CN105669622B - Artemisia anomalas extract 4 ', the preparation and application of 5,7- trihydroxyflavones - Google Patents
Artemisia anomalas extract 4 ', the preparation and application of 5,7- trihydroxyflavones Download PDFInfo
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Abstract
The present invention provides one kind and extracts 4' in artemisia anomalas, and 5,7 trihydroxyflavones and its application in antibacterials are prepared, are related to pharmaceutical technology field.The present invention use artemisia anomalas medicinal powder petroleum ether, ethyl acetate, chloroform, and n-butanol is extracted, the extract powder of each polarity is obtained after concentration, is studied by antibacterial activity, it is found that ethyl acetate and chloroform polar fraction activity are notable.Silica gel column chromatography is passed through to ethyl acetate and chloroform extract, using thin-layer chromatography point sample method, antibacterial activity test, obtain each Active fractions, high-purity flow point is identified using high performance liquid chromatography mass spectrometer (LC MS TOF), and MS, 1HNMR and 13CNMR analysis are carried out to the monomer flow point, obtain compound apiolin.The experimental method separates polar fraction by silica gel column chromatography, separation method simple and fast, has certain practical operation meaning.
Description
Technical field
Extract 4' the present invention relates to pharmaceutical technology field, in particular in artemisia anomalas, 5,7- trihydroxyflavones and it
Prepare the application in antibacterials.
Background technology
The ground drying nest of composite family mugwort artemisia anomalas Artemisia anomala S.Moore, perennial herb are planted
Thing, herb show flavonoid glycoside reaction.South of Jiangsu Province is originated in, is grown on hillside, roadside and border.It is distributed in East China, Central-South and southwestern
Each provinces and regions.Also known as Artemisia anomah S. Moore, Jin Jinu, June white (Zhejiang, Jiangxi), wild Herba Kalimeridis (Zhejiang), nine timothys (Hunan), bitter speed
Mother-in-law/hardship mother-in-law dish (Fujian), serissa serissoide/June white (Anhui, Jiangxi, Hunan), great Ye wormwood artemisias, iron oriental wormwood (Jiangsu), its nature and flavor is pungent,
It is bitter, flat.First recorded in the Northern and Southern Dynasties《Thunder God processes opinion》, main book on Chinese herbal medicine of later successive dynasties is such as《Tang materia medica》、《Classified Materia Meidca》、《Book on Chinese herbal medicine
Detailed outline》With《Chinese book on Chinese herbal medicine》Deng recording.The traditional Chinese medical science thinks that artemisia anomalas has eliminating dampness and heat, promoting blood circulation and removing blood stasis, inducing meastruation to relieve menalgia, and removing toxic substances disappears
The effect of swollen.Current research shows that artemisia anomalas also has anti-inflammatory, and anti-oxidant, anti-nitrification, platelet aggregation-against, promotees vasodilation, leads to
Cross Mutiple Targets and adjust the effect of to suppress vascular inflammation that more to have scholar to find that it also has HSV (herpes simplex virus) good by
Therapeutic effect (Zheng M.Experimental study of 472herbs with antiviral action against
the herpes simplex virus.Zhong Xi Yi Jie He Za Zhi.1990Jan；10(1):39-41,
6.Chinese.) practice of modern clinics finds that artemisia anomalas can treat acute bacillary dysentery, and diarrhea is burnt, and traumatic injury, snake stings
The diseases such as wound, early stage acute mastitis, these prompting artemisia anomalas may be inhibited to various pathogens, can prevent and treat sense
Dye.Liu Yun DS etc. studied find artemisia anomalas have good anti-mycotic efficiency previously using artemisia anomalas all herbal medicine (decocting liquid or
Concentrated tablet) carry out clinical treatment and antibacterial experiment, it is difficult to analyze the source of effective antimicrobial component of artemisia anomalas.
Previously research or clinical practice carry out clinical treatment using artemisia anomalas all herbal medicine (decocting liquid or concentrated tablet etc.)
And antibacterial experiment, it is difficult to analyze the source of effective antimicrobial component of artemisia anomalas, therefore, applicant carries out systematicness to artemisia anomalas in recent years
Separation and Extraction, then carries out Antimicrobial test with 80% ethanol extract and each polar fraction, the analysis effective antibacterial of artemisia anomalas into
The source divided, certain basis is established for next step extraction purification thing.The early-stage study of applicant is found：The different extractions of artemisia anomalas
Thing shows clinical various pathogens good bactericidal effect.Such as：(1) ethyl acetate extract to Shigella flexneri,
The minimum bactericidal concentration of Shigella dysenteriae and Streptococcusagalactiae is 6.25mg/mL, and the unrestraint of large intestine normal flora is acted on.
Wherein Shigella is the main pathogenic bacteria for causing bacillary dysentery, when bacillary dysentery is also field operation and rescue and relief work, portion
One of disease that team most easily occurs extensively.And Streptococcusagalactiae is commonly known as B group streptococcus (group B
Streptococcus, GBS), belong to conditionity pathogenic bacteria.At present, GBS infection is treated, alternative medicine is more [9].So
And under special circumstances, such as Large Scale Natural Disasters or outburst war occurs, still it is likely to occur GBS senses because lacking medicine
A large amount of outbursts of dye, pregnancy women urogenital system infection GBS, can trigger premature rupture of fetal membranes, premature labor, stillborn foetus, intrauterine infection
Etc. a series of complications of pregnancy, Maternal Infection GBS can cause puerperal endometritis, pelvic infecton or even septicemia；Neonate feels
Dye GBS can cause pneumonia, septicemia and meningitis.(2) chloroform extract has good bactericidal effect to escherichia coli,
Minimum bactericidal concentration is 6.25mg/mL, and escherichia coli is that normal post in human body intestinal canal grows bacterium, and wartime belly is subject to wound
Afterwards, if enteron aisle rupture or perforation can cause Escherichia coli to shift, so that cause abdominal cavity infection, so that whole body is sexy
Dye, can also infect because war wound causes Abwehrkraft des Koepers degradation.The 3rd affiliated hospital's data of The 2nd Army Medical College is shown：Greatly
The uncommon bacterium of intestines angstrom is the highest strain of pathogenic bacteria of nosocomial infection recall rate, accounts for the 28.3- of nosocomial infection detection bacterial strain sum
34.6%；And ratio higher shared in infection of biliary tract, recall rate is more than 50%.In recent years, abuse of antibiotics, causes big
The uncommon Bacteria drug tolorance rate of intestines angstrom remains high, and it is super that Cheng Xunmin etc. [12] detects 54 plants (43.5%) production altogether in 124 plants of escherichia coli
Wide spectrum beta-lactamase (extended-spectrum β-lactamases, ESBLs).Existing antibiosis is usually invalid, therefore there is an urgent need for
New antibiotic is developed to substitute.(3) chloroform and ethyl acetate extract there is good sterilization to make staphylococcus aureus
With minimum bactericidal concentration is 12.5mg/mL.Staphylococcus aureus is the most common polluted bacteria of open wound, and
Wartime opens the most common pathogenic bacteria of the contact scar surface of a wound.
Artemisia anomalas is grown on East China, Central-South and southwest extensively, and feed distribution region is vast, convenient material drawing.It is East China, Central-South
It is respectively the area under one's jurisdiction of Nanjing Military Command, the Guangzhou military region and Chengdu military area command with southwest.Militarily three above area faces
Extremely complicated international or domestic situation, such as on the one hand Nanjing and the Guangzhou military region need to carry out military struggle against Taiwan
Preparation, on the other hand need to face from Japan and every country in Southeast Asia to the island in China East Sea and the South Sea and seabed resources
Contention.And need to face the long-term struggle with Tibetan independence force in terms of Chengdu military area command, while strick precaution is also taken India.Therefore with
All there is the possibility of reply local war in upper three military regions.In peacetime, three above area is the occurred frequently of natural calamity
Region, such as：East China and the region occurred frequently that ALFISOL IN CENTRAL is flood, southwest is the geological disaster such as earthquake, mud-rock flow
Region occurred frequently, it is necessary to tackle may generation large-scale natural calamity.Therefore, further investigate the antibacterial activity of artemisia anomalas into
Point, efficient antibiotic of high quality and at a reasonable price is developed, when not only improving war and Large Scale Natural Disasters generation, utilizes its feed distribution
The characteristics of region is extensive, ensures that army and the people are concentrated use in the needs of the corresponding bacterium infection of antibiosis extract for treating in a short time,
Also can effectively meet the needs of usually people's general antibacterial therapy.
Application No. 201110187502.6, a kind of artemisia anomalas of Chinese patent application of Publication No. CN102309533A are always yellow
The preparation method of ketone, this method is mainly using supercritical carbon dioxide extraction method extraction artemisia anomalas powder, and ethanol is as carrying
Agent, extract are adsorbed using macroporous absorbent resin, and ethanol elution, is concentrated under reduced pressure, and freeze-drying obtains artemisia anomalas general flavone.It is prepared
Method is simple, fast, nontoxic.But this method is the preparation method for artemisia anomalas general flavone, be not easy to artemisia anomalas monomer reactivity into
Prepared by the extraction divided, because the invention is of great significance the preparation method of monomer reactivity component in artemisia anomalas.
The present inventor has been devoted to carry out artemisia anomalas systemic separation and Extraction, and completes different extracts and each polarity
The Antimicrobial test that position carries out, the source of the effective antimicrobial component of artemisia anomalas is filtered out by Antimicrobial test, and complete
The separation and identification of antibacterial effective monomer.
The content of the invention
Another object of the present invention is to provide the preparation method of three kinds of artemisia anomalas extract 4', 5,7- trihydroxyflavones.
It is an object of the invention to provide three kinds of artemisia anomalas extract 4', 5,7- trihydroxyflavones in antibacterials are prepared
Using.
Artemisia anomalas extract 4', the preparation method of 5,7- trihydroxyflavones (apiolin), carries out as steps described below：
It is immersed in after dry artemisia anomalas crude drug is crushed in 80% ethanol, 65 DEG C of seepage pressure effects, extraction terminates rear film and steams
Hair, it is stand-by after dry, obtain 80% ethanol extract dried powder；Wherein the usage ratio of artemisia anomalas crude drug and 80% ethanol is 1：
5kg/L。
80% ethanol extract dried powder 1kg is taken, is filtered with 6L petroleum ethers immersion 24h, 6L acetic acid is used after filter residue and drying
Ethyl ester immersion 24h filterings, filtrate are concentrated to give ethyl acetate extract about 6.2kg.Ethyl acetate extract dissolved with methanol after on
Silicagel column, with petroleum ether-ethyl acetate system with：10:1、5:1、2:1、1:1、1:2、1:5、1:10 each configuration 400ml are with 2d/s
(drop/sec) gradient elution, a sample is collected every 30min, and No. 8 sample that elution time is collected in 3.5h-4h is 4',
5,7- trihydroxyflavones (apiolin).
In 96 samples that elution is collected, the sample containing material of the same race in sample is mixed by thin-layer chromatography point sample method
Close, mixed sample is condensed into extracting solution by Rotary Evaporators and dry powder-shaped thing is made by SpeedVac again
(being partly jelly), all samples are stored in the EP pipes of 1.5mL, and Co60 sterilizings are stand-by.
Thin-layer chromatography point sample method, reference：Sun Yuqing, Hu Yuzhu analytical chemistry Beijing:Science Press, 2007.467~
470。
The structural formula of wherein 4', 5,7- trihydroxyflavone (apiolin) is：
Application of artemisia anomalas extract 4', the 5,7- trihydroxyflavone in antibacterials are prepared.
Wherein described bacterial strain is staphylococcus aureus (staphylococcus aureus, SA) standard type ATCC
25923rd, staphylococcus aureus (staphylococcus aureus, SA) standard type ATCC 29213.
Staphylococcus aureus (staphylococcus aureus, SA) standard type ATCC 25923, Staphylococcus aureus
Bacterium (staphylococcus aureus, SA) standard type ATCC 29213, is purchased from the limited public affairs of the safe biotechnology in Wenzhou City
Department.
Beneficial effect：
The present invention use artemisia anomalas medicinal powder petroleum ether, ethyl acetate, chloroform, and n-butanol is extracted, must after concentration
To the extract powder of each polarity, studied by antibacterial activity, find ethyl acetate and chloroform polar fraction activity significantly.It is right
Ethyl acetate and chloroform extract are by silica gel column chromatography, and using thin-layer chromatography point sample method, antibacterial activity test, obtains each activity
Flow point, high-purity flow point is identified using high performance liquid chromatography mass spectrometer (LC-MS TOF), and the monomer flow point is carried out
MS、1HNMR and13CNMR is analyzed, and obtains compound apiolin.This method goal in research is clear and definite, simple and quick, is that Chinese medicine is strange
The active ingredient follow of wormwood artemisia provides prescription method.
The experimental program be by early period to artemisia anomalas opposed polarity section antibacterial experiment result based on, to the significant chloroform of activity
Position and ethyl acetate polar fraction carry out further tracking activity, and structure mirror is carried out to the significant monomeric compound of activity
It is fixed, artemisia anomalas moderate resistance bacterium main active is gone out with Preliminary Identification.The experimental method carries out polar fraction by silica gel column chromatography
Separation, separation method simple and fast, has certain practical operation meaning.
Brief description of the drawings
No. 13 TIC collection of illustrative plates of Fig. 1 chloroform extracts
No. 8 TIC collection of illustrative plates of Fig. 2 ethyl acetate extracts
No. 8 MS collection of illustrative plates in t=14.076 of Fig. 3 ethyl acetate extracts
The TIC collection of illustrative plates of Fig. 4 apiolin reference substances
Embodiment
In conjunction with embodiment, the invention will be further described, but the implementation of the present invention is not limited to that.
In following embodiments and comparative example, staphylococcus aureus (standard type ATCC25923, purchased from the safe life in Wenzhou City
Thing Science and Technology Ltd.), staphylococcus aureus (standard type ATCC29213, purchased from the limited public affairs of the safe biotechnology in Wenzhou City
Department), escherichia coli (standard type ATCC25922, purchased from safe bio tech ltd of Wenzhou City), pseudomonas aeruginosa
(standard type ATCC27853, purchased from safe bio tech ltd of Wenzhou City), Streptococcusagalactiae (Streptococus
Agalactiae, SA, clinical type 03598276), Shigella flexneri (Shigella flexneri, SF, clinical type
03475491), Salmonella typhimurtum (Salmonella typhimurium, ST, clinical type 5002324172), hydrolyzes network albumen
Agar (Mueller-H in ton, M-H) culture medium, M-H nutrient solution M-H nutrient solutions (model C MO337, purchased from Britain
OXOID).Streptococcusagalactiae (clinical type), Shigella flexneri (clinical type), Salmonella typhimurtum (clinical type) are cured from Changhai
Clinical laboratory of institute is clinically separated the pathogenic strain of acquisition, is also purchased from institute of materia medica of the Chinese Academy of Sciences.
Embodiment 1：The preparation method of artemisia anomalas extract 4', 5,7- trihydroxyflavone (apiolin)
40kg drying artemisia anomalas crude drugs (are purchased from Bozhou City tcm and herbal slice company, the place of production：Zhejiang Linan, also known as production south
Artemisia anomala), it is immersed in after crushing in 80% ethanol, 65 DEG C of seepage pressure effects, it is thin after extraction successively using 80% ethanol 200L
Film evaporates, stand-by after dry.
80% ethanol extract dried powder about 1kg is taken, is filtered with 6L petroleum ethers immersion 24h, 6L second is used after filter residue and drying
Acetoacetic ester immersion 24h filterings, filtrate are concentrated to give ethyl acetate extract 6.2kg.Ethyl acetate extract dissolved with methanol after on
Silicagel column, with petroleum ether-ethyl acetate system with：10:1、5:1、2:1、1:1、1:2、1:5、1:10 each configuration 400ml are with 2d/s
(drop/sec) gradient elution, collects a sample every 30min, there are 96 samples.It will be contained by thin-layer chromatography point sample method of the same race
The sample mixing of material, mixed sample are condensed into extracting solution by Rotary Evaporators and pass through SpeedVac again
Dry powder-shaped thing (being partly jelly) is made, ethyl acetate extract is stored in by merging to obtain 26 samples (being shown in Table 1)
In the EP pipes of 1.5mL, Co60 sterilizings are stand-by.Identified, No. 8 sample that elution time is collected in 3.5h-4h is 4', 5,7-
Trihydroxyflavone (apiolin).
Comparative example artemisia anomalas chloroform, ethyl acetate extract
40kg dries artemisia anomalas crude drug, is immersed in after crushing in 80% ethanol, 65 DEG C of seepage pressure effects, successively using 80% second
Alcohol 200L, thin film evaporation after extraction are stand-by after dry.80% ethanol extract dried powder about 1kg is taken, with 6L petroleum ethers
24h filterings are soaked, filtrate obtains ligroin extraction after being concentrated and dried.With the immersion 24h filterings of 6L chloroforms, filter after filter residue and drying
Liquid is evaporated under reduced pressure, and is concentrated to give chloroform extract about 560g.With the immersion 24h filterings of 6L ethyl acetate, filtrate concentration after filter residue and drying
Obtain ethyl acetate extract 6.2kg.Chloroform crude extract upper silicagel column after being dissolved with methanol, using the methanol-chloroform of various concentrations
Solution：10:1、5:1、2:1、1:1、1:2、1:5、1:10 each configuration 400ml are with 2d/s (drop/sec) gradient elution, every 30min
Collect a sample, common 81 samples.Ethyl acetate extract upper silicagel column after being dissolved with methanol, with the oil of various concentrations
Ether-ethyl acetate solution：10:1、5:1、2:1、1:1、1:2、1:5、1:10 each configuration 400ml are washed with 2d/s (drop/sec) gradient
It is de-, a sample is collected every 30min, there are 96 samples.The sample containing material of the same race is mixed by thin-layer chromatography point sample method
Close, mixed sample is condensed into extracting solution by Rotary Evaporators and dry powder-shaped thing is made by SpeedVac again
81 components are obtained in (being partly jelly), chloroform extract, and merging component numbering, 16-20 components are merged into group at the same time respectively
Divide in 21,22-24 is merged into 25, and 26 are merged into 27, and 28-30 is merged into 31, and 32-34 is merged into 35, and 36 are merged into
In 37,38-40 is merged into 41, and 42-44 is merged into 45, and 46-47 is merged into 48, and 49-61 is merged into 62, and 63-68 is closed
It is incorporated in 69,70-78 and 80-81 components are merged into 79, are amounted to after merging and are obtained 26 samples.Ethyl acetate extract
96 components are obtained, it is respectively that 2-3 is merged into 4 to merge component numbering, and 5-6 is merged into 7, and 9-10 is merged into 11,12-
During 17 merge 18,19-17 is merged into 28, and during 29 merge 30,31 are merged into 32, and 33-37 is merged into 38, and 39-45 merges
In entering 46,48-50 is merged into 51, and 52-56 is merged into 57, and 58-62 is merged into 63, and 64 are merged into 65, and 67 are merged into
In 68,69 are merged into 70, and during 73-74 merges 75,76-79 is merged into 80, and 81 are merged into 82, and 83-85 is merged into 86,
87-91 and 93-96 components are merged into 92, are amounted to after merging and are obtained 26 samples.All samples are stored in the EP of 1.5mL
Guan Zhong, Co60 sterilizing are stand-by.
1 chloroform of table and ethyl acetate extract sample number into spectrum
| Sample number into spectrum | |
| Chloroform extract | 1,2,3,4,5,6,7,8,10,11,12,13,14,15,21,25,27,31,35,37,41,45,48,62,69,79 |
| Ethyl acetate extract | 1,4,7,8,11,18,28,30,32,38,46,47,51,57,63,65,66,68,70,71,72,75,80,82,86,92 |
Separation receives obtained sample and merges through thin-layer sample application method, the identical component of Rf values, chloroform polar portion and acetic acid
Ethyl ester polar fraction merges separation component and obtains 26 sample components.
No. 8 samples wherein obtained after combined ethyl acetate extract are 1 gained 4', 5,7- trihydroxyflavone of embodiment
(apiolin).
Test example 1：
Antimicrobial test
1. disk diffusion method is tested and result
Take artemisia anomalas chloroform extract and each sample 10mg of ethyl acetate extract respectively, when dissolving is separately added into cosolvent
Sodium carboxymethylcellulose 1mg, is dissolved using sterile purified water, is added to 1ml and is configured to the liquid that concentration is 1%.First will be added with
The circle scraps of paper of 50 μ l original concentration liquids (10mg/m l) are placed on (agalasisa chain on the M-H agar plates of the bacterial inoculum containing standard volume
Coccus needs to add blood culture), 35 DEG C of culture 18h, judge antibacterial according to the inhibition zone size showed around drug sensitive test paper
The power of effect, will confirm that antimicrobial sample of the inhibition zone more than 9mm (including 9mm, scraps of paper diameter 6mm) sieves in disk diffusion method
Go out samples of the by this method preliminary screening to sensitive bacteria, to treat further to measure minimum bactericidal concentration (MBC).
Disk diffusion method experimental result is as follows：
Each sample and the bacterial strain proportioning of chloroform extract have carried out 3 experiments to be averaged acquisition result, as a result
Refer to table 2.
2 chloroform extract disk diffusion method bacteriostatic test result of table
1) a diameter of 6mm of the scraps of paper, 2) antibacterial circle diameter is 9mm
Each sample and the bacterial strain proportioning of ethyl acetate extract have carried out 3 experiments to be averaged acquisition result,
The results detailed in Table 3
3 ethyl acetate extract disk diffusion method bacteriostatic test result of table
1) a diameter of 6mm of the scraps of paper, 2) antibacterial circle diameter is 9mm
4 are shown in Table by the obtained sensitive bacterial spectrum (antibacterial circle diameter >=9mm) of disk diffusion method:
4 disk diffusion method sensitive bacterial of table is composed
2. constant broth dilution method determination minimal inhibitory concentration (minimum inhibitory concentration, MIC[8]) and minimum bactericidal concentration (minimalbactericidal concentration, MBC[9])
Constant broth dilution method experimental procedure：
The pairing of antibacterial circle diameter >=9mm further measures minimum bactericidal concentration in disk diffusion method experiment, each is matched somebody with somebody
It is averaged to being carried out at the same time 3 groups of experiments.It is bacterium colony 3-5 to be checked similar with oese picking form, it is inoculated in the hydrolysis of 4-5ml
In casein (MH) meat soup, 35 DEG C of incubation 2-6h.Increase the exponential phase bacterium solution physiological saline after bacterium or the correction of MH meat soups is dense
Spend to 0.5 Maxwell than turbid standard, containing about 1~2 × 108CFU/ml.Above-mentioned bacteria suspension is subjected to 1: 100 dilution standby with MH meat soups
With.The preparation and bacterium solution inoculation for diluting antibacterials take sterile test tube 9, are arranged in a row, except the 1st pipe adds 1.6mlMH meat soups
Outside, remaining often pipe addition MH meat soup 1ml, add antibacterials stoste (such as 10mg/ml) 0.4ml in the 1st pipe and mix, then draw
1ml to the 2nd is managed, and draws the pipes of 1ml to the 3rd after mixing again, and so continuous doubling dilution draws 1ml to the 7th pipe from the 7th pipe
Discard, the 8th pipe is the growth control of positive controls not drug containing, and the 9th pipe is negative control pipe asepsis growth.Each pipe medicine at this time
Thing concentration is followed successively by 2,1,0.5,0.25,0.125,0.0625,0.03125 μ g/ml.Then above-mentioned prepare is added in every pipe
Each 1ml of inoculum, it is about 5 × 105CFU/ml to make the final bacterial concentration of every pipe.1st pipe to the 7th pipe drug concentration is respectively 1,
0.5、0.25、0.125、0.0625、0.03125、0.01562μg/ml.The dilution tube being inoculated with is stoppered plug, put 35 DEG C it is general
16~20h is incubated in blowing air incubator.Test group sample, positive controls sample and negative control group sample are put into 37 DEG C of cultures
In case, 48h is cultivated, observes result.When positive control pipe has bacterium to grow (muddiness), negative control pipe asepsis growth (transparent), is tried
Group is tested to visually observe the liquid highest dilution of asepsis growth as MIC.100 μ l are taken out of each pipe of the asepsis growth, are added respectively
Enter agar plate, put in 37 DEG C of incubators, cultivate 48h, observe bacterial growth situation, the liquid Cmin that no bacterium colony is formed is
MBC[9].MIC the results are shown in Table 5, MBC the results are shown in Table 6.
5 artemisia anomalas difference extract constant broth dilution method MIC result of the tests of table
6 artemisia anomalas difference extract constant broth dilution method MBC result of the tests of table
3. the content that pair sample for having bacteriostasis is weighed to obtain total effectively antibacterial substance in artemisia anomalas is shown in Table 7.
There are the weighing results of the sample of fungistatic effect in 7 artemisia anomalas of table
The identification of antibacterial effective monomer
1.LC/MSD TOF[10]The detection of liquid chromatography/mass spectrometry combined instrument (flight time mass spectrum)
(1) liquid phase chromatogram condition
Chromatographic column：Shiseido TYPE MG (3.0 × 100mm, 3.0 μm)；Mobile phase:Pure water：Methanol=90:10；Sample introduction
Amount：5μL；Flow velocity：0.6mL/min；Column temperature：25℃；Detection wavelength 235nm；Run time 16min.
(2) Single Quadrupole-MS conditions
Using ESI ion guns, under negative ion mode (ESI-)：Split ratio is 2 after column:1, dryer temperature：350℃；Hair
Tubule voltage：4000V；Drier flow velocity：9.0L/min；Nebulizer pressure：35psig；Cracking gas voltage：200V.
(3) TIC (total ion chromatogram) is detected
Here is the TIC collection of illustrative plates of some samples:(see Fig. 1-Fig. 4)
(4) point data MS collection of illustrative plates detects under TIC
TIC collection of illustrative plates by analyzing each sample can show that No. 8 impurity of ethyl acetate extract are less, and essentially sterling can
It is further analyzed.
The mass-spectrogram of No. 8 main peaks (T=14.076) of ethyl acetate extract is shown in Fig. 2, it can be deduced that ethyl acetate extracts
The leading ion peak of thing 8 is 269.0454, tR=14.076. is primarily formed [M-H] because being negative ion mode–Ion
Peak[11], so the M=269.0454+1.0078=270.0532 of material；(see Fig. 3)
2. by analyzing the physicochemical property combination mass spectrum of material, UV spectral results speculate compound molecule formula and structural formula
Ethyl acetate extract 8:
Physical property：Yellow needles powder (MeOH), 347~348 DEG C of fusing point, is practically insoluble in water, is partially soluble in hot wine
Essence, is dissolved in dilute KOH solution.
Chemical property：Compound on TCL plates with FeCl3-K3Fe(CN)6Reaction is in blue, is expressed as phenols；HCl-Mg
Powder reaction, which is positive, is shown to be flavone compound；
MS collection of illustrative plates：Mass spectrogram under T=14.076 show quasi-molecular ions for 269.0454. because being negative ion mode, mainly
Formed [M-H]–Quasi-molecular ions[11], therefore M=269.0454+1.0078=270.0532；
It can speculate that compound molecule formula is C with reference to above property15H10O5, it is C by comparing molecular formula15H10O5Each Huang
Ketone compounds can speculate that compound is 4', 5,7- trihydroxyflavones (apiolin).
Its structural formula is：
3. compared by the TIC collection of illustrative plates with reference substance to determine effective antibacterial material component
No. 8 TIC collection of illustrative plates (see Fig. 2) of ethyl acetate extract compare with reference substance apiolin TIC collection of illustrative plates (see Fig. 4) and can determine that
Main component in ethyl acetate extract 8 is apiolin.
Basic principle, main feature and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, various changes and modifications of the present invention are possible without departing from the spirit and scope of the present invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent defines.
Claims (1)
1.4', the preparation method of 5,7- trihydroxyflavones, it is characterised in that it is that 4' is extracted from artemisia anomalas, 5,7- trihydroxyflavones,
And carry out as steps described below：
It is immersed in after dry artemisia anomalas crude drug is crushed in 80% ethanol, 65 DEG C of seepage pressure effects, thin film evaporation after extraction, does
It is stand-by after dry, obtain 80% ethanol extract dried powder；Wherein the usage ratio of artemisia anomalas crude drug and 80% ethanol is 1kg:5L；
80% ethanol extract dried powder 1kg is taken, is filtered with 6L petroleum ethers immersion 24h, 6L ethyl acetate is used after filter residue and drying
24h filterings are soaked, filtrate is concentrated to give ethyl acetate extract 6.2kg；Ethyl acetate extract upper silicagel column after being dissolved with methanol,
With petroleum ether-ethyl acetate system with：10:1、5:1、2:1、1:1、1:2、1:5、1:10 each configuration 400ml are washed with 2d/s gradients
It is de-, a sample is collected every 30min, the sample that elution time is collected in 3.5h-4h is 4', and 5,7- trihydroxies are yellow
Ketone.
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Non-Patent Citations (5)
| Title |
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| Apigenin alleviates the symptoms of Staphylococcus aureus pneumonia by inhibiting the production of alpha-hemolysin;Jing Dong 等;《FEMS Microbiol Lett.》;20121128;第338卷(第2期);第124-131页 * |
| Variation in synergistic activity by flavone and its related compounds on the increased susceptibility of various strains of methicillin-resistant Staphylococcus aureus to -lactam antibiotics;Youichi Sato 等;《International Journal of Antimicrobial Agents》;20040930;第24卷(第3期);第28-35页 * |
| 刘寄奴化学成分研究;肖同书 等;《中草药》;20130331;第44卷(第5期);第515-518页 * |
| 刘寄奴黄酮类成分研究;温晶 等;《中国中药杂志》;20100731;第35卷(第14期);第1827-1830页 * |
| 南刘寄奴黄酮类成分的研究;张琳 等;《中国药学杂志》;20100131;第45卷(第2期);第104-107页 * |
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