CN103808819A - Method for measuring contents of main components in rhodiola rosea medicinal material - Google Patents
Method for measuring contents of main components in rhodiola rosea medicinal material Download PDFInfo
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Abstract
The invention relates to a method for measuring contents of gallic acid, salidroside, tyrosol and p-coumaric acid in a rhodiola rosea medicinal material. The method comprises the steps of preparing a gallic acid reference substance solution, a salidroside reference substance solution, a tyrosol reference substance solution, a p-coumaric acid reference substance solution and a rhodiola rosea medicinal material sample solution respectively; taking PhenomenexLunna C18 (4.6mmx250mm, 5 microns) as a chromatographic column and taking acetonitrile-0.3% glacial acetic acid solution as a flowing phase to measure the reference substance solutions and the sample solution under column temperature of 40 DEG C, a volume flow of 0.7mL/min and wavelengths of 275nm and 308nm, recording a chromatogram and calculating the contents of gallic acid, salidroside, tyrosol and p-coumaric acid in the rhodiola rosea sample solution.
Description
Technical field
The present invention relates to the detection field of Chinese medicinal material, be specifically related to the detection method of Chinese medicinal material rhodiola root.
Background technology
Rhodiola is dry root and the rhizome of Crassulaceae rhodiola Rhodiola crenulata (Hook.f.et Thoms.) H.Ohba plant, is the medicine resource of only this kind of sengreen of clearly stipulating of version Chinese Pharmacopoeia in 2010.China is the distribution center of rhodiola, and storage capacity is large, contains rhodioside, tyrosol, crenulatin, Polyphenols and polysaccharide etc. in plant.Modern pharmacological research shows that rhodiola root has anti-ageing, inoxidizability haemolysis, radioresistance isoreactivity." rhodiola root heat " is in recent years widely used rhodiola root, and its quality is the prerequisite that guarantees drug effect, has effective constituent in more bibliographical information Syrups by HPLC rhodiola root medicinal material:
At present about the report of rhodiola root quality assessment is less, the quality control of rhodiola root is only take the content of rhodioside as index, this may be that it is also different that kind is commonly used in various places because rhodiola plant source on market is complicated, and these evaluation methods exist certain defect.Set up efficient, accurately and the wide detecting and assessing method of applicability, for rhodiola root evaluation of medical materials' quality provides according to being the current problem that solves of needing.
Summary of the invention
The object of this invention is to provide the content assaying method of principal ingredient in rhodiola root medicinal material, described assay method utilizes high performance liquid chromatography (HPLC), measuring gallic acid, rhodioside, tyrosol and p-Coumaric Acid composition in rhodiola root medicinal material under dual wavelength, can detect gallic acid in rhodiola root medicinal material, rhodioside, tyrosol and p-Coumaric Acid content simultaneously, described detection method has efficiently, accurately and the beneficial effect such as applicability is wide, the quality assessment that can be rhodiola root provides foundation.
For achieving the above object, technical scheme of the present invention is:
In rhodiola root medicinal material, a content assaying method for principal ingredient, is characterized in that, comprises the steps:
1) rhodiola root medicinal material need testing solution preparation:
1. get the rhodiola root medicinal material decocting of 6～10 times of amounts and boil 1～3h, filter, obtain filtrate and filter residue for the first time, the decocting that filter residue adds 6～10 times of amounts again boils 1～3h, filters, and obtains filtrate for the second time, merge twice filtrate and be evaporated to 1/20～1/12 volume of filtrate, obtain concentrate I;
2. with macroreticular resin, concentrate I is carried out to purifying, carry out loading with the weight ratio 1: 1～3 of rhodiola root medicinal material and resin; Then first with water elution, discard water liquid, then with 15～30% ethanol elutions, collect 15～30% ethanol eluates;
3. eluent is concentrated into density 1.05～1.10, obtains concentrate II;
4. use 75～85% ethanol alcohol precipitation concentrate II12～36h, obtain supernatant, abandon precipitation;
5. by alcohol precipitation supernatant concentration, dry, pulverizing, obtain gadol extract;
6. take gadol extract, add 10～30% ethanol to dissolve and determine moltenly, every milliliter containing gadol extract 1～5mg, to obtain final product;
2) prepare respectively gallic acid reference substance solution, rhodioside reference substance solution, tyrosol reference substance solution and p-Coumaric Acid reference substance solution;
3) utilize high efficiency liquid phase chromatographic analysis method to detect gallic acid in rhodiola root medicinal material, rhodioside, tyrosol and p-Coumaric Acid content at dual wavelength simultaneously;
Chromatographic condition is: HPLC chromatographic column is Phenomenex Luna C18 post, mobile phase is acetonitrile (A)-0.1～0.5% glacial acetic acid solution (B), elution program is: 0 → 5min, and A rises to 6～12%, B from 0 linearity and drops to 94～88% from 100% linearity; 5 → 30min, A rises to 20～30%, B from 6～12% linearities and drops to 80～70% from 94～88% linearities; 30 → 35min, A rises to 35～45%, B from 20～30% linearities and drops to 65～55% from 80～70% linearities; Column temperature: 20～40 ℃; Volumetric flow rate 0.5～1.5mLmin
-1; Detect wavelength: 275nm, 308nm; Sample size 2～20 μ l;
4) according to the content of gallic acid, rhodioside, tyrosol and p-Coumaric Acid in efficient liquid phase chromatographic analysis result calculating rhodiola root medicinal material.
Further, above-mentioned content assaying method, preferably prepares need testing solution take 20% ethanol as solvent.
Further, the chromatographic condition of above-mentioned content assaying method is:
HPLC chromatographic column be Phenomenex Luna C18 post (4.6mm × 250mm, m), mobile phase is acetonitrile (A)-0.3% glacial acetic acid solution (B) to 5 μ, elution program is: 0～5min, 9%A; 5～30min, 9%～25%A, 30～35min, 25%～40%A; Column temperature: 40 ℃; Volumetric flow rate 0.7mLmin-1; Detect wavelength: 275nm, 308nm; Sample size 10 μ l.
Further, above-mentioned content assaying method, from rhodiola root medicinal material, prepare need testing solution and comprise the following steps:
1) get the rhodiola root medicinal material decocting of 6～8 times of amounts and boil 1～3h, filter, obtain filtrate and filter residue for the first time, the decocting that filter residue adds 6～10 times of amounts again boils 1～3h, filters, and obtains filtrate for the second time, merge twice filtrate and be evaporated to 1/20～1/12 volume of filtrate, obtain concentrate I;
2) with macroreticular resin, concentrate I is carried out to purifying, carry out loading with the weight ratio 1: 1～3 of rhodiola root medicinal material and resin; Then first with water elution, discard water liquid, then with 15～30% ethanol elutions, collect 15～30% ethanol eluates;
3) eluent is concentrated into density 1.05～1.10, obtains concentrate II;
4) with 80% ethanol alcohol precipitation concentrate II24～36h, obtain supernatant, abandon precipitation;
5) by alcohol precipitation supernatant concentration, dry, pulverizing, obtain gadol extract;
6) take gadol extract, add 10～30% ethanol to dissolve and determine moltenly, every milliliter containing gadol extract 1～5mg, to obtain final product.
Further, above-mentionedly from rhodiola root medicinal material, prepare comprising the following steps of need testing solution:
1) get the rhodiola root medicinal material decocting of 8 times of amounts and boil 2h, filter, obtain filtrate and filter residue for the first time, the decocting that filter residue adds 8 times of amounts again boils 2h, filters, and obtains filtrate for the second time, merges twice filtrate and be evaporated to 1/16 of filtrate to amass, and obtains concentrate I;
2) with macroreticular resin, concentrate I is carried out to purifying, carry out loading at 1: 2 with the weight ratio of rhodiola root medicinal material and resin; Then first with water elution, discard water liquid, then with 20% ethanol elution, collect 20% ethanol eluate;
3) eluent is concentrated into density 1.05～1.10, obtains concentrate II;
4) with 80% ethanol alcohol precipitation concentrate II24～32h, obtain supernatant, abandon precipitation;
5) by alcohol precipitation supernatant concentration, dry, pulverizing, obtain gadol extract;
6) take gadol extract, add 20% ethanol to dissolve and determine moltenly, every milliliter containing gadol extract 2mg, to obtain final product.
Further, rhodiola root medicinal material is rhodiola or rhodiola kirilowii Regel.
Known rhodiola root contains rhodioside, tyrosol, pyrosulfuric acid, gallic acid, cupreol, flavonoids and Coumarins composition.After rhodiola root medicinal material is extracted purifying by the present invention, wherein various compositions, particularly coumaric acid are clear and legible in chromatogram, and peak area is larger, coumaric acid content can be incorporated into quality control.Adopt content assaying method of the present invention to measure representative effective constituent in rhodiola root medicinal material, make that each composition in rhodiola root medicinal material is more clear and definite, quality control is stricter, for the quality control of rhodiola root medicinal material provides data.
Content assaying method of the present invention, utilize high performance liquid chromatography (HPLC), respectively on 275nm and 308nm dual wavelength once property to rhodiola root medicinal material in the measuring of gallic acid, rhodioside, tyrosol and p-Coumaric Acid content, only need test sample of preparation, detect the testing that once just can complete above-mentioned four kinds of compositions.
Detection method of the present invention has following beneficial effect:
1, the invention provides the method to the assay of gallic acid, rhodioside, tyrosol and p-Coumaric Acid content in rhodiola root medicinal material simultaneously;
2, the present invention provides the method for the rhodioside, gallic acid, tyrosol and the four kinds of compositions of p-Coumaric Acid that utilize in Dual-wavelength Spectrophotometric Determination rhodiola root medicinal material first; Especially detect the method for p-Coumaric Acid composition at 308nm place;
3, method provided by the invention, what adopt is Phenomenex Luna C18 post, compared with the small particle diameter HALO C18 post of prior art report, Kromasil C18 post, Thermo C18 post, YWG-C18 post, can realize separated component and the good effect of degree of separation within the relatively short time;
4, the assay technology that the application provides, compared with prior art, has reduced experiment number, and experimental implementation is easy, and experiment condition is simply controlled, has feature efficient, that accurate, applicability is wide simultaneously.
Accompanying drawing explanation
Fig. 1 is that mobile phase is followed successively by acetonitrile-0.3% glacial acetic acid, acetonitrile-water, methyl alcohol-glacial acetic acid, acetonitrile-0.1% phosphoric acid from top to bottom, and 30 ℃ of column temperatures, flow velocity are 1ml/min, and chromatographic column is Phenomenex Luna C
18the chromatogram of post.
Fig. 2 is that mobile phase is that acetonitrile-0.3% glacial acetic acid, chromatographic column are Phenomenex Luna C
18post, flow velocity are 1ml/min, and column temperature is followed successively by the chromatogram of 25 ℃, 30 ℃, 40 ℃ from top to bottom.
Fig. 3 is that mobile phase is that acetonitrile-0.3% glacial acetic acid, chromatographic column are Phenomenex Luna C
18post, column temperature are that 40 ℃, flow velocity are followed successively by 0.5,0.7,1.0 from top to bottom, the chromatogram of 1.5ml/min.
Fig. 4 is the chromatogram of gallic acid, rhodioside, tyrosol reference substance solution; Wherein in Fig. 4 A, from left to right △ △ indicates respectively gallic acid, rhodioside, the tyrosol maximum absorption band at 275nm place; Fig. 4 B is illustrated in the long uv absorption chromatogram of 200-400nm all-wave, is respectively from top to bottom gallic acid, rhodioside, tyrosol.
Fig. 5 is the chromatogram of p-Coumaric Acid reference substance solution, and wherein Fig. 5 A is illustrated in the chromatograms of the p-Coumaric Acid at 308nm place, and △ △ represents maximum absorption band; Fig. 5 B represents the long uv absorption figure of p-Coumaric Acid 200-400nm all-wave.
Fig. 6 is the chromatogram that extraction solvent is followed successively by methyl alcohol, ethanol, 80% methyl alcohol, 80% ethanol, 20% ethanol.
Fig. 7 is that chromatographic column is from top to bottom followed successively by Kromasil C
18post, Thermo C
18the chromatogram of post, Phenomenex Luna C18 post, HALO C18 post, InertsilODS2SP post, YWG-C18 post.
Wherein, mobile phase is acetonitrile-0.3% glacial acetic acid, column temperature: 40 ℃; Volumetric flow rate 1.0mLmin-1; Detect wavelength: 275nm.
Fig. 8 is the mixing reference substance of gallic acid, rhodioside, tyrosol and p-Coumaric Acid and the chromatogram of 100702 batches of rhodiola root medicinal materials;
Wherein, A1 is that wavelength is the mixing reference substance chromatogram of 275nm; A2 is that wavelength is the rhodiola root medicinal material chromatogram of 275nm; B1 is that wavelength is the mixing reference substance chromatogram of 308nm; B2 is that wavelength is the rhodiola root medicinal material chromatogram of 308nm; The 1st, gallic acid; The 2nd, rhodioside; The 3rd, tyrosol; The 4th, p-Coumaric Acid.
Fig. 9 is the assay chromatogram of 100902 batches of rhodiola root medicinal materials.
Figure 10 is the assay chromatogram of 101001 batches of rhodiola root medicinal materials.
Embodiment
The instrument using in following examples and reagent:
High performance liquid chromatograph (the Waters2695-2487 U.S.); BP-211D electronic balance (Sartorius Germany); Chromatographic column Phenomenex Luna C
18post (4.6mm × 250mm, 5 μ are m); Hydro-extractor Centrifuge5415D.
Gallic acid reference substance (lot number 110831-200803); Rhodioside reference substance (lot number 110818-200404); Tyrosol (lot number 111676-200602); Above reference substance is all purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
P-Coumaric Acid is purchased from Man Site bio tech ltd, Chengdu, lot number MUST-11122604.
Acetonitrile (on chromatographically pure starfish can biochemical company limited), water is deionized water, all the other be analyze pure.
The preparation of [embodiment 1] need testing solution
Get Tibet and produce rhodiola root (rhodiola root content >=1.0%) medicinal material 10kg, get filtrate for the first time after boiling 2h with the decocting of 8 times of weight; The decocting that filter residue adds 8 times of weight again boils 2h, gets filtrate for the second time, merges twice filtrate and is evaporated to 10L, obtains concentrate I.Concentrate I is carried out to purifying with macroreticular resin, loading ratio is 1g rhodiola root medicinal material: 2g resin; Then first with water elution, discard water liquid, then with 20% ethanol elution, collect 20% ethanol eluate.Eluent is concentrated into density 1.05～1.10, obtains concentrate II.Then by 80% ethanol alcohol precipitation 24～32 hours for concentrate II, obtain supernatant, abandon precipitation.By alcohol precipitation supernatant concentration, dry, pulverizing, obtain gadol extract.Take gadol extract, add 10～30% ethanol to dissolve and determine moltenly, every milliliter containing gadol extract 1～5mg, to obtain final product.
The testing conditions of [embodiment 2] screening gallic acid, rhodioside, tyrosol and p-Coumaric Acid content.
The screening of chromatographic condition
The flow phase system such as acetonitrile-water, acetonitrile-0.1% phosphoric acid, acetonitrile-0.3% glacial acetic acid, methanol-water, methyl alcohol-0.1% phosphoric acid are investigated, result as shown in Figure 1, through test relatively, finally determine the assay that completes above four compositions with acetonitrile-0.3% glacial acetic acid gradient elution.
Investigated 25 ℃, 30 ℃, 40 ℃ different column temperatures, result as shown in Figure 2; And 0.5,0.7,1.0, the impact of 1.5mL/min different in flow rate on each component separating, result is as shown in Figure 3; Find while being 0.7mL/min, to separate best results at 40 ℃, flow velocity.
The screening of full wavelength scanner
Gallic acid, rhodioside, tyrosol, p-Coumaric Acid reference substance solution full wavelength scanner (200～400nm) are found to other three compositions all have absorption maximum near 275nm except coumaric acid, but larger in the fluctuation of 220nm place establishment of base line, therefore select the detection wavelength of 275nm as gallic acid, rhodioside, tyrosol, as shown in Figure 4; P-Coumaric Acid obtained the maximum absorption 308nm measures wavelength as it, as shown in Figure 5.
Extract solvent screening
Investigated methyl alcohol, the ethanol of variable concentrations, as shown in Figure 6, wherein ethanol, 80% alcohol extract chromatogram peak shape are bad, do not calculate content, are below methyl alcohol, 80% methyl alcohol, 20% alcohol extract content comparison for result, and result is 20% ethanol the best.
The selection of chromatographic column
Investigate successively Kromasil C
18post, Thermo C
18post, Phenomenex Luna C
18post, HALOC18 post, InertsilODS2SP post, YWG-C18 post, result as shown in Figure 7, Phenomenex LunaC
18post separating effect the best.
The content detection of gallic acid, rhodioside, tyrosol and p-Coumaric Acid in [embodiment 3] rhodiola root medicinal material
Chromatographic condition and system flexibility:
Chromatographic column is Phenomenex Luna C
18post (4.6mm × 250mm, m), mobile phase is acetonitrile (A)-0.3% glacial acetic acid solution (B) to 5 μ, elution program is: 0～5min, 9%A; 5～30min, 9%～25%A, 30～35min, 25%～40%A; Column temperature: 40 ℃; Volumetric flow rate 0.7mLmin
-1; Detect wavelength: 275nm, 308nm; Sample size 10 μ l.Get need testing solution, sample introduction analysis under above-mentioned chromatographic condition, in sample, the degree of separation of each component to be measured and adjacent peak is all greater than 1.5, and tailing factor is between 0.95～1.05, and the number of plates is calculated and is greater than 20000 by rhodioside.Chromatogram as shown in Figure 8.
Reference substance solution preparation
Get respectively gallic acid, rhodioside, tyrosol, p-Coumaric Acid reference substance appropriate, accurately weighed, dissolve and be diluted to concentration with methyl alcohol and be respectively 391.2 μ gmL
-1, 1460.0 μ gmL
-1, 1000.8 μ gmL
-1, 517.6 μ gmL
-1reference substance stock solution.
Need testing solution preparation
Press the preparation of method described in embodiment 1.
Linear relationship test
Precision measures gallic acid, rhodioside, tyrosol, p-Coumaric Acid reference substance stock solution is in right amount in 10mL volumetric flask, dilute constant volume with methyl alcohol, shake up, obtain mixing reference substance solution, to mix again the doubly dilution of reference substance solution methyl alcohol, constant volume, obtain the mixing reference substance solution of six required concentration of typical curve, shake up centrifugal, extracting centrifugal liquid, measure by chromatographic condition sample introduction under above-mentioned chromatographic condition and system flexibility item, record chromatogram, take peak area A as ordinate, concentration C is that horizontal ordinate carries out linear regression, result shows, each composition is good linear relation within the scope of respective concentration, the range of linearity, regression equation, related coefficient is in table 1.
Table 1 range of linearity, regression equation and related coefficient
Instrument precision test
(gallic acid concentration is 3.478 μ gmL to draw reference substance mixed solution
-1, rhodioside concentration is 182.500 μ gmL
-1, tyrosol is 62.550 μ gmL
-1, p-Coumaric Acid is 12.940 μ gmL
-1), measure by chromatographic condition sample introduction under above-mentioned chromatographic condition and system flexibility item, repeat sample introduction 6 times, record peak area, calculate RSD.The RSD of the peak area of gallic acid, rhodioside, tyrosol and p-Coumaric Acid is respectively: 0.90%, 0.89%, 1.00%, 1.10%, show that instrument precision is good.
Repeatability
Get 100702 batches of rhodiola root medicinal materials by parallel six parts of the need testing solutions of preparing of method under above-mentioned need testing solution preparation, measure by chromatographic condition sample introduction under above-mentioned chromatographic condition and system flexibility item, record chromatogram, the RSD that calculates gallic acid, rhodioside, tyrosol and p-Coumaric Acid content is respectively 2.43%, 2.19%, 2.78%, 2.84%.
Result shows that this method repeatability is good.
Stability
Get rhodiola root need testing solution (lot number 100702), at ambient temperature respectively at 0,3,6,9,12,15,18h measures by chromatographic condition sample introduction under above-mentioned chromatographic condition and system flexibility item, record chromatogram, the RSD that calculates gallic acid, rhodioside, tyrosol, p-Coumaric Acid content is respectively 2.10%, 2..46%, 1.78% and 2.52%.
Result shows, need testing solution is stable in 18 hours.
Recovery test
Get totally 6 parts, rhodiola root sample (lot number 100702), every part of about 20mg, accurately weighed, put in 25mL measuring bottle, add respectively gallic acid, tyrosol, p-Coumaric Acid reference substance stock solution are appropriate, 6 parts of accurately weighed rhodioside reference substances again, add respectively in sample, constant volume after dissolving with 20% ethanol, measures by chromatographic condition sample introduction under above-mentioned chromatographic condition and system flexibility item, records chromatogram, the average recovery that calculates gallic acid, rhodioside, tyrosol, p-Coumaric Acid, the results are shown in Table 2.
Table 2 determination of recovery rates (n=6)
Sample determination
Get rhodiola root medicinal material (lot number 100702) by above-mentioned need testing solution preparation below legal system available test sample solution, by chromatographic condition under above-mentioned chromatographic condition and system flexibility item, sample introduction is measured, record chromatogram, the percentage composition that calculates gallic acid, rhodioside, tyrosol, p-Coumaric Acid, the results are shown in Table 3.
Table 3100702 batch rhodiola root medicinal material assay
The content detection of gallic acid, rhodioside, tyrosol and p-Coumaric Acid in [embodiment 4] rhodiola root medicinal material
Chromatographic condition and system flexibility:
Chromatographic column is Phenomenex Luna C
18post (4.6mm × 250mm, m), mobile phase is acetonitrile (A)-0.5% glacial acetic acid solution (B) to 5 μ, elution program is: 0～5min, 6%A; 5～30min, 6%～20%A, 30～35min, 20%～35%A; Column temperature: 20 ℃; Volumetric flow rate 0.5mLmin
-1; Detect wavelength: 275nm, 308nm; Sample size 5 μ l.
Reference substance solution preparation
Get respectively gallic acid, rhodioside, tyrosol, p-Coumaric Acid reference substance appropriate, accurately weighed, dissolve and be diluted to concentration with methyl alcohol and be respectively 391.2 μ gmL
-1, 1460.0 μ gmL
-1, 1000.8 μ gmL
-1, 517.6 μ gmL
-1reference substance stock solution.
Need testing solution preparation
By the preparation of method described in EXAMPLE l.
Sample determination
Get 100902 batches of rhodiola root medicinal materials by above-mentioned need testing solution preparation below legal system available test sample solution, by chromatographic condition under above-mentioned chromatographic condition and system flexibility item, sample introduction is measured, record chromatogram, calculate the percentage composition of gallic acid, rhodioside, tyrosol, p-Coumaric Acid, the results are shown in Table 4, chromatogram as shown in Figure 9.
Table 4100902 batch rhodiola root medicinal material assay
The content detection of gallic acid, rhodioside, tyrosol and p-Coumaric Acid in [embodiment 5] rhodiola root medicinal material
Chromatographic condition and system flexibility:
Chromatographic column is Phenomenex Luna C
18post (4.6mm × 250mm, m), mobile phase is acetonitrile (A)-0.1% glacial acetic acid solution (B) to 5 μ, elution program is: 0～5min, 12%A; 5～30min, 12%～30%A, 30～35min, 30%～45%A; Column temperature: 30 ℃; Volumetric flow rate 1.4mLmin
-1; Detect wavelength: 275nm, 308nm; Sample size 20 μ l.
Reference substance solution preparation
Get respectively gallic acid, rhodioside, tyrosol, p-Coumaric Acid reference substance appropriate, accurately weighed, dissolve and be diluted to concentration with methyl alcohol and be respectively 391.2 μ gmL
-1, 1460.0 μ gmL
-1, 1000.8 μ gmL
-1, 517.6 μ gmL
-1reference substance stock solution.
Need testing solution preparation
Press the preparation of method described in embodiment 1.
Sample determination
Get 1010041 batches of rhodiola root medicinal materials by above-mentioned need testing solution preparation below legal system available test sample solution, by chromatographic condition under above-mentioned chromatographic condition and system flexibility item, sample introduction is measured, record chromatogram, calculate the percentage composition of gallic acid, rhodioside, tyrosol, p-Coumaric Acid, the results are shown in Table 5, shown in chromatogram as shown in figure 10.
Table 5101001 batch rhodiola root medicinal material assay
Claims (5)
1. a content assaying method for principal ingredient in rhodiola root medicinal material, is characterized in that, comprises the steps:
1) rhodiola root medicinal material need testing solution preparation:
1. get the rhodiola root medicinal material decocting of 6～10 times of amounts and boil 1～3h, filter, obtain filtrate and filter residue for the first time, the decocting that filter residue adds 6～10 times of amounts again boils 1～3h, filters, and obtains filtrate for the second time, merge twice filtrate and be evaporated to 1/20～1/12 volume of filtrate, obtain concentrate I;
2. with macroreticular resin, concentrate I is carried out to purifying, carry out loading with the weight ratio 1: 1～3 of rhodiola root medicinal material and resin; Then first with water elution, discard water liquid, then with 15～30% ethanol elutions, collect 15～30% ethanol eluates;
3. eluent is concentrated into density 1.05～1.10, obtains concentrate II;
4. use 75～85% ethanol alcohol precipitation concentrate II12～36h, obtain supernatant, abandon precipitation;
5. by alcohol precipitation supernatant concentration, dry, pulverizing, obtain gadol extract;
6. take gadol extract, add 10～30% ethanol to dissolve and determine moltenly, every milliliter containing gadol extract 1～5mg, to obtain final product;
2) prepare respectively gallic acid reference substance solution, rhodioside reference substance solution, tyrosol reference substance solution and p-Coumaric Acid reference substance solution;
3) utilize high efficiency liquid phase chromatographic analysis method to detect gallic acid in rhodiola root medicinal material, rhodioside, tyrosol and p-Coumaric Acid content at dual wavelength simultaneously;
Chromatographic condition is: HPLC chromatographic column is Phenomenex Luna C18 post, mobile phase is acetonitrile (A)-0.1～0.5% glacial acetic acid solution (B), elution program is: 0 → 5min, and A rises to 6～12%, B from 0 linearity and drops to 94～88% from 100% linearity; 5 → 30min, A rises to 20～30%, B from 6～12% linearities and drops to 80～70% from 94～88% linearities; 30 → 35min, A rises to 35～45%, B from 20～30% linearities and drops to 65～55% from 80～70% linearities; Column temperature: 20～40 ℃; Volumetric flow rate 0.5～1.5mLmin
-1; Detect wavelength: 275nm, 308nm; Sample size 2～20 μ l;
4) according to the content of gallic acid, rhodioside, tyrosol and p-Coumaric Acid in efficient liquid phase chromatographic analysis result calculating rhodiola root medicinal material.
2. the assay method as described in any one in claim 1, is characterized in that, described chromatographic condition is:
HPLC chromatographic column be Phenomenex Luna C18 post (4.6mm × 250mm, m), mobile phase is acetonitrile (A)-0.3% glacial acetic acid solution (B) to 5 μ, elution program is: 0～5min, 9%A; 5～30min, 9%～25%A, 30～35min, 25%～40%A; Column temperature: 40 ℃; Volumetric flow rate 0.7mLmin
-1; Detect wavelength: 275nm, 308nm; Sample size 10 μ l.
3. content assaying method as claimed in claim 1, is characterized in that, need testing solution preparation comprises the following steps:
1) get the rhodiola root medicinal material decocting of 8 times of amounts and boil 2h, filter, filtrate and filter residue for the first time, the decocting that filter residue adds 8 times of amounts again boils 2h, filter, filtrate for the second time, merges twice filtrate and is evaporated to 1/16 volume of filtrate, acquisition concentrate I;
2) with macroreticular resin, concentrate I is carried out to purifying, carry out loading at 1: 2 with the weight ratio of rhodiola root medicinal material and resin; Then first with water elution, discard water liquid, then with 20% ethanol elution, collect 20% ethanol eluate;
3) eluent is concentrated into density 1.05～1.10, obtains concentrate II;
4) with 80% ethanol alcohol precipitation concentrate II24～32h, obtain supernatant, abandon precipitation;
5) by alcohol precipitation supernatant concentration, dry, pulverizing, obtain gadol extract;
6) take gadol extract, add 20% ethanol to dissolve and determine moltenly, every milliliter containing gadol extract 2mg, to obtain final product.
4. assay method as claimed in claim 1, it is characterized in that, described in step 2, the preparation method of gallic acid reference substance solution, rhodioside reference substance solution, tyrosol reference substance solution and p-Coumaric Acid reference substance solution comprises: take respectively appropriate gallic acid reference substance, rhodioside reference substance, tyrosol reference substance and p-Coumaric Acid reference substance, dissolving and be diluted to concentration with methyl alcohol is 391.2 μ gmL
-1gallic acid reference substance solution, 1460.0 μ gmL
-1rhodioside reference substance solution, 1000.8 μ gmL
-1tyrosol reference substance solution and 517.6 μ gmL
-1p-Coumaric Acid reference substance solution.
5. assay method as claimed in claim 1, is characterized in that, described rhodiola root medicinal material is rhodiola or rhodiola kirilowii Regel.
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| CN111337592A (en) * | 2019-12-17 | 2020-06-26 | 广西中医药大学 | Method for simultaneously determining multi-component content in sedum aizoon |
| CN112858491A (en) * | 2019-11-12 | 2021-05-28 | 北京以岭药业有限公司 | Rhodiola rosea medicinal material fingerprint spectrum determination method |
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| CN112858491A (en) * | 2019-11-12 | 2021-05-28 | 北京以岭药业有限公司 | Rhodiola rosea medicinal material fingerprint spectrum determination method |
| CN112858491B (en) * | 2019-11-12 | 2024-03-12 | 北京以岭药业有限公司 | Rhodiola rosea medicinal material fingerprint spectrum determination method |
| CN111337592A (en) * | 2019-12-17 | 2020-06-26 | 广西中医药大学 | Method for simultaneously determining multi-component content in sedum aizoon |
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