CN104447983A - Method for classifying and extracting active components of deer blood - Google Patents

Abstract

The invention belongs to the technical field of biomedicine, and in particular relates to a method for classifying and extracting active components of deer blood. The method for classifying and extracting the active components of deer blood according to the present invention is to carry out anticoagulant treatment on fresh deer blood; the anticoagulated deer blood plasma is precipitated by salt precipitation with saturated ammonium sulfate, centrifuged to remove the precipitate, and the upper layer liquid is desalted by ultrafiltration; The final upper layer liquid is properly diluted for multi-stage membrane separation, and each separated fraction is collected, further concentrated and dried, and then frozen for storage. The present invention integrates chemical rough separation and multi-stage membrane fine separation technology, and extracts the main active components of deer blood from deer blood according to the molecular weight of specific components, so as to achieve the purpose of producing a single active component, and the yield of active components can reach 85%.

Description

A kind of deer blood active ingredient classification extraction method

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a method for classifying and extracting active components of deer blood.

Background technique

In the medical books of the past dynasties, the blood or antler blood of sika deer or red deer has the functions of nourishing blood and nourishing essence, promoting blood circulation and removing blood stasis, reducing swelling and healing injuries. Deer blood was first seen in Sun Simiao's "Qian Jin Yi Fang Shi Zhi" in the Tang Dynasty, and Li Shizhen's "Compendium of Materia Medica" in the Ming Dynasty recorded the medical effects of deer blood in detail. Deer blood has an important position in the clinical practice of traditional Chinese medicine, and is also widely used among the people. Modern clinical studies have shown that deer blood has outstanding curative effects in treating palpitations, insomnia, forgetfulness, falls, rheumatism and rheumatoid diseases, and anti-aging. The pharmacological effects of deer blood are mainly because it contains Y-globulin that can improve immunity, enzymes superoxide dismutase and glutathione oxidase that have anti-aging effects, and creatine phosphokinase related to nerve and cardiovascular functions , lysophosphatidylcholine, A-hydroxybutyrate dehydrogenase and phosphocreatine kinase, as well as serum testosterone, estradiol, progesterone, cortisol and other hormones. There have been many years of domestic research on the active ingredients of deer blood, and the components and mechanisms related to the functions of deer blood have been preliminarily grasped. Currently, they are sold in the market by making decoction pieces, deer blood wine, and deer blood powder. Because people with different physical conditions have different choices of deer blood functions, taking crude deer blood products may cause more serious side effects, and only part of the active ingredients of deer blood are suitable for this group of people. However, there are no products containing single or partial deer blood active ingredients. Although there are also products in the form of deer blood oral liquid on the market, they are still made with deer blood as an ingredient. Products containing single or partial deer blood active ingredients will have broad market prospects. At present, the technology for industrial separation and purification of active components of deer blood is not yet mature. Although there is a process design for obtaining active peptides by enzymatically hydrolyzing deer blood, it is still unable to separate peptides of different active types, which can be regarded as only the primary processed products of deer blood. .

To extract and separate certain proteins from deer blood, the traditional preparation method is to firstly fractionate (roughly extract), and then subdivide (purify). Crude extraction generally uses methods such as alcohol precipitation, salt precipitation, isoelectric point precipitation and organic solvent precipitation. Commonly used neutral salts for salting out include ammonium sulfate, sodium sulfate, magnesium sulfate, polyphosphate, etc. Among them, ammonium sulfate has the best effect. There are many reagents used in the organic solvent precipitation method, ethanol is the most commonly used, followed by acetone and polyethylene glycol. Ammonium sulfate salting-out method and cold ethanol method are more methods used in actual production and application. To be applied to large-scale extraction of proteins in plasma, both have their own shortcomings. The ammonium sulfate salting-out method has poor selectivity to protein, the product needs to be desalted, and the ammonium sulfate needs to be recycled. If it cannot be recovered well, it will cause environmental pollution and increase the production cost. The specificity of the cold ethanol method is also poor, because the temperature of ethanol will cause protein denaturation, so the temperature needs to be strictly controlled during the operation. Also consider the issue of ethanol recovery in order to reduce costs. A more refined separation technique is chromatography. Commonly used are ion exchange chromatography (IEX), gel filtration chromatography (GF), affinity chromatography (AC), hydrophobic chromatography (HIC) and so on. Various chromatography methods have their own advantages and disadvantages. Generally speaking, chromatography is a mild purification method, which is easy to maintain the activity of sample molecules, high yield, and high purity. The material has a limited lifespan and needs to be continuously updated, and the cost is high. The chromatography system must prevent microbial contamination. Once the contamination is cumbersome, the virus removal effect is poor, and it is not suitable for large-scale production. Membrane separation technology is based on the effect of molecular sieve, using external force to push the components in the mixture through a specially manufactured membrane with selective permeability, so as to achieve the purpose of separation, purification and concentration. Membrane separation technology is generally used for the separation of a single component because of its molecular sieve effect, and can obtain a higher purity of the target product. At present, membrane separation technology has been widely used in many industries such as electronics industry, water treatment, environmental protection, food, pharmaceutical and biological engineering, chemical industry and textile. However, there are few studies on the simultaneous separation of multiple specific components directly using multi-stage membranes, and there is no commercial equipment available.

Due to the complex composition of deer blood, although there have been researches on enzymatic protein extraction, albumin extraction and other related processes, there have been no reports on the separation of active components in the deer blood product industry.

In this study, by integrating a variety of separation and purification technologies, after several years of technological exploration, a simple and effective method for the separation and purification of active ingredients in deer blood has been obtained.

Contents of the invention

Although there are process designs in the deer blood product industry to obtain active peptides and isolate albumin by enzymatically hydrolyzing deer blood, none of them touches the core of the market value of deer blood—active ingredients. Because there are many types of related active ingredients and relatively low content, it has always been a technical problem in the industry. The present invention integrates chemical rough separation and multi-stage membrane fine separation technology, and extracts the main active components of deer blood from deer blood according to the molecular weight of specific components, so as to achieve the purpose of producing a single active component, and the yield of active components can reach 85%.

The present invention uses ammonium sulfate precipitation for rough separation, and then utilizes self-designed multi-stage membrane (molecular weight cut-off gradually decreases) fine separation process design, designs the molecular weight cut-off size of the membrane according to the molecular weight of the main active ingredient, and deer blood multi-stage The components are separated and purified according to the difference in molecular weight, so as to obtain different active ingredients with high purity. Its technical concept is shown in Figure 1 and Figure 2 of accompanying drawing.

The method for classifying and extracting active components of deer blood according to the present invention comprises the following steps:

(1) Fresh deer blood is subjected to anticoagulant treatment; anticoagulated deer plasma is salted and precipitated with saturated ammonium sulfate, centrifuged to remove the precipitate, and the upper liquid is desalted by ultrafiltration;

(2) Appropriately dilute the desalted upper liquid to perform multi-stage membrane separation, and collect each separated component.

Each of the isolated components collected in the above step (2) can be further concentrated and dried and stored in a frozen state.

The polyethersulfone membranes with molecular weight cut-offs of 0.5kDa, 7kDa, and 10kDa can be selected as the multi-stage membrane in the present invention.

The present invention also provides a multi-stage membrane separation device, which includes a feed liquid barrel, a pump, and a membrane separation chamber arranged in sequence on the circulating liquid delivery pipeline. , the molecular weight cut-off of the membrane decreases step by step; the membranes of each stage in the membrane separation chamber are equipped with collection pipes connected to the collection tank. One operation of the device can simultaneously separate 3 kinds or add membrane sieve to separate more types of components.

The separation process of this process is smooth, and the separation rate of active components can reach more than 85%.

The separation technology adopted in the present invention first utilizes the traditional chemical rough separation technology to remove a large amount of impurities in batches very conveniently; more importantly, it uses multi-stage membrane separation technology to separate various types of active ingredients in deer blood according to molecular weight It is extracted step by step in sequence, and the yield is over 85%, and the purity is also high, which solves the long-standing problems of mixed functions and side effects of deer blood products. The multi-stage membrane separation of the present invention is a modification of the conventional multi-stage membrane device, and the membranes of different levels are solidified in the same action chamber, which can be operated continuously and has a large processing capacity. A variety of active extracts can be obtained by adding samples at one time. But it is not a single compound, so the cost of treatment is relatively low, and it does not affect the efficacy of the product.

Description of drawings

Fig. 1 is a flow chart of the extraction process of active ingredients from deer blood of the present invention.

Fig. 2 is a structural diagram of the multi-stage membrane fine separation process of the present invention.

Detailed ways

Example:

According to the following steps, different classifications of biologically active peptides with a molecular weight between 0.2kDa and 10kDa can be obtained:

1. Preparation of deer plasma: add 0.5% trisodium citrate anticoagulant to fresh deer blood, stir well, centrifuge at 4000r/min for 15min, take the supernatant, which is deer plasma, and store it at -20 Store at ℃ for later use.

2. Salting out and precipitation: add an equal volume of saturated ammonium sulfate solution drop by drop to the deer plasma while stirring, let stand at room temperature for 30 minutes, centrifuge at 4000r/min for 20 minutes, collect the precipitate, and further separate the upper liquid after desalting by ultrafiltration. 3. Multi-level membrane separation: For active substances such as unsaturated fatty acids and lyso-lecithin, which have the functions of lowering blood pressure and treating cardiovascular diseases, a polyethersulfone membrane with a molecular weight cut-off of 0.5kDa is selected; for promoting growth and development, controlling diabetes For active substances such as insulin-like growth factor 1 and growth hormone that have the effect on osteoporosis, the polyethersulfone membrane with a molecular weight cut-off of 7kDa is selected; for deer nerve growth factor that has anti-fatigue, anti-inflammatory and analgesic effects, and promotes wound healing, A polyethersulfone membrane with a molecular weight cut-off of 10 kDa was selected. In such a design, a membrane with a specific molecular weight cut-off is selected as required to form a multi-stage membrane separation system. As shown in Figure 2, the desalted upper layer liquid is properly diluted and placed in the feed liquid tank 1, and the feed liquid tank 1 is placed in the water bath 5, and the liquid in the feed liquid tank 1 is pumped into the membrane separation chamber 2 through the pump M, and separated Chamber 2 is provided with membranes 21, 22, 23, 24 with gradually decreasing molecular weight cut-offs along the direction of liquid flow, and collecting tanks 41, 42, 43, 44 respectively collect different types of If the liquid after membrane separation contains more components, it will return to the liquid tank 1 through the return pipe 3 to recover various components again to increase the recovery rate; there are gate valves on each pipeline. Carry out multi-stage membrane separation, collect each separated component and detect the content and purity, and then further concentrate and dry and store in freezer.

After specific tests, using 500 grams of deer antler blood, separated by the method of the present invention, the collected and separated lysolecithin, insulin-like growth factor 1 and deer nerve growth factor accounted for 65%, 72, 68% (assayed by enzyme labeling method), which basically meets the requirements of extraction.

Claims (4)

1. a deer blood active ingredient classification extraction method, is characterized in that comprising the following steps: (1) Fresh deer blood is subjected to anticoagulant treatment; anticoagulated deer plasma is salted and precipitated with saturated ammonium sulfate, centrifuged to remove the precipitate, and the upper liquid is desalted by ultrafiltration; (2) Appropriately dilute the desalted upper layer liquid to perform multi-stage membrane separation, collect each separated fraction, further concentrate and dry it, and freeze it for storage. 2. The method for classifying and extracting active components of deer blood according to claim 1, characterized in that step (2) utilizes a multi-stage membrane to collect each separated component. 3. The method for classifying and extracting active components of deer blood according to claim 1, wherein the multi-stage membrane is a polyethersulfone membrane with a molecular weight cut-off of 0.5kDa, 7kDa, or 10kDa. 4. The method for classifying and extracting active components of deer blood according to claim 1, characterized in that the separation device used during multi-stage membrane separation comprises a feed-liquid barrel, a pump, and a membrane separation chamber sequentially arranged on the circulating liquid delivery pipeline The membrane separation chamber is equipped with multi-stage membranes, and the molecular weight cut-off of the membranes decreases step by step along the direction of liquid flow; the membrane separation chambers are equipped with collection pipes connected to the collection tanks behind each membrane.