CN108392496A - A kind of Chinese medicine maggot fat acid-extraction - Google Patents
A kind of Chinese medicine maggot fat acid-extraction Download PDFInfo
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Abstract
The invention discloses a kind of Chinese medicine maggot fat acid-extractions, use ethyl acetate soxhlet extraction with solid-liquid ratio for 1：5, extraction time is 2h, extraction effect is best when extraction time is 1 time, the recovery rate of maggot extract is improved, and retentive activity ingredient to greatest extent, later, degumming is carried out to it, acid stripping and refining, purpose is a small amount of protein removed in maggot aliphatic acid crude extract, the colloid substances such as phosphatide and mucus, heavy metal, and remove free fatty therein, and the maggot aliphatic acid after freezing and crystallizing finally obtains concentration, through analysis, all unsaturated fatty acids in the Chinese medicine maggot fatty acid extract obtained with the technique, and it is verified by experiments, the Chinese medicine maggot fatty acid extract is to staphylococcus aureus, pneumonia diplostreptococcus and pseudomonas aeruginosa all have antibiotic property, various drug forms can be prepared into pharmaceutically acceptable carrier, clinic is facilitated to take.
Description
Technical field
The present invention relates to animal fat extractive technique fields, are extracted more particularly, to a kind of Chinese medicine maggot aliphatic acid
Technique.
Background technology
The abuse of antibiotic caused bacterial drug resistance to significantly increase in recent years, the severe infectious surface of a wound caused by drug-fast bacteria
Case increases year by year, and serious challenge is brought to clinical treatment.Exploitation is a kind of with antibacterial but also can be not only not likely to produce drug resistant effective
External used medicine becomes current field of medicaments problem in the urgent need to address.Chinese medicine is with toxic side effect is small, action target spot is more, makees
The advantages that lasting with effect is one of the effective way for treating bacterium infection.
Maggot is commonly called as maggot, is the larva of Calliphoridae insect.Because it has no drug resistance, the antibacterial action of lasting effective and safe
It is by the unique of U.S. FDA and Europe CE certifications with the apparent concern for promoting Healing increasingly to cause international medical community
It can be in the medical insect of Clinical practice.Maggot is prepared into Chinese medicine to treat infectious surgical disease by Traditional Chinese Medicine, according to
《Book on Chinese herbal medicine asks source》、《Book on Chinese herbal medicine is just read》Described in, the worm is cold in nature, after dry grind, is applied with, and can treat that shank is rotten, lip pustule, illustrates five
Contain certain Antibacterial Constituents in paddy polypide.Therefore, a kind of Chinese medicine maggot extraction process is invented to solve Chinese medicine maggot
Application problem clinically is very necessary.
Invention content
The purpose of the present invention is to provide a kind of Chinese medicine maggot fat acid-extractions.
To achieve the above object, technical scheme is as follows：
A kind of Chinese medicine maggot fat acid-extraction, which is characterized in that include the following steps：
Step S1：Chinese medicine maggot dry powder after drying is wrapped with filter paper in input Soxhlet extractor, dichloromethane is added
Alkane or petroleum ether or ethyl acetate solvent, are warming up to solvent boiling point, refluxing extraction 2h~4h, and reflow's cycle is 1~3 time, wherein
The mass ratio of Chinese medicine maggot dry powder and solvent after drying is 1:5~20；
Step S2：Extracting solution obtained by step S1 is placed in Rotary Evaporators and is evaporated under reduced pressure, recycling design, and in ventilation
Residual solvent is vapored away in cupboard, obtains Chinese medicine maggot aliphatic acid crude extract；
Step S3：Using acid refining method degumming, the Chinese medicine maggot aliphatic acid crude extract obtained by step S2 is put into container
In, it is heated with stirring to 70 DEG C~80 DEG C in a water bath, it is Chinese medicine maggot aliphatic acid coarse extraction substance that quality is added while stirring
The 1% of amount, a concentration of 75%~80% phosphoric acid continue 1min~15min, are then centrifuged for detaching to obtain liquid, take upper layer oily
Sample is degumming maggot aliphatic acid；
Step S4：It is degumming maggot aliphatic acid quality that quality is added into the degumming maggot aliphatic acid obtained by step S3
1%~1.5%, the sodium hydroxide solution of a concentration of 300g/L, after uniform stirring, be heated to 65 DEG C~70 DEG C, heat preservation
30min is then cooled to room temperature, stratification, centrifuges to obtain liquid；With remaining soap stock in deionized water cleaning liquid,
It cleans 3 times repeatedly, later, centrifuges to obtain liquid, take liquid upper layer oil sample, for the maggot aliphatic acid after refining.
Further, further include the steps that being concentrated to the maggot aliphatic acid after refining using freeze crystallization.
Further, in the step S1, using ethyl acetate as solvent, 70 DEG C, refluxing extraction 2h are warming up to, reflow's cycle
It it is 1 time, the mass ratio of Chinese medicine maggot dry powder and ethyl acetate after drying is 1:5.
Chinese medicine five cereals lacca prepared by any one Chinese medicine maggot fat acid-extraction described in claims 1 to 3
Fat acid is applied in antibacterials.
It can be seen from the above technical proposal that the present invention can prepare antibacterial ability by optimizing extractant, reaction condition
Strong maggot extract obtains concentration and the higher maggot extraction of purity by further increasing refining and concentration step
Object, for that in antimicrobial, the use of antibiotic can be replaced, be a kind of novel, without side-effects antimicrobial product.
Description of the drawings
Fig. 1 is the aliphatic acid extracted amount comparative result figure under the conditions of different feed liquid ratio；
Fig. 2~Fig. 4 is respectively using dichloromethane, petroleum ether, ethyl acetate as extracts reagent, in solid-liquid ratio 1:When 5 extraction
Aliphatic acid extraction quality with extraction time result schematic diagram；
Fig. 5~Fig. 7 is respectively using dichloromethane, petroleum ether, ethyl acetate as extracts reagent, in solid-liquid ratio 1:When 5 extraction
Aliphatic acid extraction quality with extraction time result schematic diagram；
Fig. 8 is the makings spectrogram using the aliphatic acid crude extract of optimal conditions extraction；
Fig. 9 is the comparative result figure of the OD600 values of the adipic acid solution of three kinds of methods extraction；
Figure 10 is antibacterial curve of the 1mg/ml aliphatic acid to S. aureus L-forms；
Figure 11 is antibacterial curve of the 2mg/ml aliphatic acid to Escherichia coli；
Figure 12 is 1mg/ml aliphatic acid to pneumonia diplostreptococcus antibacterial curve；
Figure 13 is antibacterial curve of the 2mg/ml aliphatic acid to pseudomonas aeruginosa.
Specific implementation mode
Below in conjunction with the accompanying drawings, the specific implementation mode of the present invention is described in further detail.
The invention discloses a kind of Chinese medicine maggot fat acid-extractions, include the following steps：
Step S1：Chinese medicine maggot dry powder after drying is wrapped with filter paper in input Soxhlet extractor, dichloromethane is added
Alkane or petroleum ether or ethyl acetate solvent, are warming up to solvent boiling point, refluxing extraction 2h~4h, and reflow's cycle is 1~3 time, wherein
The mass ratio of Chinese medicine maggot dry powder and solvent after drying is 1:5~20.
Step S2：Extracting solution obtained by step S1 is placed in Rotary Evaporators and is evaporated under reduced pressure, recycling design, and in ventilation
Residual solvent is vapored away in cupboard, obtains Chinese medicine maggot aliphatic acid crude extract.
The present invention also advanced optimizes reaction condition, to improve the quality of Chinese medicine maggot extract, respectively to material ratio
Example, extraction time, Extracting temperature etc. are optimized.
One, maggot dry powder and the solid-liquid ratio of extracts reagent optimize
The ground maggot dry powder of 10g is weighed, filter paper, which is wrapped, to be placed in Milko-Tester, presses 1 respectively:2、1:5、
1:10、1:15、1:Each organic extracts reagent is added in 20 solid-liquid ratio, and Soxhlet extractor refluxing extraction is heated with the boiling point of each reagent
After 4h, takes out maggot dry powder and weigh dry powder weight after reagent to be extracted volatilization completely, calculate different feed liquid as follows
The aliphatic acid of ratio extracts quality：
Aliphatic acid quality (g)=M-m (M：Dry weight before being extracted for maggot dry powder；m：Have after being extracted for maggot dry powder
Dry weight after the volatilization completely of machine reagent).
1 is pressed respectively:2、1:5、1:10、1:15、1:Each organic extracts reagent is added in 20 solid-liquid ratio, when solid-liquid ratio is 1:2
When, it can not effectively extract maggot aliphatic acid, thus it is speculated that reason, which may be maggot dry powder lyosoption, causes amount of solution very little, fat
Fat acid extraction efficiency is extremely low.When solid-liquid ratio is 1:5、1:10、1:15 and 1:When 20, the no significant difference of extraction effect amount of aliphatic acid
(as shown in Figure 1), therefore, in order to save reagent dosage, it is 1 to choose solid-liquid ratio:5 carry out subsequent experimental.
Two, extraction time optimizes
The ground maggot dry powder of 10g is weighed, filter paper, which is wrapped, to be placed in Milko-Tester, is 1 by solid-liquid ratio:5
Reagent is added, respectively with the extraction time Soxhlet refluxing extraction of 1h, 2h, 3h, 4h after, calculate the extraction of each aliphatic acid by the above process
Quality, as a result as in Figure 2-4.
With solid-liquid ratio 1:5 extractions, in the extraction result such as figure, after extracting 3h, the extraction matter of three kinds of reagents of different time
Amount is not much different, but most with ethyl acetate；And when extracting 2h, extraction quality and matter when extraction 4h of ethyl acetate
Amount is not much different, it is considered that the extracting method of ethyl acetate 2h should be taken more reasonable after tradeoff.
Three, extraction time optimizes
The ground maggot dry powder of 10g is weighed, filter paper, which is wrapped, to be placed in Milko-Tester, by solid-liquid ratio 1:5 add
Enter reagent, and press respectively by extraction time 2h extraction 1 time, 2 times, after 3 extraction time Soxhlet refluxing extractions, as stated above
Calculate each aliphatic acid extraction quality.
Aliphatic acid is extracted with above-mentioned condition, in result such as Fig. 5-Fig. 7 of different extraction times, ethyl acetate after extracting 1 time
Aliphatic acid extraction quality it is most, and with other reagents extraction quality have otherness；After extraction 3 times, the fat of ethyl acetate
Fat acid extraction quality is still most, but three kinds of reagents extraction mass differences are little and ethyl acetate is being extracted 1 time and extracted 3 times
Aliphatic acid mass difference it is little, consider with ethyl acetate solid-liquid ratio 1:5, it extracts 1 time, extraction 2h is maggot aliphatic acid
Extracting method, recovery rate 21.43%, than the recovery rate 18.67% of dichloromethane under same case and the extraction of petroleum ether
Rate 18.80% is all big, and has statistical significance.[recovery rate formula：Recovery rate (%)=(M-m)/M rates (justice.Carry (M：It is five
Weight before the extraction of paddy worm dry powder；m：Weight after being extracted for maggot dry powder after organic reagent volatilization completely)].
The ingredient of the aliphatic acid crude extract using the extraction of above-mentioned optimal conditions is analyzed below.
Preparation of samples before analysis：It takes 0.1g aliphatic acid to be put into 25ml pipes, is separately added into 4ml benzene-petroleum ether (v:V=1:
1), 4ml 0.4M NaOH-MEOH are put into 50 DEG C of heating water bath 30min, add the tri-distilled water of 10ml, take after acutely mediating
Supernatant nitrogen dries up, and 100ul methanol is added and redissolves, you can analyzes sample as gas chromatograph-mass spectrometer (GC-MS) loading.
GC-MS conditions：Chromatographic condition is 250 DEG C of injector temperature, split sampling, split ratio 10:1, sample size is
1ul；Analytical column is Rxi-5Sil MS 30m x 0.25mm x 0.25um, and column temperature temperature program is 60 DEG C of initial column temperature, is kept
1min, rises to 170 DEG C with 5 DEG C/min, keeps 5min, rises to 230 DEG C with 5 DEG C/min, keeps 5min.Mass Spectrometry Conditions be the sources EI from
Component, 230 DEG C of ion source temperature；250 DEG C of interface temperature；Electron energy is 70eV；Acquisition mode is scanning quality range m/z
45-600；It is qualitative with NIST standard mass spectrum library searchings.
Aliphatic acid crude extract makings spectrogram is as shown in Figure 8, wherein ingredient in figure corresponding to the peak number of numeral mark and
Its content is shown in Table 1.As shown in Table 1：It is miscellaneous also to contain saturated fatty acid etc. in aliphatic acid crude extract besides unsaturated fatty acids
Matter.
Table 1：Chinese medicine maggot fatty acid analysis
But play anti-inflammatory effect is only unsaturated fatty acid, and in order to improve the purity of unsaturated fatty acid, the present invention
Also further aliphatic acid crude extract is refined, it is further comprising the steps of：
Step S3：Using acid refining method degumming, the Chinese medicine maggot aliphatic acid crude extract obtained by step S2 is put into container
In, it is heated with stirring to 70 DEG C~80 DEG C in a water bath, it is Chinese medicine maggot aliphatic acid coarse extraction substance that quality is added while stirring
The 1% of amount, a concentration of 75%~80% phosphoric acid continue 1min~15min, are then centrifuged at 4500r/min
Liquid takes upper layer oil sample, is degumming maggot aliphatic acid.
Step S4：It is degumming maggot aliphatic acid quality that quality is added into the degumming maggot aliphatic acid obtained by step S3
1%~1.5%, the sodium hydroxide solution of a concentration of 300g/L, after uniform stirring, be heated to 65 DEG C~70 DEG C, heat preservation
30min is then cooled to room temperature, stratification, centrifuges to obtain liquid；With remaining soap in deionized water 50ml cleaning liquids
Foot cleans 3 times, later, centrifuges 10~15min with 4500r/min, centrifuge to obtain liquid, take liquid upper layer oil sample, be repeatedly
Maggot aliphatic acid after refining.
Above-mentioned steps 3 and step 4 mainly a small amount of protein in removal maggot aliphatic acid crude extract, phosphatide and glutinous
The colloid substances such as fluidity, heavy metal and removing free fatty therein, keep the maggot aliphatic acid after refining all not
Saturated fatty acid.
The present invention extracts the use of aliphatic acid primarily to as antibacterial, anti-inflammation components from Chinese medicine maggot, below
Aliphatic acid antibacterial activity after refined is detected.
One, the preparation of adipic acid solution
By the fatty acid dilution after being refined using optimal conditions to 20mg/ml, then it is added in a 96 new orifice plates
Each sodium soap salting liquid of 10ul 20mg/ml is added in 90ul culture mediums, and OD values (OD600) are surveyed under microplate reader 600nm.
Two, Bacteria Culture
It takes the bacterium that 20uL has recovered to be added in the NB culture mediums of 3ml fresh steriles, is put into constant-temperature table, 37 DEG C,
160 rotational oscillations swing 3~4h to logarithmic phase (OD values 0.6~0.8), take fresh NB culture mediums that bacterium solution is diluted to about 106cfu/ml
Suspension it is spare.
Three, antibacterial activity detects
96 sterile orifice plates are taken, in the diluted bacteria suspensions of 90uL are added in superclean bench into every hole, administration group is added
10uL 0.1M NaOH solutions are added in the adipic acid solution of concentration needed for 10uL, negative control group, and it is fresh that 10uL is added in blank group
10uL antibiotic solutions are added in culture medium, positive controls, and 4 multiple holes are set per hole, and sealed membrane sealing is placed on 37 DEG C, 5%CO2
Constant incubator in stationary culture, respectively cultivate 0,3,6,9,18h when under 570nm wavelength measure OD values (OD570).Most
All data deduct 0 moment D570 value afterwards, obtain the antibacterial action curve of aliphatic acid.
The aliphatic acid dissolution properties of three kinds of methods extraction have a certain difference, with microplate reader detection each method extraction fat
OD600 values when acid concentration is 2mg/ml are as shown in Figure 9.It can be seen that：Absorbance after the aliphatic acid dissolving of ethyl acetate extraction
It is minimum.
Three kinds of method extraction fatty acid samples and staphylococcus aureus, Escherichia coli, pneumonia diplostreptococcus, verdigris are false single
Born of the same parents bacterium is compared, antibacterial action curve such as Figure 10-figure in 37 DEG C of common incubation 18h with blank sample and 0.1M NaOH samples
13.It can be seen that：1mg/ml aliphatic acid has very strong antibacterial activity, and the fat of ethyl acetate extraction to staphylococcus aureus
Sour best results；Such as Fig. 6-2 it is found that 2mg/ml aliphatic acid is very weak to the antibacterial effect of Escherichia coli, illustrate maggot aliphatic acid
It is not strong to the function and effect of Escherichia coli；Such as Fig. 6-3 it is found that aliphatic acid has very strong antibacterial action to pneumonia diplostreptococcus, and
It is best with ethyl acetate extraction method aliphatic acid antibacterial effect；Such as Fig. 6-4 it is found that three kinds of reagent extraction aliphatic acid are all false to verdigris single
Born of the same parents' bacterium antibacterial activity is suitable.From the above results, aliphatic acid has preferable activity to the bacterium of G+ and G-, wherein to golden yellow
The effect of color staphylococcus and pneumonia diplostreptococcus is best, takes second place to pseudomonas aeruginosa, poor to the effect of Escherichia coli.And
And as can be seen that the function and effect of ethyl acetate extraction aliphatic acid all extract fat compared with other two kinds of reagents on antibacterial action curve
Fat acid effect will be got well.
Four, the minimal inhibitory concentration of doubling dilution detection aliphatic acid
96 sterile orifice plates are taken, in 180ul bacterium solutions are added in superclean bench into the first hole, are added in remaining hole
The bacterium solution of 100ul；20ul (2mg/mL) aliphatic acid is added in first hole administration group, and it is molten that 20ul 0.1M NaOH are added in negative control group
20ul antibiotic is added in liquid, positive controls, and 20ul fresh cultures are added in blank group, and 100ul, which is sucked out, after mixing is added second
Hole, and so on, 100ul liquid is removed after last hole mixing, it is final per liquid of the hole containing 100ul；Per 3, hole multiple holes.Envelope
96 orifice plates are put in stationary culture in 37 DEG C of constant temperature incubators after mouthful and for 24 hours, OD values (OD570) are then measured under 570nm wavelength.Suppression
Bacterium rate calculation formula is as follows：
Bacteriostasis rate (%)=(negative control group OD values-medicine group OD values)/negative control group OD value × 100%
Minimal inhibitory concentration (MIC) result such as table 2 of three kinds of method extraction aliphatic acid.From table 2 it can be seen that ethyl acetate
It is minimum for the MIC of S. aureus L-forms and pseudomonas aeruginosa to extract aliphatic acid, and the aliphatic acid that three kinds of extracting methods are extracted is for lung
Scorching diplostreptococcus three MIC is suitable, and the antibacterial effect for considering the aliphatic acid of ethyl acetate extraction is optimal.
Table 2：The minimal inhibitory concentration (MIC) of Chinese medicine maggot aliphatic acid
As it can be seen that the Chinese medicine maggot aliphatic acid that the present invention extracts can be applied in antibacterials, have to golden yellow Portugal
Grape coccus, pneumonia diplostreptococcus, pseudomonas aeruginosa antibacterial ability.Antibacterial ability with Chinese medicine maggot content of fatty acid increasing
Add and enhances.Therefore, in order to improve the antibacterial ability of drug, the present invention is further dense to the Chinese medicine maggot aliphatic acid after refining
Contracting, the product after concentration is added in drug, enhances antibacterial effect.
It is concentrated using freeze crystallization, the depickling maggot aliphatic acid after refining, which is placed in 0 DEG C of refrigerator, to be placed
12h, maggot fat acid crystal are constantly precipitated, and obtain the crystallization of maggot fatty-acid solid with suction method to get refined five rapidly
Paddy worm aliphatic acid.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (4)
1. a kind of Chinese medicine maggot fat acid-extraction, which is characterized in that include the following steps：
Step S1：By the Chinese medicine maggot dry powder after drying with filter paper wrap input Soxhlet extractor in, be added dichloromethane or
Petroleum ether or ethyl acetate solvent, are warming up to solvent boiling point, refluxing extraction 2h~4h, and reflow's cycle is 1~3 time, wherein dry
The mass ratio of Chinese medicine maggot dry powder and solvent afterwards is 1:5~20；
Step S2：Extracting solution obtained by step S1 is placed in Rotary Evaporators and is evaporated under reduced pressure, recycling design, and in draught cupboard
Residual solvent is vapored away, Chinese medicine maggot aliphatic acid crude extract is obtained；
Step S3：Using acid refining method degumming, the Chinese medicine maggot aliphatic acid crude extract obtained by step S2 is put into container,
Stirred in water bath is heated to 70 DEG C~80 DEG C, and it is Chinese medicine maggot aliphatic acid crude extract quality that quality is added while stirring
1%, a concentration of 75%~80% phosphoric acid, continue 1min~15min, be then centrifuged for detaching to obtain liquid, take upper layer oil sample, be
Degumming maggot aliphatic acid；
Step S4：It is degumming maggot aliphatic acid quality that quality is added into the degumming maggot aliphatic acid obtained by step S3
1%~1.5%, the sodium hydroxide solution of a concentration of 300g/L, after uniform stirring, be heated to 65 DEG C~70 DEG C, heat preservation
30min is then cooled to room temperature, stratification, centrifuges to obtain liquid；With remaining soap stock in deionized water cleaning liquid,
It cleans 3 times repeatedly, later, centrifuges to obtain liquid, take liquid upper layer oil sample, for the maggot aliphatic acid after refining.
2. Chinese medicine maggot fat acid-extraction according to claim 1, which is characterized in that further include using freezing knot
The step of crystallization concentrates the maggot aliphatic acid after refining.
3. Chinese medicine maggot fat acid-extraction according to claim 1, which is characterized in that in the step S1, with
Ethyl acetate is solvent, is warming up to 70 DEG C, refluxing extraction 2h, and reflow's cycle is 1 time, the Chinese medicine maggot dry powder after drying and second
The mass ratio of acetoacetic ester is 1:5.
4. Chinese medicine maggot fat prepared by any one Chinese medicine maggot fat acid-extraction described in claims 1 to 3
Acid is applied in antibacterials.
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| CN109464468A (en) * | 2018-11-29 | 2019-03-15 | 四川金岁方药业有限公司 | A kind of maggot powder and preparation method thereof |
| CN109394792A (en) * | 2018-12-20 | 2019-03-01 | 大连麦琪克生物技术有限公司 | A kind of preparation method of Calliphoridae insect larvae extract |
| CN112322681A (en) * | 2019-08-01 | 2021-02-05 | 大连麦琪克生物技术有限公司 | Traditional Chinese medicine maggot active peptide and preparation method and application thereof |
| CN112322681B (en) * | 2019-08-01 | 2022-09-09 | 大连麦琪克生物技术有限公司 | Traditional Chinese medicine maggot active peptide and preparation method and application thereof |
| CN114886927A (en) * | 2022-05-16 | 2022-08-12 | 西北工业大学 | Maggot active ingredient composition and preparation method and application thereof |
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