CN102879502A - Method for identifying lonicera similis hemsl high-quality medicinal materials - Google Patents
Method for identifying lonicera similis hemsl high-quality medicinal materials Download PDFInfo
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Abstract
The invention discloses a method for identifying a lonicera similis hemsl high-quality medicinal material. The method comprises the steps of measuring the medicinal material through high performance liquid chromatography, wherein the content of 5-hydroxy-7,3',4'-trimethoxy flavone in the medicinal material is over 0.3 weight percent. The invention also provides a method for detecting the lonicera similis hemsl medicinal material or extract thereof. According to the method, the compound of 5-hydroxy-7,3',4'-trimethoxy flavone is found in the lonicera similis hemsl at the first time; and meanwhile, the compound is used for identifying the index components of the lonicera similis hemsl, the lonicera similis hemsl can be effectively distinguished from other honeysuckle medicinal materials recorded in the conventional Chinese pharmacopoeia, and a novel method is provided for quality identification.
Description
Technical field
The present invention relates to the authentication method of L. similis Hemsl certified products medicinal material.
Background technology
Honeysuckle is a kind of Chinese crude drug commonly used, can clearing heat and detoxicating, wind-heat dissipating.Before 2005, all classify Caprifoliaceae honeysuckle, lonicera hypoglauca miq, mountain honeysuckle flower and hair style honeysuckle the plant origin of honeysuckle as, caused the situation of honeysuckle one medicine multi-source.But from chemical composition analysis, as the galuteolin of main medicinal ingredient, content is higher in honeysuckle, and in the honeysuckle flower of mountain content seldom, both component differences are larger.Therefore, after 2005, " Chinese pharmacopoeia stipulated, the dry flower of Caprifoliaceae honeysuckle or the flower of just opening are the certified products honeysuckle, and with all the other kinds: largeflower-like honeysuckle flower, lonicera hypoglauca miq, Lonicera confusa, fulvoushair honeysuckle flower are classified mountain honeysuckle flower plant origin as.Since then, because the source of honeysuckle is less, its price also rises gradually; And other popular medication sources of honeysuckle, because output is higher, cost is lower, the phenomenon of usefulness also just occurred replacing with mountain honeysuckle flower or other caprifoliaceae plants.
L. similis Hemsl Lonicera similes Hemsl, main flow kind for river pan honeysuckle flower, it is wide to distribute, the resource reserves greatly according to investigations, the honeysuckle that Chengdu lotus pond Chinese crude drug specialized market is sold, the honeysuckle that shop of Chinese medicines and medical institutions use in the Sichuan Province mostly is southern river pan honeysuckle flower (L. similis Hemsl), and sells to all parts of the country.In recent years, studies show that although have much, it is suitable that the green pharmacological action of L. similis Hemsl and pharmacopeia are recorded the honeysuckle kind, but river honeysuckle flower Dominant variety L. similis Hemsl is not selected in Chinese Pharmacopoeia yet at present, especially " Chinese pharmacopoeia version in 2010, strengthened discriminating and assay to Chinese crude drug specificity composition, and L. similis Hemsl still lacks relevant fundamental research, this has restricted the utilization of river pan honeysuckle flower resource greatly.
For the continuation use of giving L. similis Hemsl provides reliable basis, at first need to study material composition and the quality index of L. similis Hemsl, yet, at present rarely seen this type of research to L. similis Hemsl.
Summary of the invention
The object of the present invention is to provide the authentication method of the stronger L. similis Hemsl certified products medicinal material of a kind of specificity.
The invention provides the authentication method of L. similis Hemsl certified products medicinal material, the material of getting it filled is measured by high performance liquid chromatography, 5-hydroxyl-7,3 in the medicinal material ', 4 '-content of trimethoxy flavone is more than 0.3%w/w.
Further, 5-hydroxyl-7,3 in the medicinal material ', 4 '-content of trimethoxy flavone is at 0.3 ~ 1.2%w/w.
Further preferably, 5-hydroxyl-7,3 in the medicinal material ', 4 '-content of trimethoxy flavone is at 0.36 ~ 1.08%w/w.
Wherein, the chromatographic condition of described high performance liquid chromatography is as follows:
Chromatographic column: octadecyl silane post
Mobile phase: acetonitrile: 0.3-0.5% phosphoric acid=(20-24): (76-80)
Column temperature: 25-35 ℃
Detect wavelength: 269 ± 2nm.
Further, described mobile phase is acetonitrile: 0.4% phosphoric acid=22:78; Column temperature is 30 ℃; Detect wavelength: 269nm.
Wherein, in the described high performance liquid chromatography, the preparation method of need testing solution is as follows:
Get L. similis Hemsl, pulverize, extract with the 60-80%v/v alcohol reflux, filter, measure subsequent filtrate, after the acid hydrolysis, be concentrated into driedly, residue adds methyl alcohol makes dissolving, and constant volume namely gets need testing solution.
Further, the concentration of described ethanol is 70%v/v.
Further, in the acid hydrolysis step, used acid is hydrochloric acid.
Further preferably, the concrete operations of acid hydrolysis are: add 1mol/L hydrochloric acid, the volume ratio of hydrochloric acid and subsequent filtrate is 5:1, puts heating hydrolysis in 90 ℃ of water-baths.
The present invention also provide L. similis Hemsl medicinal material or extract detection method, it adopts HPLC to carry out, operation steps is as follows:
(1) get L. similis Hemsl, pulverize, extract with the 60-80%v/v alcohol reflux, filter, measure subsequent filtrate, after the acid hydrolysis, be concentrated into driedly, residue adds methyl alcohol makes dissolving, and constant volume namely gets need testing solution;
(2) with 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone is reference substance, the preparation reference substance solution;
(3) respectively with in need testing solution, the reference substance solution injection liquid chromatography, detect, get final product, wherein, the chromatographic condition of described high performance liquid chromatography is as follows:
Chromatographic column: octadecyl silane post
Mobile phase: acetonitrile: 0.3-0.5% phosphoric acid=(20-24): (76-80)
Column temperature: 25-35 ℃
Detect wavelength: 269 ± 2nm.
Among the present invention, in L. similis Hemsl, found first 5-hydroxyl-7,3 ', 4 '-existence of this compound of trimethoxy flavone, simultaneously, as the index components of identifying L. similis Hemsl, honeysuckle other kinds of class that L. similis Hemsl and existing Chinese Pharmacopoeia can be recorded are effectively distinguished with this compound, its specificity is strong, for its Quality Identification provides a kind of new method.
Description of drawings
Fig. 1 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone (5-hydroxyl-7,3', 4'-trimethoxyflavone) hydrogen spectrogram
Fig. 2 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone (5-hydroxyl-7,3', 4'-trimethoxyflavone) carbon spectrogram
Chromatograms when Fig. 3 mobile phase is methyl alcohol-0.1% phosphoric acid (70:30)
Chromatograms when Fig. 4 mobile phase is methyl alcohol-tetrahydrofuran-water (13:4:11)
Chromatograms when Fig. 5 mobile phase is acetonitrile-0.4% phosphoric acid (22:78)
Embodiment
Test used L. similis Hemsl medicinal material and purchase in Nanjiang County, Sichuan Province, identify through professor Ma Yuying of Chengdu University of Traditional Chinese Medicine, be caprifoliaceae plant L. similis Hemsl Lonicera similis Hemsl.
(1) take by weighing the dry flower 1.9Kg of L. similis Hemsl Lonicera similes Hemsl, the alcohol reflux with 90%, 80%, 70% extracts, and material ratio is 1: 8, filters, and decompression recycling ethanol gets medicinal extract.After measured, contain general flavone 3.39% in this extract.
(2) get above-mentioned medicinal extract, add water preparation upper prop liquid, upper column liquid concentration is counted 0.10g/ml with crude drug, upper large pore resin absorption column, successively water, concentration be 10%, 20%, 40%, 60%, 80%, the ethanol elution of 95%V/V, collect 95% ethanol position;
(3) get gained ethanol position, behind the recovery solvent, upper silica gel column chromatography (200-300 order, 350g, column volume 925ml), usefulness cyclohexane-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ system carried out gradient elution in 0: 1, and every stream part 150ml collects eluent, the thin layer monitoring merges same stream part, gets 39 ~ 59 parts, after reclaiming solvent, redissolve with methyl alcohol, go up again the SephadexLH-20 gel column, use methanol-eluted fractions, collect eluent, reclaim solvent, recrystallization, namely get 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone (35mg), the rate of transform is 0.17%, measure through HPLC, purity is 99.5%.
The condition of gradient elution is:
Hexane-ethyl acetate=20: 1 (997.5ml), cyclohexane-ethyl acetate=10: 1 (990ml), cyclohexane-ethyl acetate=8: 1 (3050ml), cyclohexane-ethyl acetate=6: 1 (970ml), cyclohexane-ethyl acetate=5: 1 (1901ml), cyclohexane-ethyl acetate=4:1 (1855ml), cyclohexane-ethyl acetate=3:1 (3755ml), cyclohexane-ethyl acetate=2:1 (1550ml), cyclohexane-ethyl acetate=1:1 (1960ml), ethyl acetate (1500ml), ethyl acetate-methyl alcohol=5:1 (2035ml), ethyl acetate-methyl alcohol=3:1 (1430ml), ethyl acetate-methyl alcohol=2:1 (1335ml), ethyl acetate-methyl alcohol=1:1 (1500ml), methyl alcohol (1500ml).
Compound identification:
The gained compound is yellow powder, mp167 ~ 169 ℃.Develop the color spray AlCl under the TLC uviol lamp
3Yellow fluorescence is arranged, and hydrochloric acid-magnesium powder reaction is positive, and ferric chloride reaction is positive.Tentatively be judged as thus flavonoids or flavonols compound.
ESI-MS?m/z:329[M+H]
+。
1H-NMR(C
5D
5N,400MHz)δ:7.07(1H,s,H-3),6.60(1H,s,H-6),6.73(1H,s,H-8),7.57(1H,s,H-2′),7.06(1H,d,J=8.6Hz,H-5'),7.66(1H,d,J=8.6Hz,H-6′),13.60(1H,s,5-OH),3.82(3H,s,-OCH
3),3.79(3H,s,-OCH
3),3.72(3H,s,-OCH
3)。
13C-NMR(C
5D
5N,100MHz)δ:164.4(C-2,s),104.9(C-3,d),182.9(C-4,s),162.7(C-5,s),98.8(C-6,d),166.0(C-7,s),93.1(C-8,d),158.2(C-9,s),106.0(C-10,s),123.2(C-1',s),110.0(C-2',d),149.6(C-3',s),153.2(C-4',s),112.20(C-5',d),120.7(C-6',d),56.5(-OCH
3),56.4(-OCH
3),56.2(-OCH
3)。Above data and document are basically identical, thus be defined as 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone.
Compound VII 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone (5-hydroxyl-7,3', 4'-trimethoxyflavone)
The authentication method of embodiment 2 L. similis Hemsl certified products medicinal materials of the present invention
5-hydroxyl-7,3 ', 4 '-trimethoxy flavone to staphylococcus aureus etc. have antibacterial action (Xing Junbo is etc., In Flower Buds of Lonicera Japonica Thunb chemical constitution study [J]. Chinese Journal of New Drugs, 2002,11 (11): 856-859), matching with antibiotic, the antiviral effect of L. similis Hemsl, is the effective constituent that its function of reflection cures mainly, at present, yet there are no the relevant report that contains this compound in the L. similis Hemsl.
For L. similis Hemsl is distinguished with honeysuckle other medicinal kinds of class that existing Chinese Pharmacopoeia records, simultaneously in order to provide a kind of L. similis Hemsl exclusive authentication method, the present invention adopt high performance liquid chromatography to various honeysuckles medicinal kind carried out 5-hydroxyl-7,3 ', 4 '-assay of trimethoxy flavone.
(1) preparation of need testing solution:
Get respectively L. similis Hemsl, honeysuckle, largeflower-like honeysuckle flower, fulvoushair honeysuckle flower, the about 1g of Lonicera confusa powder (crossing sieve No. three), accurately weighed, put in the tool plug conical flask accurate 70% ethanol 50ml, the weighed weight of adding, heating and refluxing extraction 1 hour, let cool, weighed weight is supplied the weight that subtracts mistake with 70% ethanol again, shake up, filter.Precision is measured subsequent filtrate 20ml, and the accurate 1mol/L hydrochloric acid 4ml that adds put in 90 ℃ of water-baths heating hydrolysis 2 hours, took out, and is concentrated into driedly, and residue adds methyl alcohol makes dissolving, is settled to 1ml, namely gets the need testing solution of various honeysuckle medicinal plant.
(2) preparation of reference substance solution:
Get 5-hydroxyl-7,3 ', 4 '-(embodiment 1 preparation detects through HPLC trimethoxy flavone, and purity is 99.5%, adds methyl alcohol and prepares reference substance solution.
(3) respectively need testing solution and reference substance solution are carried out HPLC and detect, calculate 5-hydroxyl-7,3 in the L. similis Hemsl ', 4 '-content of trimethoxy flavone, chromatographic condition is as follows:
Chromatographic column: Diamonsil
TMC
18HPLC chromatographic column (250 * 4.6mm, 5 μ m)
Mobile phase: acetonitrile-0.4% phosphoric acid (22:78)
Flow velocity: 1.0ml/min
Column temperature: 30 ℃
Sample size: 10 μ l
Detect wavelength: 269nm
Under above selected condition, 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone and sample in other component chromatographic peaks can substantially separate, 5-hydroxyl-7,3 ', 4 '-degree of separation that trimethoxy flavone is adjacent chromatographic peak is greater than 1.5, and peak shape is symmetrical, sharp-pointed, and theoretical cam curve is 6000.The performance of considering different instruments, chromatographic column may there are differences, thus decide number of theoretical plate with 5-hydroxyl-7,3 ', 4 '-the trimethoxy flavone meter should be not less than 4000.
Testing result is as follows:
5-hydroxyl-7,3 in each sample of table 1 ', 4 '-trimethoxy flavone assay result (n=2)
Annotate: "-" do not show and do not detect.The medicinal material sample that above-mentioned experiment is used identifies that through professor Ma Yuying of Chengdu University of Traditional Chinese Medicine 1 ~ No. 10 sample is caprifoliaceae plant L. similis Hemsl Lonicera similis Hemsl; 11 ~ No. 12 samples are caprifoliaceae plant Lonicera japonica Thunb.; 13 ~ No. 14 samples are caprifoliaceae plant largeflower-like honeysuckle flower L.macranthoides Hand.Mazz; No. 15 samples are caprifoliaceae plant fulvoushair honeysuckle flower L.fulvotomentose Hsu et S.C.Cheng; No. 16 samples are caprifoliaceae plant Lonicera confusa Lonicera confusa DC..
Above-mentioned experimental result shows:
(1) by L. similis Hemsl and pharmacopeia kind honeysuckle (honeysuckle), mountain honeysuckle flower (fulvoushair honeysuckle flower, largeflower-like honeysuckle flower, Lonicera confusa) comparative study are found, except L. similis Hemsl and pharmacopeia kind honeysuckle, all do not contain 5-hydroxyl-7 in fulvoushair honeysuckle flower, largeflower-like honeysuckle flower, the Lonicera confusa, 3 ', 4 '-trimethoxy flavone; And in honeysuckle, although contain 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone, the content of this compound is lower, does not all reach 0.2%.
The 5-hydroxyl-7,3 of (2) 10 batches of L. similis Hemsls ', 4 '-trimethoxy flavone content reaches as high as 1.08%, and is minimum 0.36%, average out to 0.68%; Each batch sample 5-hydroxyl-7,3 ', 4 '-content of trimethoxy flavone has certain difference, analyzes reason, and may be different relevant from the storage time of the place of production, collecting time and commodity medicinal material.
Add up as can be known according to above-mentioned 10 batch datas, 5-hydroxyl-7,3 in the L. similis Hemsl ', 4 '-content of trimethoxy flavone is all more than 0.3%, therefore, the present invention drafts, with 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone is as the n-compound of identifying L. similis Hemsl, and, if in the honeysuckle class medicinal material, this compounds content can judge that 0.3% when above this medicinal material is L. similis Hemsl.
The detection method of embodiment 3 L. similis Hemsl medicinal materials or extract
According to embodiment 2 as can be known, 5-hydroxyl-7 in the L. similis Hemsl medicinal material, 3 ', 4 '-trimethoxy flavone content is higher, and other pharmacopeia record do not contain this compounds in the kind or content lower, simultaneously, this compound has antibacterial action to staphylococcus aureus etc., match with antibiotic, the antiviral effect of L. similis Hemsl, the effective constituent that its function of reflection cures mainly, therefore, be index components if select this compound, can reflect to a certain extent the quality height of L. similis Hemsl medicinal material or its extract.
To sum up, the present invention is by adopting HPLC that L. similis Hemsl medicinal material or extract are carried out quality testing, and operation steps is as follows:
(1) get L. similis Hemsl, pulverize, the accurate 70% ethanol 50ml that adds, weighed weight, heating and refluxing extraction 1 hour lets cool, and weighed weight is supplied the weight that subtracts mistake with 70% ethanol again, shakes up, and filters; Precision is measured subsequent filtrate 20ml, and the accurate 1mol/L hydrochloric acid 4ml that adds put in 90 ℃ of water-baths heating hydrolysis 2 hours, took out, and is concentrated into driedly, and residue adds methyl alcohol makes dissolving, is settled to 1ml, namely gets need testing solution.
(2) get 5-hydroxyl-7,3 ', 4 '-(embodiment 1 preparation detects through HPLC trimethoxy flavone, and purity is 99.5%, adds methyl alcohol and prepares reference substance solution.
(3) respectively with in need testing solution, the reference substance solution injection liquid chromatography, detect, get final product, wherein, the chromatographic condition of described high performance liquid chromatography is as follows:
Chromatographic column: Diamonsil
TMC
18HPLC chromatographic column (250 * 4.6mm, 5 μ m)
Mobile phase: acetonitrile-0.4% phosphoric acid (22:78)
Flow velocity: 1.0ml/min
Column temperature: 30 ℃
Sample size: 10 μ l
Detect wavelength: 269nm.
The methodological study of high performance liquid chromatography in embodiment 4 authentication methods of the present invention
1 instrument and reagent
Japan Shimadzu LC-20A type high performance liquid chromatograph
Electronic analytical balance BP121S(Beijing Sartorius balance company limited)
5-hydroxyl-7,3 ', 4 '-trimethoxy flavone (the HPLC area normalization method is determined for the preparation of embodiment 1 method, purity 99.5%)
Acetonitrile, methyl alcohol are chromatographically pure (German Fisher), and water is double distilled water, and it is pure that all the other reagent are analysis.
2 5-hydroxyls-7,3 ', 4 '-purity test of trimethoxy flavone
TLC method precision take by weighing 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone 2.7mg, methyl alcohol dissolving is settled to 10ml.According to " 2010 editions one (appendix VI B) methods experiment of Chinese pharmacopoeia, draw respectively 5 μ l, 10 μ l, 20 μ l points on polyamide film, use respectively chloroform-methanol (2: 1), methanol-water (4: 1), three kinds of development systems of 75% microemulsion-formic acid (9: 1) to launch, the colour developing of 1% aluminium choride ethanolic solution all is single spot in the thin-layer chromatography; At the lower experience of ultraviolet lamp (254nm), all be the yellow-green fluorescence spot.
HPLC method precision take by weighing 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone is an amount of, makes the solution that every 1ml contains 0.984mg.Get 10 μ l and inject high performance liquid chromatograph, inspect under 269nm, occur there are no impurity peaks, purity is the 99.5%(HPLC area normalization method).
3 detect the selection of wavelength
Get 5-hydroxyl-7,3 ', 4 '-the trimethoxy flavone reference substance makes with methyl alcohol dissolving and contains 5-hydroxyl-7,3 ', 4 '-solution of trimethoxy flavone 0.984mg/ml, other gets blank methanol solution, on UV-1100 type spectrophotometer, draws ultra-violet absorption spectrum in the scanning of 200～800nm wavelength coverage.5-hydroxyl-7,3 ', 4 '-trimethoxy flavone has a maximum absorption band at 269.0nm wavelength place.
The selection of 4 mobile phases
Investigate respectively mobile phase methanol-0.1% phosphoric acid (70:30), methyl alcohol-tetrahydrofuran-water (13:4:11), acetonitrile-0.4% phosphoric acid (22:78), chromatogram is seen Fig. 3-5.The result shows when mobile phase is acetonitrile-0.4% phosphoric acid (22:78), 5-hydroxyl-7,3 ', but 4 '-trimethoxy flavone and sample in other component chromatographic peak baseline separation, appearance time is stable, suitable, so determine that acetonitrile-0.4% phosphoric acid (22:78) is mobile phase.
The chromatographic condition of determining is as follows:
Chromatographic column: Diamonsil
TMC
18HPLC chromatographic column (250 * 4.6mm, 5 μ m)
Mobile phase: acetonitrile-0.4% phosphoric acid (22:78)
Flow velocity: 1.0ml/min
Column temperature: 30 ℃
Sample size: 10 μ l
Detect wavelength: 269nm
Under above selected condition, 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone and sample in other component chromatographic peaks can substantially separate, 5-hydroxyl-7,3 ', 4 '-degree of separation that trimethoxy flavone is adjacent chromatographic peak is greater than 1.5, and peak shape is symmetrical, sharp-pointed, and theoretical cam curve is 6000.The performance of considering different instruments, chromatographic column may there are differences, thus decide number of theoretical plate with 5-hydroxyl-7,3 ', 4 '-the trimethoxy flavone meter should be not less than 4000.
The investigation of 5 linear relationships
Get above-mentioned 5-hydroxyl-7,3 ', 4 '-the trimethoxy flavone reference substance solution, add methyl alcohol and be diluted to the solution that concentration is 0.1968mg/ml.Draw respectively this solution 1 μ l, 5 μ l, 10 μ l, 15 μ l, 20 μ l, 25 μ l carry out stratographic analysis by above-mentioned chromatographic condition, measure peak area.The results are shown in Table 2.Take peak area as ordinate, 5-hydroxyl-7,3 ', 4 '-amount of trimethoxy flavone is horizontal ordinate, the drawing standard curve gets regression equation.
Table 2 linear relationship examination result
The preparation of 6 need testing solutions
6.1 extracting solvent investigates
Take by weighing respectively 4 parts in L. similis Hemsl sample, every part of about 1.0g, accurately weighed, add respectively methyl alcohol, 50% ethanol, 70% ethanol, 90% ethanol 50ml, weighed weight, refluxing extraction 1 hour.Let cool, weighed weight is supplied the weight that subtracts mistake with corresponding solution respectively again, shakes up, and filters.Precision is measured subsequent filtrate 20ml, adds 1mol/L hydrochloric acid 4ml, refluxes 1 hour, is concentrated into driedly, and residue adds methyl alcohol makes dissolving, is settled to 1ml, and get final product.With 0.45 μ m filtering with microporous membrane, draw 10 μ l before the sample introduction, the injection liquid chromatography is measured according to aforementioned chromatographic condition, calculates peak area.The method operation repetitive is twice like this.The results are shown in Table 3.
Table 3 extracts solvent and investigates (n=2)
Above result shows, the 5-hydroxyl-7,3 of 70% alcohol extract gained ', 4 '-trimethoxy flavone content is higher, therefore selects 70% ethanol as extracting solvent.
6.2 extracting method is investigated
Take by weighing respectively 2 parts in L. similis Hemsl sample, every part of about 1.0g, accurately weighed, each adds 70% ethanol 50ml, weighed weight, a ultrasonic extraction 1 hour, a refluxing extraction 1 hour.Let cool, weighed weight is supplied the weight that subtracts mistake with 70% ethanol again, shakes up, and filters.Precision is measured subsequent filtrate 20ml, and each adds 1mol/L hydrochloric acid 4ml, refluxes 1 hour, is concentrated into driedly, and residue adds methyl alcohol makes dissolving, is settled to 1ml, and get final product.With 0.45 μ m filtering with microporous membrane, draws 10 μ l before the sample introduction, the injection liquid chromatography, the aforementioned chromatographic condition of photograph is measured, 5-hydroxyl-7,3 in the calculation sample ', 4 '-content of trimethoxy flavone.The method operation repetitive is twice like this.The results are shown in Table 4.
Table 4 extracting method is investigated (n=2)
Above result shows, the 5-hydroxyl-7,3 of refluxing extraction method gained ', 4 '-the more ultrasonic extraction of trimethoxy flavone content is high, therefore selects the refluxing extraction method.
6.3 extraction time is investigated
Take by weighing respectively 3 parts in L. similis Hemsl sample, every part of about 1.0g, accurately weighed, add 70% ethanol 50ml, weighed weight, refluxing extraction is 1 hour, 2 hours, 3 hours respectively.Let cool, weighed weight is supplied the weight that subtracts mistake with 70% ethanol again, shakes up, and filters.Precision is measured subsequent filtrate 20ml, adds 1mol/L hydrochloric acid 4ml, refluxes 1 hour, is concentrated into driedly, and residue adds methyl alcohol makes dissolving, is settled to 1ml, and get final product.With 0.45 μ m filtering with microporous membrane, draws 10 μ l before the sample introduction, the injection liquid chromatography, the aforementioned chromatographic condition of photograph is measured, 5-hydroxyl-7,3 in the calculation sample ', 4 '-content of trimethoxy flavone.The method operation repetitive is twice like this.The results are shown in Table 5.
Table 5 extraction time investigation (n=2)
Above result shows, extract 1 hour, 2 hours, 3 hours 5-hydroxyls-7,3 ', 4 '-content difference of trimethoxy flavone is little, considers, and determines that extraction time is 1 hour.
6.4 being hydrolyzed sour kind investigates
Take by weighing respectively 3 parts in L. similis Hemsl sample, every part of about 1.0g, accurately weighed, add respectively 70% ethanol 150ml, weighed weight, refluxing extraction 1 hour.Let cool, weighed weight is supplied the weight that subtracts mistake with 70% ethanol again, shakes up, and filters.Precision is measured subsequent filtrate 20ml, and each adds 1mol/L hydrochloric acid 4ml, 1mol/L sulfuric acid 4ml, water 4ml, refluxes 1 hour, is concentrated into driedly, and residue adds methyl alcohol makes dissolving, is settled to 1ml, and get final product.With 0.45 μ m filtering with microporous membrane, draws 10 μ l before the sample introduction, the injection liquid chromatography, the aforementioned chromatographic condition of photograph is measured, 5-hydroxyl-7,3 in the calculation sample ', 4 '-content of trimethoxy flavone.The method operation repetitive is twice like this.The results are shown in Table 6.
The sour kind of table 6 hydrolysis is investigated (n=2)
Annotate: "-" do not show and do not detect.
Above result shows, blank without 5-hydroxyl-7,3 ', 4 '-the trimethoxy flavone chromatographic peak, it is better to add the hydrochloric acid hydrolysis effect, therefore selects hydrochloric acid hydrolysis.
6.5 concentration of hydrochloric acid is investigated
Take by weighing respectively 4 parts in L. similis Hemsl sample, every part of about 1.0g, accurately weighed, add respectively 70% ethanol 50ml, weighed weight, refluxing extraction 1 hour.Let cool, weighed weight is supplied the weight that subtracts mistake with 70% ethanol again, shakes up, and filters.Precision is measured subsequent filtrate 20ml, and each adds the hydrochloric acid 4ml that concentration is 2mol/L, 1mol/L, 0.5mol/L, 0.25mol/L, refluxes 1 hour, is concentrated into driedly, and residue adds methyl alcohol makes dissolving, is settled to 1ml, and get final product.With 0.45 μ m filtering with microporous membrane, draws 10 μ l before the sample introduction, the injection liquid chromatography, the aforementioned chromatographic condition of photograph is measured, 5-hydroxyl-7,3 in the calculation sample ', 4 '-content of trimethoxy flavone.The method operation repetitive is twice like this.The results are shown in Table 7.
Table 7 concentration of hydrochloric acid is investigated (n=2)
Above result shows, after being the hydrochloric acid hydrolysis of 0.25mol/L, 0.5mol/L with concentration, the measured 5-hydroxyl-7 of test sample, 3 ', 4 '-trimethoxy flavone content is slightly low, be that content is higher behind the hydrochloric acid hydrolysis of 1mol/L, 2mol/L with concentration, and the two difference is little, consider, select the hydrochloric acid of 1mol/L to be hydrolyzed.
6.6 the hydrochloric acid addition is investigated
Take by weighing respectively 3 parts in L. similis Hemsl sample, every part of about 1.0g, accurately weighed, add respectively 70% ethanol 50ml, weighed weight, refluxing extraction 1 hour.Let cool, weighed weight is supplied the weight that subtracts mistake with 70% ethanol again, shakes up, and filters.Precision is measured subsequent filtrate 20ml, adds 1mol/L hydrochloric acid (namely adding respectively 1mol/L hydrochloric acid 4ml, 2ml, 1ml) in 5: 1,10: 1,20: 1 ratio respectively, refluxes 1 hour, is concentrated into driedly, and residue adds methyl alcohol makes dissolving, is settled to 1ml, and get final product.With 0.45 μ m filtering with microporous membrane, draws 10 μ l before the sample introduction, the injection liquid chromatography, the aforementioned chromatographic condition of photograph is measured, 5-hydroxyl-7,3 in the calculation sample ', 4 '-content of trimethoxy flavone.The method operation repetitive is twice like this.The results are shown in Table 8.
Table 8 hydrochloric acid addition is investigated (n=2)
Above result shows that hydrolysis effect was better when the ratio row added 1mol/L hydrochloric acid by 5: 1, in conjunction with practical operation, therefore adds the 1mol/L hydrochloric acid hydrolysis by 5: 1 than being listed as (being 4ml).
6.7 hydrolysis time is investigated
Take by weighing respectively 3 parts in L. similis Hemsl sample, every part of about 1.0g, accurately weighed, add respectively 70% ethanol 50ml, weighed weight, refluxing extraction 1 hour.Let cool, weighed weight is supplied the weight that subtracts mistake with 70% ethanol again, shakes up, and filters.Precision is measured subsequent filtrate 20ml, adds 1mol/L hydrochloric acid 4ml, refluxes respectively 1 hour, 2 hours, 3 hours, is concentrated into driedly, and residue adds methyl alcohol makes dissolving, is settled to 1ml, and get final product.With 0.45 μ m filtering with microporous membrane, draws 10 μ l before the sample introduction, the injection liquid chromatography, the aforementioned chromatographic condition of photograph is measured, 5-hydroxyl-7,3 in the calculation sample ', 4 '-content of trimethoxy flavone.The method operation repetitive is twice like this.The results are shown in Table 9.
Table 9 hydrolysis time is investigated (n=2)
Above result shows, acid hydrolysis 1 hour, and the measured 5-hydroxyl-7,3 of test sample ', 4 '-trimethoxy flavone content is slightly low, and the content that is hydrolyzed 2 hours, 3 hours is higher, and the two difference is little, considers, and determines that the acid hydrolysis time is 2 hours.
According to above-mentioned test findings, determine the need testing solution preparation method: get the about 1g of L. similis Hemsl powder (crossing sieve No. three), accurately weighed, put in the tool plug conical flask, the accurate 70% ethanol 50ml that adds, weighed weight, heating and refluxing extraction 1 hour lets cool, weighed weight again, supply the weight that subtracts mistake with 70% ethanol, shake up, filter.Precision is measured subsequent filtrate 20ml, and the accurate 1mol/L hydrochloric acid 4ml that adds put in 90 ℃ of water-baths heating hydrolysis 2 hours, took out, and is concentrated into driedly, and residue adds methyl alcohol makes dissolving, is settled to 1ml, and get final product.
7 precision tests
Accurate absorption 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone reference substance solution (0.984mg/ml) 10 μ l, continuous sample introduction 5 times is measured peak area value, the results are shown in Table 10.
Table 10 precision is investigated the result
Above result shows: this method precision is good.
8 stability tests
8.1 the stability of reference substance solution
Accurate absorption 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone reference substance solution (0.984mg/ml) 10 μ l, to measure once at 0,4,8,12,24 hour respectively, measurement result sees Table 11.
The stability test of table 11 reference substance solution
Above result shows: reference substance solution is good at 24 hours internal stabilities.
8.2 the stability of need testing solution
Get the about 1.0g of L. similis Hemsl sample, accurately weighed, the preparation method processes by test sample, namely gets need testing solution.Press the test sample assay method, measured once at 0,4,8,12,24 hour respectively, measurement result sees Table 12.
The stability test of table 12 need testing solution
Measurement result shows: need testing solution is basicly stable in 24 hours.
The test of 9 reappearances
Get parallel 6 parts of L. similis Hemsl sample, every part of about 1.0g, accurately weighed, the preparation method processes according to test sample, namely gets need testing solution.Press the test sample assay method, record the sample peak area, calculate 5-hydroxyl-7,3 ', 4 '-content of trimethoxy flavone, the results are shown in Table 13.
The reappearance test (n=6) of table 13 need testing solution
Above result shows: 5-hydroxyl-7,3 in this method mensuration test sample ', 4 '-trimethoxy flavone content, reappearance is good.
10 application of sample recovery tests
Adopt the average recovery method, get respectively the about 1.0g of L. similis Hemsl sample of known content, accurately weighed, an amount of 5-hydroxyl-7,3 of each accurate adding ', 4 '-the trimethoxy flavone reference substance, press sample determination method operation, record 5-hydroxyl-7,3 in the sample ', 4 '-content of trimethoxy flavone reference substance, calculate recovery rate.The results are shown in Table 14.
Table 14 recovery test result (n=9)
Test findings shows: as a result 5-hydroxyl-7,3 ', 4 '-the average average recovery of trimethoxy flavone is 99.98%, RSD=0.96%(n=9), shows between 95% ~ 105% that this method recovery is good.
By above-mentioned experiment as can be known, the used high performance liquid chromatography of authentication method of the present invention, reliable and stable, reappearance is good.
Among the present invention, in L. similis Hemsl, found first 5-hydroxyl-7,3 ', 4 '-existence of this compound of trimethoxy flavone, simultaneously, as the index components of identifying L. similis Hemsl, other class medicinal material that L. similis Hemsl and existing Chinese Pharmacopoeia can be recorded is effectively distinguished honeysuckle, for its cultivar identification provides a kind of new method with this compound.
Claims (10)
1. the authentication method of L. similis Hemsl certified products medicinal material is characterized in that: the material of getting it filled, measure by high performance liquid chromatography, and 5-hydroxyl-7,3 in the medicinal material ', 4 '-content of trimethoxy flavone is more than 0.3%w/w.
2. authentication method according to claim 1 is characterized in that: 5-hydroxyl-7,3 in the medicinal material ', 4 '-content of trimethoxy flavone is at 0.3 ~ 1.2%w/w.
3. authentication method according to claim 2 is characterized in that: 5-hydroxyl-7,3 in the medicinal material ', 4 '-content of trimethoxy flavone is at 0.36 ~ 1.08%w/w.
4. authentication method according to claim 1, it is characterized in that: the chromatographic condition of described high performance liquid chromatography is as follows:
Chromatographic column: octadecyl silane post
Mobile phase: acetonitrile: 0.3-0.5% phosphoric acid=(20-24): (76-80)
Column temperature: 25-35 ℃
Detect wavelength: 269 ± 2nm.
5. authentication method according to claim 4, it is characterized in that: described mobile phase is acetonitrile: 0.4% phosphoric acid=22:78; Column temperature is 30 ℃; Detect wavelength: 269nm.
6. authentication method according to claim 1, it is characterized in that: in the described high performance liquid chromatography, the preparation method of need testing solution is as follows:
Get L. similis Hemsl, pulverize, extract with the 60-80%v/v alcohol reflux, filter, measure subsequent filtrate, after the acid hydrolysis, be concentrated into driedly, residue adds methyl alcohol makes dissolving, and constant volume namely gets need testing solution.
7. authentication method according to claim 6, it is characterized in that: the concentration of described ethanol is 70%v/v.
8. authentication method according to claim 6, it is characterized in that: in the acid hydrolysis step, used acid is hydrochloric acid.
9. according to claim 6 or 8 described authentication methods, it is characterized in that: the concrete operations of acid hydrolysis are: add 1mol/L hydrochloric acid, the volume ratio of hydrochloric acid and subsequent filtrate is 5:1, puts heating hydrolysis in 90 ℃ of water-baths.
10. the detection method of L. similis Hemsl medicinal material or extract is characterized in that: it adopts HPLC to carry out, and operation steps is as follows:
(1) get L. similis Hemsl, pulverize, extract with the 60-80%v/v alcohol reflux, filter, measure subsequent filtrate, after the acid hydrolysis, be concentrated into driedly, residue adds methyl alcohol makes dissolving, and constant volume namely gets need testing solution;
(2) with 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone is reference substance, the preparation reference substance solution;
(3) respectively with in need testing solution, the reference substance solution injection liquid chromatography, detect, get final product, wherein, the chromatographic condition of described high performance liquid chromatography is as follows:
Chromatographic column: octadecyl silane post
Mobile phase: acetonitrile: 0.3-0.5% phosphoric acid=(20-24): (76-80)
Column temperature: 25-35 ℃
Detect wavelength: 269 ± 2nm.
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| ARUSA CHAOVANALIKIT等: "Characterization and Quantification of Anthocyanins and Polyphenolics in Blue Honeysuckle(Lonicera caerulea L.)", 《J. AGRIC. FOOD CHEM.》 * |
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| CN114295759A (en) * | 2021-12-31 | 2022-04-08 | 成都中医药大学 | Quality detection method of asarum or asarum extract for treating migraine |
| CN114295759B (en) * | 2021-12-31 | 2023-10-24 | 成都中医药大学 | Quality detection method of herba asari or herba asari extract for treating migraine |
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