol that Hyperoside pair is made
It is spare according to product solution；
The preparation of step 2, test solution:
It takes ginseng Rong to be good for waist wine and methanol, precipitating is added, then filtering removal carbohydrate content is added chloroform, is centrifuged, discards
Chloroform liquid, removes grease-like composition；Then solvent is volatilized, water-saturated n-butanol, ultrasound is added, ammonia is added in Aspirate supernatant
Water stratification takes upper liquid to be evaporated, and methanol is added to dissolve, and obtains test solution, spare；
Step 3,
(3.1) respectively in the 2~5ul of ginsenoside mixed reference substance solution and step 2 in accurate aspiration step 1 for examination
Product 5~10ul of solution puts respectively on same silica gel g thin-layer plate, using chloroform-methanol-water as solvent, is unfolded, takes out,
It dries, with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C for spray, is placed under ultraviolet lamp 365nm wavelength and examines
Depending on in ginsenoside mixing reference substance chromatography same position, showing the spot or fluorescent spot of same color in sample chromatogram
Point；
(3.2) echinacoside, acteoside and each 2~5ul of Hyperoside reference substance solution and test solution 5 are drawn
~10ul is put respectively on same polyam ide TLC plate, using methanol-acetic acid-water as solvent, is unfolded, is taken out, dry, set
It is inspected under 365nm ultraviolet light, in sample chromatogram, at the position corresponding to the chromatogram of the reference substance, shows the fluorescent spot of same color
Point.
Preferably, above-described seed ginseng Rong is good for the thin layer measuring method of waist wine, step 1, reference substance solution
Preparation method are as follows:
(1.1) ginsenoside Rb1, ginsenoside Re and ginsenoside Rg1's reference substance are taken, adds methanol that every 1ml is made and contains
The mixed reference substance solution of 2mg ginsenoside Rb1, ginsenoside Re and ginsenoside Rg1；
(1.2) echinacoside reference substance is taken, adds methanol that the reference substance solution of every 1ml echinacoside containing 1mg is made；
(1.3) acteoside reference substance is taken, adds methanol that the reference substance solution of every 1ml acteoside containing 1mg is made；
(1.4) Hyperoside reference substance is taken, adds methanol that the reference substance solution of every 1ml Hyperoside containing 1mg is made.
Preferably, above-described seed ginseng Rong is good for the thin layer measuring method of waist wine, step 2, test solution
The preparation method comprises the following steps: ginseng Rong is taken to be good for waist wine 100ml, be evaporated to 5-10ml on water-bath, the methanol that 6~10 times of amounts are added shake up
Stand 2~3 hours afterwards, filtering removes precipitating carbohydrate content, obtains clear liquid, chloroform 40ml is added, sufficiently shakes up, from
It is centrifuged layering in scheming, discards chloroform liquid, removes grease-like composition；Then raffinate volatilizes solvent, adds water and stirs wetting,
Water saturated 10~15ml of n-butanol is added, is ultrasonically treated 30 minutes, Aspirate supernatant adds triplication ammonium hydroxide, shakes up, and places and divides
Layer, takes upper liquid to be evaporated, residue adds methanol to make to dissolve, and obtains test solution, spare.
Preferably, above-described seed ginseng Rong is good for the thin layer measuring method of waist wine, and step (3.1) is accurate respectively
Ginsenoside mixed reference substance solution 5ul in the aspiration step 1 and test solution 10ul in step 2, respectively point Yu Tongyi
On silica gel g thin-layer plate, chloroform-methanol-water that volume ratio is 13:7:2 is first placed 12 hours in refrigerator, is then taken
Out, after room temperature 2~3 hours, taking lower layer's solution is solvent, is unfolded, and takes out, dries, and is sprayed with 10% ethanol solution of sulfuric acid,
It is clear to be heated to spot development at 105 DEG C, is placed under ultraviolet lamp 365nm wavelength and inspects, in sample chromatogram, with ginseng soap
In glycosides mixing reference substance chromatography same position, the spot or fluorescence spot of same color are shown.
Preferably, above-described seed ginseng Rong is good for the thin layer measuring method of waist wine, and step (3.2) draws pine nut
Chrysanthemum glycosides, acteoside and Hyperoside reference substance solution each 2ul and test solution 10ul put thin in same polyamide respectively
On laminate, using volume ratio be 2:1:7 or the methanol-acetic acid-water of 4:1:5 is solvent, is unfolded, and takes out, dries, and sets 365nm purple
It is inspected under outer light, in sample chromatogram, at the position corresponding to the chromatogram of the reference substance, shows the fluorescence spot of same color.
The optimization of thin layer measuring method is tested:
(1) screening of ginsenoside unfolding condition: 3 kinds of ginsenosides are unfolded in the present invention simultaneously on silica gel plate, by big
Measure the solvent (composition such as petroleum ether, benzene, chloroform, ethyl acetate, n-butanol, ethyl alcohol, first alcohol and water of experiment screening difference composition
Solvent), according to after the selection result choose volume ratio be 13:7:2 chloroform-Methanol-water be solvent, discovery exhibition
Find that spot is not shown after opening；Chloroform-Methanol-water with volume ratio for 13:7:2, which prepares, is placed on refrigerator 12 hours
It takes out and directly uses afterwards, spot occur, but spot is still unintelligible；It is finally chloroform-first of 13:7:2 with volume ratio
Alcohol-water prepares be placed on refrigerator 12 hours after take out, placed 1~3 hour under room temperature, take lower layer's solution use, 3 ginseng soaps
Glycosides separating degree is good, and clear spot.
(2) screening of echinacoside, acteoside and the unfolding condition of Hyperoside:
Since the structure and its property of echinacoside, acteoside and Hyperoside are different, separation requirement is higher.This hair
It is bright to be screened by many experiments, select chloroform, ethyl acetate, n-butanol, acetone, ethyl alcohol, methanol, phosphoric acid, acetic acid and water difference
Group become solvent, screening experiment the result shows that, use methanol-acetic acid-water be solvent expansion effect it is best, especially with
Volume ratio is 2:1:7 or the methanol-acetic acid-water of 4:1:5 is solvent echinacoside, acteoside and the expansion of Hyperoside
Separating degree is good, clear spot.
(3) test sample screening experiment；The method that the present invention has screened following test sample preparation
(3.1) ginseng Rong is taken to be good for waist wine 50ml, precision is measured, is placed in separatory funnel, and the 50ml that adds diethyl ether extraction discards upper layer
Solution takes lower layer that acetonitrile is added to be settled to 200ml, and ultrasonic treatment (power 150W, frequency 20kHZ) 30 minutes lets cool, sinks again, in
Separatory funnel layering, takes clarified solution to be placed in evaporating dish, and water-bath volatilizes, and residue adds methanol to dissolve, and is settled to 10ml, filter membrane,
To obtain the final product.
(3.2) it takes ginseng Rong to be good for waist wine 50ml, after being evaporated to 10ml on water-bath, adds chloroform 40ml, be heated to reflux 1
Hour, chloroform liquid is discarded, the dregs of a decoction volatilize solvent, add water 0.5ml stirring wetting, add water-saturated n-butanol 10ml, at ultrasound
Reason 30 minutes, Aspirate supernatant adds 3 times of amount ammonium hydroxide, shakes up, and places layering, upper liquid is taken to be evaporated, and residue adds methanol to make to dissolve, and makees
For sample.
(3.3) it takes ginseng Rong to be good for waist wine 100ml, 5-10ml is evaporated on water-bath, adds chloroform 40ml, is heated to reflux
1 hour, chloroform liquid is discarded, the dregs of a decoction volatilize solvent, add water 0.5ml stirring wetting, add water-saturated n-butanol 10ml, at ultrasound
Reason 30 minutes, Aspirate supernatant adds 3 times of amount ammonium hydroxide, shakes up, and places layering, upper liquid is taken to be evaporated, and residue adds methanol to make to dissolve, and makees
For sample.
(3.4) test solution the preparation method comprises the following steps: take ginseng Rong be good for waist wine 100ml, 5-10ml is evaporated on water-bath,
It is added after the methanol that 6~10 times are measured shakes up and stands 2~3 hours, filter, remove precipitating carbohydrate content, obtain clear liquid, trichlorine is added
Methane 40ml, sufficiently shakes up, and layering is centrifuged in centrifuge, discards chloroform liquid, removes grease-like composition；Then raffinate
Solvent is volatilized, wetting is added water and stirred, water saturated 10~15ml of n-butanol is added, is ultrasonically treated 30 minutes, Aspirate supernatant adds
Triplication ammonium hydroxide, shakes up, and places layering, takes upper liquid to be evaporated, residue adds methanol to make to dissolve, and obtains test solution, spare.
The experimental results showed that the preparation method of the test solution using above (3.4), can not only remove carbohydrate and oil
The impurity components such as rouge, and ginsenoside and echinacoside, acteoside and Hyperoside etc. that ginseng Rong is good in waist wine can be retained
Active constituent, convenient for the separation of subsequent thin-layer chromatography, therefore the preparation side of the final preferably test solution of (3.4) of the present invention
Method.
The beneficial effects of the present invention are:
(1) the ginseng Rong of multiple determination established by the present invention is good for the thin layer measuring method of waist wine, is sieved by many experiments
Select the preparation of test solution, best unfolding condition and coloration method etc., the method simple and effective that the present invention establishes, cost
It is low, and have many advantages, such as that stability, consistency and controllability are good, so that it is guaranteed that benefit ginseng Rong is good for the safety and validity of waist wine,
It can effective, the strong waist wine of overall monitor benefit ginseng Rong quality.
Detailed description of the invention
Fig. 1 is the thin-layer chromatogram that present invention ginseng Rong is good for ginsenoside in waist wine.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, actual conditions are not specified in embodiment
Person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer, being can be with
Conventional products that are commercially available.
One seed ginseng Rong is good for the thin layer measuring method of waist wine, comprising the following steps:
The preparation of step 1, reference substance solution
(1.1) ginsenoside Rb1, ginsenoside Re and ginsenoside Rg1's reference substance are taken, adds methanol that every 1ml is made and contains
The mixed reference substance solution of 2mg ginsenoside Rb1, ginsenoside Re and ginsenoside Rg1；
(1.2) echinacoside reference substance is taken, adds methanol that the reference substance solution of every 1ml echinacoside containing 1mg is made；
(1.3) acteoside reference substance is taken, adds methanol that the reference substance solution of every 1ml acteoside containing 1mg is made；
(1.4) Hyperoside reference substance is taken, adds methanol that the reference substance solution of every 1ml Hyperoside containing 1mg is made.
The preparation of step 2, test solution:
It takes ginseng Rong to be good for waist wine 100ml (production of Jiangsu Hai Sheng pharmaceutcal corporation, Ltd, lot number 180101), is evaporated on water-bath
It to 5-10ml, is added after the methanol that 6~10 times are measured shakes up and stands 2~3 hours, filter, remove precipitating carbohydrate content, obtain clear
Liquid is added chloroform 40ml, sufficiently shakes up, and layering is centrifuged in centrifuge, discards chloroform liquid, remove grease type at
Point；Then raffinate volatilizes solvent, adds water and stirs wetting, and water saturated 10~15ml of n-butanol is added, and is ultrasonically treated 30 minutes,
Aspirate supernatant adds triplication ammonium hydroxide, shakes up, and places layering, takes upper liquid to be evaporated, residue adds methanol to make to dissolve, and obtains test sample
Solution, it is spare.
Step 3,
The confession in ginsenoside mixed reference substance solution 5ul and step 2 in the accurate aspiration step 1 of step (3.1) difference
Test sample solution 10ul, puts respectively on same silica gel g thin-layer plate, by chloroform-methanol-water that volume ratio is 13:7:2 in ice
It first places 12 hours, then takes out in case, after room temperature 2 hours, taking lower layer's solution is solvent, is unfolded, it takes out, dries,
With 10% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C for spray, is placed under ultraviolet lamp 365nm wavelength and inspects, supplies
In test product chromatography, in ginsenoside mixing reference substance chromatography same position, the spot or fluorescence spot of same color are shown.Such as
Shown in Fig. 1.
Step (3.2) draws echinacoside, acteoside and each 2ul of Hyperoside reference substance solution and test solution
10ul is put respectively on same polyam ide TLC plate, using volume ratio for 2:1:7 methanol-acetic acid-water as solvent, expansion takes
Out, it dries, sets and inspected under 365nm ultraviolet light, in sample chromatogram, at the position corresponding to the chromatogram of the reference substance, show identical face
The fluorescence spot of color.
Embodiment 2
The other the same as in Example 1, step (3.2) using volume ratio for 4:1:5 methanol-acetic acid-water as solvent, expansion, take
Out, it dries, sets and inspected under 365nm ultraviolet light, in sample chromatogram, at the position corresponding to the chromatogram of the reference substance, show identical face
The fluorescence spot of color.
Above embodiments are only exemplary embodiment of the present invention, are not used in the limitation present invention, protection scope of the present invention
It is defined by the claims.Those skilled in the art can within the spirit and scope of the present invention make respectively the present invention
Kind modification or equivalent replacement, this modification or equivalent replacement also should be regarded as being within the scope of the present invention.
Claims (5)
1. the thin layer measuring method that a seed ginseng Rong is good for waist wine, which comprises the following steps:
The preparation of step 1, reference substance solution
(1.1) ginsenoside Rb1 is taken, ginsenoside Re and ginsenoside Rg1's reference substance are appropriate, and it is accurately weighed respectively, it is placed in appearance
In measuring bottle, add methanol that ginsenoside mixed reference substance solution is made, it is spare；
(1.2) take the sweet reference substance of Echinacea purpurea appropriate, it is accurately weighed, it is placed in volumetric flask, adds methanol that the sweet reference substance of Echinacea purpurea is made
Solution, it is spare；
(1.3) take acteoside reference substance appropriate, it is accurately weighed, it is placed in volumetric flask, adds methanol that acteoside pair is made
It is spare according to product solution；
(1.4) take Hyperoside reference substance appropriate, it is accurately weighed, it is placed in volumetric flask, adds methanol that Hyperoside reference substance is made
Solution, it is spare；
The preparation of step 2, test solution:
It takes ginseng Rong to be good for waist wine and methanol, precipitating is added, then filtering removal carbohydrate content is added chloroform, is centrifuged, discards trichlorine
Methane liquid, removes grease-like composition；Then solvent is volatilized, water-saturated n-butanol, ultrasound is added, ammonium hydroxide point is added in Aspirate supernatant
Layer, takes upper liquid to be evaporated, methanol is added to dissolve, obtain test solution, spare；
Step 3,
(3.1) test sample respectively in the 2~5ul of ginsenoside mixed reference substance solution and step 2 in accurate aspiration step 1 is molten
5~10ul of liquid puts respectively on same silica gel g thin-layer plate, using chloroform-methanol-water as solvent, is unfolded, takes out, dry in the air
Dry, with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C for spray, is placed under ultraviolet lamp 365nm wavelength and examines
Depending on in ginsenoside mixing reference substance chromatography same position, showing the spot or fluorescent spot of same color in sample chromatogram
Point；
(3.2) draw echinacoside, acteoside and each 2~5ul of Hyperoside reference substance solution and test solution 5~
10ul is put respectively on same polyam ide TLC plate, using methanol-acetic acid-water as solvent, is unfolded, is taken out, dry, set 365nm
It is inspected under ultraviolet light, in sample chromatogram, at the position corresponding to the chromatogram of the reference substance, shows the fluorescence spot of same color.
2. the thin layer measuring method that seed ginseng Rong according to claim 1 is good for waist wine, which is characterized in that
Step 1, the preparation method of reference substance solution are as follows:
(1.1) ginsenoside Rb1, ginsenoside Re and ginsenoside Rg1's reference substance are taken, adds methanol that every 1ml is made and contains 2mg people
Join saponin(e Rb1, the mixed reference substance solution of ginsenoside Re and ginsenoside Rg1；
(1.2) echinacoside reference substance is taken, adds methanol that the reference substance solution of every 1ml echinacoside containing 1mg is made；
(1.3) acteoside reference substance is taken, adds methanol that the reference substance solution of every 1ml acteoside containing 1mg is made；
(1.4) Hyperoside reference substance is taken, adds methanol that the reference substance solution of every 1ml Hyperoside containing 1mg is made.
3. the thin layer measuring method that seed ginseng Rong according to claim 1 is good for waist wine, which is characterized in that
Step 2, test solution the preparation method comprises the following steps: take ginseng Rong be good for waist wine 100ml, 5-10ml is evaporated on water-bath, is added
Enter after the methanol that 6~10 times are measured shakes up and stand 2~3 hours, filter, remove the carbohydrate content of precipitating, obtain clear liquid, trichlorine is added
Methane 40ml, sufficiently shakes up, and layering is centrifuged in centrifuge, discards chloroform liquid, removes grease-like composition；Then raffinate
Solvent is volatilized, wetting is added water and stirred, water saturated 10~15ml of n-butanol is added, is ultrasonically treated 30 minutes, Aspirate supernatant adds
Triplication ammonium hydroxide, shakes up, and places layering, takes upper liquid to be evaporated, residue adds methanol to make to dissolve, and obtains test solution, spare.
4. the thin layer measuring method that seed ginseng Rong according to claim 1 is good for waist wine, which is characterized in that
The test sample in ginsenoside mixed reference substance solution 5ul and step 2 in the accurate aspiration step 1 of step (3.1) difference
Solution 10ul, puts respectively on same silica gel g thin-layer plate, by chloroform-methanol-water that volume ratio is 13:7:2 in refrigerator
It first places 12 hours, then takes out, after room temperature 2~3 hours, taking lower layer's solution is solvent, is unfolded, and takes out, dries, and is sprayed
With 10% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C, is placed under ultraviolet lamp 365nm wavelength and inspects, for examination
In product chromatography, in ginsenoside mixing reference substance chromatography same position, the spot or fluorescence spot of same color are shown.
5. the thin layer measuring method that seed ginseng Rong according to claim 1 is good for waist wine, which is characterized in that
Step (3.2) draws echinacoside, acteoside and Hyperoside reference substance solution each 2ul and test solution 10ul
It is put respectively on same polyam ide TLC plate, using volume ratio be 2:1:7 or the methanol-acetic acid-water of 4:1:5 is solvent, expansion,
It takes out, dries, set and inspected under 365nm ultraviolet light, in sample chromatogram, at the position corresponding to the chromatogram of the reference substance, show identical
The fluorescence spot of color.
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