Composition containing sweet-scented osmanthus extract for improving liver lipid metabolism disorder and preparation method of composition

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a composition containing a sweet-scented osmanthus extract for improving liver lipid metabolism disorder and a preparation method of the composition. According to the invention, the extraction process is optimized, active components in the sweet osmanthus and the hawthorn are extracted in a combined manner, polyphenol substances in the hawthorn and the sweet osmanthus have relatively strong oxidation resistance, and in the combined extraction process, the polyphenol components can achieve a synergistic effect and remove free radicals generated in the extraction process, so that other active components are protected from oxidative damage; an embodiment verifies that the active ingredients can effectively inhibit excessive synthesis and accumulation of liver fat and promote fatty acid oxidation and energy metabolism.

Description

Technical Field

[0001] The invention belongs to the technical field, and particularly relates to a composition containing osmanthus fragrans extract for improving liver lipid metabolism disorder and a preparation method thereof. Background Art

[0002] Hepatic lipid metabolism disorder refers to abnormal fat metabolism in the liver, which leads to excessive accumulation of fat in the liver, and then triggers a series of pathological and physiological changes. This disorder is one of the core pathological mechanisms of metabolic liver diseases (such as non-alcoholic fatty liver disease, NAFLD) and metabolic dysfunction-associated fatty liver disease (MASLD). Its pathogenesis is complex, affected by multiple factors, and closely related to multiple diseases. Therefore, comprehensive intervention for hepatic lipid metabolism disorder is of great significance for improving liver health and preventing related diseases.

[0003] Hepatic lipid metabolism disorders involve multiple pathways, such as DNL (de novo lipogenesis), FAO (fatty acid oxidation), VLDL (very low-density lipoprotein) secretion, etc. However, existing products mostly focus on the regulation of a single pathway, making it difficult to comprehensively improve hepatic lipid metabolism disorders and effectively solve the fundamental problem of lipid accumulation in the liver. Moreover, hepatic lipid metabolism disorders not only involve lipid metabolism, but are also closely related to liver inflammation and fibrosis. Existing products mostly focus on the regulation of lipid metabolism, with less intervention in inflammation and fibrosis. Summary of the invention

[0004] The purpose of the present invention is to provide a composition containing osmanthus fragrans extract for improving liver lipid metabolism disorder and a preparation method thereof, so as to achieve multi-target comprehensive regulation and effectively improve liver lipid metabolism disorder.

[0005] The present invention provides a combined extract of osmanthus and hawthorn for improving liver lipid metabolism disorder. The preparation method of the combined extract comprises the following steps: After the osmanthus is naturally dried in the shade, it is crushed and passed through a 200-mesh sieve to obtain osmanthus powder; after the hawthorn is dried, it is crushed and passed through an 80-mesh sieve to obtain hawthorn powder; the osmanthus powder and the hawthorn powder are mixed in a mass ratio of 1~1.2:0.8~1 to obtain a mixed powder; the mixed powder is soaked in pure water at a ratio of 1g:10mL of solid-liquid ratio, and then filtered after standing for 24 hours to obtain the filtrate; the filtrate is extracted by ultrasonic water bath heating, filtered, and cooled to room temperature to obtain a combined extract; the combined extract is rotary evaporated until a yellow syrupy concentrate is obtained; the concentrate is passed through a macroporous adsorption resin column, and gradient eluted with water and 95% ethanol solution, the ethanol eluate is collected, and after drying, it is frozen into blocks, and then vacuum freeze-dried into a yellow solid to obtain an osmanthus and hawthorn combined extract.

[0006] Preferably, the mass ratio of the osmanthus powder to the hawthorn powder is 1-1.2:0.8.

[0007] Preferably, the frequency of the ultrasonic water bath heating extraction is 20-40 kHz, the time is 20-50 min, and the temperature of the water bath heating is 50° C.-70° C.

[0008] Preferably, the macroporous adsorption resin column is D101 macroporous adsorption resin or AB-8 macroporous adsorption resin.

[0009] Preferably, the rotary evaporation conditions are 70° C. to 80° C. and 35 to 50 r / min.

[0010] Preferably, the vacuum freeze-drying conditions are 20 Pa and -57°C.

[0011] The present invention also provides the use of the combined extract described in the above technical solution in preparing a product for improving liver lipid metabolism disorders.

[0012] Preferably, the product comprises a buccal tablet and / or an oral liquid.

[0013] The present invention also provides an oral liquid for improving liver lipid metabolism disorders, characterized in that the oral liquid comprises osmanthus flower water, the combined extract described in the above technical scheme, purified water, sucrose, citric acid and β-cyclodextrin; the volume ratio of the osmanthus flower water, the combined extract and purified water is 1:1:8, the added amount of sucrose is 10g / 100mL, the added amount of citric acid is 0.02g / 100mL, and the added amount of β-cyclodextrin is 0.2g / 100mL.

[0014] Preferably, the osmanthus flower water is prepared by freezing and thawing fresh osmanthus and fresh hawthorn.

[0015] Beneficial effects of the present invention: The combined extract of the present invention can significantly reduce key lipid metabolism indicators such as total cholesterol (T-CHO), triglycerides (TG), sterol regulatory element binding protein-1c (SREBP-1c), triglyceride synthase (GPAT) and fatty acid synthase (FASN) in liver cells, thereby effectively inhibiting excessive synthesis and accumulation of liver fat; the combined extract of the present invention can effectively alleviate the decline of key metabolic regulatory factors such as retinol X receptor (RXR), peroxisome proliferator-activated receptor α (PPAR-α) and AMP-activated protein kinase (AMPK) in liver cells, thereby promoting fatty acid oxidation and energy metabolism.

[0016] In addition, the polyphenol components in osmanthus and hawthorn (such as verbascoside, ellagic acid, etc.) have strong antioxidant capacity, which can scavenge free radicals, reduce oxidative stress, and protect liver cells from oxidative damage; the active ingredients in the combined extracts also have anti-inflammatory effects, which can inhibit the production of inflammatory factors, reduce liver inflammation, and thus improve liver lipid metabolism disorders.

[0017] The present invention optimizes the extraction process and jointly extracts the active ingredients in osmanthus and hawthorn. The polyphenols in hawthorn and osmanthus have strong antioxidant capacity. During the joint extraction process, these polyphenols can work synergistically to remove free radicals generated during the extraction process, thereby protecting other active ingredients from oxidative damage; these high-content active ingredients ensure the high efficiency and biological activity of the extract.

[0018] When hawthorn is used alone, it may cause stomach discomfort due to its high tannic acid content. Through this combined extraction process, the tannic acid content is effectively reduced, reducing irritation to the gastric mucosa and the risk of side effects.

[0019] Ultrasonic-assisted extraction can quickly destroy plant cell walls and release active ingredients into the extract quickly. This rapid release process reduces the residence time of active ingredients in cells, thereby reducing the risk of active ingredients being degraded in cells.

[0020] The extraction process of the invention does not use organic solvents, avoids chemical residues, and meets environmental protection and safety requirements. This green extraction process not only reduces environmental pollution, but also ensures the safety of the product.

[0021] The oral solution is composed of osmanthus flower water, combined extract, purified water, sucrose, citric acid and β-cyclodextrin. The volume ratio of osmanthus flower water: osmanthus extract: purified water is 1:1:8, sucrose is added in an amount of 10% to adjust the sweetness of the taste; citric acid is added in an amount of 0.02% to adjust the acidity and protect the color; β-cyclodextrin is added in an amount of 0.2% to stabilize homogenization, cover the bitterness and prevent precipitation. DETAILED DESCRIPTION

[0022] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1 Preparation of joint extracts After the osmanthus is naturally dried in the shade, it is crushed and passed through a 200-mesh sieve to obtain osmanthus powder; after the hawthorn is dried, it is crushed and passed through an 80-mesh sieve to obtain hawthorn powder; the osmanthus powder and the hawthorn powder are mixed in a mass ratio of 1:0.8 to obtain a mixed powder; the mixed powder is soaked in pure water at a ratio of 1g:10mL of solid-liquid ratio, and then filtered after standing for 24 hours to obtain the filtrate; the filtrate is extracted by ultrasonic water bath heating at 50°C and 20kHz for 30min, filtered, and cooled to room temperature to obtain a combined extract; the combined extract is rotary evaporated until a yellow syrupy concentrate is obtained; the concentrate is passed through a macroporous adsorption resin column, and gradient eluted with water and 95% ethanol solution, the ethanol eluate is collected, and after drying, it is frozen into blocks, and then vacuum freeze-dried into a yellow solid to obtain an osmanthus and hawthorn combined extract.

[0024] Example 2 Preparation of buccal tablets Take 10 g of the combined extract prepared in Example 1 and pass it through an 80-mesh sieve for later use; mix 60 g of lactose, 20 g of microcrystalline cellulose, 5 g of cross-linked polyvinylpyrrolidone and 2 g of aspartame, pass it through an 80-mesh sieve, and mix it evenly with the sieved combined extract.

[0025] 5 g of povidone K30 was dissolved in 30 mL of purified water to prepare an adhesive solution.

[0026] Slowly add the binder solution into the powder mixture and stir well to make a soft material.

[0027] The soft material was granulated through a 16-mesh sieve to prepare wet granules.

[0028] The wet granules were dried in an oven at 60°C to a moisture content of less than 5%.

[0029] The dried granules were sieved through a 16-mesh sieve to make the granules uniform in size.

[0030] The granules were mixed with 3 g of magnesium stearate to ensure that the lubricant was evenly distributed.

[0031] The mixed granules are compressed into buccal tablets on a tablet press.

[0032] Example 3 Preparation of oral solution 1. Prepare the combined extract solution in the manner described in Example 1; 2. Fresh osmanthus flowers and fresh hawthorn are mixed in a mass ratio of 1:0.8, and the mixture is prepared through the steps of removing impurities, pre-freezing, and sublimation drying. The material thickness during pre-freezing is 1-2 cm, and the material is pre-frozen at -25°C~30°C for 2-3 hours, and the cold trap temperature is -80°C; the heating program is set as: 1 hour, -10°C; 1 hour, 5°C; 1 hour, 20°C; 4~12 hours, 35°C; when the detected material temperature reaches 35°C and remains basically unchanged, the material can be taken out and filtered to obtain osmanthus flower water.

[0033] 3. Mix osmanthus flower water, combined extract, purified water, sucrose, citric acid and β-cyclodextrin to obtain osmanthus oral liquid. The volume ratio of osmanthus flower water: osmanthus extract: purified water is 1:1:8, sucrose is added in an amount of 10% to adjust the sweetness of the taste; citric acid is added in an amount of 0.02% to adjust the acidity and protect the color; β-cyclodextrin is added in an amount of 0.2% to stabilize homogenization, cover the bitterness and prevent precipitation.

[0034] Comparative Example 1 Preparation of Osmanthus fragrans extract After the osmanthus flowers are dried naturally in the shade, they are crushed with a grinder and passed through a 200-mesh sieve to obtain osmanthus powder. Weigh 50g of osmanthus powder and put it into a conical flask. Add 500mL of pure water at a material-liquid ratio of 1g:10mL, soak for 24 hours, and then filter to collect the filtrate. Then extract the filtrate by ultrasonic water bath heating for 30min, filter, centrifuge, and cool to room temperature to obtain osmanthus extract; evaporate at 70℃ and 35r / min to obtain a yellow syrupy osmanthus concentrate; freeze the osmanthus concentrate at -20℃ to form a block, and then vacuum freeze-dry at 20Pa and -57℃ to obtain a yellow solid to obtain an osmanthus extract.

[0035] Comparative Example 2 Preparation of hawthorn extract After the hawthorn is dried, it is crushed and passed through an 80-mesh sieve to obtain hawthorn powder. Weigh 50g of 5 parts of hawthorn powder and put them into a conical flask. Add 500mL of pure water at a material-liquid ratio of 1g:10mL, soak for 24 hours, and then filter to collect the filtrate. Then extract the filtrate by ultrasonic water bath heating for 30min, filter, centrifuge, and cool to room temperature to obtain hawthorn extract; evaporate at 70℃ and 35r / min to obtain yellow syrupy hawthorn concentrate; freeze the hawthorn concentrate at -20℃ into blocks, and then vacuum freeze-dry at 20Pa and -57℃ to obtain a yellow solid to obtain hawthorn extract.

[0036] Test Example 1 The extracts prepared in Example 1 and Comparative Examples 1 and 2 were respectively used as samples, and the effective components were quantitatively analyzed by high performance liquid chromatography (HPLC). Three parallel runs were set for each group of samples. The results are shown in Table 1.

[0037] Table 1 Content of active ingredients in extracts

[0038] As shown in Table 1, the combined extract of Example 1 is superior to the separately extracted osmanthus extract and hawthorn extract in terms of the content of active ingredients (verbascoside, salidroside and ellagic acid), indicating that the combined extraction method has certain advantages and can increase the content of active ingredients in the extract.

[0039] Test Example 2 Physiological activity of different doses of osmanthus-crataw combined extract in improving liver lipid metabolism disorders 1. Preparation of Liver Organ Chip (a) Cell culture: HepG2 cells were inoculated in complete culture medium (containing 89% high glucose medium + 10% fetal bovine serum + 1% penicillin-streptomycin) and cultured at 37°C and 5% CO2 saturated humidity.

[0040] (b) Preparation of liver organ chip: 99 μL cells (1×10 6 Cells / mL) were mixed with 20 µL of 15 mg / mL fibrinogen (F8630, Sigma) and 1 µL of 100 U / mL thrombin (T7513, Sigma) in pre-warmed DPBS (C14190500BT, Gibco). The mixture was then added to the middle channel of the microfluidic chip and incubated for 40 min to allow gelation to occur.

[0041] (c) Drug induction: The mice were first administered with DMEM high-glucose medium containing different concentrations of osmanthus extract (OSF: 2 μg / mL, 20 μg / mL, 200 μg / mL) and DMEM high-glucose medium containing vitamin C (VitC: 50 μg / mL) for 24 h for pre-protection, and then modeled with DMEM high-glucose medium containing dibutyl phthalate (DBP: 50 μg / mL).

[0042] 2. Determination of lipid metabolism related indicators The extracts prepared in Example 1 and Comparative Example 1 and Comparative Example 2 were used as samples, respectively, and the total cholesterol (T-CHO) test kit (A111-1-1), triglyceride (TG) test kit (A110-1-1) of Nanjing Jiancheng Bioengineering Institute, and the ELISA detection kit of Shanghai ELISA Biotechnology Co., Ltd. were used, including: human retinol X receptor (RXR) (YJ120328), human sterol regulatory element binding protein ((SREBP)-1c) (YJ403612), peroxisome proliferator-activated receptor ((PPAR)-α) (YJ410876), 3-phosphoglycerate acyltransferase (GPAT) (3-YJ495064), AMP-activated protein kinase (AMPK) (YJ302851), fatty acid synthase (FASN) (YJ360846) were measured strictly according to the instructions of the kit. The results are shown in Tables 2 and 3, wherein ORJ represents the combined extract, OSF represents the osmanthus extract, and HE represents the hawthorn extract.

[0043] Table 2 Effects of combined extracts on lipid metabolism related indices

[0044] Table 3 Effects of osmanthus extract and hawthorn extract on lipid metabolism related indicators

[0045] As shown in Table 3, compared with the single osmanthus extract and hawthorn extract, the combined extract of the present invention can reduce the T-CHO, TG, SREBP-1c, GPAT, and FASN indicators in liver cells with lipid metabolism disorders caused by DBP to the greatest extent, and alleviate the decline of RXR, PPAR-α, and AMPK indicators; as shown in Table 2, the 200 μg / mL osmanthus-hawthorn combined extract has the best effect. This shows that the 200 μg / mL osmanthus-hawthorn combined extract has the strongest ability to repair lipid metabolism disorders in damaged liver cells.

[0046] 3. Effective dose conversion First, convert μg to mg: since 1 mg = 1000 μg, 200 μg = 200 ÷ 1000 = 0.2 mg. At this point, the concentration is 0.2 mg / mL. Suppose we consider a specific volume of human body fluid (such as blood), assuming that the total body fluid volume of the human body is about 40L (for an average-weight adult), 40L = 40000mL. If we generalize the 0.2 mg substance in 1mL to 40000mL (the hypothetical body fluid volume of the human body), then the total mass of the substance is: 0.2 mg / mL × 40000mL = 8000mg, or 8g. The best effect is achieved when an adult consumes 8g of the combined extract per day.

[0047] It can be seen from the above examples that the combined extract provided by the present invention has a good effect on improving liver lipid metabolism disorders, and can significantly reduce key lipid metabolism indicators such as total cholesterol (T-CHO), triglycerides (TG), sterol regulatory element binding protein-1c (SREBP-1c), triglyceride synthase (GPAT) and fatty acid synthase (FASN) in liver cells, thereby effectively inhibiting excessive synthesis and accumulation of liver fat; at the same time, it can also effectively alleviate the decline of key metabolic regulatory factors such as retinol X receptor (RXR), peroxisome proliferator-activated receptor α (PPAR-α) and AMP-activated protein kinase (AMPK) in liver cells, thereby promoting fatty acid oxidation and energy metabolism, realizing multi-target comprehensive regulation, and effectively improving liver lipid metabolism disorders.

[0048] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A combined extract of osmanthus fragrans and hawthorn for improving hepatic lipid metabolism disorders, characterized in that: The preparation method of the combined extract comprises the following steps: After the osmanthus flowers are naturally dried in the shade, they are crushed and passed through a 200-mesh sieve to obtain osmanthus flower powder; After drying the hawthorn, crush it and pass it through an 80-mesh sieve to obtain hawthorn powder; Mixing osmanthus powder and hawthorn powder in a mass ratio of 1-1.2:0.8-1 to obtain a mixed powder; Soak the mixed powder in pure water at a ratio of 1 g to 10 mL, let it stand for 24 hours, and then filter and collect the filtrate; The filtrate is subjected to ultrasonic water bath heating extraction, filtered, and cooled to room temperature to obtain a combined extract; The combined extract is subjected to rotary evaporation until a yellow syrupy concentrate is obtained; The concentrate was passed through a macroporous adsorption resin column and gradiently eluted with water and 95% ethanol solution. The ethanol eluate was collected, dried and frozen into blocks, and then vacuum freeze-dried into a yellow solid to obtain an osmanthus and hawthorn joint extract.

2. The combined extract according to claim 1, characterized in that The mass ratio of the osmanthus powder to the hawthorn powder is 1-1.2:0.

8.

3. The combined extract according to claim 1, characterized in that The frequency of the ultrasonic water bath heating extraction is 20-40 kHz, and the time is 20-50 min; the temperature of the water bath heating is 50° C.-70° C.

4. The combined extract according to claim 1, characterized in that The macroporous adsorption resin column is D101 macroporous adsorption resin or AB-8 macroporous adsorption resin.

5. The combined extract according to claim 1, characterized in that The rotary evaporation conditions are 70° C.-80° C. and 35-50 r / min.

6. The combined extract according to claim 1, characterized in that The vacuum freeze-drying conditions are 20 Pa and -57°C.

7. Use of the combined extract according to claim 1 in the preparation of a product for improving liver lipid metabolism disorders.

8. The use according to claim 7, characterized in that: The products include buccal tablets and / or oral liquids.

9. An oral liquid for improving liver lipid metabolism disorder, characterized in that: The oral liquid comprises osmanthus flower water, the combined extract according to claim 1, purified water, sucrose, citric acid and β-cyclodextrin; the volume ratio of the osmanthus flower water, the combined extract and purified water is 1:1:8, the added amount of sucrose is 10g / 100mL, the added amount of citric acid is 0.02g / 100mL, and the added amount of β-cyclodextrin is 0.2g / 100mL.

10. The method according to claim 1, characterized in that The osmanthus flower water is prepared by freezing and thawing fresh osmanthus and fresh hawthorn.

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