CN108354956A - Sunflower disk powder and its active ingredient are preparing the application in repairing wounded hepatocytes drug - Google Patents

Abstract

The invention discloses a sunflower pan powder and the application of its active ingredients in the preparation of a medicine for repairing damaged liver cells. The sunflower pan powder and its active ingredients of the invention have good anti-inflammatory and repairing effects on damaged liver cells caused by hyperuricemia.

Description

Application of sunflower pan powder and its active ingredients in the preparation of drugs for repairing damaged liver cells

technical field

The invention relates to the technical field of medicines, in particular to the application of sunflower dish powder and its active ingredients in the preparation of medicines for repairing damaged liver cells.

Background technique

The number of cases of hyperuricemia at home and abroad is currently on the rise. The pathogenesis of hyperuricemia is mainly due to increased uric acid production and decreased renal uric acid excretion, and the drug used for this disease often causes damage to the kidneys and liver and increases the burden on the liver and kidneys.

The sunflower disc is the flower disc of the Compositae plant sunflower (Helianthus annuuss L.), also known as sunflower receptacle, sunflower cake and sunflower house. It is flat in nature, sweet in taste, non-toxic, and returns to the liver meridian. , hemostasis, for the treatment of arthritis, mastitis, dizziness, tinnitus, abdominal pain and so on. The water decoction of sunflower disc has the effect of treating tumors, and the ethanol extract of sunflower disc has a certain antihypertensive effect. It is also used as a medicinal diet for regulating atrophic gastritis. my country is a big sunflower planting country, and the resources of sunflower discs are abundant. However, in traditional applications, the sunflower tray after removing the sunflower seeds is usually used as livestock feed to feed livestock or as a burning material for heating and cooking, which has not brought into play its greater economic benefit and medical effect, resulting in waste of resources.

Contents of the invention

In order to solve the problems in the prior art, the embodiment of the present invention provides the application of sunflower powder and its active ingredients in the preparation of a drug for repairing damaged liver cells. Described technical scheme is as follows:

On the one hand, the application of a sunflower powder and its active ingredients in the preparation of a drug for repairing damaged liver cells.

Further, the damaged liver cells are caused by hyperuricemia.

Further, the active ingredient of the sunflower dish powder is prepared by the following method:

1) Crush the sunflower disc to 100-200 mesh, add 6-10 times the mass of deionized water, stir thoroughly, adjust the pH value to 8-9 with sodium hydroxide, and stir at 45-50°C for 120-240 minutes to obtain Sunflower plate isolate protein liquid;

2) Heat the sunflower dish protein separation solution to 85-90°C, stir for 1-3h, cool to 45-55°C, add a compound protease accounting for 0.1%-0.3% of the total volume of the sunflower dish separation protein solution, and enzymolyze it for 3-5h , when the reaction is terminated, the temperature is raised to 85-90° C. within 5 minutes, and kept for 5-10 minutes to inactivate the complex protease, and the obtained enzymatic hydrolyzate is used for later use;

3) centrifuging the enzymatic solution in step 2) at a speed of 5000 rpm for 8-12 minutes, collecting the supernatant to obtain a crude sunflower protein small molecular peptide;

4) ultrafiltering the crude sunflower protein small molecular peptide solution in step 3) through a hollow fiber membrane with a cut-off molecular weight of 5000-20000 to remove the large molecular protein to obtain the sunflower disc small molecular peptide liquid;

5) Concentrate the sunflower disc small molecule peptide solution in step 4) to 1/4 to 1/2 of its original volume, and spray dry it to obtain the product.

The beneficial effect brought by the technical solution provided by the embodiment of the present invention is: the sunflower dish powder and its active ingredients of the present invention have good anti-inflammatory and repairing effects on damaged liver cells caused by hyperuricemia.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings that need to be used in the description of the embodiments.

Fig. 1 is the blank group liver cell figure in the embodiment of the present invention 2;

Fig. 2 is a diagram of liver cells of the model group in Example 2 of the present invention;

Fig. 3 is the hepatic cell diagram of sunflower dish powder active ingredient group in the embodiment of the present invention 2;

Fig. 4 is a diagram of liver cells of the alkaloid group in Example 2 of the present invention;

Fig. 5 is the liver cell figure of total flavone group in the embodiment of the present invention 2;

Figure 6 is a diagram of liver cells in the polysaccharide group in Example 2 of the present invention;

Fig. 7 is the hepatocyte figure of sunflower dish powder group in the embodiment of the present invention 2;

Fig. 8 is a diagram of liver cells in the allopurine group in Example 2 of the present invention.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the implementation manner of the present invention will be further described in detail below in conjunction with the accompanying drawings.

The preparation of embodiment 1 sunflower dish powder and active ingredient thereof

1) Crush 5 kg of sunflower discs to 100 mesh, add 30 kg of deionized water, stir thoroughly, adjust the pH value to 8 with sodium hydroxide, and stir at 45° C. for 120 minutes to obtain a sunflower disc isolated protein solution.

2) Heat the sunflower dish protein separation solution to 85°C, stir for 1 hour, cool to 45°C, add compound protease (ginger protease, snail protease, bat moth larvae protein digestion enzyme) accounting for 0.1% of the total volume of the sunflower dish protein separation solution : 0.5:1 mass ratio), enzymatic hydrolysis for 3 hours, when the reaction was terminated, the temperature was raised to 85°C within 5 minutes, and kept for 5 minutes to inactivate the complex protease, and the obtained enzymatic hydrolyzate was used for future use.

3) Centrifuge the enzymolysis solution in step 2) at a speed of 5000 rpm for 8 minutes, collect the supernatant, and obtain a crude sunflower protein small molecule peptide.

4) Ultrafiltering the crude sunflower protein small molecular peptide solution in step 3) through a hollow fiber membrane with a molecular weight cut-off of 5000 to remove macromolecular proteins to obtain a sunflower disc small molecular peptide liquid.

5) Concentrate the sunflower dish small molecule peptide solution in step 4) to 1/4 of its original volume, and spray dry to obtain the active ingredient of sunflower dish powder.

The preparation method of the bat moth larvae protein digestion enzyme comprises the following steps: remove the head of the bat moth larvae, remove the skin and fat, cancel the whole digestive tract, add 10 times the mass of phosphate buffer, homogenate, organize the slurry at 4°C, Then centrifuge at a speed of 3000rpm/min, take the supernatant and heat it to 40°C and rapidly cool it to 5°C, filter to remove fat, dialyze to remove salt, and freeze-dry to obtain.

Example 2 Sunflower dish powder and its active ingredients lowering uric acid and repairing damaged liver cells test

(1) Material

Small molecule peptide: the active ingredient of the sunflower dish powder prepared in Example 1.

Yeast paste: British Oxiod company.

Oxonic acid potassium salt: Sigma Company, USA, batch number: 101310713.

Allopurinol tablets: Shimao Tianjie Pharmaceutical (Jiangsu) Co., Ltd., batch number: 201608301.

Mouse uric acid detection kit: Nanjing Jiancheng Institute of Bioengineering.

SPF grade KM mice: male, body weight (22-25) g, provided by the Experimental Animal Center of the First Hospital of Jilin University.

(2) Modeling and drug administration

The mice were weighed before the study, 12 of them were used as the blank control group, and the rest were used as the hyperuricemia model group. Weighing was performed every morning during the experiment. In this modeling experiment, the method of gavage with yeast cream was adopted. By giving a large amount of purine substances, the uric acid value was increased and the purine metabolism was disordered, so as to achieve the effect of temporarily increasing the blood uric acid value in a short period of time. Different degrees of damage to the liver tissue. 100 mice were randomly divided into 8 groups, about 12 in each group, respectively blank group, model group, sunflower powder active ingredient group, polysaccharide group, alkaloid group, total flavonoids group, sunflower powder group and allopurin alcohol group. During the modeling period, except for the blank group (equal volume water gavage), other groups were gavaged with 20g/kg yeast extract every day, once a day, for 7 consecutive days, fasting at night on the 7th day, and early morning Take blood to measure the blood UA value, and the result of measuring the blood uric acid value shows that the blood uric acid value increases and can be administered immediately. From the 9th day, the blank group was administered intragastrically with equal volume of water every day. According to the test results of blood uric acid value, 12 animals were randomly selected as the model group, and the rest were divided into 6 groups for administration. Stomach administration, continuous administration for 7 days, fasting in the evening after 7 days, execution by dislocation of the neck the next day, and taking the liver for immunohistochemical analysis. Gavage with a high-purine diet containing yeast extract can cause transient hyperuricemia in mice, which can last for 15-20 days.

(3) Immunohistochemical immunohistochemical test to test the effect of drugs on the liver

① After the tissue is immersed in paraffin, move the tissue into a paraffin cup for embedding before embedding. Take it out from the incubator and place it on a tripod, and light an alcohol lamp under it to keep the paraffin in a dissolved state.

② Prepare the embedding frame, heat the tweezers for embedding on an alcohol lamp (to prevent the tweezers from sticking to wax), hold the wax cup in your left hand, and put the heated tweezers in the wax mouth (upright) with your right hand to let Paraffin is injected into the embedding frame along the forceps.

③Quickly tweezer the tissue block into the paraffin embedding frame, put the cut side down and put it into the bottom of the frame and flatten it gently to ensure that there are no more air bubbles.

④Paste the label on the paraffin wax surface before it solidifies. Be careful not to confuse the label with the specimen. When the wax block is cooled until there is a transparent wax film on the wax surface, immerse it in cold water to cool it quickly, otherwise crystals often form in the wax .

⑤ After the wax block is completely hardened, remove the copper frame to ensure that the wax is completely solidified, and trim it immediately.

⑥Take out the slices, stain with immunohistochemical kits, observe under the microscope to check the state of the sliced tissue cells and take pictures.

The experimental results are shown in Figures 1-8. In the blank group (Figure 1), the hepatic cells and cords of the hepatic cells in the hepatic lobules were clear, and the cytoplasm of the hepatic cells was plump. In the model group (Figure 2), hepatocytes and hepatocyte cords in some hepatic lobules were not clear, and hepatocyte atrophy could be seen with punctate necrosis and hepatocyte eosinophilia. In the alkaloid group, total flavonoid group and polysaccharide group (Fig. 4-6), punctate necrosis and eosinophilic changes were occasionally seen. In the sunflower pan powder group (Figure 7) and the sunflower pan powder active ingredient group (Figure 3), the liver cells and liver cell cords in the liver lobules were clear, the cytoplasm of the liver cells was plump, and occasionally punctate necrosis was seen. It has a good repairing effect on liver cells. In the allopurinol group (Fig. 8), hepatocytes and hepatocyte cords in some hepatic lobules were not clear, and hepatocyte atrophy was mostly seen in scattered inflammatory cell infiltration foci, punctate necrosis foci, and occasional cavitation and light staining of hepatocytes around the central vein. Allopurinol group had greater damage to liver cells. The microstructure suggested that the sunflower powder and its active ingredients had certain anti-inflammatory and repairing effects on damaged liver cells, and the best effect was also the active ingredient group of sunflower powder.

The application of sunflower dish powder and its active ingredients of the present invention in the preparation of drugs for repairing damaged liver cells, sunflower dish powder and its active ingredients can play anti-inflammatory, detumescence, pain relief and reduce blood uric acid, dissolve sodium urate, and at the same time It has a good repair effect on the damaged cells of the liver, treats both symptoms and root causes of hyperuricemia, and treats hyperuricemia fundamentally.

The serial numbers of the above embodiments of the present invention are for description only, and do not represent the advantages and disadvantages of the embodiments.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (3)

1. sunflower disk powder and its active ingredient are preparing the application in repairing wounded hepatocytes drug.

2. application as described in claim 1, which is characterized in that the wounded hepatocytes are caused by hyperuricemia.

3. application as described in claim 1, which is characterized in that the sunflower disk powder active ingredient is prepared by the following method It arrives：

1) sunflower disk is crushed to 100-200 mesh, the deionized water of 6-10 times of quality is added, is sufficiently stirred, with sodium hydroxide tune Section pH value is 8-9, is stirred 120-240 minutes at 45-50 DEG C, obtains sunflower disk protein isolate liquid；

2) sunflower disk protein isolate liquid is heated to 85-90 DEG C, stirs 1-3h, be cooled to 45-55 DEG C, addition accounts for sunflower disk separation The compound protease of protein liquid total volume 0.1%-0.3% digests 3-5h, and when reaction terminating, 85-90 DEG C is warming up in 5 minutes, It is kept for 5-10 minutes, so that compound protease is lost activity, gained enzymolysis liquid is spare；

3) enzymolysis liquid of step 2) is centrifuged 8-12 minutes under 5000 revs/min of rotating speed, collects supernatant, obtains sunflower protein The thick liquid of small-molecular peptides；

4) by the thick liquid of sunflower protein small-molecular peptides of step 3) through shut off molecular weight be 5000-20000 hollow-fibre membrane ultrafiltration, High molecular weight protein is removed, sunflower disk small-molecular peptides liquid is obtained；

5) the sunflower disk small-molecular peptides liquid of step 4) is concentrated, makes its volume concentration to original a quarter to half, Spray drying to get.