CN112641807A - Preparation method of leech extract - Google Patents
Preparation method of leech extract Download PDFInfo
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- CN112641807A CN112641807A CN202011607116.3A CN202011607116A CN112641807A CN 112641807 A CN112641807 A CN 112641807A CN 202011607116 A CN202011607116 A CN 202011607116A CN 112641807 A CN112641807 A CN 112641807A
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- 241000545744 Hirudinea Species 0.000 title claims abstract description 67
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 239000000706 filtrate Substances 0.000 claims abstract description 32
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000010438 heat treatment Methods 0.000 claims abstract description 27
- 238000003828 vacuum filtration Methods 0.000 claims abstract description 10
- 238000007789 sealing Methods 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 22
- 239000008213 purified water Substances 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000002002 slurry Substances 0.000 claims description 13
- 238000001914 filtration Methods 0.000 claims description 9
- 239000012452 mother liquor Substances 0.000 claims description 7
- 238000011049 filling Methods 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 5
- 239000004677 Nylon Substances 0.000 claims description 4
- 229920001778 nylon Polymers 0.000 claims description 4
- 239000004019 antithrombin Substances 0.000 abstract description 8
- 238000000605 extraction Methods 0.000 abstract description 4
- 239000007787 solid Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 3
- 102000007625 Hirudins Human genes 0.000 description 2
- 108010007267 Hirudins Proteins 0.000 description 2
- 241000237903 Hirudo Species 0.000 description 2
- WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 description 2
- 229940006607 hirudin Drugs 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229960004676 antithrombotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000014508 negative regulation of coagulation Effects 0.000 description 1
- 238000005325 percolation Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 201000001474 proteinuria Diseases 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229910021643 water for pharmaceutical use Inorganic materials 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/62—Leeches; Worms, e.g. cestodes, tapeworms, nematodes, roundworms, earth worms, ascarids, filarias, hookworms, trichinella or taenia
Landscapes
- Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a leech extract preparation method, which is characterized by comprising the following steps: s4, carrying out vacuum filtration on the leech intermediate to obtain a filtrate, and carrying out vacuum heating concentration to 1/8-1/3 of the original volume; s5, adding ethanol to make the ethanol concentration reach 70-92%, sealing, and standing for 20-30 h; and S6, carrying out vacuum filtration again to obtain filtrate, and concentrating the filtrate to 1/8-1/3 of the original volume. The leech extract obtained by the invention has high antithrombin content and high yield, and the extraction process is economic, stable and feasible.
Description
Technical Field
The invention relates to the technical field of leech processing, in particular to a preparation method of leech extract.
Background
A leech extract prepared from leech (leech), leech (leech) or leech (leech) of the family leech contains hirudin, histamine-like substance, heparin, antithrombotic agent and other medicinal components, wherein the hirudin is a single-chain polypeptide consisting of 66 amino acid residues and has strong anticoagulant activity. The leech extract has various pharmacological activities of reducing proteinuria, improving renal function, reducing blood fat, resisting coagulation, resisting thrombosis, resisting inflammation, resisting proliferation, resisting fibrosis and the like, can be used for treating various diseases, and is a medicament with wide application prospect.
In the prior art, the extraction process of the leech extract commonly adopts a dipping method, a percolation method, a decoction method and a reflux extraction method, the antithrombin content of the extract obtained by the methods is about 200u/g, never exceeds 210u/g, and the yield is lower.
Therefore, the skilled in the art is devoted to develop a method for preparing the leech extract solution which can improve the antithrombin content of the leech extract solution.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention provides a method for preparing a leech extract with an increased antithrombin content.
In order to achieve the aim, the invention provides a leech extract preparation method, which comprises the following steps:
s4, carrying out vacuum filtration on the leech intermediate to obtain a filtrate, and carrying out vacuum heating concentration to 1/8-1/3 of the original volume;
s5, adding ethanol to make the ethanol concentration reach 70-92%, sealing, and standing for 20-30 h;
and S6, carrying out vacuum filtration again to obtain filtrate, and concentrating the filtrate to 1/8-1/3 of the original volume.
Preferably, the leech intermediate is prepared by the following steps:
s1, filling the leech slurry into a first filter bag, then putting the first filter bag into purified water, heating to 90-120 ℃, keeping the temperature for 0.6-1.5 h, and filtering integrally when the temperature is hot to obtain a first filtrate;
s2, putting the first filter bag into purified water again, heating to 90-120 ℃, keeping the temperature for 0.3-1 h continuously, and filtering integrally when the temperature is hot to obtain a second filtrate;
s3, combining the first filtrate and the second filtrate, filling the first filtrate and the second filtrate into a second filter bag, centrifuging until no large-particle impurities are found in the centrifugal mother liquor, and collecting the centrifugal mother liquor to obtain the leech intermediate.
Preferably, the leech intermediate is leech dry powder added with 3-6 times of purified water.
Preferably, in the step S4, the vacuum degree of vacuum heating concentration is less than-0.08 Mpa, and the heating temperature is 50 to 70 ℃.
Preferably, in the step S6, the vacuum degree of vacuum heating concentration is less than-0.08 Mpa, and the heating temperature is 30 to 50 ℃.
Preferably, the amount of the purified water used in step S1 is 2 to 6 times of the weight of the leech slurry.
Preferably, the amount of the purified water used in step S2 is 1-5 times of the weight of the leech slurry.
Preferably, the first filter bag is a double-layer 200-mesh nylon gauze filter bag; the second filter bag is a 2000-mesh filter bag.
The invention has the beneficial effects that: the leech extract obtained by the invention has high antithrombin content, high yield, low alcohol concentration, low solid content and economic, stable and feasible extraction process.
Detailed Description
The present invention will be further described with reference to the following examples.
Examples 1 to 3
Preparing a leech extract by using leech slurry:
the method comprises the following steps:
s1, filling the leech slurry into a first filter bag, then putting the filter bag into purified water, heating to 90-120 ℃, keeping the temperature for 0.6-1.5 h, and filtering integrally when the temperature is hot to obtain a first filtrate. In the filtration in this step, all the heated materials including purified water, the first filter bag, and purified water are filtered together. The first filter bag is a double-layer 200-mesh nylon gauze filter bag, or the leech slurry can be directly wrapped by the double-layer 200-mesh nylon gauze and then tied by a binding belt to remove the redundant gauze. The amount of the purified water in the step is 2-6 times of the weight of the leech slurry.
And S2, putting the first filter bag into purified water again, heating to 90-120 ℃, keeping the temperature for 0.3-1 h, and filtering integrally when the temperature is hot to obtain a second filter solution. This filtration is also a process of filtering all heated materials including the first filter bag and purified water together. The amount of the purified water in the step is 1-5 times of the weight of the leech slurry.
S3, combining the first filtrate and the second filtrate, filling the first filtrate and the second filtrate into a second filter bag, centrifuging until no large-particle impurities are found in the centrifugal mother liquor, and collecting the centrifugal mother liquor to obtain the leech intermediate. No large-particle impurities in the centrifugal mother liquor mean that no particles or solid impurities are observed by naked eyes. The step adopts a filter bag (namely a second filter bag) with 2000 meshes of a flat plate centrifuge to carry out centrifugation.
S4, carrying out vacuum filtration on the leech intermediate to obtain a filtrate, and carrying out vacuum heating concentration to 1/8-1/3 of the original volume; in the step, the vacuum degree of vacuum heating concentration is less than-0.08 Mpa, and the heating temperature is 50-70 ℃.
S5, adding ethanol, wherein in the step, the ethanol with the concentration of more than 95 percent by volume is added to ensure that the concentration of the ethanol reaches 70 to 92 percent, sealing, and standing for 20 to 30 hours;
s6, carrying out vacuum filtration again to obtain filtrate, and concentrating the filtrate to 1/8-1/3 of the original volume to obtain the leech extract. In the step, the vacuum degree of vacuum heating concentration is less than-0.08 Mpa, and the heating temperature is 30-50 ℃.
The amounts of the hirudo slurries and other preparation parameters used in examples 1 to 3 are given in Table 1:
table 1 table of parameters of examples 1 to 3
| Example 1 | Example 2 | Example 3 | |
| Leech slurry quality (kg) | 5 | 5 | 5 |
| Purified water amount (kg) in step S1 | 10 | 20 | 30 |
| Heating temperature (. degree.C.) in step S1 | 90 | 100 | 120 |
| Purified water amount (kg) in step S2 | 5 | 10 | 25 |
| Heating temperature (. degree.C.) in step S2 | 90 | 100 | 120 |
| Heating temperature (. degree.C.) in step S4 | 50 | 60 | 70 |
| Ethanol concentration in step S5 | 75％ | 80％ | 92％ |
| Heating temperature (. degree.C.) in step S6 | 30 | 40 | 50 |
Examples 4 to 6
Preparing leech extract by using leech dry powder.
The method comprises the following steps: adding purified water in an amount which is 3-8 times the weight of the dried leech powder to obtain a leech intermediate.
Then the following steps are carried out:
s4, carrying out vacuum filtration on the leech intermediate to obtain a filtrate, and carrying out vacuum heating concentration to 1/8-1/3 of the original volume;
s5, adding ethanol to make the concentration of the ethanol reach 75-92%, sealing, and standing for 20-30 h;
and S6, carrying out vacuum filtration again to obtain filtrate, and concentrating the filtrate to 1/8-1/3 of the original volume.
Examples 4 to 6 hirudo powder and other preparation parameters are given in Table 2:
table 2: EXAMPLES 4 TO 6 parameter tables
The leech extract solutions obtained in examples 1 to 6 were subjected to the following tests, and the test results are shown in table 3.
Table 3 table of test results of examples 1 to 6
In the above items, the antithrombin content is detected by a determination method in determination of antithrombin activity of leech in the 'Chinese pharmacopoeia' (2015 edition), and the detection is carried out for three times to obtain an average value; the alcohol concentration is directly measured by a commercially available alcohol meter; the solid content is measured according to the total solid content measuring method 3101 in the four parts of Chinese pharmacopoeia (2015 edition), and the average value is taken in the three times of measurement; the pH value is measured according to the pH value measurement method of the four parts 0631 in the Chinese pharmacopoeia (2015 edition); the conductivity was measured by the method of measuring the conductivity of water for pharmaceutical use in accordance with 0681 in the fourth part of the pharmacopoeia of China (2015 edition).
As can be seen from the above examples, the leech extract prepared by the method of the present invention has high antithrombin content and few impurities.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (8)
1. The preparation method of the leech extract is characterized by comprising the following steps:
s4, carrying out vacuum filtration on the leech intermediate to obtain a filtrate, and carrying out vacuum heating concentration to 1/8-1/3 of the original volume;
s5, adding ethanol to make the ethanol concentration reach 70-92%, sealing, and standing for 20-30 h;
and S6, carrying out vacuum filtration again to obtain filtrate, and concentrating the filtrate to 1/8-1/3 of the original volume.
2. The method for preparing leech extract according to claim 1, wherein the leech intermediate is prepared by the following steps:
s1, filling the leech slurry into a first filter bag, then putting the first filter bag into purified water, heating to 90-120 ℃, keeping the temperature for 0.6-1.5 h, and filtering integrally when the temperature is hot to obtain a first filtrate;
s2, putting the first filter bag into purified water again, heating to 90-120 ℃, keeping the temperature for 0.3-1 h continuously, and filtering integrally when the temperature is hot to obtain a second filtrate;
s3, combining the first filtrate and the second filtrate, filling the first filtrate and the second filtrate into a second filter bag, centrifuging until no large-particle impurities are found in the centrifugal mother liquor, and collecting the centrifugal mother liquor to obtain the leech intermediate.
3. The method for preparing leech extract according to claim 1, wherein the leech intermediate is dried leech powder and 3-6 times of purified water is added to the dried leech powder.
4. The method for preparing leech extract according to claim 1, 2 or 3, wherein the leech extract is prepared by the following steps: in the step S4, the vacuum degree of vacuum heating concentration is less than-0.08 Mpa, and the heating temperature is 50-70 ℃.
5. The method for preparing leech extract according to claim 1, 2 or 3, wherein the leech extract is prepared by the following steps: in the step S6, the vacuum degree of vacuum heating concentration is less than-0.08 Mpa, and the heating temperature is 30-50 ℃.
6. The method for preparing leech extract according to claim 2, wherein the method comprises the following steps: the amount of the purified water in the step S1 is 2-6 times of the weight of the leech slurry.
7. The method for preparing leech extract according to claim 2, wherein the method comprises the following steps: the amount of the purified water in the step S2 is 1-5 times of the weight of the leech slurry.
8. The method for preparing leech extract according to claim 2, wherein the method comprises the following steps: the first filter bag is a double-layer 200-mesh nylon gauze filter bag; the second filter bag is a 2000-mesh filter bag.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011607116.3A CN112641807A (en) | 2020-12-30 | 2020-12-30 | Preparation method of leech extract |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011607116.3A CN112641807A (en) | 2020-12-30 | 2020-12-30 | Preparation method of leech extract |
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| CN112641807A true CN112641807A (en) | 2021-04-13 |
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| CN202011607116.3A Pending CN112641807A (en) | 2020-12-30 | 2020-12-30 | Preparation method of leech extract |
Country Status (1)
| Country | Link |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000119253A (en) * | 1998-08-11 | 2000-04-25 | Dai Ichi Seiyaku Co Ltd | Novel sulfonyl derivative |
| CN102139092A (en) * | 2011-03-21 | 2011-08-03 | 中山大学 | Leech freeze-dried powder injection solution for injection and preparation method thereof |
| CN107287269A (en) * | 2017-06-19 | 2017-10-24 | 山东大学 | A kind of HIRULOG preparation technology for mitigating leech bleeding adverse reaction |
-
2020
- 2020-12-30 CN CN202011607116.3A patent/CN112641807A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000119253A (en) * | 1998-08-11 | 2000-04-25 | Dai Ichi Seiyaku Co Ltd | Novel sulfonyl derivative |
| CN102139092A (en) * | 2011-03-21 | 2011-08-03 | 中山大学 | Leech freeze-dried powder injection solution for injection and preparation method thereof |
| CN107287269A (en) * | 2017-06-19 | 2017-10-24 | 山东大学 | A kind of HIRULOG preparation technology for mitigating leech bleeding adverse reaction |
Non-Patent Citations (1)
| Title |
|---|
| 罗梓河: "水蛭注射液的制备", 广东药学, no. 03, pages 19 - 20 * |
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Application publication date: 20210413 |