CN104042633A - Extraction method of Periplaneta americana L., Periplaneta americana L. extract and use of Periplaneta americana L. extract - Google Patents
Extraction method of Periplaneta americana L., Periplaneta americana L. extract and use of Periplaneta americana L. extract Download PDFInfo
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- CN104042633A CN104042633A CN201410091462.9A CN201410091462A CN104042633A CN 104042633 A CN104042633 A CN 104042633A CN 201410091462 A CN201410091462 A CN 201410091462A CN 104042633 A CN104042633 A CN 104042633A
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Abstract
The invention provides an extraction method of Periplaneta americana L. The extraction method comprises that Periplaneta americana L. is subjected to flash extraction in the presence of and water as a solvent. The invention also provides Periplaneta americana L. extract and a use thereof. The extraction method of Periplaneta americana L. has the advantages of simple processes, short extraction time and low energy consumption. The Periplaneta americana L. extract has liver damage-resistant activity obviously better than that of other extraction methods, has more comprehensive effects, effectively improves a utilization rate of a Periplaneta americana L. medicinal material, reduces a production cost and has a good industrial application prospect.
Description
Technical field
The present invention relates to extracting method and the extract obtained and purposes of periplaneta americana.
Background technology
Periplaneta americana (Periplaneta americana L.), for Insecta Blattaria Blattidae Periplaneta insecticide, be commonly called as Blatta seu periplaneta, it is used as medicine and begins to be loaded in < < Sheng Nong's herbal classic > >, its taste salty in the mouth cold in nature, poisonous, there is acid, have dissipating blood stasis, the function such as removing food stagnancy, removing toxic substances, diuretic, detumescence.Curing mainly infantile malnutrition, tonsillitis, health enclosed mass, carbuncle skin ulcer swells and ache and Scolopendra, venom etc.
Modern study discovery, periplaneta americana is used for the treatment of the various diseases such as oral ulcer, esophageal ulcer, esophageal mucosa membrane injury damage, gastrorrhagia, gastric and duodenal ulcers, exterior trauma, burn and scald, diabetic ulcer, deficiency of YIN pulmonary tuberculosis, pulmonary tuberculosis.
Current research thinks, the main effective ingredient of periplaneta americana is aminoacid, polypeptide constituents.In current preparation production technique, the extraction of total amino acids adopts heat reflow method conventionally.Wu Hongmeis etc. have been studied the extraction effect of different extracting modes to periplaneta americana main component, adopt respectively 85% ethanol to reflux or flash extraction, wherein, in flash extracting solution the content of total amino acids be respectively 5.63,5.69,5.65,5.56,5.77mg/g, in reflux extracting liquid, be respectively 5.69,5.87,5.71,5.65,5.84mg/g, both compare, basically identical (the Wu Hongmei of total amino acids content, Deng, the technique comparison of flash and reflux, extract, periplaneta americana total amino acids, Chinese experimental pharmacology of Chinese medical formulae magazine, 18 21 phases of volume in 2012).
Summary of the invention
The object of the present invention is to provide the extracting method of a kind of periplaneta americana.The present invention also provides a kind of American-cockroach-extract and uses thereof.
The invention provides the extracting method of periplaneta americana: get periplaneta americana, take water as solvent, through flash extraction, prepare; The technological parameter of described flash extraction is: extraction time 1～5min/ time, voltage 50～150V, extracts 1～4 time.
Further, the technological parameter of described flash extraction is: extraction time 3min/ time, voltage 90V, extracts 2 times.
Further, water is periplaneta americana 8～16 times.
Further, water is periplaneta americana 10 times.
The American-cockroach-extract that the present invention also provides said method to prepare.
Further, in this extract, total peptide content is more than 0.8%, and free aminoacid content is more than 2.0%.
The present invention also provides the purposes of above-mentioned American-cockroach-extract in preparing the medicine of anti-liver injury.
Further, described medicine is to reduce serum transaminase, antioxidative medicine.
Further, described serum transaminase is serum glutamic pyruvic transminase or glutamic oxaloacetic transaminase, GOT.
The present invention also provides a kind of pharmaceutical composition, and it is the preparation that contains above-mentioned American-cockroach-extract.
The extracting method of periplaneta americana of the present invention, easy and simple to handle, consuming time short, energy consumption is low, extract obtained anti-liver injury drug activity is obviously better than other extracting method, and effect is more comprehensive, has effectively improved the utilization rate of periplaneta americana medical material, reduce production cost, there is good prospects for commercial application.
Although now there are some researches show, in the American-cockroach-extract of employing backflow, flash extracting method gained, the amino acid whose content of effective ingredient is basically identical, yet, the present invention uses the flash extract of specific extraction solvent and extracting parameter gained, its antihepatitis activity is better than refluxing and warm macerating extracts, special in the most remarkable to the reducing effect of serum transaminase.
Accompanying drawing explanation
The impact of Fig. 1 American-cockroach-extract on murine liver tissue pathomorphism
The specific embodiment
The extracting method of embodiment 1 periplaneta americana
The coarse powder of periplaneta americana medical material of getting 50 ℃ of oven dry is appropriate, adds 10 times of water gagings under room temperature, carries out flash extraction, and wherein, each extraction time is 3min, and voltage is 90V, extracts 2 times, and filtration, merging filtrate, obtains American-cockroach-extract.
The extracting method of embodiment 2 periplaneta americanaes
The American-cockroach-extract of getting embodiment 1 preparation, concentrating under reduced pressure, adds appropriate dextrin, starch, prepares granule, capsule or tablet.
By test example, beneficial effect of the present invention is described below.
Test example 1
1 material and instrument
1.1 material
Periplaneta americana medical material (purchased from Tengchong In Yunnan Province, award and be accredited as Insecta Pterigota Blattaria Blattidae Periplaneta insecticide periplaneta americana Periplaneta americana L. through the first penetrating judgment of Lu of crude drug teaching and research room)
Kunming kind white mice (Da Shuo bio tech ltd, Chengdu, body weight 18～22g, male and female half and half)
(BSA, Chinese pharmaceutical biological product is identified institute to bovine serum albumin standard substance, 140619-201120)
(Chinese pharmaceutical biological product is identified institute to alanine standard substance, 140680-201002)
Bifendate drop pill (Zhejiang Medicine Co, 120801,1.5mg*250 ball)
1.2 instrument
Digital display constant temperature blender with magnetic force (Mei Ying Pu, Shanghai 88-1)
Ultraviolet-visible spectrophotometer (UV-1700 of Hitachi)
2 methods and result
It is appropriate that the preparation method of 2.1 extracts is got the coarse powder of medical material of 50 ℃ of oven dry, under room temperature, add respectively 10 times of water gagings or ethanol, press circumfluence method (1h/ time), flash extraction method (3min/ time respectively, 90V) extract with magnetic agitation extraction method (12h/ time), each 2 times, filter merging filtrate, make respectively water backflow, 95% alcohol reflux, water is flash, 95% ethanol is flash, water stirs, 95% ethanol stirs extracting solution, respectively referred to as A-F.
2.2 anti-CCl
4type acute liver damage effect research
2.2.1 for the preparation of reagent liquid, by 2.1 methods, extract, concentrating under reduced pressure the 0.5%CMC-Na of take are mixed with concentration as 1gml
-1for reagent liquid.Positive drug bifendate drop pill powder be take 0.5%CMC-Na and is mixed with the suspension that concentration is 15mg/ml.
2.2.2 100 of mices are got in administration and modeling, and male and female half and half are divided into 10 groups at random, and 10 every group, i.e. blank group, solvent control group, model control group, positive controls, extract A-F group.Except blank group ig pure water, beyond solvent control group and model control group ig0.5%CMC-Na, all the other organize equal ig relative medicine (10ml(kgd)
-1), continuous 10d.After last administration, reference literature to respectively organizing ip0.2%CCl except blank, solvent control group
4oil solution (10mlkg
-1) duplicating model.After 24h, eyeball is got blood, separation of serum; After putting to death mice, dissect and get full liver and spleen immediately.
2.2.3 index detection assay serum glutamic pyruvic transminase (ALT), glutamic oxaloacetic transaminase, GOT (AST) activity and malonaldehyde (MDA) content, superoxide dismutase (SOD) activity; Full liver and the weighed quality of spleen, calculate organ coefficient; Separated leftlobe of liver is fixed on censorship in 10% formalin.
2.2.4 date processing adopts SPSS17.0 software to carry out statistical disposition.Experimental result is with mean ± standard deviation
represent, with one factor analysis of variance, compare group difference.
2.2.5 drug effect comparison
Table 1 American-cockroach-extract on the impact of mice serum transaminase activity (
n=10)
With solvent control group comparison: ##P<0.01
With model control group comparison: * * P<0.01, under * P<0.05(with)
By the visible model group animal ALT of table 1 and AST activity, all apparently higher than blank and solvent control group, show modeling success; Compare with model group, extract C, D and bifendate all can obviously play opposing CCl
4cause the effect that serum transaminase raises, and A, E, F only present the enzyme trend of falling, and act on remarkable not; B even has the trend that increases the weight of damage.
Table 2 murine liver tissue pathomorphism standards of grading
Table 3 American-cockroach-extract on the impact of murine liver tissue pathomorphism (
n=10)
From table 3, Fig. 1, blank, solvent control group hepatic tissue structure is intact, hepatic sinusoid has no expansion, and hepatocyte size is more even, and Cytoplasm is abundant, its nucleolus.All the other groups have respectively form in various degree to change, and main damage form has hepatocyte hydropic degeneration and necrosis.Wherein model group pathological changes is the heaviest, hepatocyte obvious tumefaction, and hydropic degeneration is serious, and cell boundary is unclear, and endochylema is light to be dyed, visible significantly inflammatory infiltration and necrosis, all there is significant difference with blank, solvent control group in Injury score, shows modeling success; And C, E group only exists individually hydropic degeneration with downright bad, inflammatory infiltration is less, and scoring all exists significant difference with model group.Showing that extract C, E have comparatively significantly improves CCl
4the effect that induced mice hepatocyte pathology changes.
Table 4 American-cockroach-extract on the impact of mice organs coefficient (
n=10)
The heavy coefficient of model group animal liver obviously raises as shown in Table 4, in pathological manifestations, can correspond to hepatocyte inflammatory infiltration, edema etc.; Compare with model group, C group significantly reduces, and binding of pathological morphology microscopy result conjecture C can improve the hepatocyte hydropic degeneration that modeling causes, thereby the heavy coefficient of C treated animal liver increases limited.The variation of the heavy coefficient of each experimental group spleen can reflect that the immune state of body is different to a certain extent.First model group and solvent control group height two ends in sequence completely, visible CCl
4as foreign object, stimulate the immune system of animal has been produced to activation, cause the increase that spleen is heavy; And C, E have improved CCl
4the spleen causing heavily increases, and conjecture may be by antagonism CCl
4stimulation to body, mechanism may be relevant to antioxidation, elimination inflammatory infiltration, reparation etc.
Table 5 American-cockroach-extract on the impact of mice serum MDA content, SOD activity (
n=10)
As shown in Table 5, model group Content of MDA obviously raises, and SOD activity decreased; Bifendate and extract B, C, D, E have the effect that antagonistic Serum MDA content raises, but are not embodied aspect active improving SOD, may be removed free radicals or suppressed peroxidization by other approach, rather than enhance SOD activity.
In sum, extract C is at antagonism CCl
4induced Acute hepatic injury mice serum MDA increases (* * P<0.01), suppress that serum transaminase increases (* * P<0.01), (* * P<0.01) is heavy with liver, the heavy coefficient of spleen raises, and aspects such as (* P<0.05) all has good effect to alleviate liver tissue lesions; hepatic injury is had to certain protection and therapeutical effect; and the effect of all the other extracts or be only embodied in above-mentioned some aspect is not comprehensive.
2.3 paste-forming rates are measured
Get medicinal powder 25g, prepare extracting solution A-F by 2.1, respectively measure 100ml and put in the evaporating dish of constant weight, water-bath volatilizes solvent, and 105 ℃ of constant pressure and dries are to constant weight, and weighed quality, calculates paste-forming rate.
2.4 total peptide contents are measured
2.4.1BSA the preparation of reference substance solution
Get the about 7.5mg of BSA, with water, be settled to 50mL after accurately weighed, obtain reference substance solution (153.4mgl
-1).
2.4.2 the preparation of need testing solution
Respectively get medicinal powder 25g, by 2.1 operations, merging filtrate is settled to 500ml with coordinative solvent, as storing solution.Accurate storing solution A, each 8ml of C, E of drawing, filters, and filtrate is settled to 25ml with water, as need testing solution A, C, E; Get storing solution B, D, each 100ml of F, after decompression and solvent recovery, add water appropriate, heated and stirred, filters, and filtrate is settled to 25ml with water, shakes up, as need testing solution B, D, F.
2.4.3 determining of maximum absorption wavelength
Accurate reference substance solution, water, each 1ml of need testing solution of drawing, adds respectively Coomassie brilliant blue G-250 solution 5ml, shakes up, and in 400～800nm, scans.Result shows that reference substance and sample have absorption maximum at 595nm place, blank noiseless, λ
max=595nm.
2.4.4 the foundation of standard curve
Each accurate absorption 0,0.2,0.4,0.6,0.8,1.0ml reference substance solution, add water to 1ml, by 2.4.3 colour developing, measures A
595, take albumin concentration as abscissa, A
595for vertical coordinate production standard curve.Its regression equation is y=0.0041x+0.1500 (r=0.9995), and within the scope of 30.69～153.45 μ g, between the content of BSA and absorbance, linear relationship is good.
2.4.5 precision test
The accurate reference substance solution 1ml that draws presses 2.4.4 replication 6 times.RSD=0.157%, shows that precision is good.
2.4.6 replica test
Accurate 6 parts of the same lot reservation liquid of drawing are prepared need testing solution by 2.4.2, and the accurate above-mentioned solution 1ml of absorption also presses 2.4.4 and measures A.RSD=0.736%, shows that repeatability is good.
2.4.7 stability test
The accurate need testing solution 1ml that draws measures A by 2.4.4 colour developing and in 0,10,20,30, during 40min.RSD=1.115%, shows that need testing solution is stable in 40min.
2.4.8 average recovery test
Same batch of sample storing solution (451.7mgl of accurate absorption
-1) totally 6 parts of 2ml, respectively add reference substance solution 6ml, by 2.4.2, prepare need testing solution, and press 2.4.4 and measure A.Average recovery rate is 98.055%, RSD=2.816%.
2.4.9 need testing solution assay
Accurate each 1ml of test sample liquid that draws, measures A by 2.4.4.By standard curve, calculate total peptide content.
2.5 free aminoacid contents are measured the equal reference literature of 2.5.4～2.5.6 and are carried out.
2.5.1 the preparation of need testing solution
Accurate each 10ml of storing solution preparing by 2.4.2 that draws, adjusts pH to 4.4, and boiling water bath 5min is standing, centrifugal, gets supernatant and is settled to 25ml with coordinative solvent, obtains diluent.Accurate diluent A, each 2ml of C, E and B, D, each 4ml of F of drawing, is settled to 50ml, as need testing solution respectively.2.5.2 determining of maximum absorption wavelength
Accurate alanine reference substance solution, water, 95% ethanol, each 1ml of need testing solution of drawing, reference literature colour developing, scans in 400～800nm.Result shows that reference substance and sample have absorption maximum at 570nm place, blank, negative noiseless, λ
max=570nm.
2.5.3 the foundation of standard curve
The accurate alanine reference substance solution (15.88mgl that draws
-1) 0,0.3,0.6,0.9,1.2,1.5,1.8,2.1,2.4ml, by 2.5.2, develop the color and measure A
570.Take alanine concentration as abscissa, absorbance be vertical coordinate preparation standard curve.Its regression equation is y=0.0248x-0.0064 (r=0.9994), and within the scope of 4.764～38.112 μ g, between the content of alanine and absorbance, linear relationship is good.
2.5.4 precision test RSD=0.199%, shows that precision is good.
2.5.5 replica test RSD=1.382%, shows that repeatability is good.
2.5.6 stability test RSD=0.515%, shows that need testing solution is stable in 1h.
2.5.7 average recovery test
Same batch of sample storing solution (1061.25mgl of accurate absorption
-1) 6 parts by 2.5.1, prepare sample diluting liquid, the accurate sample diluting liquid 2ml that draws also adds alanine reference substance solution (317.6mgl
-1) 2.5ml, being settled to 50ml, the accurate above-mentioned solution 1ml of absorption presses 2.5.3 and measures A.Average recovery rate is 101.178%, RSD=2.894%.
2.5.8 need testing solution assay
Accurate each 1ml of need testing solution that draws presses 2.5.3 mensuration A, by standard curve, calculates amino acid content.
2.6 technology assessment
According to formula y=(x
1* f
1/ x
1max+ x
2* f
2/ x
2max+ x
3min* f
3/ x
3) * 100 many indexs of calculating weighting scores, carry out technology assessment.X wherein
1, x
2, x
3represent respectively total peptide content, free aminoacid content and paste-forming rate; Total peptide, free amino acid is respectively the main component in water, alcohol extract, is also possible active ingredient, is therefore leading indicator, and weight setting is f
1=f
2=0.4; And paste-forming rate is only auxiliary characteristics, weight setting is f
3=0.2.
The total peptide of table 6, free aminoacid content and paste-forming rate measurement result (n=3)
Between water extract, content difference is little as shown in Table 6, but apparently higher than ethanol extract, illustrates that take water is more suitable for the extraction of total peptide and free amino acid as solvent.The comprehensive grading of each technic index shows, the flash extraction method score of water is the highest, and in conjunction with the control with production cost and the energy that maintains of extract pharmacologically active, this method has obvious advantage.
4 discuss
CCl
4the main feature of acute hepatic injury model is the peroxidating characteristic of free radical and the adipose membrane destruction of causing thus, organelle damage, hydropic degeneration etc.The present invention shows that the flash extract of periplaneta americana water has good protective effect to above-mentioned model mice and drug effect is better than other extracts, and Content of MDA measurement result shows that it has the snperoxiaized ability that alleviates, its pharmacological mechanism may with alleviate CCl
4the hepatocyte peroxidating causing is relevant.
Assay result also shows that the total peptide content of extract just with a little less than anti-liver injury strong drug action exists certain contacting, and prerequisite need keep active.Flashly compare with magnetic agitation extraction method with backflow, the former has the highest total peptide, free aminoacid content, and the activity for composition retains this point, be undoubtedly one of safer method, both can avoid the stimulation of high temperature to heat-sensitive ingredients, thereby can shorten again the change that extraction time reduces composition, especially be suitable for the extraction of the materials such as peptide class, this is also perhaps that the flash extract drug effect of water is better than one of other reason.Make a general survey of related preparations state of art, in conjunction with production requirement, be not difficult to find, flash water extraction has potentiality and the value of formulation development.
The present invention has only carried out the comparison content from the angle of large constituents to each extract, can not disclose for a full due the material base of periplaneta americana anti-liver injury.In the situation that also accurately not knowing periplaneta americana anti-liver injury material base at present, research worker can be according to the present invention multiple Different Extraction Method extract obtained between the difference of drug activity, further launch the research to periplaneta americana anti-liver injury material base.
In sum, the extracting method of periplaneta americana of the present invention, easy and simple to handle, consuming time short, energy consumption is low, and extract obtained anti-liver injury drug activity is obviously better than other extracting method, and act on more comprehensive, the utilization rate that has effectively improved periplaneta americana medical material, has reduced production cost, has good prospects for commercial application.
Claims (10)
1. the extracting method of periplaneta americana, is characterized in that: get periplaneta americana, take water as solvent, through flash extraction, prepare; The technological parameter of described flash extraction is: extraction time 1～5min/ time, voltage 50～150V, extracts 1～4 time.
2. extracting method according to claim 1, is characterized in that: the technological parameter of described flash extraction is: extraction time 3min/ time, voltage 90V, extracts 2 times.
3. extracting method according to claim 1, is characterized in that: water is periplaneta americana 8～16 times.
4. extracting method according to claim 3, is characterized in that: water is periplaneta americana 10 times.
5. the American-cockroach-extract that described in claim 1～4 any one prepared by method.
6. American-cockroach-extract according to claim 5, is characterized in that: in this extract, total peptide content is more than 0.8%, and free aminoacid content is more than 2.0%.
7. the purposes of American-cockroach-extract in preparing the medicine of anti-liver injury described in claim 5 or 6.
8. purposes according to claim 7, is characterized in that: described medicine is to reduce serum transaminase or antioxidative medicine.
9. purposes according to claim 8, is characterized in that: described serum transaminase is serum glutamic pyruvic transminase or glutamic oxaloacetic transaminase, GOT.
10. a pharmaceutical composition, is characterized in that: it is the preparation that contains American-cockroach-extract described in claim 5 or 6.
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| CN108403723A (en) * | 2018-05-07 | 2018-08-17 | 中国科学院昆明动物研究所 | A kind of American-cockroach-extract that intervening AD and its extracting method and application |
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Application publication date: 20140917 |