CN108815205B - Cordyceps militaris extract and preparation method and application thereof - Google Patents
Cordyceps militaris extract and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a cordyceps militaris extract, and a preparation method and application thereof. The invention finds that the extract extracted from the cordyceps militaris raw material by a special method can be independently used as a medicine for treating acute kidney injury, can be further developed into a protective medicine for acute kidney injury, and has wide application prospect. Meanwhile, the preparation method of the cordyceps militaris extract is simple and is suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of effective component extraction, and particularly relates to a cordyceps militaris extract and a preparation method and application thereof.
Background
Acute Kidney Injury (AKI) is a more common acute condition in clinical families. AKI means that the absolute value of at least 2 times of serum creatinine level increase within 48h is more than or equal to 26.5umol/L (0.3mg/dL) or more than or equal to 50% higher than the original serum creatinine value; and (or) urine volume <0.5mL/kg/h for more than 6 h. After factors such as shock, severe trauma, sepsis, use of certain anti-tumor (platinum) drugs and antibiotic drugs (aminoglycosides, beta-lactams) and the like occur, Chronic Kidney Disease (CKD) can develop due to rapid development of disease conditions and untimely treatment, so that the kidney replacement therapy is relied on for life and even the patient dies. Research results at home and abroad show that the incidence rate of AKI is 3-10% in a comprehensive hospital, 30-60% in an intensive care unit and the mortality rate of critically AKI patients reaches 30-80%. Recent studies have shown that not only does AKI significantly increase a patient's short-term mortality rate whenever AKI occurs, but that whether renal function is restored or not, it may have an adverse effect on the patient's long-term prognosis (including long-term survival, incidence of chronic kidney disease). This is particularly important for the treatment of AKI.
Cordyceps militaris, also called cordyceps militaris, cordyceps militaris and the like, belongs to the ergot family and cordyceps genus in taxonomy, is a traditional Chinese medicine, and has great medicinal value. The cordyceps militaris contains various chemical components, has various medicinal effects, is similar to the pharmacological function and clinical effect of the cordyceps militaris, but has lower price than the cordyceps militaris, so that the cordyceps militaris gradually contains the substitute of the cordyceps militaris, and has great potential market.
The Cordyceps militaris extract has multiple functions, for example, Cordyceps militaris polysaccharide has the functions of resisting tumor, reducing blood sugar, resisting hepatic fibrosis and improving immunity, and cordycepin has the obvious functions of resisting bacteria, diminishing inflammation, resisting oxidation, regulating endocrine of a human body, enhancing the immunity of the human body and the like. However, no report on the treatment of acute kidney injury by cordyceps militaris extract is available at present.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a cordyceps militaris extract and a preparation method thereof. The cordyceps militaris extract prepared by the method has a remarkable treatment effect on acute kidney injury.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides a preparation method of a cordyceps militaris extract, which comprises the following steps:
(1) drying Cordyceps militaris raw material until the water content is 10-20%, and then carrying out vacuum low-temperature puffing treatment;
(2) adding the cordyceps militaris raw material subjected to vacuum low-temperature puffing treatment into an ethanol solution for ultrasonic extraction, concentrating an extracting solution, and drying to obtain a crude extract;
(3) re-dissolving the crude extract with 40-60% ethanol solution, adsorbing with macroporous resin column, sequentially eluting with low-to-high gradient ethanol solution, collecting 80% ethanol eluate, removing solvent, concentrating to obtain extract, dissolving with petroleum ether, filtering, removing solvent from filtrate, concentrating, and drying to obtain Cordyceps militaris extract.
Preferably, in the step (1), the cordyceps militaris raw materials comprise: cordyceps militaris, Cordyceps militaris fruiting body or Cordyceps militaris mycelium.
Preferably, in the step (1), the vacuum low-temperature puffing treatment conditions are as follows: the puffing temperature is 45-55 ℃; the times of vacuum pumping and discharging are 6-10 times, and the expansion time is 1-2 hours. Vacuum low temperature bulking is to place the sample in the overhead tank, through heating and pressurization, makes sample internal pressure and external pressure balanced, then reduces the pressure suddenly and puts the vacuum, makes inside moisture of material vaporize suddenly, the flash distillation makes sample cell bulking, can make the inside cell tissue clearance of sample become the increase through the evacuation of relapse, puts vacuum treatment, and the permeability becomes good, changes in and draws the solvent and contact to the extraction rate of active ingredient has been improved. And the processing temperature of the vacuum low-temperature puffing is low, the time is short, and the effective substances in the raw materials are basically not damaged.
The research of the invention finds that in the vacuum low-temperature puffing treatment process, the cordyceps militaris with too low water content is lack of flexibility and is fragile in the pressurizing process; and the swelling effect of the cordyceps militaris can be influenced when the water content is too high. Multiple tests show that the swelling effect is ideal when the water content of the cordyceps militaris raw material is 10-20%.
Preferably, in the step (2), the volume concentration of the ethanol solution is 50-60%.
Preferably, in the step (2), the ultrasonic extraction conditions are as follows: the temperature is 40-60 ℃, the time is 20-30min, and the power is 100-.
Preferably, in the step (3), the macroporous resin column is a D101 type macroporous resin column.
Preferably, in the step (3), the step of eluting with the ethanol solution with gradient concentration from low to high specifically comprises:
the column was eluted sequentially with 4 column volumes 40% ethanol, 4 column volumes 60% ethanol and 6 column volumes 80% ethanol.
In a second aspect of the invention, the cordyceps militaris extract prepared by the method is provided.
The third aspect of the invention provides an application of the cordyceps militaris extract in preparing a medicine for treating acute kidney injury.
The fourth aspect of the invention provides a medicine for treating acute kidney injury, which is prepared from the cordyceps militaris extract and pharmaceutically acceptable auxiliary materials.
The invention has the beneficial effects that:
the invention unexpectedly discovers that the extract of the cordyceps militaris raw material extracted by a special method can be independently used as a medicine for treating acute kidney injury, can be further developed into a protective medicine for acute kidney injury, and has wide application prospect. Meanwhile, the preparation method of the cordyceps militaris extract is simple and is suitable for industrial production.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples and comparative examples of the present invention, which were not specifically described, were conventional in the art and commercially available.
The cordyceps militaris sporocarp raw materials used in the embodiment and the comparative example are cordyceps militaris harvested in the same production place and at the same time, and the content of the effective components of the cordyceps militaris sporocarp raw materials is not obviously different.
Example 1:
(1) taking cordyceps militaris sporocarp as a raw material, drying 1kg of cordyceps militaris raw material until the water content is 15%, and then carrying out vacuum low-temperature puffing treatment under the following conditions: the puffing temperature is 50 ℃; the times of vacuum pumping and discharging are 8 times, and the expansion time is 1.5 hours.
(2) Adding 8 times of ethanol solution with volume concentration of 60% by weight into the raw materials subjected to vacuum low-temperature puffing treatment for ultrasonic extraction, wherein the ultrasonic extraction conditions are as follows: the temperature is 50 ℃, the time is 20min, the power is 100W, and the extracting solution is concentrated and dried to obtain a crude extract.
(3) Re-dissolving the crude extract with 2 times of ethanol solution with volume concentration of 50%, adsorbing by D101 type macroporous resin column, eluting with 40% ethanol for 4 column volumes, eluting with 60% ethanol for 4 column volumes, eluting with 80% ethanol for 6 column volumes, collecting 80% ethanol eluate, removing solvent, concentrating to obtain extract, dissolving with petroleum ether, filtering, removing solvent from filtrate, concentrating to obtain extract, vacuum drying at 60 deg.C, and pulverizing to obtain Cordyceps militaris extract.
Example 2:
(1) taking cordyceps militaris sporocarp as a raw material, drying 1kg of cordyceps militaris raw material until the water content is 10%, and then carrying out vacuum low-temperature puffing treatment under the following conditions: the puffing temperature is 45 ℃; the times of vacuum pumping and discharging are 10 times, and the expansion time is 2 hours.
(2) Adding 8 times of ethanol solution with volume concentration of 50% by weight into the raw materials subjected to vacuum low-temperature puffing treatment for ultrasonic extraction, wherein the ultrasonic extraction conditions are as follows: the temperature is 40 ℃, the time is 30min, the power is 100W, and the extracting solution is concentrated and dried to obtain a crude extract.
(3) Re-dissolving the crude extract with 2 times of ethanol solution with volume concentration of 40%, adsorbing by D101 type macroporous resin column, eluting with 40% ethanol for 4 column volumes, eluting with 60% ethanol for 4 column volumes, eluting with 80% ethanol for 6 column volumes, collecting 80% ethanol eluate, removing solvent, concentrating to obtain extract, dissolving with petroleum ether, filtering, removing solvent from filtrate, concentrating to obtain extract, vacuum drying at 60 deg.C, and pulverizing to obtain Cordyceps militaris extract.
Example 3:
(1) taking cordyceps militaris sporocarp as a raw material, drying 1kg of cordyceps militaris raw material until the water content is 20%, and then carrying out vacuum low-temperature puffing treatment under the following conditions: the puffing temperature is 55 ℃; the times of vacuum pumping and discharging are 6 times, and the expansion time is 1 hour.
(2) Adding 8 times of ethanol solution with volume concentration of 55% by weight into the raw materials subjected to vacuum low-temperature puffing treatment for ultrasonic extraction, wherein the ultrasonic extraction conditions are as follows: the temperature is 60 ℃, the time is 20min, the power is 120W, and the extracting solution is concentrated and dried to obtain a crude extract.
(3) Re-dissolving the crude extract with 2 times of ethanol solution with volume concentration of 40%, adsorbing by D101 type macroporous resin column, eluting with 40% ethanol for 4 column volumes, eluting with 60% ethanol for 4 column volumes, eluting with 80% ethanol for 6 column volumes, collecting 80% ethanol eluate, removing solvent, concentrating to obtain extract, dissolving with petroleum ether, filtering, removing solvent from filtrate, concentrating to obtain extract, vacuum drying at 60 deg.C, and pulverizing to obtain Cordyceps militaris extract.
Comparative example 1:
the method comprises the following steps of taking cordyceps militaris sporocarp as a raw material, adding 8 times of ethanol solution with volume concentration of 60% by weight, and carrying out ultrasonic extraction under the conditions of: the temperature is 50 ℃, the time is 20min, the power is 100W, the extracting solution is concentrated, dried and crushed to obtain the cordyceps militaris extract A.
Comparative example 2:
adding an ethanol solution with volume concentration of 60% and 8 times of the weight of cordyceps militaris as a raw material, performing reflux extraction for 3 times, wherein the time of 3 times is 2, 1 and 0.5 hours respectively, concentrating an extracting solution until the relative density is 1.01 (measured at 50 ℃), adsorbing by a D101 type macroporous resin column, sequentially eluting 4 column volumes by 40% ethanol, 4 column volumes by 60% ethanol and 6 column volumes by 80% ethanol, collecting 80% ethanol eluent, concentrating to obtain an extract, performing vacuum drying at 60 ℃, and crushing to obtain a cordyceps militaris extract B.
Test example 1: influence of cordyceps militaris extract on glycerin-induced acute kidney injury of mice
(1) An experimental animal ICR mouse, a clean grade, a male and a body weight of 22-26 g.
(2) Modeling, grouping and administering
After adaptive feeding of mice, the mice were randomized and divided into a blank group, a model group, a uremia clearance (commercially available) positive drug control group (3.25g/kg), a test 1 group (100 mg/kg of the cordyceps militaris extract prepared in example 1), a test 2 group (100 mg/kg of the cordyceps militaris extract A prepared in comparative example 1), a test 3 group (100 mg/kg of the cordyceps militaris extract B prepared in comparative example 2), and 10 mice per group.
After the animals are not fasted for 18h, 50% glycerol is injected into the two hind legs by an intramuscular injection method, the total injection amount is 8ml/kg, and the injection dose of each hind leg is half. Wherein the glycerol is diluted to 50% with physiological saline. The blank group was injected with an equal amount of physiological saline. After the molding is finished, the animals continue to be deprived of water for 8 hours, and then freely eat and drink water and start to take the medicine. 1 time per day for 3 consecutive days. The blank and model groups were dosed with the same amount of purified water. The administration is carried out by intragastric administration.
(3) Blood sample collection, processing and indicator determination
After 24h of the last administration, the animals were anesthetized, and the abdominal aorta was sampled and anticoagulated whole blood was collected. Then, the animals are sacrificed, two kidneys are taken, kidney tissues are fixed by 10% formaldehyde for 24h, dehydrated by conventional alcohol, embedded by paraffin, cut into tissue sections with the diameter of 5 mu m, stained by hematoxylin-eosin (HE), and the number of casts in the renal tubule lumen is observed by a light mirror.
Centrifuging anticoagulated whole blood, centrifuging at 5000r/min for 10min to obtain blood plasma, and freezing for storage. The creatinine content in the blood plasma was measured by the sarcosine oxidase method.
(4) Results of the experiment
The influence of the cordyceps militaris extract on the blood creatinine of the mice with acute renal injury induced by glycerol is shown in table 1, and the influence on the number of the renal tubule types of the mice with acute renal injury induced by glycerol is shown in table 2.
Table 1: influence of cordyceps militaris extract on blood creatinine of mice with acute kidney injury induced by glycerol
| Group of | Creatinine (mu mol/L) |
| Blank group | 74.12±21.32 |
| Model set | 143.54±52.60 |
| Urine toxin yang-clearing control group | 124.22±38.57 |
| Test 1 group | 92.26±18.35 |
| Test 2 groups | 136.40±34.82 |
| Test 3 groups | 120.25±30.20 |
Table 2: effect of Cordyceps militaris extract on number of renal tubule types of mice with acute renal injury induced by glycerol
| Group of | Number of tubes |
| Blank group | 0 |
| Model set | 17.24±5.80 |
| Urine toxin yang-clearing control group | 15.26±5.24 |
| Test ofGroup 1 | 7.02±2.40 |
| Test 2 groups | 16.10±4.68 |
| Test 3 groups | 14.96±5.73 |
The number of the canaliculi is reniformis is one of indexes for evaluating the damage of the kidney tissue structure, and the cordyceps militaris extract achieves statistical difference on the reduction of the serum creatinine and also achieves statistical difference on the indexes of the pathological damage of the kidney. The cordyceps militaris extract has an unexpected technical effect on treating acute kidney injury.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (1)
1. Application of Cordyceps militaris extract in preparing medicine for treating increase in the number of renal tubule types of mice with acute renal injury induced by glycerol; the cordyceps militaris extract is prepared by the following method:
(1) taking cordyceps militaris sporocarp as a raw material, drying 1kg of cordyceps militaris raw material until the water content is 15%, and then carrying out vacuum low-temperature puffing treatment under the following conditions: the puffing temperature is 50 ℃; the times of vacuum pumping and discharging are 8 times, and the expansion time is 1.5 hours;
(2) adding 8 times of ethanol solution with volume concentration of 60% by weight into the raw materials subjected to vacuum low-temperature puffing treatment for ultrasonic extraction, wherein the ultrasonic extraction conditions are as follows: concentrating the extractive solution at 50 deg.C for 20min with power of 100W, and drying to obtain crude extract;
(3) re-dissolving the crude extract with 2 times of ethanol solution with volume concentration of 50%, adsorbing by D101 type macroporous resin column, eluting with 40% ethanol for 4 column volumes, eluting with 60% ethanol for 4 column volumes, eluting with 80% ethanol for 6 column volumes, collecting 80% ethanol eluate, removing solvent, concentrating to obtain extract, dissolving with petroleum ether, filtering, removing solvent from filtrate, concentrating to obtain extract, vacuum drying at 60 deg.C, and pulverizing to obtain Cordyceps militaris extract.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101508715A (en) * | 2009-04-01 | 2009-08-19 | 江苏省农业科学院 | Extraction and purification process for cordycepin in cordyceps militaris link |
| CN106317148A (en) * | 2016-07-29 | 2017-01-11 | 河北省科学院生物研究所 | Method for extracting cordycepin from cordyceps militaris |
| KR20180068516A (en) * | 2016-12-14 | 2018-06-22 | 주식회사 바이오아라 | Method for extracting functional material from cordyceps militaris |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101508715A (en) * | 2009-04-01 | 2009-08-19 | 江苏省农业科学院 | Extraction and purification process for cordycepin in cordyceps militaris link |
| CN106317148A (en) * | 2016-07-29 | 2017-01-11 | 河北省科学院生物研究所 | Method for extracting cordycepin from cordyceps militaris |
| KR20180068516A (en) * | 2016-12-14 | 2018-06-22 | 주식회사 바이오아라 | Method for extracting functional material from cordyceps militaris |
Non-Patent Citations (3)
| Title |
|---|
| "蛹虫草中虫草素的提取及纯化工艺研究";李学军等;《生物技术》;20121015;第22卷(第5期);第75-78页 * |
| "蛹虫草防治高尿酸血症的实验研究";胡芳等;《中成药》;20130831;第35卷(第8期);第1779-1782页 * |
| 膨化技术在中药材加工领域的应用;贾淑珍等;《农产品加工》;20120515(第05期);第75-76页 * |
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