CN101474251B - Litchi grass total flavonoids extract and its preparation method and use - Google Patents

Abstract

The invention provides a method for preparing a common sage herb general flavone extract, comprising the steps that: after being pulverized, the overground part of common sage herb is heated in an enhydrous alcoholic solution and is processed by the operation of reflux extraction, a common sage herb concentrated solution is obtained by concentration, and the alcoholic solution is recycled; the common sage herb concentrated solution is led through a pretreated nonpolarity or low polarity macroporous absorbent resin column; and the macroporous absorbent resin column is bathed and then is eluted by eluent, and parts of eluted solutions are collected and concentrated to be dry, thereby gaining the common sage herb general flavone extract. The invention also provides the common sage herb general flavone extract prepared by the preparation method and the application of the common sage herb general flavone extract in preparing medicines for curing the hepatopathy or health products; and the common sage herb general flavone extract of the invention has the advantages of high content of general flavone in the common sage herb general flavone extract, obvious function of liver protection, high preparation efficiency, simple and safe operation, less equipment investment, short production period and easy industrial production.

Description

Litchi grass total flavonoids extract and its preparation method and application

technical field

The present invention relates to the technical field of substance extraction, more specifically, to the technical field of extraction of total flavonoids from lychee grass, in particular to the total flavonoids extract from lychee grass and its preparation method and application, which has a significant hepatoprotective effect.

Background technique

my country is an area with a high incidence of liver disease, and hepatitis patients account for more than 10% of the total population. At present, anti-hepatitis drugs commonly used in clinic can be roughly divided into three categories: antiviral drugs, immunomodulatory drugs and hepatoprotective drugs.

Litchigrass (Salvia plebeia R.Br.) is the aboveground part of the genus Salvia in the Labiatae family, also known as toad grass, snow primrose, holly and so on. Annual or biennial erect herbaceous plant, distributed in other regions except the northwest, mainly produced in Jiangsu, Zhejiang, Anhui and other provinces. The whole herb tastes bitter, pungent, and cool in nature. It has the functions of clearing heat, detoxifying, cooling blood and diuresis. For sore throat, bronchitis, nephritis edema, external treatment of mastitis, etc. Modern pharmacology shows that lychee grass has the functions of relieving asthma, relieving cough, antiviral, antibacterial, and antioxidant.

The study of phytochemical constituents showed that the chemical constituents of Lychee Grass are flavonoids, terpenoids and caffeic acid, among which flavonoids are the main constituents. Flavonoids widely exist in nature and belong to some secondary metabolites produced by plants in the long-term natural selection process. Flavonoids have: anti-oxidation, scavenging oxygen free radicals; regulating cardiovascular system; anti-cancer, anti-cancer effects; anti-inflammatory immunity and anti-aging effects; effects on the endocrine system; Phlegm, diarrhea, antispasmodic, improve memory, anti-allergic, blood circulation, choleretic and liver protection. The flavonoids in Lychee Grass are mainly high psylloids, high psyllogens, nepetalin, eupatorium flavonoids and luteolin and so on. High psyllogens and high psyllogens have hepatoprotective effects; high psyllogens can inhibit the growth of liver cancer cells and human KB cells;

The traditional purification methods of flavonoids include extraction method and polyamide column chromatography, etc., but the operation is troublesome, and the solvents have different degrees of toxicity. Therefore, it is necessary to provide a high-efficiency preparation method of total flavonoids of lychee grass with hepatoprotective effect, which is simple, safe and easy to industrialized production, so as to provide a basis for further development and utilization of lychee grass

Contents of the invention

The main purpose of the present invention is to address the above existing problems and deficiencies, to provide a total flavonoids extract from Lychee Grass, which has a high content of total flavonoids, has a significant hepatoprotective effect, and is highly efficient in preparation and easy to operate. , safety, less investment in equipment, short production cycle, and easy industrial production.

In order to achieve the above object, in the first aspect of the present invention, a method for preparing total flavonoids of Lychee Grass is provided, which is characterized in that it comprises the steps of:

1) After the ground part of litchi grass is crushed, it is extracted by heating and refluxing with water-containing alcohol solution, concentrated to obtain the concentrated solution of litchi grass, and the alcohol solution is recovered at the same time;

2) passing the lychee grass concentrate through a pretreated non-polar or weakly polar macroporous adsorption resin column;

3) Washing the macroporous adsorption resin column with water, and then eluting with an eluent, collecting part of the eluted solution, and concentrating to dryness to obtain the total flavonoids extract of Lychee Grass. Among them, water washing is to remove residual solution and other impurities in the resin.

The aerial parts of litchi above can be dried before being crushed, or directly crushed without drying.

Preferably, the number of extractions in step 1) is 3 times, and the alcohol solution is methanol solution or ethanol solution. Of course, the number of extractions is not limited to 3 times, it can be 2 times, or 4 times. Generally speaking, the more times, the more total flavonoids can be extracted, but the total flavonoids extracted in the next few times will decrease. Fewer and fewer, and multiple extractions can easily cause the volume of the extract to be too large, generally 3 times is more appropriate.

The extraction of the total flavonoids extract of Litchi Grass adopts an orthogonal test, and the best method is: after the aboveground part of Litchi Grass is dried and crushed, it is placed in the extraction with 50% ethanol solution at a weight-to-volume ratio of 1:20 (g/ml). Reflux extraction at 70°C for 3 times, 1 hour each time.

Preferably, in step 1), centrifugation or filtration is performed before the concentration or after the lychee concentrate is obtained. The centrifugation or filtration therein is to remove obvious impurities therein.

Preferably, in the step 2), the macroporous adsorption resin column is a polystyrene type or cross-linked acrylonitrile non-polar or weakly polar resin column. HZ820 and AB8 are preferred, and HZ820 is most preferred. Of course, it can also be other suitable non-polar or weak polar resin columns, such as cross-linked acrylonitrile-like non-polar or weak polar resin columns.

Preferably, in the step 2), the flow rate of the lychee grass concentrate is 0.5-5 BV/h. Preferably 1.5-4.5BV/h, more preferably 2.5BV/h.

Preferably, in the step 3), the eluent is alcohol solution or ketone solution, and the concentration of the eluent is 10-95%.

Preferably, in the step 3), the flow rate of the water washing is 5-8BV/h, and the flow rate of the eluent is 2-8BV/h. The flow rate of the eluent is preferably 4BV/h.

Preferably, in the step 3), the part of the post-elution solution is 0.5-3 BV/h of the post-elution solution. The concentration of the eluent is preferably 50-70%.

In the second aspect of the present invention, there is provided an extract of total flavonoids from Lychee Grass, which is characterized in that it is prepared by the above-mentioned preparation method.

In the third aspect of the present invention, there is provided the use of the above-mentioned total flavonoids extract of Lychee Grass in the preparation of medicines or health care products for treating liver diseases. Wherein the liver disease may be liver damage, hepatitis, fatty liver or cirrhosis.

The beneficial effects of the present invention are specifically as follows:

1. The present invention adopts water-containing alcohol solution to carry out multiple reflux extractions on the crushed lychee grass aerial parts, after filtration and concentration, the resin column is adsorbed, and then eluted with an eluent, the eluted solution is collected, and dried to obtain the total flavonoids of lychee grass The total flavonoids content of the extract is not less than 60%, the equipment investment is small, the production cycle is short, the preparation is efficient, the operation is simple, safe, low in cost, and easy for industrial production;

2. The total flavonoids extract of Lychee Grass obtained in the present invention has been proved by pharmacological tests to have significant effects of protecting the liver, protecting the liver, and treating liver diseases, which provides a basis for further development and utilization of Lychee Grass.

Description of drawings

Fig. 1 is the impact figure (HE × 400) of the total flavonoids extract of Lychee Grass of the present invention on the pathological changes of acute liver injury in mice induced by _CCl , (wherein A: normal group; B: model group; C: positive control group ; D: high-dose group; E: middle-dose group; F: low-dose group).

Detailed ways

The above-mentioned features mentioned in the present invention, or the features mentioned in the embodiments can be combined arbitrarily. All the features disclosed in the specification of this case can be used in combination with any combination, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, usually follow the conventional conditions or the conditions suggested by the manufacturer. All percentages and parts are by weight unless otherwise indicated.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the method of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Example 1

Weigh 250g of lychee aerial part, after drying and pulverizing, add 5000ml of 50% ethanol solution, and reflux extraction at 70°C for 3 times, each time for 1 hour. The extracts were combined, left to stand overnight, centrifuged at 4000rpm/min for 10 minutes, and concentrated under reduced pressure to obtain 650ml of Lychee grass concentrate.

Take a certain amount of pretreated HZ-820 macroporous resin, put it into a glass column with an inner diameter of 2.6cm, make the bed volume 160ml, and slowly pass 650ml of lychee grass concentrate through the resin bed at a flow rate of 2.5BV/h. Then use 1000ml of water to wash away the remaining solution in the column, and then use a concentration of 70% ethanol solution to flow through the resin column with a flow rate of 4BV/h, collect the solution after elution between 0.5～3BV, concentrate to dryness under reduced pressure, reduce After pressing and drying, 7.75 g of tan powder was obtained, and the yield was 3.1% (calculated as crude drug) of the total flavonoids extract of Lychee Grass, and the total flavonoids content was 63.5%.

Example 2

Weigh 250g of lychee aerial part, after drying and pulverizing, add 5000ml of 50% ethanol solution, and reflux extraction at 70°C for 3 times, each time for 1 hour. The extracts were combined and concentrated to dryness under reduced pressure to obtain 62 g of Litchi grass extract. Weigh 50g of Lychee Grass extract, add 1000ml of water, ultrasonically treat for 20 minutes, after fully dissolving, obtain Lychee Grass concentrated solution, centrifuge at 4000rpm/min for 10 minutes.

Take a certain amount of pretreated AB-8 macroporous resin, put it into a 3.4cm glass column, make the bed volume 350ml, and make 1000ml litchi grass concentrate slowly pass through the resin bed at a flow rate of 2.5BV/h. Then use 2000ml of water to wash away the remaining solution in the column, and then use a 70% ethanol solution to flow through the resin column at a flow rate of 4BV/h, collect the eluate between 0.5 and 3BV, concentrate to dryness under reduced pressure, and depressurize. After drying, 5.85 g of tan powder was obtained, and the yield was 2.9% (calculated as crude drug) of the total flavonoids extract of Lychee Grass, and the total flavonoids content was 61.2%.

For the total flavonoids extract of Lychee Grass obtained in the above examples, the content determination of the total flavonoids contained in it is determined by the NaNO ₂ -Al(NO ₃ ) ₃ -NaOH complex colorimetric method, with rutin as the external standard , found that the content of total flavonoids is not less than 60%.

Example 3

1 Materials and reagents

The total flavonoids extract of Lychee Grass obtained in Example 1; Carboxymethylcellulose sodium (CMC-Na); CCl ₄ (analytical pure); Bifendate; ALT, AST kit (Nanjing Jiancheng Biotechnology Company); Kunming 60 breeding mice, both male and female (Shanghai Jiuting Experimental Animal Center).

2 Experimental methods

60 Kunming mice were divided into 6 groups, each group was half male and half male, and mice of the same sex were randomly selected. That is, the normal control group, the model group, the positive control group (bifendate group) and the high, medium and low dosage groups of the total flavonoids extract of Litchi grass. The medicines were all made of 0.8% sodium carboxymethylcellulose into a suspension, administered orally once a day, for 7 consecutive days, the normal group and the model group were given corresponding volumes of vehicle, and the normal group was intraperitoneally injected with peanut oil 0.1% after the last administration. ml/10g, and the remaining 5 groups were injected with 0.1% CCl ₄ 0.1ml/10g. After fasting for 16 hours, the eyeballs were picked to take blood, and the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, and the same part of the left lobe of the liver was taken, fixed, paraffin-embedded, sectioned, and stained with HE. Morphological changes were observed under a light microscope.

3 Experimental results

Table 1 The protective effect of the total flavonoids extract of Lychee Grass on _CCl4 -induced acute liver injury in mice (x±s, n=10)

Group Dose (mg/kg) AST(U/L) ALT(U/L) normal control group - 28.5±7.8 30.0±13.5 Model control group - 70.4±34.9 ^## 244.2±81.5 ^### Positive control group 150 38.6±11.4 ^** 36.5±17.2 ^*** High dose group 300 36.7±10.8 ^*** 35.9±18.4 ^*** Middle dose group 150 40.5±13.2 ^** 80.3±26.8 ^*** low dose group 50 41.2±13.0 ^** 81.8±32.9 ^***

Compared with normal group:

^## means P<0.01, ^### means P<0.001; compared with the model group: ^** means P<0.01, ^*** means P<0.001.

The results in Table 1 show that after the mice were injected intraperitoneally with CCl ₄ , their serum ALT and AST activities were significantly increased, and after the mice were pre-administered the total flavonoids extract of Lychee Grass, the serum ALT and AST values were compared with those of the model group. decreased, and the high-dose group decreased significantly.

The results of histopathological examination of the liver are shown in Figure 1: the liver cells in the normal control group were normal in structure, neatly arranged, uniform in size, with clear boundaries, and no pathological changes were seen; the liver volume of the mice in the model group increased, and the liver cells had focal necrosis and More punctate necrosis, hepatic lobular structural damage, disordered arrangement, unclear boundaries of hepatocytes, bright balloon-like cytoplasm, and a large number of inflammatory cell infiltration in the interstitium; hepatocyte necrosis in each dose group of total flavonoids of lychee grass There are varying degrees of relief.

Example 4

The total flavonoids extract of Lychee Grass obtained in Example 2 replaces the total flavonoids extract of Lychee Grass obtained in Example 1 in Example 3, and other conditions are the same as in Example 3, and similar results are obtained, that is, after the mice are intraperitoneally injected with CCl ₄ , The serum ALT and AST activities were significantly increased, and after the mice were pre-administered the total flavonoids extract of Litchi grass, the serum ALT and AST values were reduced compared with the model group, and the high-dose group decreased significantly; the liver pathological tissue The results of medical examination also showed that the degree of liver cell necrosis in each dose group of total flavonoids extract of Lychee grass was alleviated to varying degrees.

Therefore, the present invention uses water-containing alcohol solution to carry out multiple reflux extractions on the crushed litchi grass shoots, after filtration and concentration, the resin column is adsorbed, and then eluted with an eluent, the eluted solution is collected, and dried to obtain the total flavonoids of litchi grass After determination, the total flavonoid content in the extract is not less than 60%. At the same time, the above pharmacological experiments prove that the total flavonoid extract of Lychee Grass extracted by the present invention has obvious protective effect on acute liver injury in mice induced by _CCl4 .

In summary, the total flavonoids extract of Lychee Grass total flavonoids of the present invention has a high content of total flavonoids, has a significant hepatoprotective effect, and is highly efficient in preparation, simple in operation, safe, low in cost, and easy for industrial production.

It should be noted that all documents mentioned in this application are incorporated by reference in this application, just as if each document was individually cited as a reference. In addition, it should be understood that what is described above are the specific embodiments of the present invention and the applied technical principles. After reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention without departing from the present invention. The spirit and scope of these equivalent forms also fall within the scope of the present invention.

Claims (9)

1. a method for preparing total flavonoids of Lychee Grass extract, is characterized in that, comprises steps: 1) After crushing the aboveground part of litchi grass, heat and reflux extract with aqueous alcohol solution, concentrate to obtain litchi grass concentrated solution, and recover the alcohol solution at the same time; 2) passing the lychee grass concentrate through a pretreated non-polar or weakly polar macroporous adsorption resin column, the macroporous adsorption resin column is HZ-820 macroporous resin or AB-8 macroporous resin; 3) Washing the macroporous adsorption resin column with water, and then eluting with an eluent, collecting part of the eluted solution, and concentrating to dryness to obtain the total flavonoids extract of Lychee Grass. 2. the method for preparing the total flavonoids extract of Lychee Grass according to claim 1, is characterized in that, in step 1), the number of times of said extraction is 3 times, and said alcoholic liquid is methanol solution or ethanol solution. 3. The method for preparing the total flavonoids extract of Lychee Grass according to claim 1, characterized in that, in step 1), centrifugation or filtration is carried out before said concentration or after obtaining said Lychee Grass concentrate. 4. The method for preparing the total flavonoids extract of Lychee Grass according to claim 1, characterized in that, in the step 2), the flow rate of the Lychee Grass concentrated solution is 0.5～5BV/h. 5. the method for preparing total flavonoids extract of Lychee Grass according to claim 1, is characterized in that, in described step 3), described eluent is alcohol liquid or ketone liquid, and the concentration of described eluent is 10-95%. 6. the method for preparing the total flavonoids of Lychee Grass extract according to claim 1, is characterized in that, in described step 3), the flow velocity of described washing is 5～8BV/h, and the flow velocity of described eluent is 2～8BV/h. 7. The method for preparing the total flavonoids extract of Lychee Grass according to claim 1, characterized in that, in the step 3), the part of the eluted solution is the eluted solution between 0.5～3BV/h . 8. An extract of total flavonoids from Lychee Grass, characterized in that it is prepared by the preparation method described in any one of claims 1-7. 9. the total flavonoids extract of Lychee Grass described in claim 8 is used in the purposes in the medicine or the health care products for the preparation of treatment of liver disease.