CN103435678A - Preparation method of maslinic acid and application of maslinic acid in preparation of antibacterial agent - Google Patents
Preparation method of maslinic acid and application of maslinic acid in preparation of antibacterial agent Download PDFInfo
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Abstract
The invention provides a preparation method of maslinic acid and an application of maslinic acid in preparation of an antibacterial agent. According to the preparation method, powerful antibacterial agents (medicines) extracted and separated from akebia medicinal plants widely distributed in China are adopted, the preparation process is simple and has low cost and low contamination, the plant source is rich, the extraction is convenient, and when the fruits of the plants are extracted, the plants are not damaged and can be used for long term, so that the preparation method is environment-friendly and has good economic benefit. The monomeric compound product is stable and easy to store. Maslinic acid has wide antibacterial spectrum and high bacteriostatic activity, is much more likely to be developed into effective and safe food or vegetable and fruit antibacterial agents or new antibacterial agents capable of preventing and treating diseases caused by bacterial infection, and has a better market prospect.
Description
Technical field:
The invention belongs to antibiotic anticorrosion and medical technical field, be specifically related to a kind of preparation method of Crategolic acid and the application in preparing antiseptic-germicide thereof.
Background technology:
The bacterial invasion of food or fruits and vegetables is important channels of human infection's disease, and the resistance problem of pathogenic bacteria also forms more and more significant threat to people's health.Develop that the antiseptic-germicide of effective, safe food or fruits and vegetables and new anti-infection drug have reality always and demand widely.Crategolic acid is a kind of pentacyclic triterpene acid, is that its chemical structure is as shown in figure below Chinese style (I) to the mankind and the safety low-poison natural compounds of environment:
The bibliographical information Crategolic acid is present in Fructus oleae europaeae, hop, cloves, red date, Thinlear Adina Fruit, peppermint, hawthorn, in the vegetable materials such as pomegranate and mouse tail, especially be found in the olive Marc oil high level (Wang Bo is arranged, Chou Wenwei, explain gorgeous China, Yang Fan, Tang Jie, the progress of Crategolic acid, life science, 2009, 21(2): 264-269), and in recent years be found its be present in Akebia threeleaf akebia axis tissue in (Mimaki Y., Kuroda M., Yokosuka A., Harada H., Fukushima M., Sashida Y.Triterpenes and Triterpene Saponins from the Stems of Akebia trifoliata.Chemical & Pharmaceutical Bulletin, 2003,51:960-965), but there is not yet relevant its, be present in the report in Three Akebia Decne Species fruit or other Three Akebia Decne Species cauline leafs.
Research in the last few years shows, many pharmacologically actives such as that Crategolic acid has is anticancer, anti-oxidant, anti-inflammatory, anti-diabetes B, but Crategolic acid itself there is not yet the report document that it has anti-microbial activity.
Summary of the invention:
First purpose of the present invention is, a kind of new source of the Crategolic acid (maslinic acid) of finding based on the inventor and significant anti-microbial activity thereof, provide Crategolic acid (maslinic acid) or its pharmaceutically useful salt or the application of esterified derivative in preparing antiseptic-germicide.
Crategolic acid provided by the invention, confirm through external pharmacological evaluation, and it all has general potent restraining effect to gram-positive microorganism and negative bacterium.For example, its restraining effect to Bacillus thuringiensis is even also strong than positive control Kanamycin Sulfate, its restraining effect to bacterial strains such as streptococcus aureus, intestinal bacteria, dysentery bacterium and Salmonellass also approaches with positive reference substance or is suitable, therefore can be used for preparing antiseptic-germicide, the antiseptic-germicide of the bacterium such as especially anti-Bacillus thuringiensis, streptococcus aureus, intestinal bacteria, dysentery bacterium and Salmonellas.Prepare the antibacterials that the antiseptic-germicide of effective, the safe food such as fruits and vegetables or new prevention and treatment are subject to bacterium to infect associated diseases as be applied to, the application potential quality is extensive.Gram-positive microorganism and the negative bacterium of the potent inhibition of Crategolic acid energy provided by the invention include but not limited to the streptococcus aureus, Bacillus thuringiensis, intestinal bacteria, Salmonellas and the dysentery bacterium that exemplify in following examples.
Crategolic acid of the present invention or its pharmaceutically useful salt or esterified derivative, substantive Antibacterial Constituents wherein is the Crategolic acid molecule.The pharmaceutically useful salt of described Crategolic acid, its antibiotic essence is can be converted into the bioactive molecule Crategolic acid under the physiological conditions such as hydrochloric acid in gastric juice and work in tissue juice or people's digestive tube.The esterified derivative of described Crategolic acid refers to 2 in the Crategolic acid molecule, 3-position dihydroxyl is by the derivative compound of the 28-position carboxyl in esterifying organic acid or molecule and alcohol compound esterification, described esterified derivative can be 2, the partial esterification of one or two group in 3-position dihydroxyl and three functional groups of 28-position carboxyl, it can also be the full esterification of these three groups, these esterified derivatives based on the Crategolic acid molecular skeleton can be converted into easily the bioactive molecule Crategolic acid in tissue juice or people's digestive tube under the physiological conditions such as hydrochloric acid in gastric juice or intestines alkali, its essence is also that Crategolic acid plays anti-microbial effect, thereby belong to strict protection scope of the present invention.Wherein respectively with 2, the organic acid of 3-position dihydroxyl and the carboxyl functional group esterification of 28-position and Organic Alcohol can be any forms of related esters bond energy hydrolysis under the physiology acid-base condition, preferably C1 is to small molecular organic acid and the alcohol that can strengthen the characteristic such as whole macromolecule water-solubility of C4, and C6 is to the various organic acids or the Organic Alcohol that contain phenyl ring of C10.
Therefore, Crategolic acid of the present invention or its pharmaceutically useful salt or esterified derivative can be applied in preparing antiseptic-germicide.
Described antiseptic-germicide is preferably the antiseptic-germicide of gram-positive microorganism or Gram-negative bacteria.Further preferably, the antiseptic-germicide that the antiseptic-germicide of described gram-positive microorganism is streptococcus aureus or Bacillus thuringiensis; The antiseptic-germicide that the antiseptic-germicide of described Gram-negative bacteria is intestinal bacteria, dysentery bacterium or Salmonellas.
Preferably, the antiseptic-germicide that described antiseptic-germicide is food or fruits and vegetables or new prevention and treatment are subject to bacterium to infect the antibacterials of associated diseases.
The esterified derivative of described Crategolic acid preferably Crategolic acid 2, the esterified derivative that one or two in 3-position dihydroxyl and three functional groups of 28-position carboxyl or three groups and organic acid or Organic Alcohol esterification obtain.Further preferably, described organic acid or Organic Alcohol are organic acid or the Organic Alcohol containing phenyl ring of C6 to C10, or C1 is to straight or branched type small molecular organic acid or the Organic Alcohol of C4.
Crategolic acid of the present invention or its pharmaceutically useful salt or esterified derivative can be combined with pharmaceutically auxiliary material commonly used or carrier, prepare there is the Crategolic acid bacteriostatic activity can be used for suppressing the bacterial invasion of food or fruits and vegetables and medicine or the pharmaceutical composition that the control pathogenetic bacteria infects relative disease.This medicine or pharmaceutical composition can adopt the formulations such as wettable powder, tablet, granule, capsule, oral liquid, dripping pill, injection, aerosol; Also can adopt the known controlled release of modern pharmaceutical circle or slow release formulation or nanometer formulation.
Therefore, second purpose of the present invention is to provide a kind of antiseptic-germicide, it is characterized in that the Crategolic acid as active ingredient that comprises significant quantity or its pharmaceutically useful salt or its esterified derivative, and vehicle or the pharmaceutical carrier of preparation permission.
The 3rd purpose of the present invention is to provide a kind of preparation method of Crategolic acid, it is characterized in that, described Crategolic acid is stem, leaf or the fruit from akebi (Akebia quinata (Thumb.) Decne.), Caulis Akebiae (Akebia trifolia (Thumb.) Koidz.Var.australis (Diels) Rehd) or long order akebi (Akebia longeracemosa Matsumura), or in the fruit of threeleaf akebia (Akebia trifolia (Thumb.) Koidz.), the preparation separation obtains.
Described stem, leaf or fruit, can be dry product, can be also fresh goods, is preferably dry product.
Preferably, its concrete steps are:
A, prepare total medicinal extract: by stem, leaf or the fruit of akebi, Caulis Akebiae or long order akebi, or the fruit of threeleaf akebia is pulverized the rear aqueous ethanolic solution lixiviate of using, the concentrated ethanol of removing of extracting solution, obtain total medicinal extract crude extract, total medicinal extract crude extract is suspended in water, successively, with sherwood oil, ethyl acetate extraction, acetic acid ethyl ester extract obtains the total medicinal extract of ethyl acetate after concentrated;
B, separation and purification: the total medicinal extract of ethyl acetate, through the purification on normal-phase silica gel column chromatography, be take chloroform/methanol as eluent, successively from volume ratio 98:2,95:5,90:10,85:15, the 80:20 gradient elution, collect the cut that chloroform/methanol 80:20 elutes, then through the purification on normal-phase silica gel column chromatography, take chloroform/methanol as eluent, from volume ratio 98:2,95:5 is the eluent gradient wash-out successively, collects the cut of chloroform/methanol 95:5 wash-out, take methyl alcohol as solvent carries out repeatedly recrystallization, obtain the pure compound Crategolic acid.
The 4th purpose of the present invention is to provide stem, leaf or the fruit of akebi, Caulis Akebiae or long order akebi, or the application of the fruit of threeleaf akebia in preparing Crategolic acid.
The present invention adopts the Akebia medicinal plant more extensively distributed from China to extract and separates potent antiseptic-germicide (medicine) Crategolic acid, its preparation process is easy, low-cost, low to be polluted, its plant origin is abundant, it is convenient to extract, and can also make plant itself be utilized for a long time without destruction when adopting fruit to be extracted, environmentally friendly and economic benefit is preferably arranged, and this monomeric compound product is stable, easy to store.Crategolic acid bacteriostatic activity spectrum is wide, and bacteriostatic activity is high, is further development of most probably the antiseptic-germicide of effective, safe food or fruits and vegetables and new anti-infection drug, and market-oriented prospect is better.
Embodiment
Following examples are to further illustrate of the present invention, rather than limitation of the present invention, and the simple modifications that essence according to the present invention is carried out the present invention all belongs to the scope of protection of present invention.
Embodiment 1: the preparation of Crategolic acid in the Trilobed Caulis Akebiae fruit dry product
1.1 instrument and reagent
Concentrating under reduced pressure adopts the Tokyo N-1000 of physics and chemistry company Rotary Evaporators, the circulating cooling tank of CCA-1110 and SB-1000 electric-heated thermostatic water bath; HPLC adopts the Japanese Shimadzu LC-20AT of company type liquid chromatograph, SPD-M20A detector and Shim-PackPRC-ODS chromatographic column (particle diameter 5 μ m, aperture 12nm, 250mm * 20mm); Half preparation of middle pressure adopts Shanghai Li Sui Science and Technology Ltd. (Dr Flash-S) separation and purification system; Electrospray ionization mass spectrum (ESIMS) adopts Applied biosystems MDS SCIEX API2000LC/MS/MS instrument, take methyl alcohol as solvent direct injection mensuration;
1h NMR spectrum and
13cNMR spectrum adopts Bruker Avance600 nuclear magnetic resonance analyser, and take tetramethylsilane as interior mapping fixed.Coloration method adopts 10% ethanol solution of sulfuric acid or sulfuric acid Vanillin to process post-heating colour developing or iodine vapor colour developing.
1.2 plant origin and evaluation
Pick up from Hunan Province for extraction vegetable material threeleaf akebia (Akebia trifolia (Thumb.) Koidz.) in September, 2009 domestic, identified by South China Botanical Garden Chinese Academy of Sciences Xing Fuwu researcher.
1.3 extract and separate
Sample (the Trilobed Caulis Akebiae fruit dry product weighs 0.5 kilogram) is used under volume fraction 95% ethanol room temperature and is extracted three times after pulverizing, and united extraction liquid concentrating under reduced pressure is removed organic solvent ethanol, obtains total medicinal extract crude extract.Total medicinal extract crude extract is suspended in 500ml water, and then, with being extracted with ethyl acetate after isopyknic Petroleum ether extraction 3 times, acetic acid ethyl acetate extract obtains the total medicinal extract of ethyl acetate (44g) through concentrating under reduced pressure again.The total medicinal extract of ethyl acetate is dissolved by the chloroform/methanol (100mL) of 1:1, add purification on normal-phase silica gel (80-100 order) to mix sample with weight ratio 1:1.5, volatilized solvent, dry column-packing (200-300 order, 800 grams), the dry method loading, use successively chloroform/methanol=98:2,95:5,90:10,85:15,80:20,70:30,60:40,1:1,0:100v/v is the eluent gradient wash-out, according to thin layer plate, detect, each stream part is collected 9 component F1 – F9 from small to large successively according to the difference of polarity; Cut by F5(chloroform/methanol 80:20 wash-out, this component be take chloroform/methanol 85:15 and is carried out positive TLC detection as developping agent, and spray the heating colour developing with 10% sulfuric acid-ethanol, principal constituent presents the incarnadine spot of Rf=0.8) again through purification on normal-phase silica gel column chromatography (200-300 order, 150 grams) separation and purification, with chloroform/methanol=98:2,95:5,90:10v/v is eluent gradient wash-out (each gradient elution 500ml, every 20ml is collected as a component), detect and collect and the appropriate elutriant that merges according to the positive thin layer plate, obtain 6 component F5-1-F5-6; F5-3(chloroform/methanol 95:5 wash-out part) take methyl alcohol as solvent carries out repeatedly recrystallization, obtain colourless (white) crystal powder powder compound 1(15mg, Crategolic acid).
1.4 the Structural Identification of Crategolic acid
Compound 1 is white crystalline powder shape thing, molecular formula C
30h
48o
4; ESI-MS (neg.) m/z471[M-H]
–, 975[2M-H]
–; ESI-MS (pos.) m/z511[M+Na]
+, 999[2M+Na]
+;
1h(C
5d
5n, 600MHz) and
13c NMR (C
5d
5n, 151MHz) data displays is as shown in table 1 below:
Table 1: NMR data (the in C of compound 1
5d
5n)
Above wave spectrum related data and document (Zhao Min, Wang Yanan, Xiao Xuebin, Qian Wenhui, Wang great Cheng, Zhang Mingjun, Deng Xuming, the research of rabdosiaexcisa chemical composition, the time precious traditional Chinese medical science traditional Chinese medicines, 2011,22(12): 2881-2882) the compound Crategolic acid maslinic acid of report is consistent, can determine that accordingly obtained compound 1 is for Crategolic acid.
Embodiment 2: the preparation of Crategolic acid in the Trilobed Caulis Akebiae fruit fresh goods
2.1 instrument and reagent: with embodiment 1
2.2 plant origin and evaluation: with embodiment 1
2.3 extract and separate
Sample (the Trilobed Caulis Akebiae fruit fresh goods weighs 0.5 kilogram) is smashed to pieces rear with extracting three times under volume fraction 95% ethanol room temperature, and united extraction liquid concentrating under reduced pressure is removed organic solvent ethanol, obtains total medicinal extract crude extract.Total medicinal extract crude extract is suspended in 500ml water, and then, with being extracted with ethyl acetate after isopyknic Petroleum ether extraction 3 times, acetic acid ethyl acetate extract obtains the total medicinal extract of ethyl acetate (12g) through concentrating under reduced pressure again.The total medicinal extract of ethyl acetate is dissolved by the chloroform/methanol (50mL) of 1:1, add purification on normal-phase silica gel (80-100 order) to mix sample with weight ratio 1:1.5, volatilized solvent, dry column-packing (200-300 order, 300 grams), the dry method loading, use successively chloroform/methanol=98:2,95:5,90:10,85:15,80:20,70:30,60:40,1:1,0:100v/v is the eluent gradient wash-out, according to thin layer plate, detect, each stream part is collected 9 component F1 – F9 from small to large successively according to the difference of polarity; Cut by F5(chloroform/methanol 80:20 wash-out, this component be take chloroform/methanol 85:15 and is carried out positive TLC detection as developping agent, and spray the heating colour developing with 10% sulfuric acid-ethanol, principal constituent presents the incarnadine spot of Rf=0.8) again through purification on normal-phase silica gel column chromatography (200-300 order, 90 grams) separation and purification, with chloroform/methanol=98:2,95:5,90:10v/v is eluent gradient wash-out (each gradient elution 300ml, every 15ml is collected as a component), detect and collect and the appropriate elutriant that merges according to the positive thin layer plate, obtain 6 component F5-1-F5-6; F5-3(chloroform/methanol 95:5 wash-out part) take methyl alcohol as solvent carries out repeatedly recrystallization, obtain colourless (white) crystal powder powder compound 2(6mg, Crategolic acid).
2.4 the Structural Identification of compound 2: with embodiment 1, compound 2 is Crategolic acid through Structural Identification.
Embodiment 3: the preparation of Crategolic acid in the Caulis Akebiae fruit dry product
3.1 instrument and reagent: with embodiment 1
3.2 plant origin and evaluation: akebi (Akebia quinata (Thumb.) Decne.)
3.3 extract and separate
Sample (the Caulis Akebiae fruit dry product weighs 0.5 kilogram) is used under volume fraction 95% ethanol room temperature and is extracted three times after pulverizing, and united extraction liquid concentrating under reduced pressure is removed organic solvent ethanol, obtains total medicinal extract crude extract.Total medicinal extract crude extract is suspended in 500ml water, and then, with being extracted with ethyl acetate after isopyknic Petroleum ether extraction 3 times, acetic acid ethyl acetate extract obtains the total medicinal extract of ethyl acetate (44g) through concentrating under reduced pressure again.The total medicinal extract of ethyl acetate is dissolved by the chloroform/methanol (100mL) of 1:1, add purification on normal-phase silica gel (80-100 order) to mix sample with weight ratio 1:1.5, volatilized solvent, dry column-packing (200-300 order, 800 grams), the dry method loading, use successively chloroform/methanol=98:2,95:5,90:10,85:15,80:20,70:30,60:40,1:1,0:100v/v is the eluent gradient wash-out, according to thin layer plate, detect, each stream part is collected 9 component F1 – F9 from small to large successively according to the difference of polarity; Cut by F5(chloroform/methanol 80:20 wash-out, this component be take chloroform/methanol 85:15 and is carried out positive TLC detection as developping agent, and spray the heating colour developing with 10% sulfuric acid-ethanol, principal constituent presents the incarnadine spot of Rf=0.8) again through purification on normal-phase silica gel column chromatography (200-300 order, 150 grams) separation and purification, with chloroform/methanol=98:2,95:5,90:10v/v is eluent gradient wash-out (each gradient elution 500ml, every 20ml is collected as a component), detect and collect and the appropriate elutriant that merges according to the positive thin layer plate, obtain 6 component F5-1-F5-6; F5-3(chloroform/methanol 95:5 wash-out part) take methyl alcohol as solvent carries out repeatedly recrystallization, obtain colourless (white) crystal powder powder compound 3(15mg, Crategolic acid).
3.4 the Structural Identification of compound 3: with embodiment 1, compound 3 is Crategolic acid through Structural Identification.
Embodiment 4: the preparation of Crategolic acid in Caulis Akebiae fruit dry product
4.1 instrument and reagent: with embodiment 1
4.2 plant origin and evaluation: Caulis Akebiae (Akebia trifolia (Thumb.) Koidz.Var.australis (Diels) Rehd)
4.3 extract and separate
Sample (Caulis Akebiae fruit dry product weighs 0.5 kilogram) is used under volume fraction 95% ethanol room temperature and is extracted three times after pulverizing, and united extraction liquid concentrating under reduced pressure is removed organic solvent ethanol, obtains total medicinal extract crude extract.Total medicinal extract crude extract is suspended in 500ml water, and then, with being extracted with ethyl acetate after isopyknic Petroleum ether extraction 3 times, acetic acid ethyl acetate extract obtains the total medicinal extract of ethyl acetate (44g) through concentrating under reduced pressure again.The total medicinal extract of ethyl acetate is dissolved by the chloroform/methanol (100mL) of 1:1, add purification on normal-phase silica gel (80-100 order) to mix sample with weight ratio 1:1.5, volatilized solvent, dry column-packing (200-300 order, 800 grams), the dry method loading, use successively chloroform/methanol=98:2,95:5,90:10,85:15,80:20,70:30,60:40,1:1,0:100v/v is the eluent gradient wash-out, according to thin layer plate, detect, each stream part is collected 9 component F1 – F9 from small to large successively according to the difference of polarity; Cut by F5(chloroform/methanol 80:20 wash-out, this component be take chloroform/methanol 85:15 and is carried out positive TLC detection as developping agent, and spray the heating colour developing with 10% sulfuric acid-ethanol, principal constituent presents the incarnadine spot of Rf=0.8) again through purification on normal-phase silica gel column chromatography (200-300 order, 150 grams) separation and purification, with chloroform/methanol=98:2,95:5,90:10v/v is eluent gradient wash-out (each gradient elution 500ml, every 20ml is collected as a component), detect and collect and the appropriate elutriant that merges according to the positive thin layer plate, obtain 6 component F5-1-F5-6; F5-3(chloroform/methanol 95:5 wash-out part) take methyl alcohol as solvent carries out repeatedly recrystallization, obtain colourless (white) crystal powder powder compound 4(15mg, Crategolic acid).
4.4 the Structural Identification of compound 4: with embodiment 1, compound 4 is Crategolic acid through Structural Identification.
Embodiment 5: the preparation of Crategolic acid in Caulis Akebiae fruit fresh goods
5.1 instrument and reagent: with embodiment 1
5.2 plant origin and evaluation: Caulis Akebiae (Akebia trifolia (Thumb.) Koidz.Var.australis (Diels) Rehd)
5.3 extract and separate
Sample (Caulis Akebiae fruit fresh goods weighs 0.5 kilogram) is smashed to pieces rear with extracting three times under volume fraction 95% ethanol room temperature, and united extraction liquid concentrating under reduced pressure is removed organic solvent ethanol, obtains total medicinal extract crude extract.Total medicinal extract crude extract is suspended in 500ml water, and then, with being extracted with ethyl acetate after isopyknic Petroleum ether extraction 3 times, acetic acid ethyl acetate extract obtains the total medicinal extract of ethyl acetate (12g) through concentrating under reduced pressure again.The total medicinal extract of ethyl acetate is dissolved by the chloroform/methanol (50mL) of 1:1, add purification on normal-phase silica gel (80-100 order) to mix sample with weight ratio 1:1.5, volatilized solvent, dry column-packing (200-300 order, 300 grams), the dry method loading, use successively chloroform/methanol=98:2,95:5,90:10,85:15,80:20,70:30,60:40,1:1,0:100v/v is the eluent gradient wash-out, according to thin layer plate, detect, each stream part is collected 9 component F1 – F9 from small to large successively according to the difference of polarity; Cut by F5(chloroform/methanol 80:20 wash-out, this component be take chloroform/methanol 85:15 and is carried out positive TLC detection as developping agent, and spray the heating colour developing with 10% sulfuric acid-ethanol, principal constituent presents the incarnadine spot of Rf=0.8) again through purification on normal-phase silica gel column chromatography (200-300 order, 90 grams) separation and purification, with chloroform/methanol=98:2,95:5,90:10v/v is eluent gradient wash-out (each gradient elution 300ml, every 15ml is collected as a component), detect and collect and the appropriate elutriant that merges according to the positive thin layer plate, obtain 6 component F5-1-F5-6; F5-3(chloroform/methanol 95:5 wash-out part) take methyl alcohol as solvent carries out repeatedly recrystallization, obtain colourless (white) crystal powder powder compound 5(6mg, Crategolic acid).
5.4 the Structural Identification of compound 5: with embodiment 1, compound 5 is Crategolic acid through Structural Identification.
Embodiment 6: the preparation of Crategolic acid in akebi cauline leaf dry product
6.1 instrument and reagent: with embodiment 1
6.2 plant origin and evaluation: akebi (Akebia quinata (Thumb.) Decne.)
6.3 extract and separate
Sample (akebi cauline leaf dry product weighs 0.5 kilogram) is used under volume fraction 95% ethanol room temperature and is extracted three times after pulverizing, and united extraction liquid concentrating under reduced pressure is removed organic solvent ethanol, obtains total medicinal extract crude extract.Total medicinal extract crude extract is suspended in 500ml water, and then, with being extracted with ethyl acetate after isopyknic Petroleum ether extraction 3 times, acetic acid ethyl acetate extract obtains the total medicinal extract of ethyl acetate (33g) through concentrating under reduced pressure again.The total medicinal extract of ethyl acetate is dissolved by the chloroform/methanol (100mL) of 1:1, add purification on normal-phase silica gel (80-100 order) to mix sample with weight ratio 1:1.5, volatilized solvent, dry column-packing (200-300 order, 800 grams), the dry method loading, use successively chloroform/methanol=98:2,95:5,90:10,85:15,80:20,70:30,60:40,1:1,0:100v/v is the eluent gradient wash-out, according to thin layer plate, detect, each stream part is collected 9 component F1 – F9 from small to large successively according to the difference of polarity; Cut by F5(chloroform/methanol 80:20 wash-out, this component be take chloroform/methanol 85:15 and is carried out positive TLC detection as developping agent, and spray the heating colour developing with 10% sulfuric acid-ethanol, principal constituent presents the incarnadine spot of Rf=0.8) again through purification on normal-phase silica gel column chromatography (200-300 order, 150 grams) separation and purification, with chloroform/methanol=98:2,95:5,90:10v/v is eluent gradient wash-out (each gradient elution 500ml, every 20ml is collected as a component), detect and collect and the appropriate elutriant that merges according to the positive thin layer plate, obtain 6 component F5-1-F5-6; F5-3(chloroform/methanol 95:5 wash-out part) take methyl alcohol as solvent carries out repeatedly recrystallization, obtain colourless (white) crystal powder powder compound 6(10mg, Crategolic acid).
6.4 the Structural Identification of compound 6: with embodiment 1, compound 6 is Crategolic acid through Structural Identification.
Embodiment 7: the anti-microbial activity of compound Crategolic acid detects
7.1 experiment bacterial species
Gram-positive microorganism: streptococcus aureus (Staphyloccocus aureus), Bacillus thuringiensis (Bacillus thuringiensis);
Gram-negative bacteria: intestinal bacteria (Escherichia coli), Salmonellas (Salmonella enterica), dysentery bacterium (Shigella dysenteriae).
7.2 experimental drug
Positive reference substance: Kanamycin Sulfate (kanamincy sulfate)
Negative control product: MeOH
By above experimental example, prepared by Crategolic acid maslinic acid
7.3 experimental technique:
Crategolic acid maslinic acid, Kanamycin Sulfate are become respectively to the solution of 1mg/ml by methyl alcohol (MeOH) preparation.Adopt resazurin color reaction method, adopt 96 porocyte culture plates to be measured the minimal inhibitory concentration (MIC) of five kinds of bacteriums, detailed process is as follows.
At first the resazurin indicator of 100 μ g/mL is joined in the 11st row hole of 96 porocyte culture plates, then the resazurin solution of 7.5mL100 μ g/mL and 5mL are contained to bacterium (10
6cfu/mL, OD=0.07) nutrient solution mix, then respectively toward the mixture that adds the above-mentioned resazurin of 100 μ L and inoculum in 1-10 row and the 12nd each culture hole be listed as.Add the specimen that 100 μ L concentration are 1mg/mL again in first hole of every row, mix, therefrom draw 100 μ L solution and get in second hole, the rest may be inferred, until 100 μ L are finally removed in the tenth hole.The change in concentration of specimen is: 500 μ g/mL, 250 μ g/mL, 125 μ g/mL, 62.5 μ g/mL, 31.2 μ g/mL, 15.6 μ g/mL, 7.8 μ g/mL, 3.9 μ g/mL, 1.9 μ g/mL and 0.9 μ g/mL.Then culture plate is placed in the incubator of 37 ℃; in the specimen hole, weak blueness does not become the pink expression inhibition; blueness becomes pink and means not have inhibition; last does not become the minimum inhibition concentration that in peach hole, sample concentration is specimen (MIC) by blueness; until the 12nd row nutrient solution becomes pink (about 5-6 hour) by blueness, observe MIC value (the Rahman M.& of compound Crategolic acid maslinic acid to every kind of bacterium; Gray A., Phytochemistry, 2005,66:1601-1606).
7.4 experimental data is referring to table 2:
Table 2: the minimum inhibition concentration (MIC) of compound Crategolic acid maslinic acid to five kinds of bacteriums, μ g/mL
7.5 experiment conclusion:
This experiment shows, Crategolic acid maslinic acid (comprises streptococcus aureus to gram-positive microorganism, Bacillus thuringiensis etc.) and Gram-negative bacteria (comprise intestinal bacteria, Salmonellas, dysentery bacterium etc.) all there is significant restraining effect, its restraining effect to Bacillus thuringiensis is even also strong than positive reference substance Kanamycin Sulfate, it is to streptococcus aureus, intestinal bacteria, the restraining effect of the bacterial strain such as dysentery bacterium and Salmonellas also very approaches with positive reference substance and is suitable, embody the anti-microbial activity that Crategolic acid has strong effect wide-spectrum, thereby there is stronger exploitation potential quality, be expected to develop for the preparation of effectively, the food antiseptic-germicide of safety or new bacterial-infection resisting class medicine, the application potential quality is extensive.
Claims (10)
1. Crategolic acid or its pharmaceutically useful salt or the application of its esterified derivative in preparing antiseptic-germicide.
2. application according to claim 1, is characterized in that, the antiseptic-germicide that described antiseptic-germicide is gram-positive microorganism or Gram-negative bacteria.
3. application according to claim 2, is characterized in that, the antiseptic-germicide that the antiseptic-germicide of described gram-positive microorganism is streptococcus aureus or Bacillus thuringiensis; The antiseptic-germicide that the antiseptic-germicide of described Gram-negative bacteria is intestinal bacteria, dysentery bacterium or Salmonellas.
4. application according to claim 1, is characterized in that, the antiseptic-germicide that described antiseptic-germicide is fruits and vegetables or food, or prevent and treat to be subject to bacterium to infect the antibacterials of associated diseases.
5. application according to claim 1, it is characterized in that, the esterified derivative of described Crategolic acid is 2 of Crategolic acid, the esterified derivative that one or two in 3-position dihydroxyl and three functional groups of 28-position carboxyl or three groups and organic acid or Organic Alcohol esterification obtain.
6. application according to claim 5, is characterized in that, described organic acid or Organic Alcohol are organic acid or the Organic Alcohol containing phenyl ring of C6 to C10, or C1 is to straight or branched type small molecular organic acid or the Organic Alcohol of C4.
7. an antiseptic-germicide, is characterized in that, the Crategolic acid as active ingredient that comprises significant quantity or its pharmaceutically useful salt or its esterified derivative, and vehicle or the pharmaceutical carrier of preparation permission.
8. the preparation method of a Crategolic acid, it is characterized in that, described Crategolic acid is stem, leaf or the fruit from akebi (Akebia quinata (Thumb.) Decne.), Caulis Akebiae (Akebia trifolia (Thumb.) Koidz.Var.australis (Diels) Rehd) or long order akebi (Akebia longeracemosa Matsumura), or in the fruit of threeleaf akebia (Akebia trifolia (Thumb.) Koidz.), the preparation separation obtains.
9. preparation method according to claim 8, is characterized in that, its concrete steps are:
A, prepare total medicinal extract: by stem, leaf or the fruit of akebi, Caulis Akebiae or long order akebi, or the fruit of threeleaf akebia is pulverized the rear aqueous ethanolic solution lixiviate of using, the concentrated ethanol of removing of extracting solution, obtain total medicinal extract crude extract, total medicinal extract crude extract is suspended in water, successively, with sherwood oil, ethyl acetate extraction, acetic acid ethyl ester extract obtains the total medicinal extract of ethyl acetate after concentrated;
B, separation and purification: the total medicinal extract of ethyl acetate, through the purification on normal-phase silica gel column chromatography, be take chloroform/methanol as eluent, successively from volume ratio 98:2,95:5,90:10,85:15, the 80:20 gradient elution, collect the cut that chloroform/methanol 80:20 elutes, then through the purification on normal-phase silica gel column chromatography, take chloroform/methanol as eluent, from volume ratio 98:2,95:5 is the eluent gradient wash-out successively, collects the cut of chloroform/methanol 95:5 wash-out, take methyl alcohol as solvent carries out repeatedly recrystallization, obtain the pure compound Crategolic acid.
10. stem, leaf or the fruit of akebi, Caulis Akebiae or long order akebi, or the application of the fruit of threeleaf akebia in preparing Crategolic acid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103755772A (en) * | 2014-02-19 | 2014-04-30 | 淄博丽德医药技术有限公司 | Preparation process of natural high-purity (2alpha, 3beta) 2,3-dyhydroxy oleanolic-12-alkene-28-acid |
| CN104068449A (en) * | 2014-06-17 | 2014-10-01 | 福建农林大学 | Separation and purification method for dictyophora indusia fisscher antibacterial active monomer |
-
2013
- 2013-08-27 CN CN2013103803430A patent/CN103435678A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| IVONE LUCIA ACEBEY-CASTELLON ET AL.: "Triterpenoid Saponins from Symplocos lancifolia", 《JOURNAL OF NATURAL PRODUCTS》 * |
| YOSHIHIRO MIMAKI ET AL.: "Triterpenes and Triterpene Saponins from the Stems of Akebia trifoliate", 《CHEM. PHARM. BULL. 》 * |
| 王博等: "山楂酸的研究进展", 《生命科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103755772A (en) * | 2014-02-19 | 2014-04-30 | 淄博丽德医药技术有限公司 | Preparation process of natural high-purity (2alpha, 3beta) 2,3-dyhydroxy oleanolic-12-alkene-28-acid |
| CN103755772B (en) * | 2014-02-19 | 2016-01-20 | 山东丽德医药技术有限公司 | The preparation technology of a kind of natural high-purity (2 α, 3 β) 2,3-dihydroxyl olea-12-alkene-28-acid |
| CN104068449A (en) * | 2014-06-17 | 2014-10-01 | 福建农林大学 | Separation and purification method for dictyophora indusia fisscher antibacterial active monomer |
| CN104068449B (en) * | 2014-06-17 | 2016-08-24 | 福建农林大学 | A kind of isolation and purification method of Cultivation of Dictyophora bacteriostatic activity monomer |
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