CN103819575B - The purposes of a kind of extracting method of Ground Beetle polysaccharide, the polysaccharide of extraction and this polysaccharide - Google Patents
The purposes of a kind of extracting method of Ground Beetle polysaccharide, the polysaccharide of extraction and this polysaccharide Download PDFInfo
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Abstract
An extracting method for Ground Beetle polysaccharide, step is as follows: (1) degreasing: pulverized by female for dry Ground Beetle body, sieve, add solvent degreasing, backflow, suction filtration, and it is stand-by that filter residue volatilizes solvent; (2) supersound process; (3) ferment treatment: adjustment pH, then uses combinative enzyme hydrolysis, is then warming up to 85 DEG C of enzymes that go out, suction filtration, collect filtrate, concentrated, obtains concentrated extracting solution; (4) alcohol precipitation: step (3) gained concentrated extracting solution is added ethanol, places and within 24 hours, separates out Ground Beetle Crude polysaccharides precipitation, suction filtration, and with 95wt% ethanol and washing with acetone twice, 50 ~ 70 DEG C of vacuum-dryings, obtain Ground Beetle polysaccharide.Present invention demonstrates that this polysaccharide is applied in anticoagulant.The present invention has that extraction conditions gentleness, extraction yield are high, with low cost, simple to operate, advantages of environment protection.
Description
Technical field
The present invention relates to biomedicine technical field, be specifically related to a kind of extracting method of Ground Beetle polysaccharide, the Ground Beetle polysaccharide utilizing the method to extract, and the purposes of this polysaccharide in platelet aggregation-against.
Background technology
Chinese medicine Ground Beetle is the ground such as the dry body of Corydiidae insect eupolyphoge sinensis or Ji eupolyphoge sinensis, main product Fujian, Hebei, Guangxi, Guangdong, Shandong, and having removing blood stasis, the effects such as reunion of fractured tendons and bones, is a kind of important traditional Chinese medicine.Modern pharmacological research shows that Ground Beetle has the effects such as thrombolysis, anticoagulation, antitumor, adjusting blood lipid.Its main component comprises the materials such as various active albumen, amino acid, unsaturated fatty acids, trace element, alkaloid and liposoluble vitamin, and rarely has report to the extraction of Ground Beetle polysaccharide and purposes.
The extracting method of existing polysaccharide mainly contains water extraction and alcohol precipitation method, microwave method, acid-base method etc.But the shortcomings such as equipment requirements is high, soda acid destroys original polysaccharide structures, aftertreatment is loaded down with trivial details that microwave method, acid-base method etc. have.
Now, also there is a kind of ferment treatment method.Such as there is bibliographical information to utilize papoid to process Ground Beetle, and then polysaccharide is extracted, but utilize polysaccharide content in the Eupolyphaga seu steleophaga extract after Papain ferment treatment lower.
And present Ground Beetle is in traditional Chinese medicine, and purposes is: broken hemostasis, continuous muscles and bones, for injured bone, hemostasis through closing, abdominal mass lump in the abdomen.
Therefore, await providing one more advanced, rational extracting method, to improve Ground Beetle Polyose extraction amount, and functional study is carried out to the polysaccharide extracted.
Summary of the invention
Technical problem to be solved by this invention is to provide the purposes of a kind of extracting method of Ground Beetle polysaccharide, the polysaccharide of extraction and this polysaccharide, thus eliminates defect in above-mentioned background technology.
For solving the problems of the technologies described above, technical scheme of the present invention is:
An extracting method for Ground Beetle polysaccharide, step is as follows:
(1) degreasing: pulverized by female for dry Ground Beetle body, sieve, add solvent degreasing, reflux 2 ~ 5 times, each 1 ~ 3 hour, suction filtration, it is stand-by that filter residue volatilizes solvent;
(2) supersound process: be soaked in the distilled water of 30 ~ 50 times of weight by step (1) gained filter residue, with 200 ~ 400W power ultrasonic process 15 ~ 45 minutes, obtains solid-liquid mixture;
(3) ferment treatment: it is 3 ~ 6 that the solid-liquid mixture of step (2) gained is adjusted pH, then use prozyme temperature 45 ~ 65 DEG C, solid-liquid ratio is 1:20 ~ 1:40 Water Under solution 40 ~ 90 minutes, then 85 DEG C of enzymes that go out are warming up to, suction filtration, collects filtrate, concentrated, obtain concentrated extracting solution, solid-liquid ratio here refers to that solid masses (g) compares with the volume (ml) of liquid;
(4) alcohol precipitation: step (3) gained concentrated extracting solution is added ethanol, makes ethanol ultimate density in system be 80wt%, places and within 24 hours, separates out Ground Beetle Crude polysaccharides precipitation, suction filtration, with 95wt% ethanol and washing with acetone twice, 50 ~ 70 DEG C of vacuum-dryings, obtain Ground Beetle polysaccharide.
Improve as one, in described step (1), after being pulverized by female for dry Ground Beetle body, cross 40 mesh sieves.
Improve as one, in described step (1), solvent is sherwood oil, and consumption is 10 times of weight of solid after sieving.
Improve as one, in described step (2), filter residue is soaked in supersound process in the distilled water of 40 times of volumes, and ultrasonic power is 300W, and ultrasonic time is 30 minutes.
Improve as one, in described step (3), utilize HAc-NaAc buffered soln to adjust pH.
Improve as one, in described step (3), control pH is 4.5, and temperature is 45 DEG C, and soaking time is 90 minutes.
Improve as one, in described step (3), prozyme is stomach en-, papoid and trypsinase, and its weight is respectively 1.5% ~ 2.0%, 1.5% ~ 2.0% and 1.5% ~ 2.0% of filter residue weight.
Contriver finds under study for action, the Ground Beetle Crude polysaccharides obtained is carried out platelet aggregation-against experiment outside animal body, in body, can suppress significantly by the platelet aggregation of ADP, AA, PAF, collagen, thrombin induction, and done relevant animal In vitroandin vivotrial, prove that the Ground Beetle Crude polysaccharides that the present invention obtains can be applied in anticoagulant.
In order to this purposes is better described, contriver is described as follows by pharmacological evaluation and result, and following Ground Beetle polysaccharide all adopts preparation method provided by the invention to prepare.
Be below Ex vivo animal experiment:
Male and healthy rabbit, 12h fasting before experiment.Sodital auricular vein injecting anesthetic, arteria carotis communis gets blood, with 3.8% Sodium Citrate anti-freezing (volume ratio 9:1).Centrifugation platelet rich plasma (PRP) and platelet poor plasma (PPP), adjusting PRP concentration with PPP is 5 × 108cell/mL.Platelet aggregation instrument returns to zero with PPP, and PRP, in 37 DEG C of incubations 5 minutes, adds ADP(or AA or PAF or collagen or zymoplasm) solution, magneton stirs curve of platelet aggregation and MA in lower record 5min.Each experiment repetition 6 times.
Male and healthy rabbit, 12h fasting before experiment.Sodital auricular vein injecting anesthetic, arteria carotis communis gets blood, with 3.8% Sodium Citrate anti-freezing (volume ratio 9:1).Centrifugation platelet rich plasma (PRP) and platelet poor plasma (PPP), adjusting PRP concentration with PPP is 5 × 108cell/mL.Platelet aggregation instrument returns to zero with PPP, the Ground Beetle polysaccharide normal saline solution that concentration is 10mg/L, 20mg/L, 40mg/L, 80mg/L, 160mg/L is added respectively in PRP, in 37 DEG C of incubations 5 minutes, add ADP(or AA or PAF or collagen or zymoplasm) solution, magneton stirs curve of platelet aggregation and MA in lower record 5min.Each experiment repetition 6 times.The results are shown in Table 1.
Result calculates: inhibiting rate (%)=[(1-medicine group aggregation rate/blank group aggregation rate)] × 100
Male and healthy rabbit, 12h fasting before experiment.Sodital auricular vein injecting anesthetic, arteria carotis communis gets blood, with 3.8% Sodium Citrate anti-freezing (volume ratio 9:1).Centrifugation platelet rich plasma (PRP) and platelet poor plasma (PPP), adjusting PRP concentration with PPP is 5 × 108cell/mL.Platelet aggregation instrument returns to zero with PPP, the acetylsalicylic acid 0.8%DMSO solution that concentration is 10mg/L, 20mg/L, 40mg/L, 80mg/L, 160mg/L is added respectively in PRP, in 37 DEG C of incubations 5 minutes, add ADP(or AA or PAF or collagen or zymoplasm) solution, magneton stirs curve of platelet aggregation and MA in lower record 5min.Each experiment repetition 6 times.The results are shown in Table 1.
Result calculates: inhibiting rate (%)=[(1-medicine group aggregation rate/blank group aggregation rate)] × 100
Data show, Ground Beetle polysaccharide can suppress by the platelet aggregation of ADP, AA, PAF, collagen, thrombin induction in vitro significantly.
Table 1 Ground Beetle polysaccharide and acetylsalicylic acid In Vitro Anti platelet aggregation ability
n
N=6, x ± s, * P < 0.01, compares with blank.
Blank group aggregation rate (%): ADP is 40.3 ± 3.3; AA is 68.4 ± 3.5; PAF is 72.9 ± 5.2; Collagen is 54.2 ± 3.2; Zymoplasm is 33.2 ± 2.7.
Be below interior animal experiment:
Acetylsalicylic acid is dissolved in the Na of 100mmol/L before use
2cO
3in, be adjusted to 7.0 with HCl.Ground Beetle polysaccharide is dissolved in physiological saline.Male Wistar rat, is divided into 6 groups at random, often organizes 6.A group: injection equal-volume physiological saline.B group: using the dosage of 10mg/kg injection aspirin solution as positive control.C-F group: with the dosage of 10mg/kg injection Ground Beetle polysaccharide normal saline solution.By above-mentioned grouping respectively by tail vein injection administration.Heart extracting blood after 0.5h.PRP and PPP preparation and platelet aggregation measure same experiment in vitro.The results are shown in Table 2.
Data show, namely Ground Beetle polysaccharide administration 0.5h obviously suppresses the rat platelet aggregation of ADP, AA, PAF, collagen and thrombin induction.Acetylsalicylic acid equally obviously suppresses the rat platelet aggregation of AA, collagen and thrombin induction in vivo, but the platelet aggregation unrestraint effect to ADP and PAF induction.Research is reached a conclusion: in the body of Ground Beetle polysaccharide, platelet aggregation inhibitory activity is similar to its external activity result, can obviously suppress by the platelet aggregation of ADP, AA, PAF, collagen, thrombin induction.
Platelet aggregation-against ability in table 2 Ground Beetle polysaccharide and acetylsalicylic acid body
N=6, x ± s, * P < 0.01, compares with blank group.
Blank group aggregation rate (%): ADP is 45.7 ± 7.2; AA is 59.2 ± 4.4; PAF is 62.3 ± 5.9; Collagen is 50.2 ± 4.4; Zymoplasm is 30.2 ± 1.8.
Beneficial effect of the present invention is as follows:
The ferment treatment that the present invention adopts is compared other method and is had that extraction conditions gentleness, extraction yield are high, with low cost, simple to operate, advantages of environment protection.
Especially, contriver has the high feature of protein content according to Ground Beetle as animal drugs, have employed specific prozyme, prozyme is stomach en-, papoid and trypsinase, its weight is respectively 1.5% ~ 2.0%, 1.5% ~ 2.0% and 1.5% ~ 2.0% of filter residue weight, and auxiliary extract polysaccharide in the mode of supersonic synergic, compare ordinary hot formulation extraction yield and significantly improve, the program is contriver through a large amount of screening experiment repeatedly, pays creative work and just draw.
Forefathers mainly concentrate on the suppression aspect of Ground Beetle glycoprotein to tumour cell to the pharmacological research of Ground Beetle, contriver surprisingly finds under study for action, and the Ground Beetle polysaccharide utilizing the method to extract can obviously suppress by the platelet aggregation of ADP, AA, PAF, collagen, thrombin induction.And thrombotic diseases is found in each system of clinical human now, the especially heart, cerebrovascular thrombotic diseases, accounted for first of the dead reason of Chinese people stomatosis, and sickness rate has the trend of increase.Thrombocyte is cell minimum in blood, participate in hemostasis and thrombosis, therefore the research and development of medicament for resisting platelet aggregation has good economic benefit and social value.
Embodiment
The technique means realized to make the present invention, creation characteristic, reaching object and effect is easy to understand, below in conjunction with specific embodiment, setting forth the present invention further.
Embodiment 1
An extracting method for Ground Beetle polysaccharide, step is as follows:
(1) degreasing: female for dry Ground Beetle body is pulverized, cross 40 mesh sieves, add solvent degreasing, reflux 2 times, each 1 hour, suction filtration, it is stand-by that filter residue volatilizes solvent, and solvent is sherwood oil, and consumption is 10 times of weight of solid after sieving;
(2) supersound process: step (1) gained filter residue 100g is soaked in the distilled water of 3000g, with 200W power ultrasonic process 15 minutes, obtains solid-liquid mixture;
(3) ferment treatment: being utilized by the solid-liquid mixture of step (2) gained HAc-NaAc buffered soln to adjust pH is 3, then the prozyme be mixed into 1.5g stomach en-, 1.5g papoid and 1.5g trypsinase is at temperature 45 C, solid-liquid ratio is 1:20 ~ 1:40 Water Under solution 40 minutes, then 85 DEG C of enzymes that go out are warming up to, suction filtration, collect filtrate, concentrated, obtain concentrated extracting solution;
(4) alcohol precipitation: step (3) gained concentrated extracting solution is added ethanol, makes ethanol ultimate density in system be 80wt%, places and within 24 hours, separates out Ground Beetle Crude polysaccharides precipitation, suction filtration, with 95wt% ethanol and washing with acetone twice, 50 DEG C of vacuum-dryings, obtain Ground Beetle polysaccharide.
Finally obtain Ground Beetle polysaccharide 14.93g.
Embodiment 2
An extracting method for Ground Beetle polysaccharide, step is as follows:
(1) degreasing: female for dry Ground Beetle body is pulverized, cross 40 mesh sieves, add solvent degreasing, reflux 5 times, each 3 hours, suction filtration, it is stand-by that filter residue volatilizes solvent, and solvent is sherwood oil, and consumption is 10 times of weight of solid after sieving;
(2) supersound process: step (1) gained filter residue 100g is soaked in the distilled water of 5000ml, with 400W power ultrasonic process 45 minutes, obtains solid-liquid mixture;
(3) ferment treatment: being utilized by the solid-liquid mixture of step (2) gained HAc-NaAc buffered soln to adjust pH is 6, then use the prozyme of 2.0g stomach en-, 2.0g papoid and 2.0g trypsinase composition temperature 65 DEG C, solid-liquid ratio is 1:20 ~ 1:40 Water Under solution 100 minutes, then 85 DEG C of enzymes that go out are warming up to, suction filtration, collect filtrate, concentrated, obtain concentrated extracting solution;
(4) alcohol precipitation: step (3) gained concentrated extracting solution is added ethanol, makes ethanol ultimate density in system be 80wt%, places and within 24 hours, separates out Ground Beetle Crude polysaccharides precipitation, suction filtration, with 95wt% ethanol and washing with acetone twice, 70 DEG C of vacuum-dryings, obtain Ground Beetle polysaccharide.
Finally obtain Ground Beetle polysaccharide 15.53g.
Embodiment 3
An extracting method for Ground Beetle polysaccharide, step is as follows:
(1) degreasing: female for dry Ground Beetle body is pulverized, cross 40 mesh sieves, add solvent degreasing, reflux 3 times, each 2 hours, suction filtration, it is stand-by that filter residue volatilizes solvent, and solvent is sherwood oil, and consumption is 10 times of weight of solid after sieving;
(2) supersound process: step (1) gained filter residue 100g is soaked in the distilled water of 4000ml, with 300W power ultrasonic process 30 minutes, obtains solid-liquid mixture;
(3) ferment treatment: being utilized by the solid-liquid mixture of step (2) gained HAc-NaAc buffered soln to adjust pH is 4.5, then the prozyme be mixed into 1.5g stomach en-, 1.5g papoid and 2.0g trypsinase is at temperature 45 C, solid-liquid ratio is 1:20 ~ 1:40 Water Under solution 90 minutes, then 85 DEG C of enzymes that go out are warming up to, suction filtration, collect filtrate, concentrated, obtain concentrated extracting solution;
(4) alcohol precipitation: step (3) gained concentrated extracting solution is added ethanol, makes ethanol ultimate density in system be 80wt%, places and within 24 hours, separates out Ground Beetle Crude polysaccharides precipitation, suction filtration, with 95wt% ethanol and washing with acetone twice, 60 DEG C of vacuum-dryings, obtain Ground Beetle polysaccharide.
Finally obtain Ground Beetle polysaccharide 17.21g.
Embodiment 4
An extracting method for Ground Beetle polysaccharide, step is as follows:
(1) degreasing: female for dry Ground Beetle body is pulverized, cross 40 mesh sieves, add solvent degreasing, reflux 4 times, each 1.5 hours, suction filtration, it is stand-by that filter residue volatilizes solvent, and solvent is sherwood oil, and consumption is 10 times of weight of solid after sieving;
(2) supersound process: step (1) gained filter residue 100g is soaked in the distilled water of 4000ml, with 350W power ultrasonic process 40 minutes, obtains solid-liquid mixture;
(3) ferment treatment: being utilized by the solid-liquid mixture of step (2) gained HAc-NaAc buffered soln to adjust pH is 3.8, then the prozyme be mixed into 1.8g stomach en-, 1.5g papoid and 2.0g trypsinase is at temperature 50 C, solid-liquid ratio is 1:20 ~ 1:40 Water Under solution 80 ~ 100 minutes, then 85 DEG C of enzymes that go out are warming up to, suction filtration, collect filtrate, concentrated, obtain concentrated extracting solution;
(4) alcohol precipitation: step (3) gained concentrated extracting solution is added ethanol, makes ethanol ultimate density in system be 80wt%, places and within 24 hours, separates out Ground Beetle Crude polysaccharides precipitation, suction filtration, with 95wt% ethanol and washing with acetone twice, 55 DEG C of vacuum-dryings, obtain Ground Beetle polysaccharide.
Finally obtain Ground Beetle polysaccharide 16.89g.
Embodiment 5
(1) female for Ground Beetle body is clean, oven drying at low temperature, pulverizes, and crosses 40 mesh sieves, adds 10 times amount sherwood oils (60-90 DEG C), backflow twice, each 2 hours, suction filtration while hot, and it is stand-by that filter residue volatilizes solvent;
(2) (1) gained filter residue 100g is accurately taken, add 3000ml distilled water, with 300W power ultrasonic process 45 minutes, add 2.0g stomach en-, 1.5g papoid, the mixing of 2.0g trypsinase, pH is regulated to be 5.5 with HAc-NaAc buffered soln, temperature control 45 DEG C keeps 60 minutes, be warming up to 85 DEG C of enzymes that go out, suction filtration, rotary evaporation concentrated filtrate, concentrated solution adds ethanol adjustment makes determining alcohol be 80%, placement is spent the night, and suction filtration obtains polysaccharide precipitation, and precipitation respectively washes twice with 95% ethanol and acetone, 60 DEG C of vacuum-dryings, pulverize to obtain Ground Beetle polysaccharide 16.13g.
(3) (1) gained filter residue 100g is accurately taken, add 4000ml distilled water, with 300W power ultrasonic process 30 minutes, add 1.5g stomach en-, 1.5g papoid, the mixing of 2.0g trypsinase, pH is regulated to be 3.0 with HAc-NaAc buffered soln, temperature control 55 DEG C keeps 90 minutes, be warming up to 85 DEG C of enzymes that go out, suction filtration, rotary evaporation concentrated filtrate, concentrated solution adds ethanol adjustment makes determining alcohol be 80%, placement is spent the night, and suction filtration obtains polysaccharide precipitation, and precipitation respectively washes twice with 95% ethanol and acetone, 60 DEG C of vacuum-dryings, pulverize to obtain Ground Beetle polysaccharide 16.43g.
Comparative example 1
Use conventional methods extraction: accurately take embodiment 5 step (1) gained filter residue 100g, add 4000ml distilled water, boil 120 minutes, repeat extraction twice, suction filtration, rotary evaporation concentrated filtrate, concentrated solution adds ethanol adjustment makes determining alcohol be 80%, and placement is spent the night, suction filtration obtains polysaccharide precipitation, precipitation respectively washes twice with 95% ethanol and acetone, and 60 DEG C of vacuum-dryings, pulverize to obtain Ground Beetle polysaccharide 9.72g.
Comparative example 2
Simple employing stomach en-extracts: accurately take embodiment 5 step (1) gained filter residue 100g, add 4000ml distilled water, with 300W power ultrasonic process 30 minutes, add 4.5g stomach en-, pH is regulated to be 4.5 with HAc-NaAc buffered soln, temperature control 45 DEG C keeps 90 minutes, be warming up to 85 DEG C of enzymes that go out, suction filtration, rotary evaporation concentrated filtrate, concentrated solution adds ethanol adjustment makes determining alcohol be 80%, placement is spent the night, and suction filtration obtains polysaccharide precipitation, and precipitation respectively washes twice with 95% ethanol and acetone, 60 DEG C of vacuum-dryings, pulverize to obtain Ground Beetle polysaccharide 11.93g.
Comparative example 3
Simple employing Papain enzyme extraction: accurately take embodiment 5 step (1) gained filter residue 100g, add 4000ml distilled water, with 300W power ultrasonic process 15 minutes, add 6.0g papoid, pH is regulated to be 4.5 with HAc-NaAc buffered soln, temperature control 45 DEG C keeps 90 minutes, be warming up to 85 DEG C of enzymes that go out, suction filtration, rotary evaporation concentrated filtrate, concentrated solution adds ethanol adjustment makes determining alcohol be 80%, placement is spent the night, and suction filtration obtains polysaccharide precipitation, and precipitation respectively washes twice with 95% ethanol and acetone, 60 DEG C of vacuum-dryings, pulverize to obtain Ground Beetle polysaccharide 12.21g.
Comparative example 4
Simple employing trypsinase extracts: accurately take embodiment 5 step (1) gained filter residue 100g, add 4000ml distilled water, with 300W power ultrasonic process 15 minutes, add the mixing of 4.0g trypsinase, pH is regulated to be 4.5 with HAc-NaAc buffered soln, temperature control 45 DEG C keeps 90 minutes, be warming up to 85 DEG C of enzymes that go out, suction filtration, rotary evaporation concentrated filtrate, concentrated solution adds ethanol adjustment makes determining alcohol be 80%, placement is spent the night, and suction filtration obtains polysaccharide precipitation, and precipitation respectively washes twice with 95% ethanol and acetone, 60 DEG C of vacuum-dryings, pulverize to obtain Ground Beetle polysaccharide 10.61g.
Comparative example 5
Do not adopt supersound process:
Accurately take embodiment 5 step (1) gained filter residue 100g, add 4000ml distilled water, add 1.5g stomach en-, 1.5g papoid, the mixing of 2.0g trypsinase, pH is regulated to be 4.5 with HAc-NaAc buffered soln, temperature control 45 DEG C keeps 90 minutes, be warming up to 85 DEG C of enzymes that go out, suction filtration, rotary evaporation concentrated filtrate, concentrated solution adds ethanol adjustment makes determining alcohol be 80%, and placement is spent the night, suction filtration obtains polysaccharide precipitation, precipitation respectively washes twice with 95% ethanol and acetone, and 60 DEG C of vacuum-dryings, pulverize to obtain Ground Beetle polysaccharide 13.13g.
Comparative example 6
Accurately take embodiment 5 step (1) gained filter residue 100g, add 4000ml distilled water, with 300W power ultrasonic process 30 minutes, add 1.5g papoid, 1.5g PRONASE A, 1.5g neutral protease mixes, pH is regulated to be 4.5 with HAc-NaAc buffered soln, temperature control 45 DEG C keeps 90 minutes, be warming up to 85 DEG C of enzymes that go out, suction filtration, rotary evaporation concentrated filtrate, concentrated solution adds ethanol adjustment makes determining alcohol be 80%, placement is spent the night, suction filtration obtains polysaccharide precipitation, precipitation respectively washes twice with 95% ethanol and acetone, 60 DEG C of vacuum-dryings, pulverize to obtain Ground Beetle polysaccharide 11.92g.
Relatively can be learnt by above embodiment and comparative example:
1, under equal conditions, the material adopting prozyme (stomach en-, papoid, trypsinase) to process comparatively adopts the material of single enzyme process, and the Ground Beetle polysaccharide of extraction is more.
If 2 do not adopt supersound process, can find out that the Ground Beetle polysaccharide quality of extraction declines greatly.
3, other prozyme is adopted, effective not as multiplex-enzyme extraction provided by the invention.Contriver thinks: other prozymes, due to the protein that can not degrade specifically in Ground Beetle, causes extraction effect poor.
The present invention is not limited to above-mentioned embodiment, and all are based on technical conceive of the present invention, and done structural improvement, all falls among protection scope of the present invention.
Claims (8)
1. an extracting method for Ground Beetle polysaccharide, is characterized in that: step is as follows:
(1) degreasing: pulverized by female for dry Ground Beetle body, sieve, add solvent degreasing, reflux 2 ~ 5 times, each 1 ~ 3 hour, suction filtration, it is stand-by that filter residue volatilizes solvent;
(2) supersound process: be soaked in the distilled water of 30 ~ 50 times of weight by step (1) gained filter residue, with 200 ~ 400W power ultrasonic process 15 ~ 45 minutes, obtains solid-liquid mixture;
(3) ferment treatment: it is 3 ~ 6 that the solid-liquid mixture of step (2) gained is adjusted pH, then use prozyme temperature 45 ~ 65 DEG C, solid-liquid ratio is 1:20 ~ 1:40 Water Under solution 40 ~ 90 minutes, then 85 DEG C of enzymes that go out are warming up to, suction filtration, collect filtrate, concentrated, obtain concentrated extracting solution; Described prozyme is stomach en-, papoid and trypsinase, and its weight is respectively 1.5% ~ 2.0%, 1.5% ~ 2.0% and 1.5% ~ 2.0% of filter residue weight;
(4) alcohol precipitation: step (3) gained concentrated extracting solution is added ethanol, ethanol ultimate density in system is made to be 80wt%, place and within 24 hours, separate out Ground Beetle Crude polysaccharides precipitation, suction filtration, with 95wt% ethanol and washing with acetone twice, 50 ~ 70 DEG C of vacuum-dryings, obtain Ground Beetle polysaccharide.
2. the extracting method of a kind of Ground Beetle polysaccharide as claimed in claim 1, is characterized in that: in described step (1), crosses 40 mesh sieves after being pulverized by female for dry Ground Beetle body.
3. the extracting method of a kind of Ground Beetle polysaccharide as claimed in claim 1, it is characterized in that: in described step (1), solvent is sherwood oil, and consumption is 10 times of weight of solid after sieving.
4. the extracting method of a kind of Ground Beetle polysaccharide as claimed in claim 1, it is characterized in that: in described step (2), filter residue is soaked in supersound process in the distilled water of 40 times of volumes, ultrasonic power is 300W, and ultrasonic time is 30 minutes.
5. the extracting method of a kind of Ground Beetle polysaccharide as claimed in claim 1, is characterized in that: in described step (3), utilizes HAc-NaAc buffered soln to adjust pH.
6. the extracting method of a kind of Ground Beetle polysaccharide as claimed in claim 1, it is characterized in that: in described step (3), control pH is 4.5, and temperature is 45 DEG C, and soaking time is 90 minutes.
7. a Ground Beetle polysaccharide, adopts extracting method as described in any one of claim 1-6 to extract and obtains.
8. the application of Ground Beetle polysaccharide in anticoagulant as claimed in claim 7.
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| CN107325197A (en) * | 2017-07-01 | 2017-11-07 | 浦江县泰如食品科技有限公司 | The extracting method of ground bettle polysaccharide |
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| CN108324771A (en) * | 2018-04-19 | 2018-07-27 | 李蕊 | A kind of pharmaceutical composition and preparation method and application for treating post-abortion irregular menstruation |
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| 五种海藻多糖体外抗血小板聚集作用的观察;刘志峰等;《中国海洋药物》;20010430(第02期);第36-38页 * |
| 地鳖虫多糖提取工艺的优化研究;罗志文等;《安徽农业科学》;20121231;第40卷(第2期);第13979页及第13982页 * |
| 当归多糖对凝血和血小板聚集的影响;杨铁虹等;《中药材》;20020531;第25卷(第05期);第344-345页 * |
| 酶解法提取地鳖虫多糖工艺的优化;罗志文等;《黑龙江农业科学》;20120831(第8期);第102-104页 * |
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