CN110196290A - Ellagic acid content assaying method in a kind of Rosa roxburghii Tratt - Google Patents
Ellagic acid content assaying method in a kind of Rosa roxburghii Tratt Download PDFInfo
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- CN110196290A CN110196290A CN201910262949.1A CN201910262949A CN110196290A CN 110196290 A CN110196290 A CN 110196290A CN 201910262949 A CN201910262949 A CN 201910262949A CN 110196290 A CN110196290 A CN 110196290A
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Abstract
The present invention provides ellagic acid content assaying method in a kind of Rosa roxburghii Tratt, this method include the preparation of reference substance solution, the preparation of test sample acid hydrating solution, test sample dissociate ellagic acid solution preparation, determine chromatographic condition, determine measuring method.The present invention detects ellagic acid content in Rosa roxburghii Tratt, and detection method is accurate, high sensitivity, reproducible, as a result reliably, provides foundation for the control of Rosa roxburghii Tratt quality of medicinal material and evaluation, facilitates the further development and utilization to Rosa roxburghii Tratt.
Description
Technical field
The present invention relates to ellagic acid content assaying methods in a kind of Rosa roxburghii Tratt, belong to the neck of drugs and health care products cosmetic art
Domain.
Background technique
Rosa roxburghii Tratt (Rosa Roxburghii Tratt) is a kind of rosaceous plant of dual-purpose of drug and food, is distributed mainly on China
Localities In Southwest.In recent years, cultivated area of the Rosa roxburghii Tratt in Guizhou is in the trend obviously risen, and fruit is processed at beverage, fruit
The products such as dried meat, cake, it is deep to be liked by the majority of consumers.Single Roxburgh Rose Fruit, roxburgh rose root and roxburgh rose leaf are recorded in version in 2003 " in Guizhou Province
Medicinal material, Ethnic crude drugs quality standard ".Single Roxburgh Rose Fruit has digestion-promoting spleen-invigorating, and astringing to arrest diarrhea and other effects, roxburgh rose root is for treating dyspepsia abdomen
Bitterly, it has a toothache, chronic cough, diarrhea, with inferior disease, roxburgh rose leaf can be used for stomach strengthening and digestion promoting.
Have in document report Single Roxburgh Rose Fruit containing ellagic acid and 3 kinds of ellagic acid glucosides [Zeng F F, Ge Z W,
Jarukitt L,et al.Antioxidant and tyrosinase inhibitory activity of Rosa
roxburghii fruit and identification of main bioactive phytochemicals by UPLC-
Triple-TOF/MS [J] .Int J FoodSci Tech, 2017 (52): 897-905.], ellagic acid (ellagic acid) is again
It is benzoaric acid, is the dimerization derivative of gallic acid, it has anti-oxidant, anticancer change/antimutagenesis, also has both anti-
Bacterium, antivirus action, are widely used in the fields such as food, medicine, medical treatment and cosmetics, and price is more expensive.
Natural ellagic acid is present in (such as pomegranate leaf, chestnut in various fruits (Cranberry, strawberry, raspberry etc.) and various plants
Son etc.), but free ellagic acid is all relatively low in content wherein, existing ellagic acid is less in a free form in nature,
Mainly exist in the form of Ellagitannins.
Currently, having no the report in relation to extracting ellagic acid method and technique from Rosa roxburghii Tratt, and not to tan flower in Rosa roxburghii Tratt
The research of acid content measuring method is reported, it is difficult to be controlled quality of the Rosa roxburghii Tratt as medicinal material and its prescribed preparation, not can guarantee clinic
The safety and validity of medication limit the further development and utilization to Rosa roxburghii Tratt.
Summary of the invention
The object of the present invention is to provide ellagic acid content assaying methods in a kind of Rosa roxburghii Tratt.The present invention is to tan flower in Rosa roxburghii Tratt
Acid content is detected, and detection method is accurate, and high sensitivity is reproducible, as a result reliably, is controlled and is commented for Rosa roxburghii Tratt quality of medicinal material
Valence provides foundation, facilitates the further development and utilization to Rosa roxburghii Tratt.
Technical solution of the present invention: ellagic acid content assaying method in a kind of Rosa roxburghii Tratt, the survey to ellagic acid content in Rosa roxburghii Tratt
Surely it is:
The preparation of reference substance solution: precision weighs reference substance ellagic acid, adds flowing phased soln that solution is made, and 0.45 μm excessively micro-
Hole filter membrane, is made reference substance solution, saves in 4 DEG C；
The preparation of test sample acid hydrating solution: taking Rosa roxburghii Tratt active component powder, and the methanol of acidification is added, and flows back, and concentration adds
Enter dimethyl sulfoxide dissolution, with flowing phase dilution, cross 0.45 μm of filtering with microporous membrane, take filtrate to get；
The preparation of the free ellagic acid solution of test sample: taking Rosa roxburghii Tratt active component powder, and methanol solution is added, and room temperature is extracted,
It is ultrasonically treated, is concentrated after suction filtration, dimethyl sulfoxide dissolution is added, with flowing phase dilution, cross 0.45 μm of filtering with microporous membrane, take filter
Liquid to get；
Chromatographic condition: chromatographic column: 2 C of ACE Excel18- Amide (2 μm, 2.1mm × 100mm)；Mobile phase: 0.1% first
Sour water is A phase, and acetonitrile is B phase, gradient elution: flow velocity: 0.2mL/min；Detection wavelength: 254nm, sample volume: 3 μ L；Column temperature 40
℃；
Measuring method: accurate absorption reference substance solution, test sample acid hydrating solution and the free ellagic acid of test sample are molten respectively
Liquid measures peak area under above-mentioned chromatographic condition respectively, calculate to get.
In a kind of Rosa roxburghii Tratt above-mentioned in ellagic acid content assaying method, the test sample acid hydrating solution is in the steps below
It is prepared: taking Single Roxburgh Rose Fruit, roxburgh rose root and roxburgh rose leaf natural air drying, crushed, cross 40 meshes, accurately weighed 5.0g, addition contains
The methanol 50mL of 1.2mol/L hydrochloric acid, 85 DEG C of reflux 8h are concentrated to dryness with Rotary Evaporators, and residue is dissolved with dimethyl sulfoxide, fixed
It is dissolved in 100mL volumetric flask, as mother liquor.By Single Roxburgh Rose Fruit and 25 times of phase dilution of the flowing of roxburgh rose root mother liquor, roxburgh rose leaf mother liquor is taken
With flowing 50 times of phase dilution, through 0.45 μm of filtering with microporous membrane to get.
In a kind of Rosa roxburghii Tratt above-mentioned in ellagic acid content assaying method, the test sample dissociates ellagic acid solution by following
Prepared by step: taking Single Roxburgh Rose Fruit, roxburgh rose root and roxburgh rose leaf natural air drying, crush, cross 40 meshes, accurately weighed 5.0g is added
90% methanol solution 50mL, room temperature extract 8h, are ultrasonically treated 30min, filter, are concentrated to dryness with Rotary Evaporators, and residue is with two
The dissolution of first sulfoxide, constant volume is in 100mL volumetric flask, as mother liquor.By the mother liquor of Single Roxburgh Rose Fruit, roxburgh rose root and roxburgh rose leaf with flowing
25 times of phase dilution, through 0.45 μm of filtering with microporous membrane to get.
In a kind of Rosa roxburghii Tratt above-mentioned in ellagic acid content assaying method, the reference substance solution is prepared: taking ellagic acid
Appropriate reference substance, accurately weighed, ellagic acid reference substance adds mobile phase that the solution of 0.4mg/mL is made, and crosses 0.45 μm of miillpore filter,
It is saved backup in 4 DEG C.
In a kind of Rosa roxburghii Tratt above-mentioned in ellagic acid content assaying method, the gradient elution program is；0~5min, 3%
→ 6%B；5~10min, 6% → 16%B；10~25min, 16% → 21%B；25~35min, 21% → 30%B.
Inventor has carried out a large amount of experiment, is the research of detection method of the present invention below
Experimental example: detection method research
1 material
1.1 instrument
Agilent1290UPLC (Agilent company, the U.S.), chromatographic column ACEExcel2C18-Amide(2μm,2.1mm
×100mm)；UV-2700 type ultraviolet specrophotometer (Shimadzu, Japan)；KQ-300DE ultrasonic cleaner (Kunshan
Ultrasonic instrument Co., Ltd, city)；TB-215D type electronic analytical balance (Beijing Sai Duolisi instrument system Co., Ltd).
1.2 medicinal materials, reference substance and reagent
Single Roxburgh Rose Fruit, roxburgh rose root and roxburgh rose leaf pick up from Guizhou Province Longli County, and every part of sample is by Guizhou medical university pharmacognosy
Teaching and research room associate professor Long Qingde is accredited as the fruit, rhizome and blade of rosaceous plant Rosaroxburghiitratt；Control
Product: ellagic acid (lot number: 1013A023, for assay), ascorbic acid (lot number: 105N032, for assay) by
Beijing Suo Laibao Science and Technology Ltd provides purity >=98%.1,1- diphenyl -2- trinitrophenyl-hydrazine (DPPH, chromatographically pure, batch
Number: W12A9E55779), 2,2- connection nitrogen-two (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts (ABTS, ultrapure, lot number:
K18N8M48402 it) is provided by Shanghai Yuan Ye Biotechnology Co., Ltd, purity is >=98%；(chromatographically pure, Germany are silent for acetonitrile
Gram company), formic acid (chromatographically pure, Tianjin Ke Miou chemical reagent Co., Ltd), methanol, dehydrated alcohol (analyze pure, Chinese medicines group
Chemical reagent Co., Ltd)；Dimethyl sulfoxide (analyzes pure, Tianjin Ke Miou chemical reagent Co., Ltd), hydrochloric acid (excellent pure grade,
Sinopharm Chemical Reagent Co., Ltd.), pure water (Hangzhou Wahaha Co., Ltd).
2 methods and result
2.1 ellagic acid assays
2.1.1 reference substance solution
Take ellagic acid reference substance appropriate, accurately weighed, ellagic acid reference substance adds mobile phase that the solution of 0.4mg/mL, mistake is made
0.45 μm of miillpore filter, saves backup in 4 DEG C.
2.1.2 the processing of medicinal material
It respectively by Single Roxburgh Rose Fruit, roxburgh rose root and the roxburgh rose leaf natural air drying after picking, crushes, it is spare to cross 40 meshes.
2.1.3 the preparation of test sample acid hydrating solution
5.0g Single Roxburgh Rose Fruit, roxburgh rose root and the roxburgh rose leaf for taking " 2.1.2 " item to handle respectively, it is accurately weighed.The first of acidification is added
Alcohol (hydrochloric acid containing 1.2mol/L) 50mL, 85 DEG C of reflux 8h, then be concentrated to dryness with Rotary Evaporators, residue is dissolved with dimethyl sulfoxide,
Constant volume is in 100mL volumetric flask, as mother liquor.By Single Roxburgh Rose Fruit and 25 times of phase dilution of the flowing of roxburgh rose root mother liquor, take roxburgh rose leaf female
Liquid with flowing 50 times of phase dilution, through 0.45 μm of filtering with microporous membrane to get.
2.1.4 the preparation of the free ellagic acid solution of test sample
5.0g Single Roxburgh Rose Fruit, roxburgh rose root and the roxburgh rose leaf for taking " 2.1.2 " item to handle respectively, it is accurately weighed.90% methanol is added
Solution 50mL, 24 DEG C of extraction 8h of room temperature are ultrasonically treated 30min, and suction filtration is concentrated to dryness with Rotary Evaporators again, residue diformazan Asia
Sulfone dissolution, constant volume is in 100mL volumetric flask, as mother liquor.Use mobile phase dilute in the mother liquor of Single Roxburgh Rose Fruit, roxburgh rose root and roxburgh rose leaf
Release 25 times, through 0.45 μm of filtering with microporous membrane to get.
2.1.5 chromatographic condition
Chromatographic column: ACEExcel2C18-Amide(2μm,2.1mm×100mm)；Mobile phase: 0.1% formic acid water (A)-acetonitrile
(B), gradient elution (0~5min, 3% → 6%B；5~10min, 6% → 16%B；10~25min, 16% → 21%B；25~
35min, 21% → 30%B)；Column temperature: 40 DEG C；Flow velocity: 0.2mL/min；Detection wavelength: 254nm, sample volume: 3 μ L.UPLC color
Spectrogram is shown in Fig. 1.Under the chromatographic condition, baseline is steady, and the peak shape of ellagic acid is good, and separating degree > 1.5.
2.1.6 methodological study
2.1.6.1 linear relationship
Precision measures ellagic acid reference substance stock solution 1.0,2.5,4.0,5.5,7.0,8.5,10.0mL under " 2.1.1 " item,
It is respectively placed in 20mL volumetric flask, adds mobile phase to scale, shake up the control series product solution to get various concentration.Respectively into
3 μ L of sample.With the mass concentration (X) of each reference substance for abscissa, corresponding integrating peak areas value (Y) is ordinate, draws standard
Curve, and establish regression equation.Y=136.97X-2249.2 (r=0.9996) is obtained, shows ellagic acid in 19.96-199.60
There is good linear relationship in μ g/mL concentration range.
2.1.6.2 instrument precision is tested
Taking the concentration prepared under " 2.1.6.1 " item is the ellagic acid reference substance solution of 20 μ g/mL, presses " 2.1.5 " Xiang Xiase
The measurement of spectral condition sample introduction, repeats sample introduction 6 times, records peak area.As a result, the RSD of ellagic acid peak area is 1.35% (n=6), table
Bright instrument precision is good.
2.1.6.3 stability test
The Single Roxburgh Rose Fruit solution for taking " 2.1.3 " item to handle, interval 0,8,12,24,48h are surveyed by chromatographic condition under " 2.1.5 " item
1 time fixed, the RSD of ellagic acid peak area is 0.97%, and test sample is dissolved in basicly stable in 48h as the result is shown.
2.1.6.4 repetitive test
Single Roxburgh Rose Fruit sample 5.0g under " 2.1.2 " item is taken, it is accurately weighed.6 parts are prepared in parallel by method under " 2.1.3 " item, often
A sample presses chromatographic condition sample introduction under " 2.1.5 " item, carries out assay.The RSD of ellagic acid content is 2.17% as the result is shown,
Show that this method repeatability is good.
2.1.6.5 sample recovery rate is tested
The Single Roxburgh Rose Fruit sample of accurately weighed 1g known content, it is accurate respectively that 1mL ellagic acid reference substance solution is added, it presses
The method of " 2.1.3 " prepares 9 parts of test solutions containing free ellagic acid in parallel.It is surveyed by chromatographic condition sample introduction under " 2.1.5 " item
It is fixed, it records peak area and calculates sample recovery rate.It the results are shown in Table 1, the average recovery rate of ellagic acid is that 99.88%, RSD is
1.43%.
2.1.7 sample size measures
Precision weighs Single Roxburgh Rose Fruit, roxburgh rose root and roxburgh rose leaf sample under " 2.1.2 " item, respectively by " 2.1.3 " item and " 2.1.4 "
Lower section legal system available test sample solution, each sample is 3 parts parallel, with mobile phase dilute sample solution according to a certain percentage, and presses
It is measured according to chromatographic condition under " 2.1.5 " item.Standard curve is substituted by peak area, calculates the tan flower that dissociates in each Rosa roxburghii Tratt sample
The content of sour and total ellagic acid, the results are shown in Table 2.
1 ellagic acid sample recovery rate of table investigates result (n=9)
1 Result of recovery tests of ellagic acid (n=9) of Tab
2 sample size measurement result (n=3) of table
2 Results of sample determination (n=3) of Tab
The measurement of 2.2 antioxidant activities
2.2.1 the preparation of reference substance solution
(1) VC reference substance solution: it is appropriate that precision weighs VC reference substance, 60% ethyl alcohol is added is prepared into mass concentration and be respectively
3, the contrast solution of 6,12,24,48,96 μ g/mL series of concentrations, the positive control for DPPH radicals scavenging test.It is accurate
Weigh that VC reference substance is appropriate, be added 70% ethyl alcohol be prepared into 10,20,40,80,160,320 μ g/mL series of concentrations control it is molten
Liquid, the positive control for ABTS radicals scavenging test.(2) DPPH standard solution: accurately weighed DPPH standard items are appropriate, add
Enter dehydrated alcohol, is prepared into the DPPH stock solution of 1mmol/L；The DPPH of 0.1mmol/L is obtained with dehydrated alcohol dilution stock solution
Standard solution, it is spare.(3) ABTS standard solution: accurately weighed ABTS standard items are appropriate, and 70% ethyl alcohol of addition is prepared into concentration and is
The standard solution of 7mmol/L, it is spare.
2.2.2 the preparation of test solution
Accurate Single Roxburgh Rose Fruit, roxburgh rose root and each 0.5g of roxburgh rose leaf powder respectively, are placed in stuffed conical flask, and precision is added 60%
Ethanol solution 15mL, close plug, weighed quality are ultrasonically treated (power: 300W, frequency: 45kHz) 30min, take out, after letting cool again
Secondary weighed quality, the quality of less loss is supplied with 60% ethanol solution, is shaken up, and filtration takes subsequent filtrate to be concentrated to dryness and pierced
The operatic circle, roxburgh rose root and roxburgh rose leaf yield are respectively 40.4%, 14.0% and 26.4%.Extract is diluted to 3 with 60% ethyl alcohol,
6, the series of concentrations of 12,24,48,96 μ g/mL is tested for DPPH radicals scavenging；Test solution is dilute with 70% ethyl alcohol
It is interpreted as the series of concentrations of 10,20,40,80,160,320 μ g/mL, is tested for ABTS radicals scavenging.
2.2.3 the test of DPPH free radical is removed
Take 0.1mmol/L DPPH standard solution 4.0mL and sample solution/VC reference substance of different quality concentration molten respectively
Liquid 2.0mL, oscillation mix, and after solution dark place is stood 30min, its absorbance is measured under 517nm wavelength.It is calculated according to formula
DPPH free radical scavenging activity: DPPH free radical scavenging activity (%)=(A0-AX+AX0)/A0× 100, in formula: Ax is 2.0mL test sample
Absorbance after solution or VC reference substance solution and DPPH radical reaction；AX0It is molten for 2.0mL test solution or VC reference substance
The absorbance of liquid+4.0mL dehydrated alcohol；A0 is 60% second of DPPH standard solution+2.0mL that 4.0mL concentration is 0.1mmol/L
The absorbance of alcohol.And it returns to zero by blank of pure water.Each sample replication 3 times, is averaged.Using GraphPad
6.0 software of Prism calculates each sample to the median elimination concentration (IC of DPPH free radical50), all data are with mean ± SD table
Show.It is examined using the t of 6.0 software of GraphPad Prism respectively to the IC of VC and Rosa roxburghii Tratt different parts50Value is compared two-by-two
Compared with.Each position sample free radical scavenging the results are shown in Table 3.
Table 3 Rosa roxburghii Tratt, 3 medicinal part extracts are to free radical scavenging activity (n=3)
Tab 3 Scavenging activity of 3 medicinal parts extract of
R.roxburghii to DPPH free radicals and ABTS free radicals (n=3)
Note: between same index difference group, if letter is identical, no significant difference (P > 0.05) is represented；If word
It is female different, then it is statistically significant (P < 0.05) to represent difference
2.2.4 the test of ABTS free radical is removed
The ABTS standard solution 5.0mL that concentration is 7mmol/L under " 2.2.1 " item is taken, the mistake that concentration is 140mmol/L is added
88.0 μ L of potassium sulfate, react 12~16h for dark place at room temperature, with 70% ethyl alcohol that ABTS standard is molten then at 734nm wavelength
It is (0.70 ± 0.02) that liquid, which is diluted to absorbance, spare.Accurately pipette the series of the different quality concentration prepared under " 2.2.1 " item
Concentration VC reference substance solution or test solution 0.1mL, the ABTS standard solution 3.9mL after being separately added into above-mentioned dilution are mixed,
6min is reacted at room temperature, its absorbance (A is measured at 734nm wavelengthE)；ABTS standard solution 3.9mL is accurately pipetted simultaneously,
70% ethanol solution 0.1mL is added, its absorbance (A is measured at 734nm wavelengthB), and it is clear that ABTS free radical is calculated by formula
Except rate: ABTS free radical scavenging activity (%)=(AB-AE)/AB×100.Each sample carries out 3 repetitions, is averaged, and uses
6.0 software of GraphPad Prism calculates each sample to the median elimination concentration (IC of ABTS free radical50), all data with
Mean ± SD is indicated.It is examined using the t of 6.0 software of GraphPad Prism respectively to the IC of VC and Rosa roxburghii Tratt different parts50Be worth into
Row compares two-by-two.Each sample, which removes ABTS free radical activity, the results are shown in Table 3.
3 discuss
Chemical composition content measurement in the Rosa roxburghii Tratt that " integration of drinking and medicinal herbs ethnic drug-Rosa roxburghii Tratt " reports existing literature has carried out total
Knot summarizes the content assaying method of the ingredients such as vitamin in document, flavones, SOD, amino acid, polysaccharide, but not yet finds Rosa roxburghii Tratt
The relevant report of middle ellagic acid assay.Granatum is one of raw material sources of ellagic acid, has there is multinomial patent report at present
The method that road extracts ellagic acid from granatum.This research is on the basis of establishing good chromatographic condition to Single Roxburgh Rose Fruit, Rosa roxburghii Tratt
Ellagic acid carries out content analysis in root and roxburgh rose leaf.Originally the experimental results showed that, ellagic acid content is apparently higher than Rosa roxburghii Tratt in roxburgh rose leaf
Root and Single Roxburgh Rose Fruit.Ellagic Acid in Granati Cortex content reported in the literature is about that [Fan Gaofu, Fu Entao, Tang Jie wait .RP- to 32mg/g
HPLC method measures content [J] Anhui Science and Technology College journal of ellagic acid in granatum and juice of my pomegranate, 2016,30 (5): 67- simultaneously
70.], and ellagic acid content of dissociating in roxburgh rose leaf is higher than Ellagic Acid in Granati Cortex content.According to the literature, ellagic acid and ellagic acid
A variety of glucosides collectively reside in Rosa roxburghii Tratt.Ellagic acid glucosides in medicinal material can make glycosidic bond after strong acid and high temperature collective effect
Ellagic acid glucosides can be converted to ellagic acid by fracture, the process.Therefore, medicinal material is using ellagic acid as control after sour water solution
Its total amount can be measured.The discovery of this experimental studies results, total ellagic acid content more non-water of the same position of Rosa roxburghii Tratt after sour water solution
The free ellagic acid content that solution obtains is compared and increases 2 times or more.
It was found that DPPH and ABTS radicals scavenging test in Single Roxburgh Rose Fruit and roxburgh rose leaf IC50With the IC of positive control VC50Phase
As the IC of roxburgh rose root50It is apparently higher than VC.The strong and weak sequence of the antioxidant activity of different parts are as follows: roxburgh rose leaf > Single Roxburgh Rose Fruit > thorn
Pears root.
Either free ellagic acid or total ellagic acid content, roxburgh rose leaf, Single Roxburgh Rose Fruit and roxburgh rose root Rosa roxburghii Tratt difference
It is successively reduced in position, and roxburgh rose leaf, Single Roxburgh Rose Fruit and roxburgh rose root antioxidant activity successively die down.Due to polyphenol such as ellagic acids
Constituents have good Antioxidation in vitro, therefore speculate the strong and weak and corresponding Polyphenols of the antioxidant activity of different parts
The height of content is related.The content of ellagic acid glucosides constituents is higher in Rosa roxburghii Tratt, and the ellagic acid in Rosa roxburghii Tratt is prepared using sour water solution
It is a kind of feasible method.
In conclusion the present invention detects ellagic acid content in Rosa roxburghii Tratt, detection method is accurate, high sensitivity, repeats
Property it is good, as a result reliably, provide foundation for the control of Rosa roxburghii Tratt quality of medicinal material and evaluation, facilitate further exploitation to Rosa roxburghii Tratt and sharp
With.
Detailed description of the invention
Fig. 1 is experimental example UPLC chromatogram of the present invention；
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated, but is not intended as the foundation limited the present invention.
Embodiment 1: ellagic acid content assaying method in a kind of Rosa roxburghii Tratt, specific measuring method are as follows:
The preparation of reference substance solution: taking ellagic acid reference substance appropriate, accurately weighed, and ellagic acid reference substance adds mobile phase to be made
The solution of 0.4mg/mL is crossed 0.45 μm of miillpore filter, is saved backup in 4 DEG C.
The preparation of test sample acid hydrating solution: taking Single Roxburgh Rose Fruit, roxburgh rose root and roxburgh rose leaf natural air drying, crushes, and crosses 40 meshes,
The methanol 50mL of the hydrochloric acid containing 1.2mol/L is added in accurately weighed 5.0g, and 85 DEG C of reflux 8h are concentrated to dryness with Rotary Evaporators, residual
Slag is dissolved with dimethyl sulfoxide, and constant volume is in 100mL volumetric flask, as mother liquor.Single Roxburgh Rose Fruit and roxburgh rose root mother liquor mobile phase is dilute
Release 25 times, take roxburgh rose leaf mother liquor flow 50 times of phase dilution, through 0.45 μm of filtering with microporous membrane to get.
The preparation of the free ellagic acid solution of test sample: the free ellagic acid solution of the test sample is made in the steps below
It is standby: to take Single Roxburgh Rose Fruit, roxburgh rose root and roxburgh rose leaf natural air drying, crush, cross 40 meshes, it is molten that 90% methanol is added in accurately weighed 5.0g
Liquid 50mL, room temperature extract 8h, are ultrasonically treated 30min, filter, are concentrated to dryness with Rotary Evaporators, and residue is dissolved with dimethyl sulfoxide,
Constant volume is in 100mL volumetric flask, as mother liquor.The mother liquor of Single Roxburgh Rose Fruit, roxburgh rose root and roxburgh rose leaf is used into 25 times of phase dilution of flowing,
Through 0.45 μm of filtering with microporous membrane to get.
Chromatographic condition: chromatographic column: ACE Excel 2C18-Amide(2μm,2.1mm×100mm)；Mobile phase: 0.1% first
Sour water (A)-acetonitrile (B), gradient elution (0~5min, 3% → 6%B；5~10min, 6% → 16%B；10~25min, 16%
→ 21%B；25~35min, 21% → 30%B)；Column temperature: 40 DEG C；Flow velocity: 0.2mL/min；Detection wavelength: 254nm, sample introduction
Amount: 3 μ L.UPLC chromatogram is shown in Fig. 1.Under the chromatographic condition, baseline is steady, and the peak shape of ellagic acid is good, and separating degree > 1.5.
Measuring method: accurate absorption reference substance solution, test sample acid hydrating solution and the free ellagic acid of test sample are molten respectively
Liquid measures peak area under above-mentioned chromatographic condition respectively, calculate to get.
The gradient elution program is；0~5min, 3% → 6%B；5~10min, 6% → 16%B；10~25min,
16% → 21%B；25~35min, 21% → 30%B.
Claims (5)
1. ellagic acid content assaying method in a kind of Rosa roxburghii Tratt, it is characterised in that: the measurement to ellagic acid content in Rosa roxburghii Tratt is:
The preparation of reference substance solution: precision weighs reference substance ellagic acid, adds flowing phased soln that solution is made, and crosses 0.45 μm of micropore filter
Reference substance solution is made in film, saves in 4 DEG C；
The preparation of test sample acid hydrating solution: taking Rosa roxburghii Tratt active component powder, and the methanol of acidification is added, and flows back, and concentration is added two
First sulfoxide dissolution, with flowing phase dilution, cross 0.45 μm of filtering with microporous membrane, take filtrate to get；
The preparation of the free ellagic acid solution of test sample: taking Rosa roxburghii Tratt active component powder, and methanol solution is added, and room temperature is extracted, ultrasound
It handles, is concentrated after suction filtration, dimethyl sulfoxide dissolution is added, with flowing phase dilution, cross 0.45 μm of filtering with microporous membrane, take filtrate, i.e.,
?；
Chromatographic condition: chromatographic column: 2 C of ACE Excel18- Amide, 2 μm, 2.1mm × 100mm；Mobile phase: 0.1% formic acid water
For A phase, acetonitrile is B phase, gradient elution: flow velocity: 0.2mL/min；Detection wavelength: 254nm, sample volume: 3 μ L；40 DEG C of column temperature；
Measuring method: it is accurate respectively to draw the free ellagic acid solution of reference substance solution, test sample acid hydrating solution and test sample,
Under above-mentioned chromatographic condition, measure peak area respectively, calculate to get.
2. ellagic acid content assaying method in a kind of Rosa roxburghii Tratt according to claim 1, it is characterised in that: the test sample
Sour hydrating solution is prepared in the steps below: being taken Single Roxburgh Rose Fruit, roxburgh rose root and roxburgh rose leaf natural air drying, is crushed, crosses 40 meshes, essence
Close weighed 5.0g, is added the methanol 50mL of the hydrochloric acid containing 1.2mol/L, and 85 DEG C of reflux 8h are concentrated to dryness, residue with Rotary Evaporators
It is dissolved with dimethyl sulfoxide, constant volume is in 100mL volumetric flask, as mother liquor.By Single Roxburgh Rose Fruit and roxburgh rose root mother liquor flowing phase dilution
25 times, take roxburgh rose leaf mother liquor flow 50 times of phase dilution, through 0.45 μm of filtering with microporous membrane to get.
3. ellagic acid content assaying method in a kind of Rosa roxburghii Tratt according to claim 1, it is characterised in that: the test sample
Free ellagic acid solution is prepared in the steps below: being taken Single Roxburgh Rose Fruit, roxburgh rose root and roxburgh rose leaf natural air drying, is crushed, crosses 40 mesh
90% methanol solution 50mL, 10-30 DEG C of extraction 8h is added in sieve, accurately weighed 5.0g, is ultrasonically treated 30min, filters, with rotation
Evaporimeter is concentrated to dryness, and residue is dissolved with dimethyl sulfoxide, and constant volume is in 100mL volumetric flask, as mother liquor.By Single Roxburgh Rose Fruit, Rosa roxburghii Tratt
The mother liquor of root and roxburgh rose leaf with flowing 25 times of phase dilution, through 0.45 μm of filtering with microporous membrane to get.
4. ellagic acid content assaying method in a kind of Rosa roxburghii Tratt according to claim 1, it is characterised in that: the reference substance is molten
Liquid is prepared: taking ellagic acid reference substance appropriate, accurately weighed, ellagic acid reference substance adds mobile phase that the molten of 0.4mg/mL is made
Liquid is crossed 0.45 μm of miillpore filter, is saved backup in 4 DEG C.
5. ellagic acid content assaying method in a kind of Rosa roxburghii Tratt according to claim 1, it is characterised in that: the gradient is washed
De- program is；0~5min, 3% → 6%B；5~10min, 6% → 16%B；10~25min, 16% → 21%B；25~
35min, 21% → 30%B.
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