Black platycodon root composite concentrated solution as well as preparation method and application thereof

Abstract

The invention relates to the technical field of platycodon grandiflorum preparation, in particular to a black platycodon grandiflorum composite concentrated solution as well as a preparation method and application thereof. The preparation method comprises the following steps: steaming fresh platycodon grandiflorum, drying after steaming, and steaming and drying for 9 times in total to obtain black platycodon grandiflorum; the preparation method comprises the following steps: mixing black platycodon grandiflorum with water, carrying out water extraction, and concentrating the extract liquid after water extraction to obtain the black platycodon grandiflorum extract. Fresh platycodon grandiflorum is steamed and dried for nine times, part of saponin is hydrolyzed and converted into secondary sapogenin which is more easily absorbed by a human body, meanwhile, effective components are highly concentrated, the black platycodon grandiflorum with an excellent effect is prepared, the black platycodon grandiflorum is prepared into the black platycodon grandiflorum concentrated solution, the taste of the black platycodon grandiflorum can be improved, and the black platycodon grandiflorum has the effects of improving the immunity of the human body and improving the immunity of the human body. In-vivo and in-vitro experiments show that the black platycodon grandiflorum concentrated solution provided by the invention has the effects of improving gastric ulcer and protecting gastric mucosa.

Description

Technical Field

[0001] The present invention relates to the technical field of platycodon preparation, and particularly relates to a black platycodon compound concentrated solution, a preparation method thereof and an application thereof. Background Art

[0002] Platycodon grandiflorum Platycodon grandiflorus (Jacq.) A. DC. is a plant of the genus Platycodon in the family Campanulaceae ( Campanuaceae ), and is widely grown in China, Japan and eastern Siberia. It is one of the plants in China that can be used as both medicine and food. The main components of platycodon grandiflorum are oleanolic acid-type pentacyclic triterpenoid saponins, polysaccharides, flavonoids, polyynes, steroids, phenolic acids, fatty acids, etc. A large number of studies have shown that platycodon grandiflorum has the effects of protecting the liver, anti-inflammatory, anti-tumor, antioxidant, reducing blood lipid, reducing blood sugar, regulating immunity, inhibiting gastric juice secretion and anti-ulcer. Although platycodon grandiflorum has excellent pharmacological effects, due to its unique taste and bitter taste, it lacks consumer preference. Therefore, attempts have been made to develop various types of products to make it easier to consume. However, in the process of removing the bitter taste, most of the main physiological substances are destroyed or reduced, and the water-soluble components are eluted, destroying the unique food and shape of platycodon grandiflorum, making it difficult to develop products with excellent effects.

[0003] Therefore, how to remove the bitter taste of platycodon grandiflorum while retaining its excellent effects has become an urgent technical problem in this field. Summary of the Invention

[0004] The purpose of the present invention is to provide a black platycodon compound concentrated solution, a preparation method thereof and an application thereof to solve the problems existing in the prior art.

[0005] In order to achieve the above invention purpose, the present invention provides the following technical solutions: The present invention provides a preparation method of a black platycodon extract, comprising the following steps: Steaming fresh platycodon grandiflorum, and drying after steaming. Steaming and drying are carried out 9 times in total to obtain black platycodon grandiflorum; Mixing the black platycodon grandiflorum with water, performing water extraction, and concentrating the extractive solution after water extraction to obtain a black platycodon extract.

[0006] Optionally, the temperature of the steaming is 110-120°C, and the time is 40-60 min; the temperature of the drying is 55-60°C, and the time is 6-8 h.

[0007] Optionally, the ratio of the black platycodon grandiflorum to water is 1:15-20 (g:mL); the temperature of the water extraction is 80-90°C, and the time is 8-12 h; the concentration is to concentrate the extractive solution to 30-35% of the original mass.

[0008] Optionally, the fresh platycodon grandiflorum is three-year-old roots or above.​​

[0009] The present invention also provides black bellflower extract prepared by the above preparation method.

[0010] The present invention also provides the application of the above-mentioned black bellflower extract in the preparation of food with gastric protective function.

[0011] The present invention also provides a black bellflower compound concentrate with stomach-protecting function, comprising the following components in parts by weight: 38-42 parts of the above-mentioned black bellflower extract, 8-12 parts of apple and pear juice, 18-22 parts of compound extract, 5-7 parts of honey, 0.4-0.5 parts of sodium alginate, and 16.5-30.6 parts of water.

[0012] Optionally, the preparation method of the complex extract includes: mixing yam, jujube, kudzu root, ginger, wolfberry and water, performing water extraction, and concentrating the extract after water extraction to obtain the complex extract.

[0013] Optionally, the mass ratio of the yam, jujube, kudzu root, ginger, and wolfberry is 3:2:2:1:2; the total mass of the yam, jujube, kudzu root, ginger, and wolfberry to the amount of water is 1:10~15 (g:mL); the water extraction temperature is 100℃ and the time is 6~8h; the concentration is to concentrate the extract to 1 / 4 of its original volume.

[0014] The present invention also provides a method for preparing the above-mentioned black bellflower compound concentrate, comprising the following steps: mixing black bellflower extract, apple pear juice, compound extract, honey, sodium alginate and water to obtain black bellflower compound concentrate.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention involves steaming and drying fresh bellflower nine times, causing some saponins to hydrolyze and transform into secondary saponins that are more easily absorbed by the human body. At the same time, the active ingredients are highly concentrated, resulting in black bellflower with excellent efficacy. Black bellflower concentrate can be prepared to improve the taste of black bellflower. Furthermore, in vivo and in vitro experiments have shown that the black bellflower concentrate provided by this invention can improve gastric ulcers and protect the gastric mucosa. Attached Figure Description

[0016] Figure 1 Diagram showing the preparation process of black bellflower; Figure 2 The negative ion currents (TIC diagrams) for Platycodon grandiflorus and Platycodon grandiflorus are shown, where (a) is the negative ion current (TIC diagram) for Platycodon grandiflorus and (b) is the negative ion current (TIC diagram) for Platycodon grandiflorus. Figure 3The positive ion currents (TIC plots) for Platycodon grandiflorus and Platycodon niger are shown, where (a) is the positive ion current (TIC plot) for Platycodon grandiflorus and (b) is the positive ion current (TIC plot) for Platycodon niger. Figure 4 Image showing the scratching effect in GES-1 cells; Figure 5 A graph showing the wound healing efficiency in GES-1 cells; Figure 6 The images show the gastric mucosal injury experiment in ICR mice. A: Blank group (no model); B: Negative control group (0.5% CMC); C: Positive control group (sucralfate); D: Experimental group (Platycodon grandiflorus extract); E: Experimental group (Platycodon grandiflorus complex). Figure 7 HE staining images of mouse gastric tissue are shown. A: Blank group (no model established); B: Negative control group (0.5% CMC); C: Positive control group (sucralfate); D: Experimental group (Platycodon grandiflorus extract); E: Experimental group (Platycodon grandiflorus compound concentrate). Figure 8 This is a graph showing the influence of gastric mucosal damage indicators and inflammation. Note: P <0.05 is considered a significant difference. P <0.01 indicates a highly significant difference. P <0.001 indicates a highly significant difference; where A: the effect of TNF-α inflammatory markers in the blood; B: the effect of IL-1β inflammatory markers in the blood; C: the effect of ADH (alcohol dehydrogenase) factor in the blood; D: the effect of GAS (gastrin) in the blood. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] All raw materials used in this invention can be obtained commercially or prepared using existing technologies.

[0023] This invention provides a method for preparing black bellflower extract, comprising the following steps: Fresh bellflower roots are steamed and then dried. This process is repeated nine times to obtain black bellflower roots. Black bellflower root is mixed with water and extracted with water. The extract is then concentrated to obtain black bellflower root extract.

[0024] This invention involves washing and drying fresh bellflower root, then steaming it in a high-pressure sterilizer. After steaming, the dried product is removed. This process is repeated nine times to obtain black bellflower root.

[0025] In this invention, the steaming temperature is 110~120℃ (e.g., 110℃, 115℃, or 120℃, etc.), and the time is 40~60min (e.g., 40min, 45min, 50min, 55min, or 60min, etc.); the drying temperature is 55~60℃ (e.g., 55℃, 56℃, 58℃, or 60℃, etc.), and the time is 6~8h (e.g., 6h, 7h, or 8h, etc.).

[0026] This invention involves crushing black bellflower root and adding it to purified water for water extraction. The resulting extract is then concentrated at 90°C to obtain black bellflower root extract.

[0027] In this invention, the ratio of black bellflower root to water is 1:15~20 (g:mL), for example, it can be 1:15 (g:mL), 1:16 (g:mL), 1:17 (g:mL), 1:18 (g:mL), 1:19 (g:mL) or 1:20 (g:mL), etc.; the water extraction temperature is 80~90℃, for example, it can be 80℃, 85℃ or 90℃, etc., and the time is 8~12h, for example, it can be 8h, 9h, 10h, 11h or 12h, etc.; the concentration is to concentrate the extract to 30~35% of the original mass, preferably 30%, 32%, 34% or 35%, etc.

[0028] In this invention, the fresh lisianthus root is three years old or older.

[0029] The present invention also provides black bellflower extract prepared by the above preparation method.

[0030] The present invention also provides the application of the above-mentioned black bellflower extract in the preparation of food with gastric protective function.

[0031] The present invention also provides a black bellflower compound concentrate with stomach-protecting function, comprising the following components in parts by weight: 38-42 parts of the above-mentioned black bellflower extract, 8-12 parts of apple and pear juice, 18-22 parts of compound extract, 5-7 parts of honey, 0.4-0.5 parts of sodium alginate, and 16.5-30.6 parts of water.

[0032] The black bellflower compound concentrate of the present invention comprises 38 to 42 parts of black bellflower extract, for example, 38 parts, 39 parts, 40 parts, 41 parts or 42 parts.

[0033] The black lisianthus compound concentrate of the present invention includes 8 to 12 parts of apple and pear juice, for example, 8, 9, 10, 11 or 12 parts.

[0034] The black bellflower compound concentrate of the present invention comprises 18 to 22 parts of compound extract, for example, 18, 19, 20, 21 or 22 parts.

[0035] The black lisianthus compound concentrate of the present invention includes 5 to 7 parts of honey, for example, 5 parts, 6 parts or 7 parts.

[0036] The black bellflower compound concentrate of the present invention includes 0.4 to 0.5 parts of sodium alginate, for example, 0.4 parts or 0.5 parts.

[0037] The black bellflower compound concentrate of the present invention includes 16.5 to 30.6 parts of water, for example, 16.5 parts, 20 parts, 24.5 parts or 30.6 parts, etc.

[0038] In this invention, the preparation method of the complex extract includes: mixing yam, jujube, kudzu root, ginger, wolfberry and water, performing water extraction, and concentrating the extract after water extraction to obtain the complex extract.

[0039] In this invention, the mass ratio of yam, jujube, kudzu root, ginger, and wolfberry is 3:2:2:1:2; the ratio of the total mass of yam, jujube, kudzu root, ginger, and wolfberry to the amount of water is 1:10~15 (g:mL), for example, it can be 1:10 (g:mL), 1:11 (g:mL), 1:12 (g:mL), 1:13 (g:mL), 1:14 (g:mL), or 1:15 (g:mL), etc.; the water extraction temperature is 100℃, and the time is 6~8h; the concentration is to concentrate the extract to 1 / 4 of its original volume.

[0040] In some embodiments of the present invention, the method for preparing apple and pear juice includes: washing and cutting apples and pears into pieces, juicing them, filtering the apple and pear juice, concentrating it to 1 / 4 of its original volume, sterilizing it at 70-90°C (for example, 70°C, 80°C or 90°C, etc.) or adding a small amount of lemon juice to prevent browning of the apple and pear juice.

[0041] The present invention also provides a method for preparing the above-mentioned black bellflower compound concentrate, comprising the following steps: mixing black bellflower extract, apple pear juice, compound extract, honey, sodium alginate and water to obtain black bellflower compound concentrate.

[0042] In some embodiments of the present invention, the black bellflower compound concentrate is packaged in portions of 12-15g.

[0043] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1 (1) Wash fresh platycodon roots that are more than three years old, put them in a drying oven at 60°C for 18 hours, then steam them in a high-pressure sterilizer at 120°C for 60 minutes. After steaming, take them out and dry them at 60°C for 8 hours. Repeat the above steps nine times. Figure 1 ), to obtain black bellflower; (2) After crushing 1000g of black bellflower, add purified water at a ratio of 1:20 (g:ml) and extract at 95℃ for 12 hours. The extract is then concentrated at 90℃ to obtain about 300g of black bellflower extract.

[0045] Example 2 (1) Wash the apples and pears, cut them into pieces, juice them, filter them and concentrate them to 1 / 4 of the original volume. Sterilize them at 90℃ for 5 minutes and add 0.5g of vitamin C to prevent browning of the apple and pear juice.

[0046] (2) Mix 300g of yam, 200g of jujube (pitted), 200g of kudzu root, 100g of ginger and 200g of wolfberry and add purified water at a ratio of 1:15 (g:ml). Soak in hot water at 100℃ for 8 hours, filter and concentrate to 1 / 4 of the original volume to obtain the complex extract.

[0047] Example 3 The components of the black bellflower compound concentrate are weighed out by weight as follows: 40 parts black bellflower extract, 10 parts apple and pear juice, 20 parts compound extract, 6 parts honey, 0.5 parts sodium alginate, and 24.5 parts purified water. By weight, black bellflower extract, apple and pear juice, compound extract, honey, sodium alginate and purified water are mixed to obtain black bellflower compound concentrate.

[0048] Test Example 1 10g each of dried Platycodon grandiflorus and prepared black Platycodon grandiflorus from Example 1 were pulverized and added to 100ml of 50% ethanol. The mixture was sonicated at 60℃ and 240W. After sonication, the mixture was filtered through filter paper. The filtrate was concentrated to 20ml and then freeze-dried to obtain extracts of Platycodon grandiflorus and black Platycodon grandiflorus. The extracts were pretreated and then analyzed by chromatography-mass spectrometry (LC-MS (Thermo, Vanquish, QExactive HF)); C18 column (Zorbax Eclipse C18 (1.8μm)). 2.1mm 100mm).

[0049] The chromatographic separation conditions were: column temperature 30 ℃; flow rate 0.3 mL / min; Mobile phase composition A: 0.1% formic acid aqueous solution, B: pure acetonitrile; The injection volume was 2 μL, and the autosampler temperature was 4 °C. Positive ion mode: heater temperature 325 ℃; sheath gas flow rate: 45 arb; auxiliary gas flow rate: 15 arb; purge gas flow rate: 1 arb; electrospray voltage: 3.5 kV; capillary temperature: 330 ℃; S-Lens RF Level: 55%.

[0050] Negative ion mode: heater temperature 325 ℃; sheath flow rate: 45 arb; auxiliary flow rate: 15 arb; purge flow rate: 1 arb; electrospray voltage: 3.5 KV; capillary temperature: 330℃; S-Lens RF Level: 55%.

[0051] Scanning modes: Full scan (m / z 100~1500) and data-dependent secondary mass spectrometry (dd-MS2, TopN = 5); Resolution: 120,000 (primary mass spectrometry) & 60,000 (secondary mass spectrometry). Collision mode: High-energy collision dissociation (HCD).

[0052] Composition analysis: Platycodon grandiflorus and Platycodon grandiflorus were extracted with 50% ethanol and then analyzed by chromatography-mass spectrometry. Figures 2-3 The positive and negative ion currents (TIC diagrams) of Platycodon grandiflorus and Platycodon grandiflorus were used to detect a total of 291 components. Among them, 86 substances in Platycodon grandiflorus were not detected in Platycodon grandiflorus (Table 1), and 126 new active ingredients were detected in Platycodon grandiflorus (Table 2). The relative contents of citric acid, mannitol, 4,4-bis(tert-butylperoxy)valerate butyl ester, chlorogenic acid, sucrose, L-tryptophan, D-glutamine, and L-leucine were relatively high in Platycodon grandiflorus. The relative contents of mannitol, mannitol-heptulose, alpha-ketoglutarate, and 7-(2-C-ethynyl-β-D-furanoribosyl)-7H-pyrrolo[2,3-D]pyrimidine-4-amine were relatively high in Platycodon grandiflorus. The components of black bellflower, such as akebia saponin D, caffeic acid, naringenin, daphnetin, (+)-catechin, dihydroquercetin, sennaol, isoflavone, protocatechuic aldehyde, methyl ferulic acid, p-hydroxycinnamic acid, 6,7-dihydroxy-4-methylcoumarin, leucine, and oxidized resveratrol, effectively scavenge free radicals that damage the gastric mucosa and inhibit inflammatory pathways such as NF-κB, reducing the levels of pro-inflammatory factors such as TNF-α and IL-6, thereby directly alleviating oxidative stress and inflammatory damage to the mucosa. Perilla frutescens and 6-shogaol have direct inhibitory effects on Helicobacter pylori. Paeonol and scopolamine can relieve mucosal discomfort through anti-inflammatory and antispasmodic effects. Components such as alfalfa acid, isoliquiritigenin, daidzein, and catalpol can stimulate mucus secretion, promote epithelial cell proliferation and migration, thereby accelerating ulcer healing and strengthening the mucosa's own defense capabilities.

[0053] Table 1. Platycodon components not detected in black platycodon.

[0054] Table 2. Newly generated components in Platycodon grandiflorus compared to Platycodon grandiflorus.

[0055] Test Example 2 In vitro cell scratching: Human gastric mucosal epithelial cells (GES-1) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture, and were placed in an incubator at 37 ℃ and 5% CO2. Under a microscope, the cells were observed to be adherent cells.

[0056] (1) After digesting and counting the cells in the control group and experimental group, take 8×10⁸ cells. 5 Each part is 35mm 2 Incubate overnight in a petri dish; (2) Draw a line on the bottom of the plate with a marker, remove the culture medium, and use a 10μL pipette tip perpendicular to the marker to remove the cells in the plate; (3) Rinse with PBS to remove the scribbled cells, process them, and perform 2 replicates per group. The experimental groups are as follows: Control group: Added normal serum-free culture medium Control group: serum-free culture medium containing 1 ng / mL TGF-β1 was added. Experimental group 1: Serum-free culture medium with 10 μg / mL black bellflower complex added Experimental group 2: Serum-free culture medium with 10 μg / mL black bellflower extract added. (4) Incubate in a 37°C incubator.

[0057] (5) Take photos at 0h and 48h of the scratch. When taking photos, select the intersection of the line drawn by the marker and the cell scratch as the observation point for fixed-point observation.

[0058] Scratch effect: GES-1 cells were scratched using a black bellflower complex and black bellflower extract, and photographs were taken at 0h and 48h to assess their in vitro healing effects on cell mucosal damage. Black bellflower extract and concentrated black bellflower complex were used in the experimental groups, with TGF-β1 serving as the positive control. Figure 4 This refers to the effect of drug treatment on the growth of gastric mucosal cells. The cell growth effect in the control group and the experimental group was significantly higher than that in the blank group. The effect of the experimental group compared with the blank group was significant, and the healing effect of the experimental group was slightly higher than that of the positive control group. Figure 5 The results indicate the wound healing efficiency of the blank group, control group, and experimental group. After 48 hours, TGF-β1 showed a 285% cell growth efficiency relative to the blank group, black bellflower extract showed a 327% cell growth efficiency relative to the blank group, and black bellflower compound concentrate showed a 340% cell growth efficiency compared to the blank group.

[0059] Test Example 3 In vivo gastric mucosal injury experiment: To investigate the preventive effect of black bellflower compound concentrate on gastric ulcers, this experiment used male ICR mice weighing 20±2g, sourced from Changchun Yisi Experimental Animal Technology Co., Ltd. Every effort was made to minimize animal suffering during the experiment. Animals were divided into five groups: a control group (no modeling, 0.2ml / 20g of 0.5% carboxymethyl cellulose), a negative control group (0.2ml / 20g of 0.5% carboxymethyl cellulose), a positive control group (sucralfate, 100mg / kg in 0.5% CMC), and an experimental group (black bellflower extract and black bellflower compound concentrate, 250mg / kg in 0.5% CMC). Animals were fed a balanced diet and allowed free access to water. After a week of acclimatization, the animals were fasted for one day. Model negative control, model positive control, and time group were induced. Before inducing hemorrhage, 0.5% carboxymethyl cellulose, sucralfate, black bellflower extract, and black bellflower compound concentrate were administered by gavage twice over 24 hours, and orally again one hour before HCl / ethanol administration. Blank group and model negative and positive control groups were also used as references. Gastric hemorrhage lesions were induced by intragastric gavage with an HCl / ethanol mixture containing 150mM HCl and 60% ethanol, and blood was collected one hour later. After blood collection, the animals were euthanized by cervical dislocation, the stomach was quickly removed, the intact stomach was exposed, the pylorus was ligated, an appropriate amount of 10% formalin solution was instilled, and the stomach was fixed for 20 minutes. The stomach was then opened along its greater curvature, placed on the paraffin palate, and photographed. The ulcer area and ulcer inhibition rate were calculated using ImageJ software.

[0060] The ulcer inhibition rate (%) of each group was calculated using the following formula: ×100% In the formula: An is the ulcer area of ​​the experimental group; A0 is the ulcer area of ​​the negative control group of the model.

[0061] Experimental results: An in vivo gastric mucosal injury experiment was conducted using ICR mice. The results were... Figure 6 As shown in Table 3, the gastric mucosa of mice in the blank group was smooth and intact, without damage, while the gastric mucosa of mice in the negative control group showed large areas of linear congestion, hemorrhage, and erosion ulcers, with an area of ​​2057.98 mm. 2 Compared with the negative control group, the positive control group and the black bellflower compound concentrate group significantly reduced gastric mucosal damage, as evidenced by narrowing of the bleeding band and reduction of erosion area. The ulcer area of ​​the positive control group was 56.79 mm. 2 The inhibition rate of gastric mucosal damage was 72.65%, and the ulcer area of ​​both black bellflower extract and black bellflower compound extract was 204.94 mm. 2 and 241.47mm 2The inhibition rates of gastric mucosal damage were 90.04% and 88.27%, respectively, which were stronger than those of the positive control group of sucralfate.

[0062] Table 3. Gastric mucosal injury inhibition rate

[0063] Test Example 4 HE staining: (1) Tissue dehydration: The tissue was dehydrated by gradient alcohol treatment: 75% alcohol (4h) - 85% alcohol (2h) - 90% alcohol (1.5h) - 95% alcohol (1h) - anhydrous ethanol I (0.5h) - anhydrous ethanol II (0.5h).

[0064] (2) Tissue transparency: The tissue block must be transparent after being dehydrated by alcohol. The transparent agent (xylene) can be mixed with the dehydrating agent and paraffin at the same time. After it replaces the dehydrating agent, the paraffin can be smoothly penetrated into the tissue. Anhydrous ethanol: xylene (1:1) (10 min) - xylene I (10 min) - xylene II (10 min).

[0065] (3) Paraffin impregnation: The transparent tissue block was impregnated with paraffin in three cylinders (60℃) in sequence: Paraffin I (60℃) (1h) - Paraffin II (60℃) (1h) - Paraffin III (60℃) (1h).

[0066] All three experimental steps above were completed in a biological tissue dehydrator (automatic program). (4) Embedding: Embedding is the process of encasing a tissue block impregnated with wax in a paraffin block. The temperature of the wax used for embedding should be slightly higher than the impregnation temperature to ensure that the tissue block and the embedding paraffin are completely integrated.

[0067] (5) Slicing and baking: The embedded paraffin blocks are sliced ​​using a Leica pathological microtome. The sliced ​​tissues are placed in a 40°C water bath and spread out. The slide is tilted and inserted into the water to retrieve the slices, so that the slices adhere to the appropriate position on the slide. The slides are then baked in a 60°C oven for 3 hours.

[0068] (6) Dewaxing of sections: Place the paraffin sections in xylene I (15 min) - xylene II (15 min) - xylene III (15 min) - anhydrous ethanol I (5 min) - anhydrous ethanol II (5 min) - 95% ethanol (5 min) - 90% ethanol (5 min) - 80% ethanol (5 min) - 70% ethanol (5 min) in sequence, and then rinse with distilled water for 5 min.

[0069] (7) Counterstaining the nucleus: Stain the section with Mayer's hematoxylin (the staining background is clean and differentiation is not required) for 5 min, then stain with PBS for 5 min, and rinse with tap water for 30 sec.

[0070] (8) Staining with eosin: Stain the sections with 1% water-soluble eosin solution for 5 min, and then rinse with tap water for 30 sec.

[0071] (9) Dehydration and transparency: 95% alcohol for 30 seconds; 95% alcohol for 1 minute; anhydrous ethanol I and II for 5 minutes each; xylene I and II for 5 minutes each.

[0072] (10) Mounting: After air drying, mount with neutral resin and finally examine under a microscope.

[0073] After removing the mouse stomach, the tissue was stained with hematoxylin and eosin (HE) through a process involving fixation, washing, dehydration, clearing, acetic acid immersion, embedding, and sectioning. Experimental results... Figure 7 The results showed that in the control group, the surface of the glandular gastric mucosa was composed of a single layer of columnar epithelium with normal epithelial cell morphology and structure. The tubular structures formed by epithelial indentations were called gastric pits. The gastric glands were tubular glands composed of chief cells and parietal cells, which were abundant and regularly arranged. The muscular layer was uniformly stained, and the muscle fibers had normal morphology and structure, with no obvious inflammatory cell infiltration. In the control group, the gastric mucosa was uneven in thickness, with extensive erosion, extensive shedding of mucosal epithelium, extensive necrosis of gastric glands (black arrows), necrosis and shedding of glandular epithelium, destruction of the original structure, focal minor hemorrhage (green arrows), and a small number of scattered lymphocyte infiltrations (red arrows). Occasionally, slight dilation of gastric glands was observed (yellow arrows), and the cytoplasm showed increased basophilicity. In the positive control group, the gastric mucosa surface was a single layer of columnar epithelium with a small amount of mucosal epithelial shedding (brown arrows). The tubular structures formed by the epithelial indentation were called gastric pits. The gastric glands were tubular glands composed of chief cells and parietal cells, which were abundant and regularly arranged. The submucosa showed multifocal edema, loose connective tissue, and a small number of scattered lymphocytes infiltrating (red arrows). In the black bellflower extract experimental group, the gastric mucosa surface consisted of a single layer of columnar epithelium with normal epithelial cell morphology and structure. The tubular structures formed by epithelial indentations were called gastric pits. The gastric glands were tubular glands composed of chief cells and parietal cells, abundant in number and regularly arranged. A small number of gastric glands showed slight dilation (yellow arrows). No obvious inflammatory cell infiltration was observed. In the black bellflower compound concentrated solution experimental group, the glandular gastric mucosa surface consisted of a single layer of columnar epithelium with normal epithelial cell morphology and structure. The tubular structures formed by epithelial indentations were called gastric pits. The gastric glands were tubular glands composed of chief cells and parietal cells, abundant in number and regularly arranged. Occasionally, slight dilation of gastric glands was observed (yellow arrows). No obvious inflammatory cell infiltration was observed.

[0074] Test Example 5 Comparison of serum IL-1β, TNF-α, ADH, and GAS levels in rats of different groups: The collected rat plasma from each group was centrifuged at 2000 prm for 15 minutes. The supernatant was then diluted 5 times with sample diluent and the experiment was conducted according to the instructions of the kit (Lunchangshuo Biotechnology).

[0075] like Figure 8 As shown, compared with the control group, the levels of TNF-α and IL-6 in the blank group were significantly higher than those in the model group, and the levels in the negative control group of the model group were significantly higher than those in the positive control group and the experimental group. ADH activity was higher in the model group than in the blank group, and the ADH activity in the positive control group and the experimental group was higher than that in the negative control group. This is because the components in sucralfate, black bellflower extract, and black bellflower complex stimulate the release of ADH factors, enhancing the stomach's ability to metabolize alcohol and accelerating alcohol clearance, thus playing a protective role. GAS activity in the model group was higher than that in the blank group. This is because the modeling agents (alcohol and hydrochloric acid) stimulated the release of gastrin. The gastrin secretion in the bellflower extract and black bellflower complex concentrate was lower than that in the negative and positive controls, thus playing a protective role against gastric mucosal damage.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing black bellflower extract, characterized in that, Includes the following steps: Fresh bellflower roots are steamed and then dried. This process is repeated nine times to obtain black bellflower roots. Black bellflower root is mixed with water and extracted with water. The extract is then concentrated to obtain black bellflower root extract.

2. The preparation method according to claim 1, characterized in that, The steaming temperature is 110~120℃ and the time is 40~60min; the drying temperature is 55~60℃ and the time is 6~8h.

3. The preparation method according to claim 1, characterized in that, The ratio of black bellflower root to water is 1:15~20 (g:mL); the water extraction temperature is 80~90℃ and the time is 8~12h; the concentration is to concentrate the extract to 30~35% of its original mass.

4. The preparation method according to claim 1, characterized in that, The fresh bellflower roots must be three years old or older.

5. The black bellflower extract prepared by the preparation method according to any one of claims 1 to 4.

6. The use of the black bellflower extract according to claim 5 in the preparation of food with gastric protective function.

7. A concentrated extract of black bellflower root with gastric protective function, characterized in that, The composition comprises the following components in parts by weight: 38-42 parts of black bellflower extract as described in claim 5, 8-12 parts of apple and pear juice, 18-22 parts of complex extract, 5-7 parts of honey, 0.4-0.5 parts of sodium alginate, and 16.5-30.6 parts of water.

8. The black bellflower compound concentrate according to claim 7, characterized in that, The preparation method of the complex extract includes: mixing yam, jujube, kudzu root, ginger, wolfberry and water, performing water extraction, and then concentrating the extract to obtain the complex extract.

9. The black bellflower compound concentrate according to claim 8, characterized in that, The mass ratio of yam, jujube, kudzu root, ginger, and wolfberry is 3:2:2:1:2; the total mass of yam, jujube, kudzu root, ginger, and wolfberry to the amount of water is 1:10~15 (g:mL); the water extraction temperature is 100℃ and the time is 6~8h; the concentration is to concentrate the extract to 1 / 4 of its original volume.

10. The method for preparing the black bellflower compound concentrate according to any one of claims 7 to 9, characterized in that, Includes the following steps: The black bellflower extract, apple and pear juice, complex extract, honey, sodium alginate and water were mixed to obtain a black bellflower complex concentrate.

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