CN109085258A - A kind of quality determining method of Ficus lyrata - Google Patents
A kind of quality determining method of Ficus lyrata Download PDFInfo
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- CN109085258A CN109085258A CN201810748960.4A CN201810748960A CN109085258A CN 109085258 A CN109085258 A CN 109085258A CN 201810748960 A CN201810748960 A CN 201810748960A CN 109085258 A CN109085258 A CN 109085258A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The invention discloses a kind of quality determining methods of Ficus lyrata, including carry out thin-layer qualitative identification to psoralen and bergapten, and be measured to its content using efficient liquid phase.The thin-layer identification method specificity is strong, and negative noiseless, which has stronger specificity, durability, and accuracy, reproducibility, stability can reach the requirement of research and production, can effectively ensure that the stability and controllability of product quality.
Description
Technical field
The invention belongs to technical field of traditional Chinese medicines, and in particular to a kind of quality determining method of Ficus lyrata.
Background technique
Ficus lyrata derives from Moraceae ficus species Ficus lyrata Ficus pandurata Hance, is used as medicine with root, leaf, property
Temperature, sweet in flavor, micro-pungent have effects that dispelling wind and eliminating dampness, removing toxicity for detumescence, promoting the circulation of qi, promoting blood circulation for regulating menstruation, channels sootheing and network vessel quickening, treat malaria, promoting lactation.
It is civil often with the form of therapy traumatic injury of Ficus lyrata simple or polypharmacy, irregular menstruation, have a pain in the back, jaundice, acute mastitis,
Malaria, mazoitis, stomachache, pertussis, venomous snake bite, damp and hot middle resistance, the card etc. of nauseous loss of appetite.The study found that Ficus lyrata pair
There is certain curative effect in bacterial prostatitis, and have for hepatic injury and preferable inhibit to repair effect, it is seen that its have compared with
High medical value.
Ficus lyrata chemical component mainly includes triterpenes, Coumarins, flavonoids, phenolic acid class etc..Huiqing Lv et al.
Separation identifies psoralen and bergapten from the alcohol extract of Ficus lyrata, but has no its Qualitive test and assay side
The report in face also has no the relevant report of quality of medicinal material detection method.
Summary of the invention
In order to overcome the above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a kind of quality testing sides of Ficus lyrata
Method, 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 achieved through the following technical solutions:
A kind of quality determining method of Ficus lyrata, including following methods:
(1) the thin-layer chromatography Qualitive test of psoralen:
Ficus lyrata root 0.5g is taken, ethyl acetate 15-25ml is added, is ultrasonically treated 10-20 minutes, filtration, filtrate is evaporated, and residue adds second
Acetoacetic ester 1ml makes to dissolve, as test solution；Psoralen reference substance separately is taken, adds ethyl acetate that every 1ml is made containing 2mg's
Solution, as reference substance solution；It is tested according to the thin-layered chromatography Pharmacopoeia of the People's Republic of China four general rules 0502 of version in 2015,
Each 4 μ l of above-mentioned test solution, reference substance is drawn respectively, is put respectively on same silica gel g thin-layer plate, oneself is positive with volume ratio
Alkane-ethyl acetate=2-4:1 is solvent, is unfolded, and takes out, dries, and sprays with 10% potassium hydroxide methanol solution, sets ultraviolet lamp
It is inspected under 365nm；The results show that sample chromatogram at the position corresponding to the chromatogram of the reference substance, shows identical fluorescence spot；
(2) the thin-layer chromatography Qualitive test of bergapten:
0.5 g of Ficus lyrata root is taken, methanol 15-25ml is added, is ultrasonically treated 10-20min, filtration, filtrate is evaporated, and residue adds methanol
1ml makes to dissolve, as test solution；Bergapten reference substance separately is taken, adds methanol that the solution that every 1ml contains 2 mg is made, is made
For reference substance solution；It tests according to the thin-layered chromatography Pharmacopoeia of the People's Republic of China four general rules 0502 of version in 2015, inhales respectively
Each 4 μ l of above-mentioned test solution, reference substance solution is taken, is put respectively in same using sodium carboxymethylcellulose as the silica G of binder
It is solvent by n-hexane-ethyl acetate=2-4:1 of volume ratio on lamellae, is unfolded, takes out, dry, set ultraviolet lamp 365
It is inspected under nm, the results show that sample chromatogram shows identical fluorescence spot on the corresponding position of chromatography identical as reference substance；
(3) assay of psoralen and bergapten:
It is measured using high performance liquid chromatography, specific method and steps are as follows:
Chromatographic condition and system suitability are filler with octadecylsilane chemically bonded silica；It is 55:45's with volume ratio
Methanol-water is mobile phase；Detection wavelength is 246nm, flow velocity 1.0ml/min, 30 DEG C of column temperature, number of theoretical plate by psoralen,
Bergapten calculates, and 3000 should be not less than by calculating；
Precision weighs psoralen reference substance respectively, bergapten reference substance is 10.2mg for the preparation of reference substance solution, sets
In 25ml measuring bottle, scale is dissolved and is settled to methanol, and precision measures 1ml and sets in 10ml measuring bottle, adds methanol dilution to scale, shake
It is even, as reference substance solution；
Ficus lyrata root is ground into most coarse powder by the preparation of test solution, takes 1.0 g of medicinal material coarse powder, accurately weighed, sets 100 ml
In conical flask, methanol 50ml is added in precision, and weighed weight ultrasonic extraction 20-40 minutes, is let cool, then weighed weight, is mended with methanol
The weight of sufficient less loss, shakes up, filtration, take subsequent filtrate to get；
Measuring method difference is accurate to draw hybrid standard product solution and each 10 μ l of test solution, injects liquid chromatograph, measures,
To obtain the final product.
In the thin-layer chromatography Qualitive test of psoralen, the present invention has investigated influence of the different solvents to chromatographic behavior:
It is unfolded by n-hexane-ethyl acetate (4:1) solvent of volume ratio, discovery untested compound spot and R f value is about 0.125, adjacent
The separating degree of spot and untested compound spot basically reaches requirement, but map background color is deep, makes to compound spots to be measured
At a degree of interference.It is unfolded by the solvent of n-hexane-ethyl acetate (2:1) of volume ratio, untested compound spot and R f
Value about 0.5 in sample chromatogram, on position corresponding with reference substance spot chromatography, shows the spot of same color, in thin layer figure
Principal spot is clearly easily debated, and separating degree is good, and adjacent spots are noiseless.Therefore selecting volume ratio is the exhibition of n-hexane-ethyl acetate 2-4:1
Open system, preferably volume ratio are n-hexane-ethyl acetate=2:1 development system.
In the thin-layer chromatography Qualitive test of bergapten, the present invention has investigated different solvents to the shadow of chromatographic behavior
It rings: being unfolded by n-hexane-ethyl acetate (4:1) solvent of volume ratio, discovery untested compound spot and R f value is about 0.125,
Adjacent spots and the separating degree of untested compound spot basically reach requirement, but map background color is deep, to untested compound spot
Point causes a degree of interference.It is unfolded by the solvent of n-hexane-ethyl acetate (2:1) of volume ratio, untested compound spot
Point Rf value about 0.5 in sample chromatogram, on position corresponding with reference substance spot chromatography, shows the spot of same color, thin layer
Principal spot is clearly easily debated in figure, and separating degree is good, and adjacent spots are noiseless.Therefore selecting volume ratio is n-hexane-ethyl acetate 2-4:1
Development system, preferred volume ratio be n-hexane-ethyl acetate=2:1 development system.
By reference to the spectrum testing result discovery of the HPLC full figure of pertinent literature and Ficus lyrata root, in root only psoralen with
Bergapten content is higher, and other compositions content can almost be disregarded.Therefore, the present invention selects psoralen and bergamot
Lactone is reasonably that two kinds of compound stabilities are good as index components, reduces the introducing of systematic error, improves measurement knot
The accuracy and reproducibility of fruit guarantee the stability and quality controllability of Ficus lyrata quality.
Compared with prior art, the present invention having the following beneficial effects:
The present invention establishes the quality determining method of Ficus lyrata for the first time, including carries out thin layer to psoralen and bergapten and determine
Property identify, and its content is measured using efficient liquid phase.The thin-layer identification method specificity is strong, and negative noiseless, this contains
Quantity measuring method has stronger specificity, durability, and accuracy, reproducibility, stability can reach research and production
It is required that can effectively ensure that the stability and controllability of product quality.
Detailed description of the invention
Fig. 1 is that the thin layer of psoralen in Ficus lyrata identifies chromatogram；Wherein 1-2 be respectively as follows: psoralen reference substance,
Ficus lyrata root；
Fig. 2 is that the thin layer of bergapten in Ficus lyrata identifies chromatogram；Wherein 1-2 be respectively as follows: bergapten reference substance,
Ficus lyrata root；
Fig. 3 is that psoralen and bergapten reference substance HPLC scheme (0~10min, 5% methanol~55% methanol；10~
20min, 55% methanol~95% methanol)；
Fig. 4 is that psoralen and bergapten reference substance HPLC scheme (0~10min, 5% methanol~55% methanol；10~
20min, -45% water of 55% methanol)；
Fig. 5 is that psoralen and bergapten reference substance HPLC scheme (0~15min, -45% water of 55% methanol)；
Fig. 6 is Ficus lyrata sample HPLC figure.
Specific embodiment
Further illustrate that the present invention, following embodiment are the specific embodiment party of the present invention below by specific embodiment
Formula, but embodiments of the present invention are not limited by following embodiments.
Embodiment 1:
A kind of quality determining method of Ficus lyrata, including following methods:
1 instrument and reagent
1.1 instrument Agilent, 1100 type high performance liquid chromatograph, BS224S type electronic balance (Beijing Sai Duolisi instrument system
Unite Co., Ltd, and d=0.0001 is g)；KQ3200E ultrasonic cleaning machine for medical purpose (Kunshan Ultrasonic Instruments Co., Ltd.)；HH-S4
Digital display thermostat water bath (Medical Instruments factory of Jintan City)；East-A type directly-heated type Constant Temp. Oven (Guangzhou east electric heating dry
Dry instrument factory).
1.2 reagent Ficus lyrata medicinal materials are to adopt certainly in Jiangmen City of Guangdong Province, teaching and research through Traditional Chinese Medicine University Of Guangzhou's Pharmacognosy Studies
Room Huang Haibo professor is accredited as the dry root of Moraceae ficus species Ficus lyrata Ficus pandurata Hance；Psoralen pair
According to product (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 110878-200102)；Bergapten reference substance (Chengdu Man Sitesheng
Object Science and Technology Ltd., lot number: MUST-17053003)；Silica gel g thin-layer plate (Haiyang Chemical Plant, Qingdao)；Methanol is chromatographically pure；
Remaining reagent is that analysis is pure, and water is Watson distilled water.
Thin-layer chromatography Qualitive test
The thin-layer chromatography Qualitive test of 2.1 psoralens:
Ficus lyrata root 0.5g is taken, ethyl acetate 20ml is added, is ultrasonically treated 15 minutes, filtration, filtrate is evaporated, and residue adds ethyl acetate
1ml makes to dissolve, as test solution；Psoralen reference substance separately is taken, adds ethyl acetate that solution of every 1ml containing 2mg is made, is made
For reference substance solution；It tests according to the thin-layered chromatography Pharmacopoeia of the People's Republic of China four general rules 0502 of version in 2015, inhales respectively
Each 4 μ l of above-mentioned test solution, reference substance is taken, is put respectively on same silica gel g thin-layer plate, using volume ratio as n-hexane-acetic acid
Ethyl ester=2:1 is solvent, is unfolded, and takes out, dries, and sprays with 10% potassium hydroxide methanol solution, sets and examine under ultraviolet lamp 365nm
Depending on；The results show that sample chromatogram at the position corresponding to the chromatogram of the reference substance, shows identical fluorescence spot, as shown in Figure 1.
The thin-layer chromatography Qualitive test of 2.2 bergaptens:
0.5 g of Ficus lyrata root is taken, methanol 20ml is added, is ultrasonically treated 15min, filtration, filtrate is evaporated, and it is molten that residue adds methanol 1ml to make
Solution, as test solution；Bergapten reference substance separately is taken, adds methanol that the solution that every 1ml contains 2 mg is made, as reference substance
Solution；It is tested according to the thin-layered chromatography Pharmacopoeia of the People's Republic of China four general rules 0502 of version in 2015, draws above-mentioned confession respectively
Each 4 μ l of test sample solution, reference substance solution, puts respectively on the silica gel g thin-layer plate in same using sodium carboxymethylcellulose as binder,
It is solvent by n-hexane-ethyl acetate=2:1 of volume ratio, is unfolded, takes out, dry, set and inspected under 365 nm of ultraviolet lamp,
The results show that sample chromatogram is at the position corresponding to the chromatogram of the reference substance, same color fluorescence spot is shown, as shown in Figure 2.
The assay of psoralen and bergapten:
3.1 chromatographic conditions and system suitability are filler with octadecylsilane chemically bonded silica；With volume ratio for 55:
45 methanol-water is mobile phase；Detection wavelength is 246nm, flow velocity 1.0ml/min, and 30 DEG C of column temperature, number of theoretical plate presses psoralea corylifolia
Element, bergapten calculate, and 3000 should be not less than by calculating；
Precision weighs psoralen reference substance, bergapten reference substance 10.2mg respectively for the preparation of 3.2 reference substance solutions, sets
In 25ml measuring bottle, scale is dissolved and is settled to methanol, and precision measures 1ml and sets in 10ml measuring bottle, adds methanol dilution to scale, shake
It is even, as reference substance solution；
Ficus lyrata root is ground into most coarse powder by the preparation of 3.3 test solutions, takes each 1.0 g of medicinal material coarse powder, accurately weighed, is set
In 100 ml conical flasks, methanol 50ml is added in precision, and weighed weight ultrasonic extraction 30 minutes, is let cool, then weighed weight, uses first
Alcohol supplies the weight of less loss, shakes up, filtration, take subsequent filtrate to get；
3.4 measuring methods difference is accurate to draw reference substance solution and each 10 μ l of test solution, injects liquid chromatograph, measures,
To obtain the final product.
System suitability:
3.5 linear relationships are investigated
Accurate reference substance solution of drawing is appropriate respectively, is diluted to 1.02 ug/mL, 2.04 ug/mL, 4.08 ug/mL, 8.16
The serial mixed standard solution of ug/mL, 16.32 ug/mL, 32.64 ug/mL, and the accurate standard for drawing above-mentioned each concentration is molten
Liquid 10uL injects liquid chromatograph, records chromatographic peak area.With sample introduction concentration (μ gml -1) for abscissa (X), peak area is
Ordinate (Y), carries out linear regression, and psoralen regression equation is Y=76.48X-29.35, r=0.9997, psoralea corylifolia
Plain concentration is in good linear relationship with peak area within the scope of 1.02 ~ 32.64 μ g/ml；Bergapten Y=46.61X-
18.33, r=0.9997, bergapten concentration is in good line with peak area within the scope of 1.02 ~ 32.64 μ g/ mL
Sexual intercourse.
3.6 precision tests: precision draws 10 μ l of reference substance solution, repeats sample introduction 6 times, calculates the mean value of each peak area
And RSD value, psoralen RSD value are 0.75%, bergapten RSD value is 0.67%, the results showed that instrument precision is good
It is good.
3.7 stability tests: taking the test solution of same lot number, injects respectively in 0,1,2,4,6,8,12 h high
Effect liquid phase chromatogram instrument simultaneously calculates each chromatographic peak area, and as a result the RSD value of psoralen chromatographic peak area is 1.13%, in bergamot
The RSD value of ester chromatographic peak area is 1.06%, shows that test solution is stablized in 12 hours.
3.8 repetitive tests: taking the sample about 0.5g of same lot number, totally 6 parts, operate by sample solution preparation method,
High performance liquid chromatograph is injected, is measured in accordance with the law, as a result the RSD value of psoralen chromatographic peak area is 0.85%, bergapten color
The RSD value of spectral peak area is 0.65%.
The test of 3.9 sample recovery rates: precision weighs 6 parts of Ficus lyrata coarse powder (0.513mgg-1) for having predicted content, often
Part about 0.5g, it is accurate respectively that different amounts of psoralen, bergapten reference substance is added, it is grasped by sample solution preparation method
Make, is measured according to above-mentioned chromatographic condition, and calculate the rate of recovery and RSD, as a result the average recovery rate of psoralen is 100.2%,
RSD=1.13%(n=6)；The average recovery rate of bergapten is that 99.8%, RSD value is 1.12%(n=6), rate of recovery symbol
It closes and requires.
Durability is investigated:
4.0 different wave lengths are investigated
Precision weighs psoralen reference substance, bergapten reference substance 10.0mg respectively, sets in 50ml measuring bottle, is dissolved with methanol
And be settled to scale, precision measures 1ml and sets in 50ml measuring bottle, add methanol dilution to shake up to scale, as prepare liquid, successively with
Ultraviolet specrophotometer carries out all-wave length (200~400nm) scanning.The results show that psoralen standard solution exists
There is most strong absorption at 244.60nm；Majudin standard solution has most strong absorption at 248.80nm.Further test, choosing
The wavelength of 246nm is taken to investigate, the results show that psoralen and majudin have preferable absorption under this wavelength condition,
And close to respective most strong absorption, therefore select 246nm as the investigation of optimal absorption wavelength progress follow-up test.
4.1 mobile phase composition ratio investigate test
Select methanol-water as mobile phase investigation object, the standard solution being mixed with psoralen and majudin
It is tested:
Test one, mobile phase is with 0~10min, 5% methanol~55% methanol；10~20min, 55% methanol~95% methanol gradient
Variation, the characteristic peak of psoralen and majudin fails to efficiently separate out under this condition as the result is shown.
Test two, mobile phase is with 0~10min, 5% methanol~55% methanol；10~20min, -45% water of 55% methanol
Variation, clearly separating degree, but its appearance compared with as the result is shown although psoralen has with the characteristic peak of both majudins
Time is partially long, easily causes reagent waste and environmental disruption.
Test three, based on test one with test two as a result, adjusting mobile phase with 0~15min, -45% water of 55% methanol
Isocratic variation, the results show that the characteristic peak separating degree of both psoralen and majudin better than both of the aforesaid test,
And appearance time is short, is conducive to avoid unnecessary reagent waste, it is both economically and environmentally beneficial.
To sum up, therefore it is preferred that the isocratic elution method of -45% water of 55% methanol carries out following liquid phase analysis with 0~15min.
Criticize the assay of Ficus lyrata test sample
3 batches of Ficus lyrata coarse powder are taken to be measured respectively, the results are shown in Table 1:
Table 1
。
Claims (2)
1. a kind of quality determining method of Ficus lyrata, which is characterized in that including following methods:
(1) the thin-layer chromatography Qualitive test of psoralen:
Ficus lyrata root 0.5g is taken, ethyl acetate 15-25ml is added, is ultrasonically treated 10-20 minutes, filtration, filtrate is evaporated, and residue adds second
Acetoacetic ester 1ml makes to dissolve, as test solution；Psoralen reference substance separately is taken, adds ethyl acetate that every 1ml is made containing 2mg's
Solution, as reference substance solution；It is tested according to the thin-layered chromatography Pharmacopoeia of the People's Republic of China four general rules 0502 of version in 2015,
Each 4 μ l of above-mentioned test solution, reference substance is drawn respectively, is put respectively on same silica gel g thin-layer plate, oneself is positive with volume ratio
Alkane-ethyl acetate=2-4:1 is solvent, is unfolded, and takes out, dries, and sprays with 10% potassium hydroxide methanol solution, sets ultraviolet lamp
It is inspected under 365nm；The results show that sample chromatogram at the position corresponding to the chromatogram of the reference substance, shows identical fluorescence spot；
(2) the thin-layer chromatography Qualitive test of bergapten:
0.5 g of Ficus lyrata root is taken, methanol 15-25ml is added, is ultrasonically treated 10-20min, filtration, filtrate is evaporated, and residue adds methanol
1ml makes to dissolve, as test solution；Bergapten reference substance separately is taken, adds methanol that the solution that every 1ml contains 2 mg is made, is made
For reference substance solution；It tests according to the thin-layered chromatography Pharmacopoeia of the People's Republic of China four general rules 0502 of version in 2015, inhales respectively
Each 4 μ l of above-mentioned test solution, reference substance solution is taken, is put respectively in same using sodium carboxymethylcellulose as the silica G of binder
It is solvent by n-hexane-ethyl acetate=2-4:1 of volume ratio on lamellae, is unfolded, takes out, dry, set ultraviolet lamp 365
It is inspected under nm, the results show that sample chromatogram is at the position corresponding to the chromatogram of the reference substance, shows same color fluorescence spot；
(3) assay of psoralen and bergapten:
It is measured using high performance liquid chromatography, specific method and steps are as follows:
Chromatographic condition and system suitability are filler with octadecylsilane chemically bonded silica；It is 55:45's with volume ratio
Methanol-water is mobile phase；Detection wavelength is 246nm, flow velocity 1.0ml/min, 30 DEG C of column temperature, number of theoretical plate by psoralen,
Bergapten calculates, and should all be not less than 3000；
Precision weighs psoralen reference substance, bergapten reference substance 10.2mg respectively for the preparation of reference substance solution, sets 25ml
In measuring bottle, scale is dissolved and is settled to methanol, precision measures 1ml and sets in 10ml measuring bottle, and add methanol dilution to shake up to scale,
As reference substance solution；
Ficus lyrata root is ground into most coarse powder by the preparation of test solution, takes 1.0 g of medicinal material coarse powder, accurately weighed, sets 100 ml
In conical flask, methanol 50ml is added in precision, and weighed weight ultrasonic extraction 20-40 minutes, is let cool, then weighed weight, is mended with methanol
The weight of sufficient less loss, shakes 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, measurement to get.
2. a kind of quality determining method of Ficus lyrata according to claim 1, which is characterized in that (1) or (2)) in,
It is solvent by n-hexane-ethyl acetate=2:1 of volume ratio.
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| CN112903868A (en) * | 2021-02-04 | 2021-06-04 | 中山市中医院 | Method for measuring contents of various chemical components in compound guava preparation |
| CN112903868B (en) * | 2021-02-04 | 2022-06-28 | 中山市中医院 | Method for measuring contents of various chemical components in compound guava preparation |
| CN113456695A (en) * | 2021-08-11 | 2021-10-01 | 中山市中医院 | Application of ficus microcarpa petroleum ether part in preparation of medicine for preventing or treating liver cancer |
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Application publication date: 20181225 |