Preparation method of clove total flavonoids and application of clove total flavonoids in medicines or foods

Abstract

The invention relates to a preparation method of total flavonoids of clove and application of the total flavonoids of clove in medicines or foods. Comprising the following steps: crushing and sieving diced and dried clove raw materials; adding 4-6 times of 30-60% ethanol, and soaking for 2-4 hours; adding 10-20 times of 70-90% ethanol solution into the obtained material, quickly reducing the pressure to 0.03-0.05 MPa at the temperature of 50-60 DEG C in a thermostatic bath, extracting, separating filtrate and filter residues, re-extracting, and combining the filtrate; and concentrating the obtained filtrate at 60 DEG C under reduced pressure, pre-freezing in a refrigerator at-20 DEG C, and freeze-drying to obtain the total flavonoids of clove. Compared with a water extraction or alcohol extraction method, the method has the advantages of low extraction temperature, short time, reduction of the consumption of organic solvents, improvement of the extraction rate, reduction of the impurity content, and facilitation of the separation and preservation of effective components. The clove general flavone has an improvement effect on gastric mucosal lesion, and can effectively relieve pathological lesion induced by alcohol, promote gastric mucus secretion, relieve inflammation and improve acute gastric mucosal lesion caused by alcohol.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a method for preparing total flavonoids of cloves and application of the total flavonoids in preparing medicines or foods for protecting gastric mucosal damage. Background Art

[0002] In recent years, although the medical community has conducted extensive research on the pathogenesis of alcoholic gastric injury, current treatment methods still have limitations, such as limited therapeutic effects and large side effects. Therefore, the development of efficient and low-toxic prevention and treatment methods is of great significance to improving the quality of life of patients.

[0003] Flavonoids have attracted more and more attention from scholars in recent years due to their strong biological activity. Their efficacy is manifested in antibacterial and antioxidant activities. The main methods for extracting flavonoids from plants include ultrasonic extraction, microwave extraction, ethanol reflux, etc.

[0004] In modern medicine and food industry, natural plant extracts are valued for their safety and multi-effects. Eugenia caryophyllata As a plant with multiple pharmacological activities, Thunb. has been shown to contain a variety of bioactive compounds, such as eugenol and eugenol, which have antioxidant, anti-inflammatory, and antibacterial properties. These properties make total flavonoids of cloves have potential application prospects in the prevention and treatment of gastric diseases.

[0005] Chinese patent document CN112294936A discloses a culture medium and preparation method for preparing gastric fermentation products, the culture medium is composed of the following components: 10-15 parts of sea buckthorn pulp, 10-15 parts of clove extract, 10-15 parts of yam powder, 3-5 parts of bamboo mushroom, tree tongue ganoderma and thin skin fibroblast extract, 1-2 parts of turmeric powder, 3-6 parts of oligosaccharide, 80-100 parts of water. The prepared Hericium erinaceus fermentation product has a significant protective effect on gastritis and gastric ulcer induced by ethanol. The preparation method of clove extract in the invention is as follows: after the clove is crushed, 20-30 times the weight of water is added, and the clove is shaken and extracted in a shaker at 40-50°C for 10-14 hours, and the supernatant is obtained after centrifugation. After the supernatant is concentrated under reduced pressure, it is vacuum freeze-dried to obtain a clove water extract.

[0006] Chinese patent document CN115006491B discloses a Chinese medicine composition for treating chronic atrophic gastritis and its preparation method, wherein the Chinese medicine composition is made of the following raw materials: 20-40 parts of dendrobium, 1-10 parts of clove, 5-20 parts of pinellia, 15-30 parts of qianjiecao, 30-50 parts of jasmine, 10-25 parts of atractylodes, 6-18 parts of codonopsis and 12-28 parts of licorice. The preparation is completed by the following steps: adding 40-50°C water to qianjiecao, jasmine and licorice for 20-30 min, distilling and extracting to obtain volatile oil and filter residue one; crushing dendrobium, clove, pinellia, atractylodes and codonopsis, adding the filter residue one, then adding ethanol and soaking for 20-50h, then percolating to obtain percolate and filter residue two, adding water to the filter residue two and microwave extraction to obtain an extract; mixing the extract, the percolate and the volatile oil, concentrating and drying. The Chinese medicine composition obtained by the method works together by inhibiting or killing Helicobacter pylori, protecting and repairing gastric mucosa, and regulating pepsin levels, thereby improving the treatment of chronic atrophic gastritis. Although cloves are added to the invention, they are not the main component of the original Chinese medicine juice, and are targeted at chronic atrophic gastritis. Summary of the invention

[0007] In view of this, in order to solve the above technical problems, the total flavonoids of cloves were prepared by the reduced pressure internal boiling method, which effectively reduced the extraction time, increased the extraction efficiency, and stabilized the effective ingredients. The prepared total flavonoids of cloves had a significant protective effect on ethanol-induced acute gastric mucosal damage.

[0008] The present invention is achieved through the following technical solutions.

[0009] The method for preparing total flavonoids of cloves disclosed by the present invention comprises the following steps.

[0010] (1) Dry the clove raw material, crush it, and pass it through an 80-mesh sieve to obtain clove powder. Take a certain amount of clove powder, add 4-6 times the amount of 30-60% ethanol, and soak it for 2-4 hours.

[0011] (2) Add 10-20 times the amount of the material obtained in step (1) into a 70-90% ethanol solution, place in a thermostatic bath at 50-60°C, rapidly reduce the pressure to 0.03-0.05 MPa, boil the solution, and extract for 5-10 minutes. Separate the filtrate from the residue, extract the residue again, and combine the filtrate.

[0012] (3) The filtrate obtained in step (2) is concentrated under reduced pressure at 60°C, the concentrate is pre-frozen in a -20°C refrigerator, and then freeze-dried to obtain total flavonoids of cloves.

[0013] The invention discloses an application of the total clove flavonoids in preparing medicine or food for preventing and treating acute alcoholic gastric mucosal damage.

[0014] The acute gastric mucosal injury mentioned in the present invention particularly refers to the acute gastric mucosal injury caused by taking a large amount of alcohol at one time.

[0015] Preferably, the concentration of the total flavonoids of clove in the drug for preventing and treating acute alcoholic gastric mucosal injury is 100 mg / kg bw.

[0016] The present invention has no special requirements on the dosage form of the drug, the auxiliary materials of the drug and the preparation method of the drug, and those familiar to those skilled in the art can be adopted.

[0017] The active ingredient of the medicine or functional food for preventing and treating gastric mucosal damage of the present invention is mainly total flavonoids of cloves, which can be added with one or more excipients acceptable in pharmaceutical preparations, and can also be compounded with other active ingredients to exert a synergistic effect in resisting acute alcoholic gastric mucosal damage.

[0018] The present invention has conducted a series of studies on the prevention and treatment of acute alcoholic gastric mucosal damage by total flavonoids of cloves, and confirmed that total flavonoids of cloves have a protective effect on gastric mucosal damage, can effectively alleviate alcohol-induced pathological damage, promote gastric mucus secretion, relieve inflammation, and improve acute gastric mucosal damage caused by alcohol.

[0019] The advantages of the present invention are: the total flavonoids of cloves for preventing and treating alcoholic gastric mucosal damage of the present invention, by the reduced pressure internal boiling method, compared with the traditional water extraction or alcohol extraction method, has a low extraction temperature, a short extraction time, reduces the consumption of organic solvents, and improves the extraction rate and reduces the impurity content, which is conducive to the separation and preservation of effective components. The present invention has found through animal experiments that the total flavonoids of cloves has an improving effect on gastric mucosal damage, can effectively alleviate alcohol-induced pathological damage, promote gastric mucus secretion, relieve inflammation, and improve acute gastric mucosal damage caused by alcohol. New medical or functional food uses have been discovered for the total flavonoids of cloves. The raw materials of the present invention are natural products, have high safety, and are widely available, and can be suitable for industrial production and market promotion applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the effect of the total flavonoids of cloves provided by the present invention on the gastric tissue morphology of mice with acute gastric mucosal injury.

[0021] Figure 2 This is a bar graph of the total flavonoids of cloves provided by the present invention on the gastric mucosal injury index of mice with acute gastric mucosal injury. Note: * P < 0.05, ** P < 0.01.

[0022] Figure 3 This is a HE-stained section diagram of the effect of the total flavonoids of clove provided by the present invention on the gastric tissue of mice with acute gastric mucosal injury.

[0023] Figure 4This is a PAS-stained section diagram of the effect of the total flavonoids of clove provided by the present invention on the gastric mucus secretion of mice with acute gastric mucosal injury.

[0024] Figure 5 This is a bar graph showing the effect of the total flavonoids of cloves provided by the present invention on the mucus area ratio of gastric tissue sections in mice with acute gastric mucosal injury. Note: * P < 0.05, ** P < 0.01.

[0025] Figure 6 This is a bar graph showing the effect of the total flavonoids of cloves provided by the present invention on the activity of glutathione peroxidase (GSH-Px) in gastric tissue of mice with acute gastric mucosal injury. Note: * P < 0.05, ** P < 0.01.

[0026] Figure 7 This is a bar graph showing the effect of the total flavonoids of cloves provided by the present invention on the secretion of pepsin in gastric tissue of mice with acute gastric mucosal injury. Note: * P < 0.05, ** P < 0.01.

[0027] Figure 8 This is a bar graph showing the effect of the total flavonoids of cloves provided by the present invention on the level of interleukin-6 (IL-6) in gastric tissue of mice with acute gastric mucosal injury. Note: * P < 0.05, ** P < 0.01.

[0028] Fig. 9 This is a bar graph showing the effect of the total flavonoids of cloves provided by the present invention on the level of tumor necrosis factor-α (TNF-α) in gastric tissue of mice with acute gastric mucosal injury. Note: * P < 0.05, ** P < 0.01. DETAILED DESCRIPTION

[0029] The present invention will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are merely illustrative and are by no means intended to limit the present invention and its application or use. All other embodiments obtained by a person of ordinary skill in the art without creative work based on the embodiments of the present invention are within the scope of protection of the present invention. Example 1

[0030] The total flavonoids of cloves for preventing and treating alcoholic gastric mucosal damage of the present embodiment comprises the following steps.

[0031] (1) Dry the clove raw material, crush it, and pass it through an 80-mesh sieve to obtain clove powder. Take a certain amount of clove powder, add 5 times the amount of 50% ethanol and soak it for 3 hours.

[0032] (2) The material obtained in step (1) was added with 15 times the amount of 80% ethanol solution, and the temperature was 55°C in a constant temperature bath, and the pressure was rapidly reduced to 0.04 MPa, and the solution was boiled inside and extracted for 8 minutes. The filtrate and residue were separated, and the residue was extracted again, and the filtrate was combined.

[0033] (3) The filtrate obtained in step (2) is concentrated under reduced pressure at 60°C, the concentrate is pre-frozen in a -20°C refrigerator, and then freeze-dried to obtain total flavonoids of cloves. Example 2

[0034] The method for preparing total flavonoids of cloves described in this embodiment comprises the following steps.

[0035] (1) Dry the clove raw material, crush it, and pass it through an 80-mesh sieve to obtain clove powder. Take a certain amount of clove powder, add 4 times the amount of 30% ethanol and soak it for 4 hours.

[0036] (2) The material obtained in step (1) was added with 20 times the amount of 70% ethanol solution, and the temperature was 50°C in a constant temperature bath, and the pressure was rapidly reduced to 0.05 MPa, and the solution was boiled inside and extracted for 10 minutes. The filtrate and residue were separated, and the residue was extracted again, and the filtrate was combined.

[0037] (3) The filtrate obtained in step (2) is concentrated under reduced pressure at 60°C, the concentrate is pre-frozen in a -20°C refrigerator, and then freeze-dried to obtain total flavonoids of cloves. Example 3

[0038] The method for preparing total flavonoids of cloves described in this embodiment comprises the following steps.

[0039] (1) Dry the clove raw material, crush it, and pass it through an 80-mesh sieve to obtain clove powder. Take a certain amount of clove powder, add 6 times the amount of 60% ethanol and soak it for 2 hours.

[0040] (2) The material obtained in step (1) was added with 10 times the amount of 90% ethanol solution, and the temperature was 60°C in a constant temperature bath, and the pressure was rapidly reduced to 0.03 MPa, and the solution was boiled inside and extracted for 5 minutes. The filtrate and residue were separated, and the residue was extracted again, and the filtrate was combined.

[0041] (3) The filtrate obtained in step (2) is concentrated under reduced pressure at 60°C, the concentrate is pre-frozen in a -20°C refrigerator, and then freeze-dried to obtain total flavonoids of cloves. Example 4

[0042] This example is an evaluation of the activity of total flavonoids of clove in preventing and treating acute alcoholic gastric mucosal injury.

[0043] 2.1 Establishment and drug administration of mouse model of acute alcoholic gastric mucosal injury.

[0044] SPF male Kunming mice (8 weeks old, body weight 20 ± 2 g) were used as the research subjects. The feeding conditions were as follows: room temperature 22 ± 1℃, 12 h / 12 ​​h light / dark cycle, free food and water, and acclimatization for 7 days. The mice were randomly divided into 3 groups, namely normal group (NC), model group (MC), and total flavonoids of clove group (ECE, 100 mg / kg bw). The mice in each group were gavaged with the test substance at 10 mL / kg, and the normal group and model group were gavaged with normal saline. The mice were gavaged once a day. After gavage for 14 days, the mice were weighed, and all animals were strictly fasted for 24 h (water was not allowed), and the test substance was also prohibited during this period. Each mouse in the model group and total flavonoids of clove group was gavaged with 0.1 mL / 10 g 70% ethanol. One hour later, the mice were anesthetized, the eyeballs were removed, and the blood was collected, and the mice were killed by cervical dislocation. The abdominal cavity was opened to remove the stomach, liver and other organs, washed with physiological saline, dried with filter paper, weighed and recorded. The stomach tissue was fixed with 4% paraformaldehyde, and the remaining tissue was stored at -80°C.

[0045] 2.2 Determination of gastric mucosal injury and inhibition rate.

[0046] The mouse stomach was cut open along the greater curvature of the stomach, and the residual in the stomach was flushed away with normal saline. The gastric mucosa was unfolded and photographed. The length and width of the bleeding point or bleeding strip were measured with a vernier caliper. The scoring criteria are shown in Table 1.

[0047] Table 1 Scoring criteria for gastric mucosal injury index .

[0048] Evaluation index: Gastric mucosal injury index = bleeding point score + erosion length score + erosion width score × 2.

[0049] Damage inhibition rate (%) = (damage index of model group - damage index of polysaccharide group) / damage index of model group.

[0050] 2.3 Pathological observation of gastric tissue by HE staining.

[0051] Hematoxylin and stained (HE) was used to observe the morphology of gastric tissue. About 10 mm × 2 mm tissue was cut from the same part of the gastric mucosal injury of the mouse, fixed in 10% neutral formalin solution, dehydrated, and embedded in paraffin. The tissue wax block was cut into sections with a thickness of 4 μm, and the pathological changes of gastric tissue were observed under an optical microscope (200 times) after HE staining.

[0052] Dehydration, embedding and sectioning of gastric tissue: After the gastric tissue is fixed, it is dehydrated with methanol twice, each time for 40-60 min, dehydrated with anhydrous ethanol twice, each time for 40-60 min, dehydrated with xylene for 20-40 min, embedded in paraffin, and 4 μm paraffin sections are prepared. The sections are baked at 65°C for one hour for later use.

[0053] The HE staining method is as follows.

[0054] 1) Dewaxing and hydration of paraffin sections: sequentially place the sections in environmentally friendly dewaxing solution I for 20 min, environmentally friendly dewaxing solution II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 75% alcohol for 5 min, and wash with tap water.

[0055] Frozen section rewarming and fixation: Take the frozen sections out of the -20℃ refrigerator and restore them to room temperature. Fix them with tissue fixative for 15 minutes and then rinse them with running water.

[0056] 2) Pretreatment: The sections were treated in high-definition constant stain pretreatment solution for 1 min.

[0057] 3) Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 min, wash with tap water, differentiate with differentiation solution, wash with tap water, reblue with bluing solution, and rinse with running water.

[0058] 4) Eosin staining: Dehydrate the sections in 95% alcohol for 1 min and then stain them in eosin solution for 15 s.

[0059] 5) Dehydration and sealing: Place the slices in anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, anhydrous ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene I for 2 min, xylene II for 2 min to make them transparent, and seal the slices with neutral gum.

[0060] 6) Microscope examination, image acquisition and analysis.

[0061] 2.4 Pathological observation of gastric tissue by PAS staining.

[0062] The fixed gastric tissue sections were taken and PAS staining was used to observe the gastric mucus secretion.

[0063] The PAS staining method is as follows.

[0064] 1) Sequentially place the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 75% alcohol for 5 min, and then wash with tap water.

[0065] 2) Stain the sections in PAS staining solution B for 10-15 min, wash with tap water, and wash twice with distilled water.

[0066] 3) Immerse the sections in PAS staining solution A for 25-30 min, protect from light, and rinse in running water for 5 min.

[0067] 4) Stain the sections with PAS staining solution C for 30 seconds, wash with tap water, differentiate with hydrochloric acid solution, wash with tap water, blue with ammonia solution, and rinse with running water.

[0068] 5) Dehydration and sealing: The sections were placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene for 5 min, and xylene II for 5 min to make them transparent, and then sealed with neutral gum.

[0069] 6) Microscope examination, image acquisition and analysis.

[0070] 2.5 Determination of biochemical indices of gastric tissue.

[0071] Using pre-cooled PBS buffer as the homogenate, take an appropriate amount of gastric tissue and make a 10% tissue homogenate in an ice bath. Centrifuge at 4°C and 3000 rpm for 10 min, collect the tissue supernatant, and detect the contents of TNF-α, IL-6, pepsin and GSH-Px according to the operating instructions of the kit.

[0072] 2.6 Experimental results

[0073] 2.6.1 Effect of total flavonoids of clove on acute alcoholic gastric mucosal injury and injury score.

[0074] like Figure 1 As shown in the figure, macroscopic observation revealed that the gastric mucosa of the normal group mice was intact, with normal morphology and color, and no abnormalities such as bleeding and ulcers were observed. However, the gastric mucosa of the model group mice had obvious bleeding and ulcers, with thick bleeding stripes and bleeding spots. The bleeding stripes and bleeding spots in the ECE group were significantly reduced. Figure 2 The injury results showed that the injury score of the model group was significantly higher than that of the normal group, indicating that the model of acute alcoholic gastric mucosal injury was successful, while the injury score of the ECE group was significantly lower than that of the model group. In summary, total flavonoids of cloves can effectively prevent gastric mucosal injury caused by alcohol.

[0075] 2.6.2 Effect of total flavonoids of clove on gastric tissue morphology in mice with acute alcoholic gastric mucosal injury.

[0076] like Figure 3HE sections showed that the gastric mucosa of normal mice was smooth, with intact glandular structure, and no bleeding or submucosal edema. However, the glandular structure of gastric tissue in the model group was disordered, with a large number of shed epithelial cells and obvious inflammatory infiltration. The gastric mucosal injury in the ECE group was significantly improved, with significantly reduced gastric mucosal epithelial shedding, congestion and glandular structure disorder, indicating that total flavonoids of cloves have a certain auxiliary protective effect on gastric mucosal injury.

[0077] 2.6.3 Effect of total flavonoids of clove on gastric mucus secretion in mice with acute alcoholic gastric mucosal injury.

[0078] PAS staining was used to observe the secretion of gastric mucus. Figure 4 As shown, the neutral mucus substance appears purple-red and the cell nucleus appears light blue. Figure 5 As shown in the figure, the amount of mucus secretion in the model group was much less than that in the normal group, while the mucus secretion in the ECE group was significantly increased, indicating that total flavonoids of cloves can play a gastric protective role by improving mucus secretion.

[0079] 2.6.4 Effect of total flavonoids from clove on the secretion of GSH-Px, Pepsin, IL-6 and TNF-α.

[0080] like Figure 6 As shown in Figure 2, the model (MC) group significantly reduced GSH-Px activity, leading to oxidative stress; Figure 7 As shown, the MC group increased the secretion of pepsin, stimulated the gastric mucosa, and caused gastric mucosal damage; in addition, Figure 7 and 8 As shown in the results, the MC group had significantly increased levels of IL-6 and TNF-α, and the level of inflammation was increased. The ECE group had a significant correction effect on GSH-Px activity, Pepsin secretion, and IL-6 and TNF-α levels, suggesting that ECE can protect gastric mucosal damage from multiple aspects such as anti-oxidation, anti-inflammatory and regulating attack factors.

[0081] In summary, the total flavonoids of cloves described in the present invention have a protective effect on gastric mucosal damage, can effectively alleviate alcohol-induced pathological damage, promote gastric mucus secretion, relieve inflammation, and improve acute gastric mucosal damage caused by alcohol. In addition, it is worth noting that the total flavonoids of cloves described in the present invention are derived from "food and medicine homology", have high safety and a wide range of sources, and the extraction process is simple, which can be suitable for industrial production and market promotion and application.

[0082] Although the embodiments of the present invention have been shown and described above, it should be understood that these embodiments are only examples and do not constitute a limitation of the present invention. Under the premise of not departing from the basic principles and purposes of the present invention, those skilled in the art may make corresponding changes, modifications, substitutions and variations to the above embodiments. Example 5

[0083] The total clove flavonoids obtained in Example 1 were added with conventional auxiliary materials and prepared into capsules using conventional pharmaceutics methods. Example 6

[0084] The total clove flavonoids obtained in Example 1 were added with conventional auxiliary materials and prepared into tablets using conventional pharmaceutics methods. Example 7

[0085] The total flavonoids of clove obtained in Example 1 were added with conventional auxiliary materials and prepared into granules by conventional methods in pharmaceutical pharmacy. Example 8

[0086] The total clove flavonoids obtained in Example 1 were added with conventional auxiliary materials and prepared into pills using conventional pharmaceutics methods. Example 9

[0087] The total clove flavonoids obtained in Example 1 were added with conventional auxiliary materials and prepared into capsules using conventional pharmaceutics methods. Example 10

[0088] The total clove flavonoids obtained in Example 1 are added to an oral liquid in a certain proportion by conventional technology to prepare a health-care oral liquid with the function of protecting the gastric mucosa. Embodiment 11

[0089] The total clove flavonoids obtained in Example 1 are added to chewable tablets in a certain proportion by conventional processes to prepare health-care chewable tablets with the function of protecting gastric mucosa. Example 12

[0090] The total clove flavonoids obtained in Example 1 are added into capsules in a certain proportion by conventional processes to prepare health-care capsules with the function of protecting gastric mucosa.

Claims

1. A method for preparing total flavonoids of cloves, characterized in that The following steps are involved: (1) Grinding the dried clove raw material and passing it through an 80-mesh sieve to obtain clove powder; (2) Take a certain amount of clove powder, add 4-6 times the amount of 30-60% ethanol and soak for 2-4 hours; (3) Add 10-20 times the amount of 70-90% ethanol solution to the material obtained in step (2), place in a thermostatic bath at 50-60°C, quickly reduce the pressure to 0.03-0.05 MPa, boil inside, extract for 5-10 minutes, separate the filtrate from the residue, extract the residue again, and combine the filtrate; (4) The filtrate obtained in step (3) is concentrated under reduced pressure at 60°C, the concentrate is pre-frozen in a -20°C refrigerator, and then freeze-dried to obtain total flavonoids of cloves.

2. Use of the total clove flavonoids according to claim 1 in the preparation of medicines or foods for preventing and treating acute alcoholic gastric mucosal damage.

3. The use of the total flavonoids of cloves according to claim 1 in the preparation of a medicine or food for preventing and treating acute alcoholic gastric mucosal damage, characterized in that The concentration of total flavonoids of clove in the preparation of the medicine for preventing and treating acute alcoholic gastric mucosal injury is 100 mg / kg bw.

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