CN108169403A - A kind of quality determining method of eight-treasure soup formula particle - Google Patents
A kind of quality determining method of eight-treasure soup formula particle Download PDFInfo
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- CN108169403A CN108169403A CN201711351716.6A CN201711351716A CN108169403A CN 108169403 A CN108169403 A CN 108169403A CN 201711351716 A CN201711351716 A CN 201711351716A CN 108169403 A CN108169403 A CN 108169403A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of quality determining methods of eight-treasure soup formula particle, including ginseng, Radix Paeoniae Alba, honey-fried licorice root, the thin-layer chromatography Qualitive test and Paeoniflorin of Radix Angelicae Sinensis and Rhizoma Chuanxiong, ferulic acid and glycyrrhizic acid content detection, the present invention establishes ginseng in eight-treasure soup formula particle, Radix Paeoniae Alba, honey-fried licorice root, Radix Angelicae Sinensis, the thin-layer chromatography Qualitive test of Rhizoma Chuanxiong, and simultaneously to Paeoniflorin in eight-treasure soup formula particle, ferulic acid and glycyrrhizic acid have carried out assay, the detection method has stronger specificity, durability, its accuracy, reproducibility, stability, it can effectively ensure that the stability and controllability of product quality.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of quality determining method of eight-treasure soup formula particle.
Background technology
By ginseng, rhizoma atractylodis macrocephalae, Poria cocos, honey-fried licorice root, prepared rehmannia root, Rhizoma Chuanxiong, Radix Angelicae Sinensis, Radix Paeoniae Alba, totally eight taste Chinese medicines are made bazhen decoction.
The sweet temperature blood-enrich of ginseng, cultivated land in side, Rhizoma Atractylodis Macrocephalae, Poria cocos invigorating spleen to remove dampness, ginseng of helping others QI invigorating tonifying spleen；Radix Angelicae Sinensis, Radix Paeoniae Alba blood-nourishing joint venture,
Help cultivated land help the moon blood；Rhizoma Chuanxiong blood-activating and qi-promoting, honey-fried licorice root and middle QI invigorating, mediation property of medicine, perfect square have effects that invigorating qi and benefiting blood, use
In diseases such as qi-blood deficiency, sallow complexion, loss of appetite, limbs fatigue, menorrhalgia.
Bazhen Pills is made of to add appropriate water pill with refined honey after each bulk pharmaceutical chemicals are crushed in pharmacopeia, and Bazhen granule is ought
Return, Rhizoma Chuanxiong, rhizoma atractylodis macrocephalae first with 95% and 50% ethyl alcohol extract after, the dregs of a decoction with other flavour of a drug decoction be made.And the eight of the present invention
The preparation method of precious soup formula particle is to make extraction solvent with water, extract is concentrated plus appropriate amount of auxiliary materials drying after make pellet and
Into.Due to the difference of preparation method, material base different from, and Bazhen granule have Radix Codonopsis with composition in Bazhen Pills,
And be ginseng in eight-treasure soup formula particle, the saponin component in ginseng is with Codonopsis pilosula polysaccharide constituents to the shadow of thin layer discrimination condition
Difference is rung, therefore, thin layer discriminating is not particularly suited for eight-treasure soup formula particle specified in pharmacopeia, and differentiates not comprehensive.
In addition, the assay item of Bazhen Pills and Bazhen granule only determines the content of Paeoniflorin, index components in pharmacopeia
It is single, it is impossible to which that more comprehensively the quality of reflection prescription entirety, quality standard controllability are too low, it is difficult to monitor the inherent matter of product
Amount, therefore, it is necessary to a kind of quality determining method for eight-treasure soup formula particle be provided, product quality can be effectively ensured
Stability and controllability.
Invention content
The purpose of the present invention is to provide a kind of quality determining method of eight-treasure soup formula particle, this method specificity is good,
Durability is strong, and stability is good, can effectively ensure that the stability and controllability of product quality.
The present invention is to be achieved through the following technical solutions：
A kind of quality determining method of eight-treasure soup formula particle, which is characterized in that including thin-layer chromatography Qualitive test and contain
Amount detection：
(1) the thin-layer chromatography Qualitive test of ginseng：
This product 6g is taken, it is finely ground, add methanol 50ml, be heated to reflux 1-2 hours, filter, filtrate is evaporated, and residue adds water 5ml to make
Dissolving adds water saturated n-butanol 50ml, is ultrasonically treated 20-40 minutes, and filtration, each enriching ammonium hydroxide 50ml ammonia of filtrate is washed, ammonia
It washes 2-4 times, divides and take n-butanol layer, merge, be evaporated, residue adds methanol 2ml to make dissolving, as test solution；Separately take ginseng pair
According to medicinal material 3g, add water 50ml, boil 20-40 minutes, be concentrated into and closely do, add methanol 50ml, be made in the same way of control medicinal material solution；It takes
Ginsenoside Rb1's reference substance, ginsenoside Re's reference substance, ginsenoside Rg1's reference substance and ginsenoside Rf's reference substance, add methanol
Every 1ml respectively mixed solutions containing 0.5mg are made, as reference substance solution；According to thin-layered chromatography《Chinese Pharmacopoeia》Version one in 2010
VI B of portion's annex test, draw above-mentioned 10 μ l of test solution, 10 μ l of control medicinal material solution and 2 μ l of reference substance solution put respectively in
On same silica gel g thin-layer plate, with volume ratio chloroform-acetate-methanol-water=15：40：22：10 in 10 DEG C with decentralization
The lower floor's solution put is solvent, is unfolded, and takes out, dries, spray with 10% ethanol solution of sulfuric acid, being heated to spot at 105 DEG C shows
Color is clear, puts and is inspected under 365nm ultraviolet lamps；In test sample chromatography, in position corresponding with control medicinal material and reference substance chromatography
On, respectively show same color fluorescence spot；
(2) the thin-layer chromatography Qualitive test of Radix Paeoniae Alba：
This product 2g is taken, it is finely ground, add methanol 50ml, be ultrasonically treated 20-40 minute, filter, filtrate is evaporated, and residue adds water 50ml
Making dissolving, filter, filtrate is extracted with ether 50ml, discards ether solution, and aqueous is extracted with n-butanol 50ml, is divided and is taken n-butanol layer,
It is evaporated, residue adds methanol 1ml to make dissolving, as test solution；Paeoniflorin reference substance separately is taken, adds methanol 1ml containing the molten of 1mg
Liquid, as reference substance solution；According to thin-layered chromatography《Chinese Pharmacopoeia》One VI B of the annex experiment of version in 2010, draws above-mentioned for examination
2 μ l of product solution, 5 μ l of reference substance solution are put respectively on same silica gel g thin-layer plate, with volume ratio acetate-methanol-water=
Be solvent at 100: 17: 13, is unfolded, and takes out, dries, spray with 5% concentrated sulfuric acid vanillic aldehyde solution, be heated to spot at 105 DEG C and show
Color is clear, is inspected under fluorescent lamp；In test sample chromatography, on position corresponding with reference substance chromatography, the spot of same color is shown
Point；
(3) the thin-layer chromatography Qualitive test of honey-fried licorice root：
This product 3g is taken, it is finely ground, add 7% 45% ethanol solution 50ml of sulfuric acid, be heated to reflux 1-2 hours, filter, let cool,
Filtrate adds the petroleum ether of 60~90 DEG C of boiling range to shake extraction 1-3 times, each 20ml, collects petroleum ether liquid, is evaporated, residue adds methanol
1ml makes dissolving, as test solution；Another extracting liquorice hypo acid reference substance, adds methanol that solution of the 1ml containing 1mg is made, as control
Product solution；According to thin-layered chromatography《Chinese Pharmacopoeia》Above-mentioned 8 μ l of test solution, right is drawn in one VI B of the annex experiment of version in 2010
According to 3 μ l of product solution, put respectively on same silica GF254 lamellae, with 30~60 DEG C of petroleum ether-toluene-second of volume ratio boiling range
Acetoacetic ester-glacial acetic acid=10: be solvent at 20: 7: 0.5 is unfolded, and takes out, dries, put and inspected under 254nm ultraviolet lamps；For examination
In product chromatography, on position corresponding with reference substance chromatography, the spot of same color is shown；
(4) the thin-layer chromatography Qualitive test of Rhizoma Chuanxiong, Radix Angelicae Sinensis
This product 6g is taken, it is finely ground, add 1% sodium bicarbonate solution 50ml, be ultrasonically treated 20-40 minute, centrifuge, supernatant is taken to use
Dilute hydrochloric acid adjusts pH value to 2~3, with ether shaking extraction 1-3 times, each 20ml, merges ether solution, volatilizes, residue adds methanol
1ml makes dissolving, as test solution；Ferulic acid reference substance separately is taken, adds methanol that solution of the 1ml containing 1mg is made, as reference substance
Solution；According to thin-layered chromatography《Chinese Pharmacopoeia》Above-mentioned 10 μ l of test solution, right is drawn in one VI B of the annex experiment of version in 2010
According to 10 μ l of product solution, put respectively on same silica gel g thin-layer plate, with hexamethylene-dichloromethane-ethyl acetate-formic acid (2.5:1:
1:0.1) it is solvent, is unfolded, takes out, dry, put and inspected under 365nm ultraviolet lamps；1% ferric trichloride and 1% iron cyanogen are sprayed again
Change the mixed liquor that potassium is 1: 1 in mass ratio, observed under daylight；In test sample chromatography, on position corresponding with reference substance chromatography,
The spot of aobvious same color；
(5) assay：
It is measured using high performance liquid chromatography, specific method and step are as follows：
Chromatographic condition is with system suitability using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase
A, using 0.05% phosphoric acid solution as Mobile phase B, gradient elution shown according to the form below；Detection wavelength is 240nm, and theoretical cam curve presses Chinese herbaceous peony
Medicine glycosides peak, which calculates, should be not less than 2000；
The preparation of reference substance solution takes Paeoniflorin, ferulic acid and ammonium glycyrrhetate reference substance appropriate, accurately weighed, adds methanol point
Be not made solution of every 1ml containing 100 μ g of Paeoniflorin, ferulic acid and ammonium glycyrrhetate, 40 μ g, 50 μ g to get；
The preparation of test solution takes this product 1.0g, finely ground, accurately weighed, puts in conical flask with cover, and precision adds in methanol
50ml, close plug, weighed weight are ultrasonically treated 15-20 minutes, let cool, then weighed weight, the weight of less loss is supplied with methanol, is shaken
It is even, filtration, take subsequent filtrate to get；
Measuring method is accurate respectively to draw reference substance solution and each 10 μ l of test solution, injects liquid chromatograph, measures, i.e.,
.
Eight-treasure soup formula particle of the present invention by ginseng, rhizoma atractylodis macrocephalae, Poria cocos, honey-fried licorice root, prepared rehmannia root, Rhizoma Chuanxiong, Radix Angelicae Sinensis,
Radix Paeoniae Alba is 1-5 in mass ratio:1-5:1-5:1-5:1-5:1-5:1-5:1-5 is made, and preparation method includes the following steps：
Ginseng, rhizoma atractylodis macrocephalae, Poria cocos, honey-fried licorice root, prepared rehmannia root, Rhizoma Chuanxiong, Radix Angelicae Sinensis, Radix Paeoniae Alba totally eight taste medicine materical crude slice are taken, decoction is secondary, often
It is secondary to add 8-12 times to measure water, it decocts 1-2 hours, collecting decoction, filtration, filtrate is concentrated into relative density 1.05~1.08, obtains clear cream,
By clear cream plus maltodextrin, dried cream powder is collected in spray drying, by dried cream powder plus maltodextrin mixing, pelletize to get.
Preferably, the preparation method of the eight-treasure soup formula particle, includes the following steps：
Ginseng, rhizoma atractylodis macrocephalae, Poria cocos, honey-fried licorice root, prepared rehmannia root, Rhizoma Chuanxiong, Radix Angelicae Sinensis, Radix Paeoniae Alba totally eight taste medicine materical crude slice are taken, decoction is secondary, often
Secondary plus 10 times of amount water, decoct 1 hour, collecting decoction, and filtration, filtrate is concentrated into relative density 1.05~1.08, obtains clear cream, will be clear
Cream adds maltodextrin, and dried cream powder is collected in spray drying, by dried cream powder plus maltodextrin mixing, pelletize to get.
Ginseng has effects that blood-enrich, and ginsenoside Rb1, ginsenoside Re, ginsenoside Rg1 and ginsenoside Rf are
Main active in ginseng, during ginseng thin layer of the present invention differentiates, the preparation of test solution is anti-by saturation n-butanol filtrate
Multiple ammonia is washed, and can be removed flavones ingredient, be avoided impacting saponin(e clear spot degree.
The content of Paeoniflorin, ferulic acid and glycyrrhizic acid is higher in eight-treasure soup formula particle, and is main active constituent.
《Chinese medicine pharmacopeia》The content of Paeoniflorin is only determined in version Bazhens Pills in 2015 and Bazhen granule, index components are single, it is impossible to compared with
The quality of comprehensive reflection prescription entirety, therefore, the present invention selects three kinds of Paeoniflorin, ferulic acid and glycyrrhizic acid ingredients as index
Ingredient by the optimization of chromatographic condition, while determines the content of three kinds of index components, improves the accuracy and again of measurement result
Existing property ensures the stability and quality controllability of eight-treasure soup formula granular mass.
Compared with prior art, the present invention it has the advantages that：
It is qualitative that the present invention establishes ginseng in eight-treasure soup formula particle, Radix Paeoniae Alba, honey-fried licorice root, Radix Angelicae Sinensis, the thin-layer chromatography of rhizome of chuanxiong
Differentiate, and assay has been carried out to Paeoniflorin, ferulic acid and glycyrrhizic acid in eight-treasure soup formula particle simultaneously, detection method tool
Have stronger specificity, a durability, accuracy, reproducibility, stability, can effectively ensure that product quality stability and can
Control property.
Description of the drawings
Fig. 1 is that the thin layer of ginseng differentiates chromatogram；Wherein 1-3 is eight-treasure soup formula particle, and 4 be ginseng control medicinal material, and 5 are
Ginseng reference substance, 6 negative sample for the eight-treasure soup formula particle of shortage of staff's ginseng；
Fig. 2 is that the thin layer of Radix Paeoniae Alba differentiates chromatogram；Wherein 1-3 is eight-treasure soup formula particle, and 4 be Paeoniflorin, and 5 is lack Radix Paeoniae Alba
Eight-treasure soup formula particle negative sample；
Fig. 3 is that the thin layer of honey-fried licorice root differentiates chromatogram；Wherein 1-3 is eight-treasure soup formula particle, and 4 be enoxolone, and 5 be scarce
The negative sample of the eight-treasure soup formula particle of honey-fried licorice root；
Fig. 4 is the thin layer discriminating chromatogram of Rhizoma Chuanxiong, Radix Angelicae Sinensis；Wherein 1-3 is eight-treasure soup formula particle, and 4 be Ligusticum chuanxiong Hort, 5
For Radix Angelicae Sinensis medicinal material, 6 be lack Rhizoma Chuanxiong, Radix Angelicae Sinensis eight-treasure soup formula particle negative sample；
Fig. 5 is mixing reference substance HPLC collection of illustrative plates；
Fig. 6 lacks Radix Paeoniae Alba negative sample HPLC collection of illustrative plates for eight-treasure soup formula particle；
Fig. 7 lacks Rhizoma Chuanxiong, Radix Angelicae Sinensis negative sample HPLC collection of illustrative plates for eight-treasure soup formula particle；
Fig. 8 lacks Radix Glycyrrhizae negative sample HPLC collection of illustrative plates for eight-treasure soup formula particle；
Fig. 9 is eight-treasure soup formula particulate samples HPLC collection of illustrative plates.
Specific embodiment
It is further illustrated the present invention below by specific embodiment, following embodiment is the specific embodiment party of the present invention
Formula, but embodiments of the present invention are not limited by following embodiments.
Embodiment 1：
A kind of quality determining method of eight-treasure soup formula particle, which is characterized in that including thin-layer chromatography Qualitive test and contain
Amount detection：
(1) the thin-layer chromatography Qualitive test of ginseng：
This product 6g is taken, it is finely ground, add methanol 50ml, be heated to reflux 1 hour, filter, filtrate is evaporated, and it is molten that residue adds water 5ml to make
Solution adds water saturated n-butanol 50ml, is ultrasonically treated 30 minutes, and filtration, each enriching ammonium hydroxide 50ml ammonia of filtrate is washed, and ammonia is washed 4 times,
Divide and take n-butanol layer, merge, be evaporated, residue adds methanol 2ml to make dissolving, as test solution；Ginseng control medicinal material 3g separately is taken,
Add water 50ml, boil 30 minutes, be concentrated into and closely do, add methanol 50ml, be made in the same way of control medicinal material solution；Take ginsenoside Rb1
Reference substance, ginsenoside Re's reference substance, ginsenoside Rg1's reference substance and ginsenoside Rf's reference substance add methanol that every 1ml is made each
Mixed solution containing 0.5mg, as reference substance solution；According to thin-layered chromatography《Chinese Pharmacopoeia》One VI B of the annex examination of version in 2010
Test, draw above-mentioned 10 μ l of test solution, 10 μ l of control medicinal material solution and 2 μ l of reference substance solution put respectively it is thin in same silica G
On laminate, with volume ratio chloroform-acetate-methanol-water=15：40：22：10 in 10 DEG C of lower floor's solution arranged below
For solvent, it is unfolded, takes out, dry, spray with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C, puts
It is inspected under 365nm ultraviolet lamps；In test sample chromatography, on position corresponding with control medicinal material and reference substance chromatography, show respectively
The fluorescence spot of same color.
Methodology validation：
1.1 specificities are investigated
Draw respectively eight-treasure soup formula particle test solution, ginseng control medicinal material solution, ginseng reference substance solution and
Negative sample solution point sample is unfolded on same silica gel g thin-layer plate, takes out, dries, put and inspected under ultraviolet lamp (365nm), real
Test the result is shown in Figure 1.As seen from Figure 1, in the test solution chromatography of eight-treasure soup formula particle, with ginseng control medicinal material, ginseng
The spot of same color is shown on the corresponding position of reference substance chromatography, and negative sample is noiseless, illustrates mirror of this method to ginseng
It Ju You not specificity.
The investigation of 1.2 different temperatures
The results show that under the conditions of room temperature (22.5 DEG C) and low temperature (5.7 DEG C), the test solution color of eight-treasure soup formula particle
In spectrum, the spot of same color is shown on position corresponding with ginseng control medicinal material, ginseng reference substance chromatography.Experimental result table
Bright, temperature differentiates without influence the thin layer of eight-treasure soup formula particle ginseng, illustrates that the thin-layer identification method durability is good.
The investigation of 1.3 different humidities
The results show that under the conditions of high humidity (75%) and low humidity (20%), the test solution chromatography of eight-treasure soup formula particle
In, the spot of same color is shown on position corresponding with ginseng control medicinal material, ginseng reference substance chromatography.The experimental results showed that
Moisturt register differentiates without influence eight-treasure soup formula particle ginseng thin layer, illustrates that the thin-layer identification method durability is good.
The investigation of 1.4 different point sample amounts
Investigate different 5 μ l of point sample amount of eight-treasure soup formula particle test solution and ginseng control medicinal material, 10 μ l, 15 μ l,
Whether 20 μ l and different 1 μ l of point sample amount, 2 μ l, 3 μ l, the 4 μ l of ginseng reference substance solution make a significant impact thin-layer chromatography,
The results show that in the test solution chromatography of eight-treasure soup formula particle, corresponding to ginseng control medicinal material, ginseng reference substance chromatography
Position on show same color spot.When the point sample amount of eight-treasure soup formula particle test solution is 10 μ l, ginseng comparison medicine
When the point sample amount of material solution is 10 μ l, ginseng reference substance solution point sample amount is when being 2 μ l, clear spot and without serious hangover.Therefore,
The point sample amount of eight-treasure soup formula particle test solution is 10 μ l, and the point sample amount of ginseng control medicinal material solution is 10 μ l, ginseng pair
Point sample amount according to product solution is 2 μ l.
The investigation of 1.5 different manufacturers silica gel g thin-layer plates
Eight-treasure soup formula particle test solution, ginseng control medicinal material solution and ginseng reference substance solution point are drawn respectively
In different manufacturers silica gel g thin-layer plate (subsidiary factory of Haiyang Chemical Plant, Qingdao, Qingdao Pu Ke separation materials Co., Ltd, laboratory self-control)
On,
The results show that subsidiary factory of Haiyang Chemical Plant, Qingdao, Qingdao Pu Ke separation materials Co., Ltd, the homemade silica gel in laboratory
G lamellaes do not make significant difference to the thin layer discriminating of eight-treasure soup formula particle ginseng, illustrate the durability of the thin-layer identification method very
It is good.
(2) the thin-layer chromatography Qualitive test of Radix Paeoniae Alba：
This product 2g is taken, it is finely ground, add methanol 50ml, be ultrasonically treated 30 minutes, filtration, filtrate is evaporated, and residue adds water 50ml to make
Dissolving, filtration, filtrate are extracted with ether 50ml, discard ether solution, and aqueous is extracted with n-butanol 50ml, is divided and is taken n-butanol layer, is steamed
Dry, residue adds methanol 1ml to make dissolving, as test solution；Paeoniflorin reference substance separately is taken, adds methanol 1ml containing the molten of 1mg
Liquid, as reference substance solution；According to thin-layered chromatography《Chinese Pharmacopoeia》One VI B of the annex experiment of version in 2010, draws above-mentioned for examination
2 μ l of product solution, 5 μ l of reference substance solution are put respectively on same silica gel g thin-layer plate, with volume ratio acetate-methanol-water=
Be solvent at 100: 17: 13, is unfolded, and takes out, dries, spray with 5% concentrated sulfuric acid vanillic aldehyde solution, be heated to spot at 105 DEG C and show
Color is clear, is inspected under fluorescent lamp；In test sample chromatography, on position corresponding with reference substance chromatography, the spot of same color is shown
Point.
Methodology validation：
2.1 specificities are investigated
Eight-treasure soup formula particle test solution, Paeoniflorin reference substance solution and negative sample solution point sample are drawn respectively
In on same silica gel g thin-layer plate, it is unfolded, takes out, dry, spray with 5% concentrated sulfuric acid vanillic aldehyde solution, spot is heated at 105 DEG C
Colour developing is clear, is inspected under fluorescent lamp, experimental result is shown in Fig. 2.From Figure 2 it can be seen that the test solution color of eight-treasure soup formula particle
In spectrum, the spot of same color is shown, and negative sample is noiseless in the corresponding position with Paeoniflorin chromatography, illustrate this method dialogue
The discriminating of Chinese herbaceous peony has specificity.
The investigation of 2.2 different temperatures
The results show that under the conditions of room temperature (22.5 DEG C) and low temperature (5.7 DEG C), the test solution color of eight-treasure soup formula particle
In spectrum, the spot of same color is shown on position corresponding with Paeoniflorin reference substance chromatography.The experimental results showed that temperature is to eight delicacies
The thin layer of soup formula particle Radix Paeoniae Alba differentiates without influence, illustrates that the thin-layer identification method durability is good.
The investigation of 2.3 different humidities
The results show that under the conditions of high humidity (75%) and low humidity (20%), the test solution chromatography of eight-treasure soup formula particle
In, the spot of aobvious same color on position corresponding with Paeoniflorin reference substance chromatography.The experimental results showed that moisturt register is to eight delicacies
Soup formula particle Radix Paeoniae Alba thin layer differentiates without influence, illustrates that the thin-layer identification method durability is good.
The investigation of 2.4 different point sample amounts
Investigate 2,3,5, the 8 μ l of different point sample amounts of eight-treasure soup formula particle test solution and Paeoniflorin reference substance solution
3,5,8,10 μ l of different point sample amounts whether thin-layer chromatography is made a significant impact, the results show that the confession of eight-treasure soup formula particle
In test sample solution chromatography, the spot of same color is shown on position corresponding with Paeoniflorin reference substance chromatography.Work as eight-treasure soup formula
When the point sample amount of particle test solution is 2 μ l, the point sample amount of Paeoniflorin reference substance solution is 5 μ l, clear spot and do not trail.
Therefore, the point sample amount of eight-treasure soup formula test solution is 2 μ l, and the point sample amount of Paeoniflorin reference substance solution is 5 μ l.
The investigation of 2.5 different manufacturers silica gel g thin-layer plates
It is thin in different manufacturers silica G that eight-treasure soup formula particle test solution, Paeoniflorin reference substance solution point are drawn respectively
Laminate
In (subsidiary factory of Haiyang Chemical Plant, Qingdao, Qingdao Pu Ke separation materials Co., Ltd, laboratory self-control), the results show that
Subsidiary factory of Haiyang Chemical Plant, Qingdao, Qingdao Pu Ke separation materials Co., Ltd, the homemade silica gel g thin-layer plate in laboratory, to bazhen decoction
The thin layer discriminating of granule Radix Paeoniae Alba does not make significant difference, and illustrates that the durability of the thin-layer identification method is fine.
The investigation of 2.6 different solvents
The unfolding condition that Radix Paeoniae Alba thin layer differentiates in pharmacopeia patent medicine is mostly chloroform-acetate-methanol-formic acid (40：5：
10：0.2), under identical sample-pretreating method, eight-treasure soup formula particle of the invention by chloroform-ethyl acetate-
Methyl alcohol-formic acid (40：5：10：0.2) after solvent expansion, the separating degree of Paeoniflorin and adjacent spots is poor, with ethyl acetate-first
Alcohol-water=100: after be unfolded at 17: 13 for solvent, the separating degree of Paeoniflorin and adjacent spots is preferable, favorable reproducibility, and it is cloudy
Property it is noiseless, therefore the present invention select with volume ratio acetate-methanol-water=100: is solvent at 17: 13.
(3) the thin-layer chromatography Qualitive test of honey-fried licorice root：
This product 3g is taken, it is finely ground, add 7% 45% ethanol solution 50ml of sulfuric acid, be heated to reflux 1 hour, filter, let cool, filter
Liquid adds the petroleum ether of 60~90 DEG C of boiling range to shake extraction 2 times, each 20ml, collects petroleum ether liquid, is evaporated, residue adds methanol 1ml
Make dissolving, as test solution；Another extracting liquorice hypo acid reference substance, adds methanol that solution of the 1ml containing 1mg is made, as reference substance
Solution；According to thin-layered chromatography《Chinese Pharmacopoeia》One VI B of the annex experiment of version in 2010, draws above-mentioned 8 μ l of test solution, control
3 μ l of product solution are put respectively on same silica GF254 lamellae, with 30~60 DEG C of petroleum ether-toluene-acetic acid of volume ratio boiling range
Ethyl ester-glacial acetic acid=10: be solvent at 20: 7: 0.5 is unfolded, and takes out, dries, put and inspected under 254nm ultraviolet lamps；Test sample
In chromatography, on position corresponding with reference substance chromatography, the spot of same color is shown；
Methodology validation：
3.1 specificities are investigated
Eight-treasure soup formula particle test solution, enoxolone reference substance solution and negative sample solution point are drawn respectively
Sample is in same silica G F254On lamellae, it is unfolded, takes out, dry, put and inspected under ultraviolet lamp (254nm), experimental result is shown in figures
3.As seen from Figure 3, in the test solution chromatography of eight-treasure soup formula particle, show identical in the corresponding position with enoxolone chromatography
The spot of color, and negative sample is noiseless, illustrates that this method has specificity to the discriminating of honey-fried licorice root.
The investigation of 3.2 different temperatures
The results show that under the conditions of room temperature (22.5 DEG C) and low temperature (5.7 DEG C), the test solution color of eight-treasure soup formula particle
In spectrum, the spot of same color is shown on position corresponding with enoxolone reference substance chromatography.The experimental results showed that temperature is to eight
The thin layer of precious soup formula particle honey-fried licorice root differentiates without influence, illustrates that the thin-layer identification method durability is good.
The investigation of 3.3 different humidities
The results show that under the conditions of high humidity (75%) and low humidity (20%), the test solution chromatography of eight-treasure soup formula particle
In, the spot of aobvious same color on position corresponding with enoxolone reference substance chromatography.The experimental results showed that moisturt register is to eight
The thin layer of precious soup formula particle honey-fried licorice root differentiates without influence, illustrates that the thin-layer identification method durability is good.
The investigation of 3.4 different point sample amounts
Investigate the difference of 3,5,8,10 μ l of eight-treasure soup formula particle test solution and enoxolone reference substance solution
Whether 1,2,3,4 μ l of sample amount make a significant impact thin-layer chromatography, the results show that the test solution color of eight-treasure soup formula particle
In spectrum, the spot of same color is shown on position corresponding with Paeoniflorin reference substance chromatography.When eight-treasure soup formula particle test sample
When the point sample amount of solution is 8 μ l, the point sample amount of enoxolone reference substance solution is 3 μ l, clear spot and without serious hangover.Cause
This, the point sample amount of eight-treasure soup formula test solution is 8 μ l, and the point sample amount of enoxolone reference substance solution is 3 μ l.
The investigation of 3.5 different manufacturers silica gel g thin-layer plates
Eight-treasure soup formula particle test solution, enoxolone reference substance solution point are drawn respectively in different manufacturers silica G
On lamellae (subsidiary factory of Haiyang Chemical Plant, Qingdao, Qingdao Pu Ke separation materials Co., Ltd, laboratory self-control), the results show that green
Island subsidiary factory of marine chemical industry factory, Qingdao Pu Ke separation materials Co., Ltd, the homemade silica gel g thin-layer plate in laboratory, match bazhen decoction
The thin layer discriminating of square particle honey-fried licorice root does not make significant difference, and illustrates that the durability of the thin-layer identification method is fine.
The investigation of 3.6 different solvents
Pharmacopeia Radix Glycyrrhizae and the patent medicine containing Radix Glycyrrhizae, mostly with acetic ether-methanoic acid-glacial acetic acid-water (15：1：1：2) it is expansion
Agent carries out thin layer discriminating to Radix Glycyrrhizae, and sample pre-treatments are mostly extracted repeatedly by n-butanol, complex.The eight delicacies of the present invention
Soup formula particle is under identical sample-pretreating method, with acetic ether-methanoic acid-glacial acetic acid-water (15：1：1：2) it is expansion
After agent is unfolded, negative sample has apparent interference.The present invention by glycyrrhizic acid by hydrolysis after, with 30~60 DEG C of oil of boiling range
Ether-
Toluene-ethyl acetate-glacial acetic acid (10: 20: 7: 0.5) carries out thin layer discriminating, spot for solvent to enoxolone
Clearly, specificity is strong.
(4) the thin-layer chromatography Qualitive test of Rhizoma Chuanxiong, Radix Angelicae Sinensis
This product 6g is taken, it is finely ground, add 1% sodium bicarbonate solution 50ml, be ultrasonically treated 30 minutes, centrifugation takes supernatant with dilute
Salt acid for adjusting pH value, with ether shaking extraction 3 times, each 20ml, merges ether solution, volatilizes, residue adds methanol 1ml to make to 2~3
Dissolving, as test solution；Ferulic acid reference substance separately is taken, adds methanol that solution of the 1ml containing 1mg is made, as reference substance solution；
According to thin-layered chromatography《Chinese Pharmacopoeia》One VI B of the annex experiment of version in 2010, it is molten to draw above-mentioned 10 μ l of test solution, reference substance
10 μ l of liquid are put respectively on same silica gel g thin-layer plate, with hexamethylene-dichloromethane-ethyl acetate-formic acid (2.5:1:1:0.1)
For solvent, it is unfolded, takes out, dry, put and inspected under 365nm ultraviolet lamps；1% ferric trichloride is sprayed again and 1% potassium ferricyanide is pressed
The mixed liquor that mass ratio is 1: 1, observed under daylight；In test sample chromatography, on position corresponding with reference substance chromatography, show identical
The spot of color.
The investigation of 4.1 different solvents
Ferulic acid differentiates often with benzene-chloroform-glacial acetic acid (6 in pharmacopeia：1：0.5), hexamethylene-dichloromethane-acetic acid
Ethyl ester-formic acid (4：1：1：0.1), toluene-ethyl acetate-formic acid (20：10：1) it is solvent.Since benzene has severe toxicity, it keeps away
Exempt to use benzene-chloroform-glacial acetic acid (6：1：0.5) as solvent.The eight-treasure soup formula particle of the present invention is in identical sample
Under conditions of product pre-treatment, using toluene-ethyl acetate-formic acid (20：10：1) when, feminine gender significantly interferes with.With hexamethylene-two
Chloromethanes-acetic ether-methanoic acid (4：1：1：0.1) separating effect of solvent is preferable, but has interference at ferulic acid spot, passes through
Adjustment ratio, the results showed that with hexamethylene-dichloromethane-ethyl acetate-formic acid (2.5：1：1：0.1) when for solvent, separation
Preferably, and negative noiseless, therefore, present invention selection is with volume ratio hexamethylene-dichloromethane-ethyl acetate-formic acid=2.5：
1：1：0.1 is solvent.
(5) assay：
It is measured using high performance liquid chromatography, specific method and step are as follows：
Chromatographic condition is with system suitability using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase
A, using 0.05% phosphoric acid solution as Mobile phase B, gradient elution shown according to the form below；Detection wavelength is 240nm, and theoretical cam curve presses Chinese herbaceous peony
Medicine glycosides peak, which calculates, should be not less than 2000；
The preparation of reference substance solution takes Paeoniflorin, ferulic acid and ammonium glycyrrhetate reference substance appropriate, accurately weighed, adds methanol point
Be not made solution of every 1ml containing 100 μ g of Paeoniflorin, ferulic acid and glycyrrhizic acid, 40 μ g, 50 μ g to get；
The preparation of test solution takes this product 1.0g, finely ground, accurately weighed, puts in conical flask with cover, and precision adds in methanol
50ml, close plug, weighed weight are ultrasonically treated 15 minutes, let cool, then weighed weight, the weight of less loss is supplied with methanol, is shaken up,
Filtration, take subsequent filtrate to get；
Measuring method is accurate respectively to draw reference substance solution and each 10 μ l of test solution, injects liquid chromatograph, measures, i.e.,
.
The every 1 gram of particle of this product containing Radix Paeoniae Alba in terms of Paeoniflorin, containing Rhizoma Chuanxiong and Radix Angelicae Sinensis in terms of ferulic acid, containing Radix Glycyrrhizae with glycyrrhizic acid
Meter must not distinguish less than 4mg, 0.2mg, 1.5mg.
Methodology validation
4 specificities are investigated
According to the content assaying method that experiment is established, Paeoniflorin, ferulic acid and ammonium glycyrrhetate in sample can be preferable
Separation, and three kinds of negative samples are noiseless, as a result see such as 5-9.
5 sample pretreatment process are investigated
5.1 extracting mode
Take this product (lot number：1410812) it is in right amount, finely ground, about 1.0g is taken, it is accurately weighed, it puts in conical flask with cover, precision adds
Enter 50ml methanol, close plug, weighed weight is ultrasonically treated (power 200W, frequency 25kHz) 1 hour (being heated to reflux 1 hour), puts
It is cold, then weighed weight, the weight of less loss is supplied with methanol, is shaken up, is filtered, take subsequent filtrate to get.Experimental result is shown in Table 1.
The different extracting mode eight-treasure soup formula particle Paeoniflorins of table 1, ferulic acid, glycyrrhizic acid content measurement result
Brief summary：The experimental results showed that eight-treasure soup formula particle uses refluxing extraction 1 hour, is ultrasonically treated 1 hour and extracts
When, the content of the Paeoniflorin after supersound process is higher, and the content of glycyrrhizic acid is relatively low, two kinds of extracting modes to the content of ferulic acid without
It significantly affects, considers using supersound process.
5.2 extraction time
Take this product (lot number：1410812) it is in right amount, finely ground, about 1.0g is taken, it is accurately weighed, it puts in conical flask with cover, precision adds
Enter 50ml methanol, close plug, weighed weight is ultrasonically treated (power 200W, frequency 25kHz) 15 minutes (30 minutes, 45 minutes), puts
It is cold, then weighed weight, the weight of less loss is supplied with methanol, is shaken up, is filtered, take subsequent filtrate to get.Experimental result is shown in Table 2.
The different extraction time eight-treasure soup formula particle Paeoniflorins of table 2, ferulic acid, glycyrrhizic acid content measurement result
Brief summary：The experimental results showed that when eight-treasure soup formula particle is using being ultrasonically treated 15 minutes, 30 minutes, 45 minutes, when
Processing time, the content of Paeoniflorin can reduce instead more than 15 minutes, ferulic acid, glycyrrhizic acid content become substantially without apparent
Change.Therefore, the sonication treatment time prepared by test solution is 15 minutes.
5.3 Extraction solvent
Take this product (lot number：1410812) it is in right amount, finely ground, about 1.0g is taken, it is accurately weighed, it puts in conical flask with cover, precision adds
Enter 50ml methanol (75% methanol, 50% methanol), close plug, weighed weight, 15 points of supersound process (power 200W, frequency 25kHz)
Clock is let cool, then weighed weight, and the weight of less loss is supplied with methanol (75% methanol, 50% methanol), is shaken up, and filtration takes continuous filter
Liquid to get.Experimental result is shown in Table 3.
3 different solvents eight-treasure soup formula particle Paeoniflorin of table, ferulic acid, glycyrrhizic acid content measurement result
Brief summary：The experimental results showed that when eight-treasure soup formula particle is using methanol, 75% methanol, the extraction of 50% methanol eddy,
50% methanol is conducive to the dissolution of glycyrrhizic acid, and 100% methanol is conducive to the dissolution of Paeoniflorin and ferulic acid.Consider, it will be for
Extraction solvent prepared by test sample solution is methanol.
In conclusion the preparation method of test solution is：Take this product appropriate, it is finely ground, about 1.0g is taken, it is accurately weighed, it puts
In conical flask with cover, precision adds in methanol 50ml, close plug, and weighed weight is ultrasonically treated 15 minutes, lets cool, then weighed weight, uses
Methanol supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get.
6 system suitabilities
6.1 linear relationships are investigated
Take Paeoniflorin, ferulic acid, ammonium glycyrrhetate mixed reference substance solution respectively into 1,2,4,6,8,10,12,14 μ l, by upper
It states chromatographic condition to be measured, sample introduction, records each reference substance peak area, each reference substance content is returned with integrating peak areas value
Return processing.The experimental results showed that Paeoniflorin sample size is in good line with peak area when in the range of 0.1059~1.4827 μ g
Sexual intercourse, regression equation are Y=975.1X+16.77 (R2=0.999)；Ferulic acid sample size is in 0.0419~0.5868 μ g models
Peak area is in good linear relationship when enclosing interior, and regression equation is：Y=3254X-2.776 (R2=1)；Ammonium glycyrrhetate sample size exists
Peak area is in good linear relationship when in 0.1047~0.7330 μ g ranges, and regression equation is：Y=586.0X+28.45 (R2=
0.999)。
6.2 Precision Experiment
Paeoniflorin, ferulic acid, ammonium glycyrrhetate mixed reference substance solution are taken, repeats sample introduction six times by above-mentioned chromatographic condition, note
Record Paeoniflorin, ferulic acid, ammonium glycyrrhetate peak area.Experimental result is shown in Table 4.
4 eight-treasure soup formula particle Paeoniflorin of table, ferulic acid, glycyrrhizic acid content measure Precision Experiment result
Brief summary：From more than experimental result as it can be seen that the RSD of Paeoniflorin peak area is 0.39%, the RSD of forulic acid peak area is
0.18%, the RSD of ammonium glycyrrhetate peak area is 0.67%.It can be seen that instrument precision is preferable.
6.3 stability experiment
A eight-treasure soup formula particle test solution is made by test solution preparation method, by above-mentioned chromatographic condition point
Not in 0,6,12,15,18,24 hour sample introduction, record Paeoniflorin, ferulic acid, enoxolone ammonium peak area.Experimental result is shown in Table 5.
5 eight-treasure soup formula particle solution stability experiment measurement result of table
Brief summary：RSD points of the peak area of Paeoniflorin in eight-treasure soup formula particle, ferulic acid, glycyrrhizic acid was measured in 24 hours
It Wei 1.58%, 1.99%, 1.11%.The experimental results showed that test solution was stablized in 24 hours.
6.4 repeated experiment
Take this product (lot number：1401418) it is in right amount, finely ground, about 1.0g is taken, it is accurately weighed, totally six parts, put conical flask with cover
In, it is made according to above-mentioned test solution preparation method, by Paeoniflorin, ferulic acid, glycyrrhizic acid in above-mentioned chromatographic condition determination sample
The content of ammonium.Experimental result is shown in Table 6.
6 eight-treasure soup formula particle solution repeated experiment measurement result of table
Brief summary：The experimental results showed that Paeoniflorin, ferulic acid, glycyrrhizic acid content measure knot in six parts of eight-treasure soup formula particles
The RSD of fruit is respectively 1.15%, 1.70%, 1.74%, so this sample size assay method repeatability is preferably.
6.5 sample-adding recovery experiments
Take the eight-treasure soup formula particle (lot number of known content：1410812) it is in right amount, finely ground, take about 0.5g, (content：Chinese herbaceous peony
Glycosides 4.51mg/g, ferulic acid 0.29mg/g, glycyrrhizic acid 2.05mg/g, it is accurately weighed, it totally six parts, puts in conical flask with cover, respectively
Precision adds in Paeoniflorin, ferulic acid, ammonium glycyrrhetate mixed reference substance solution, and (methanol dissolves, and concentration is respectively 150.1314 μ g/
Ml, 9.0736 μ g/ml, 61.4676 μ g/ml) 15ml, according to the preparation method of test solution prepare sample solution to get.It presses
Above-mentioned chromatographic condition is measured, and is calculated.Experimental result is shown in Table 7-9.
Paeoniflorin content measures rate of recovery experimental result in 7 eight-treasure soup formula particle of table
Ferulaic acid content measures rate of recovery experimental result in 8 eight-treasure soup formula particle of table
Glycyrrhizic acid content measures rate of recovery experimental result in 9 eight-treasure soup formula particle of table
Brief summary：From experimental data as it can be seen that this eight-treasure soup formula particle Paeoniflorin, ferulic acid, glycyrrhizic acid assay side
The method rate of recovery is preferable, average recovery rate is respectively 98.72%, 97.99%, 99.79%, RSD 1.19%, 1.73%,
2.99%.It can be seen that the content assaying method that Paeoniflorin, ferulic acid, glycyrrhizic acid measure simultaneously in the eight-treasure soup formula particle
Accurately, reliably.
7 durabilities are investigated
7.1 different chromatographic columns are investigated
Take a eight-treasure soup formula particle test solution, by above-mentioned chromatographic condition, investigate Waters, Agilent,
The common brand chromatographic columns of Kromasil tri- are to Paeoniflorin, ferulic acid, glycyrrhizic acid content measurement result in eight-treasure soup formula particle
Influence, experimental result is shown in Table 10.
Influence of the different chromatographic columns of table 10 to index components content measurement result each in eight-treasure soup formula particle
Brief summary：The experimental results showed that three kinds of brand chromatographic columns measure RSD difference to Paeoniflorin, ferulic acid, glycyrrhizic acid content
It is 1.57%, 1.41%, 1.39%, it can be seen that, different brands chromatographic column is to Paeoniflorin in eight-treasure soup formula, ferulic acid, sweet
Oxalic acid content measurement result does not make significant difference, and the content assaying method durability is preferable.
7.2 different instruments are investigated
A eight-treasure soup formula particle test solution is taken, by above-mentioned chromatographic condition, investigates Agilent and Waters two
A common brand high performance liquid chromatograph is to Paeoniflorin in eight-treasure soup formula particle, ferulic acid, glycyrrhizic acid content measurement result
It influences, experimental result is shown in Table 11.
Influence of the different instruments of table 11 to index components content measurement result each in eight-treasure soup formula particle
Brief summary：The experimental results showed that the RSD that different instruments measure Paeoniflorin, ferulic acid, glycyrrhizic acid content is
0.02%th, 0.69%, 0.43%, it can be seen that, different brands instrument is to Paeoniflorin, ferulic acid, glycyrrhizic acid in eight-treasure soup formula
Assay result does not make significant difference, and the content assaying method durability is preferable.
The investigation of 7.3 different mobile phases
Pharmacopeia Bazhen Pills to Paeoniflorin in Bazhen granule with only carrying out assay, so can be by Chinese herbaceous peony by isocratic elution
Medicine glycosides detaches well.The present invention measures Paeoniflorin, ferulic acid and glycyrrhizic acid simultaneously, to flow phase system and gradient into
Optimization is gone, by comparing methanol-water, acetonitrile-water, methanol-phosphoric acid, acetonitrile-phosphoric acid, acetonitrile-glacial acetic acid flow phase system, most
Select flow phase system of the acetonitrile-phosphoric acid as bazhen decoction distribution side particle content measuring eventually, under the chromatography system Paeoniflorin,
Ferulic acid, the peak type of glycyrrhizic acid are preferable, and chromatographic column has preferable reservation to target chromatographic peak.By comparing various concentration acid
The influence of (0.05% phosphoric acid, 0.1% phosphoric acid, 0.2% phosphoric acid) to separating degree and peak type, finds when acid concentration increase, peak type
And separating degree does not have significant change.Consider influence of the high concentrated acid to chromatographic column consumptive material, finally choose -0.05% phosphoric acid of acetonitrile and make
For mobile phase.
Claims (3)
1. a kind of quality determining method of eight-treasure soup formula particle, which is characterized in that including thin-layer chromatography Qualitive test and content
Detection：
（1）The thin-layer chromatography Qualitive test of ginseng：
This product 6g is taken, it is finely ground, add methanol 50ml, be heated to reflux 1-2 hours, filter, filtrate is evaporated, and residue adds water 5ml to make dissolving,
Add water saturated n-butanol 50ml, be ultrasonically treated 20-40 minutes, filtration, each enriching ammonium hydroxide 50ml ammonia of filtrate is washed, and ammonia washes 2-4
It is secondary, divide and take n-butanol layer, merge, be evaporated, residue adds methanol 2ml to make dissolving, as test solution；Separately take ginseng control medicinal material
3g adds water 50ml, boils 20-40 minutes, is concentrated into and closely does, adds methanol 50ml, be made in the same way of control medicinal material solution；Take ginseng soap
Glycosides Rb1 reference substances, ginsenoside Re's reference substance, ginsenoside Rg1's reference substance and ginsenoside Rf's reference substance, add methanol to be made often
The each mixed solutions containing 0.5mg of 1ml, as reference substance solution；According to thin-layered chromatography《Chinese Pharmacopoeia》One annex of version in 2010
VI B is tested, and is drawn above-mentioned 10 μ l of test solution, 10 μ l of control medicinal material solution and 2 μ l of reference substance solution and is put respectively in same silicon
On glue G lamellaes, with volume ratio chloroform-acetate-methanol-water=15：40：22：10 in 10 DEG C of lower floors arranged below
Solution is solvent, is unfolded, and takes out, dries, spray with 10% ethanol solution of sulfuric acid, and it is clear to be heated to spot development at 105 DEG C, puts
It is inspected under 365nm ultraviolet lamps；In test sample chromatography, on position corresponding with control medicinal material and reference substance chromatography, show respectively
The fluorescence spot of same color；
（2）The thin-layer chromatography Qualitive test of Radix Paeoniae Alba：
This product 2g is taken, it is finely ground, add methanol 50ml, be ultrasonically treated 20-40 minute, filter, filtrate is evaporated, and residue adds water 50ml to make
Dissolving, filtration, filtrate are extracted with ether 50ml, discard ether solution, and aqueous is extracted with n-butanol 50ml, is divided and is taken n-butanol layer, is steamed
Dry, residue adds methanol 1ml to make dissolving, as test solution；Paeoniflorin reference substance separately is taken, adds methanol 1ml containing the molten of 1mg
Liquid, as reference substance solution；According to thin-layered chromatography《Chinese Pharmacopoeia》One VI B of the annex experiment of version in 2010, draws above-mentioned for examination
2 μ l of product solution, 5 μ l of reference substance solution are put respectively on same silica G lamellae, with volume ratio acetate-methanol-water=
Be solvent at 100: 17: 13, is unfolded, and takes out, dries, spray with 5% concentrated sulfuric acid vanillic aldehyde solution, spot development is heated at 105 DEG C
Clearly, it is inspected under fluorescent lamp；In test sample chromatography, on position corresponding with reference substance chromatography, the spot of same color is shown
；
（3）The thin-layer chromatography Qualitive test of honey-fried licorice root：
This product 3g is taken, it is finely ground, add 7% 45% ethanol solution 50ml of sulfuric acid, be heated to reflux 1-2 hours, filter, let cool, filtrate adds
Petroleum ether shaking extraction 1-3 times, each 20ml that 60~90 DEG C of boiling range, collects petroleum ether liquid, is evaporated, residue adds methanol 1ml to make
Dissolving, as test solution；Another extracting liquorice hypo acid reference substance, adds methanol that solution of the 1ml containing 1mg is made, molten as reference substance
Liquid；According to thin-layered chromatography《Chinese Pharmacopoeia》One VI B of the annex experiment of version in 2010, draws above-mentioned 8 μ l of test solution, reference substance
3 μ l of solution are put respectively on same silica GF254 lamellae, with 30~60 DEG C of petroleum ether-toluene-acetic acid second of volume ratio boiling range
Ester-glacial acetic acid=10: be solvent at 20: 7: 0.5 is unfolded, and takes out, dries, put and inspected under 254nm ultraviolet lamps；Test sample chromatography
In, on position corresponding with reference substance chromatography, show the spot of same color；
（4）The thin-layer chromatography Qualitive test of Rhizoma Chuanxiong, Radix Angelicae Sinensis
This product 6g is taken, it is finely ground, add 1% sodium bicarbonate solution 50ml, be ultrasonically treated 20-40 minute, centrifuge, take supernatant with dilute salt
Acid for adjusting pH value, with ether shaking extraction 1-3 times, each 20ml, merges ether solution, volatilizes, residue adds methanol 1ml to make to 2~3
Dissolving, as test solution；Ferulic acid reference substance separately is taken, adds methanol that solution of the 1ml containing 1mg is made, as reference substance solution；
According to thin-layered chromatography《Chinese Pharmacopoeia》One VI B of the annex experiment of version in 2010, it is molten to draw above-mentioned 10 μ l of test solution, reference substance
10 μ l of liquid are put respectively on same silica gel g thin-layer plate, with volume ratio hexamethylene-dichloromethane-ethyl acetate-formic acid=2.5:1:
1:0.1 is solvent, is unfolded, and takes out, dries, put and inspected under 365nm ultraviolet lamps；1% ferric trichloride and 1% potassium ferricyanide are sprayed again
In mass ratio be 1: 1 mixed liquor, observed under daylight；In test sample chromatography, on position corresponding with reference substance chromatography, phase is shown
With the spot of color；
（5）Assay：
It is measured using high performance liquid chromatography, specific method and step are as follows：
Chromatographic condition is with system suitability using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A,
With
0.05% phosphoric acid solution be Mobile phase B, gradient elution shown according to the form below；Detection wavelength is 240nm, and theoretical cam curve presses Chinese herbaceous peony
Medicine glycosides peak, which calculates, should be not less than 2000；
The preparation of reference substance solution takes Paeoniflorin, ferulic acid and ammonium glycyrrhetate reference substance appropriate, accurately weighed, and methanol is added to distinguish
Be made solution of every 1ml containing 100 μ g of Paeoniflorin, ferulic acid and ammonium glycyrrhetate, 40 μ g, 50 μ g to get；
The preparation of test solution takes this product 1.0g, finely ground, accurately weighed, puts in conical flask with cover, and precision adds in methanol
50ml, close plug, weighed weight are ultrasonically treated 15-20 minutes, let cool, then weighed weight, the weight of less loss is supplied with methanol, is shaken
It is even, filtration, take subsequent filtrate to get；
Measuring method is accurate respectively to draw reference substance solution and each 10 μ l of test solution, injects liquid chromatograph, measure to get.
A kind of 2. quality determining method of eight-treasure soup formula particle according to claim 1, which is characterized in that the eight delicacies
Soup formula particle is 1-5 in mass ratio by ginseng, rhizoma atractylodis macrocephalae, Poria cocos, honey-fried licorice root, prepared rehmannia root, Rhizoma Chuanxiong, Radix Angelicae Sinensis, Radix Paeoniae Alba:1-5:1-
5:1-5:1-5:1-5:1-5:1-5 is made, and preparation method includes the following steps：
Ginseng, rhizoma atractylodis macrocephalae, Poria cocos, honey-fried licorice root, prepared rehmannia root, Rhizoma Chuanxiong, Radix Angelicae Sinensis, Radix Paeoniae Alba totally eight taste medicine materical crude slice are taken, decoction is secondary, adds every time
8-12 times is measured water, is decocted 1-2 hours, collecting decoction, and filtration, filtrate is concentrated into relative density 1.05~1.08, obtains clear cream, will be clear
Cream adds maltodextrin, and dried cream powder is collected in spray drying, by dried cream powder plus maltodextrin mixing, pelletize to get.
A kind of 3. quality determining method of eight-treasure soup formula particle according to claim 2, which is characterized in that the eight delicacies
Soup formula particle is 1 in mass ratio by ginseng, rhizoma atractylodis macrocephalae, Poria cocos, honey-fried licorice root, prepared rehmannia root, Rhizoma Chuanxiong, Radix Angelicae Sinensis, Radix Paeoniae Alba:1:1:1:1:
1:1:1 is made, and preparation method includes the following steps：
Ginseng, rhizoma atractylodis macrocephalae, Poria cocos, honey-fried licorice root, prepared rehmannia root, Rhizoma Chuanxiong, Radix Angelicae Sinensis, Radix Paeoniae Alba totally eight taste medicine materical crude slice are taken, decoction is secondary, adds every time
10 times of amount water, decoct 1 hour, collecting decoction, and filtration, filtrate is concentrated into relative density 1.05~1.08, obtains clear cream, by clear cream plus
Dried cream powder is collected in maltodextrin, spray drying, by dried cream powder plus maltodextrin mixing, pelletize to get.
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| CN112697949A (en) * | 2020-12-09 | 2021-04-23 | 浙江金城阜通制药有限公司 | Thin-layer identification method for Baoyuan decoction, similar formula extract and preparation thereof |
| CN112697949B (en) * | 2020-12-09 | 2022-05-27 | 浙江金城阜通制药有限公司 | Thin-layer identification method for Baoyuan decoction, similar formula extract and preparation thereof |
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