Preparation method of honeysuckle extract and use for preventing and treating obesity

Abstract

The application discloses a preparation method of honeysuckle extract, which comprises the following steps: (1) weighing a proper amount of honeysuckle medicinal materials, soaking in water, decocting and extracting, filtering, and concentrating to obtain a first honeysuckle extract; and optionally (2) adding ethanol to the first honeysuckle extract to remove polysaccharides to obtain a second honeysuckle extract. The honeysuckle water extract (LW) of the application can significantly reduce the body weight and peripheral fat tissue weight of HFD-induced obese mice and improve the energy metabolism rate of the mice, and it is further found that the honeysuckle extract prepared by a specific process can also reduce the lipid droplet content in 3T3-L1 cells.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for preparing honeysuckle extract and its use in preventing and treating obesity. Background Technology

[0002] Obesity is a common metabolic disorder in clinical practice, and its incidence is increasing year by year worldwide. The "World Obesity Atlas" predicts that by 2035, the number of overweight people worldwide will account for 51% of the total population, including an estimated 252 million adolescents worldwide by 2023. The appearance of obesity symptoms is often accompanied by other diseases such as type 2 diabetes, hyperlipidemia, and cardiovascular diseases. For mild to moderate obesity (BMI less than 30), interventions such as diet control and increased exercise are often used; for moderate obesity (BMI between 30 and 35), drug treatment is often used; and for severe and morbid obesity (BMI greater than 35), surgical treatment is used. Commonly recommended drugs in clinical practice include naltrexone, phentermine, orlistat, and liraglutide, all of which are FDA-approved. Among these, only orlistat is approved by the my country Food and Drug Administration for the treatment of obesity. However, the above-mentioned drugs are not the best treatment for obesity, as they each have varying degrees of toxic side effects. For example, naltrexone can cause nausea, vomiting, and gastrointestinal discomfort; phentermine can cause dry mouth, insomnia, constipation, and urticaria; and orlistat can cause oily spots, increased flatulence, headache, anxiety, and fatigue. Therefore, further research and development to find safe and effective drugs for treating obesity is of great significance.

[0003] Mountain honeysuckle is the dried flower bud or newly opened flower of Lonicera japonica, Lonicera rubrum, Lonicera sinensis, or Lonicera fulva, all belonging to the Caprifoliaceae family. It has the effects of clearing heat and detoxifying, and dispersing wind-heat. It is often used to treat carbuncles, boils, and febrile diseases.

[0004] The prior art has not found a method for preparing honeysuckle extract or its use in preventing and treating obesity as claimed in this invention. Summary of the Invention

[0005] Based on this, the present invention provides a method for preparing honeysuckle extract, the method comprising the following steps:

[0006] (1) Weigh an appropriate amount of honeysuckle medicinal material, soak it in water, decoct and extract, filter, concentrate, and obtain the first honeysuckle extract; and

[0007] Optionally (2) ethanol is added to the first honeysuckle extract to remove polysaccharides, resulting in a second honeysuckle extract.

[0008] Furthermore, the method for removing polysaccharides includes the following steps:

[0009] (a) Add ethanol to the first honeysuckle extract to obtain the first alcohol-containing solution, let stand, and obtain the first supernatant;

[0010] (b) The first supernatant is recovered from ethanol, concentrated, and a concentrated solution is obtained;

[0011] (c) Add ethanol to the concentrate to obtain a second alcohol-containing solution, allow it to stand, and obtain a second supernatant; and

[0012] (d) The second supernatant was recovered with ethanol and concentrated to obtain the second honeysuckle extract.

[0013] Furthermore, the weight of the honeysuckle medicinal material is 1 to 20g, for example, about 5g or about 10g.

[0014] Furthermore, the volume / mass (ml / g) ratio of the water to the honeysuckle medicinal material is 1 to 10, for example, about 6.

[0015] Furthermore, the soaking time is 1 to 3 hours, for example, about 2 hours.

[0016] Furthermore, the decoction is prepared by simmering over a low flame.

[0017] Furthermore, the decoction is prepared 1 to 3 times, for example, 2 times.

[0018] Furthermore, the simmering time is 60–120 minutes per batch, for example, about 90 minutes per batch.

[0019] Furthermore, this concentration is a vacuum concentration.

[0020] Furthermore, the concentration was reduced under reduced pressure until no alcoholic odor remained.

[0021] Furthermore, the relative density of this first honeysuckle extract at 80°C is 1.00–1.30, for example, about 1.16.

[0022] Furthermore, in step (a), the ethanol is anhydrous ethanol.

[0023] Further, in step (a), the concentration of ethanol in the first alcohol-containing liquid is 10% to 30%, for example, about 20%.

[0024] Furthermore, in step (b), the recovery is a depressurized recovery.

[0025] Further, in step (b), the concentration is reduced pressure concentration.

[0026] Further, in step (b), the reduced pressure concentration is carried out until there is no alcohol odor.

[0027] Furthermore, in step (b), the concentrate is a concentrated extract.

[0028] Furthermore, in step (c), the ethanol is anhydrous ethanol.

[0029] Further, in step (c), the concentration of ethanol in the second alcohol-containing liquid is 50% to 70%, for example, about 60%.

[0030] Further, in step (d), the concentration is reduced pressure concentration.

[0031] Further, in step (d), the reduced pressure concentration is carried out until there is no alcohol odor.

[0032] According to another aspect of the present invention, a first honeysuckle extract and / or a second honeysuckle extract prepared by the above preparation method are provided.

[0033] According to another aspect of the present invention, the use of the above-mentioned first honeysuckle extract and / or second honeysuckle extract in the preparation of medicaments, foods and / or health products for the prevention and / or treatment of obesity is provided.

[0034] Furthermore, this obesity is caused by dietary habits, genetics, metabolism, and / or fat cell abnormalities.

[0035] Furthermore, the abnormality is an increase in the number of adipocytes and / or adipocyte hypertrophy.

[0036] Furthermore, the effects of the first and / or second honeysuckle extracts in preventing and / or treating obesity are achieved through one or more of the following mechanisms: reducing lipid accumulation in cells, reducing adipocyte diameter, reducing body weight, reducing the relative weight of peripheral adipose tissue, and increasing energy metabolism levels.

[0037] Furthermore, the adipocytes are selected from one or more of the following: inguinal white adipose tissue cells, epididymal white adipose tissue cells, and brown adipose tissue cells.

[0038] Furthermore, the peripheral adipose tissue includes inguinal white adipose tissue and / or epididymal white adipose tissue.

[0039] Furthermore, this increase in energy metabolism occurs through one or more of the following mechanisms: increased oxygen consumption, increased carbon dioxide excretion, and increased heat production by the body.

[0040] The beneficial effects of this invention are:

[0041] The honeysuckle water extract (LW) of the present invention can significantly reduce the body weight and peripheral adipose tissue weight of HFD-induced obese mice and increase their energy metabolism rate. Furthermore, it was found that honeysuckle extract processed by a specific process can also reduce the lipid droplet content in 3T3-L1 cells. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0043] Figure 1 This diagram illustrates the effects of honeysuckle aqueous extract (LW) on energy metabolism in HFD-induced DIO mice. In the diagram, a represents the oxygen consumption of the mice; b represents the carbon dioxide exhalation of the mice; and c represents the heat generated by the mice (n=3).

[0044] Figure 2 This is a schematic diagram showing the results of H&E staining of mouse adipose tissue.

[0045] Figure 3 This diagram illustrates the effects of different processing methods on lipid accumulation in 3T3-L1 adipocytes by extracts of honeysuckle. In the diagram, a represents Oil Red O staining results; b represents quantitative results. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0048] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0049] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0050] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0051] As described in the background section, currently available drugs are not the best treatments for obesity, as they each have varying degrees of toxic side effects. To address these issues, this invention provides a method for preparing honeysuckle extract, which includes the following steps:

[0052] (1) Weigh an appropriate amount of honeysuckle medicinal material, soak it in water, decoct and extract, filter, concentrate, and obtain the first honeysuckle extract; and

[0053] Optionally (2) ethanol is added to the first honeysuckle extract to remove polysaccharides, resulting in a second honeysuckle extract.

[0054] In a preferred embodiment, the method for removing polysaccharides includes the following steps:

[0055] (a) Add ethanol to the first honeysuckle extract to obtain the first alcohol-containing solution, let it stand, and obtain the first supernatant;

[0056] (b) The first supernatant is recovered from ethanol, concentrated, and a concentrated solution is obtained;

[0057] (c) Add ethanol to the concentrate to obtain a second alcohol-containing solution, allow it to stand, and obtain a second supernatant; and

[0058] (d) The second supernatant was recovered with ethanol and concentrated to obtain the second honeysuckle extract.

[0059] In this invention, when mass, ratio, time, number of times, density, concentration, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1–20” is disclosed, the described range should be interpreted as including ranges “1–20”, “1–15”, “1–10”, “1–5”, “5–20”, “5–15”, “5–10”, “10–20”, “10–15”, “15–20”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0060] In a preferred embodiment, the honeysuckle medicinal material weighs 1 to 20g, for example, about 5g or about 10g.

[0061] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25; "about 10" includes 10 ± 5%, or from 9.5 to 10.5.

[0062] In a preferred embodiment, the volume / mass (ml / g) ratio of the water to the honeysuckle medicinal material is 1 to 10, for example, about 6.

[0063] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0064] In a preferred embodiment, the soaking time is 1 to 3 hours, for example, about 2 hours.

[0065] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes 2 ± 5%, or from 1.9 to 2.1.

[0066] In a preferred embodiment, the simmering is performed over a low flame.

[0067] In a preferred embodiment, the decoction is performed 1 to 3 times, for example, 2 times.

[0068] In a preferred embodiment, the simmering time is 60 to 120 minutes per cycle, for example, about 90 minutes per cycle.

[0069] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 90" includes ±5% of 90, or from 85.5 to 94.5.

[0070] In a preferred embodiment, the concentration is a vacuum concentration.

[0071] In a preferred embodiment, the reduced pressure concentration is carried out until there is no alcohol odor.

[0072] In a preferred embodiment, the first honeysuckle extract has a relative density of 1.00 to 1.30, for example, about 1.16, at 80°C.

[0073] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.16" includes ±5% of 1.16, or from 1.102 to 1.218.

[0074] In a preferred embodiment, in step (a), the ethanol is anhydrous ethanol.

[0075] In a preferred embodiment, in step (a), the concentration of ethanol in the first alcohol-containing liquid is 10% to 30%, for example, about 20%.

[0076] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20%" includes 20% ± 5%, or from 19% to 21%.

[0077] In a preferred embodiment, in step (b), the recovery is a depressurized recovery.

[0078] In a preferred embodiment, in step (b), the concentration is a vacuum concentration.

[0079] In a preferred embodiment, in step (b), the reduced pressure concentration is carried out until there is no alcohol odor.

[0080] In a preferred embodiment, in step (b), the concentrate is a concentrated extract.

[0081] In a preferred embodiment, in step (c), the ethanol is anhydrous ethanol.

[0082] In a preferred embodiment, in step (c), the concentration of ethanol in the second alcohol-containing liquid is 50% to 70%, for example, about 60%.

[0083] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60%" includes 60% ± 5%, or from 57% to 63%.

[0084] In a preferred embodiment, in step (d), the concentration is a vacuum concentration.

[0085] In a preferred embodiment, in step (d), the reduced pressure concentration is carried out until there is no alcohol odor.

[0086] According to another aspect of the present invention, a first honeysuckle extract and / or a second honeysuckle extract prepared by the above preparation method are provided.

[0087] According to another aspect of the present invention, the use of the above-mentioned first honeysuckle extract and / or second honeysuckle extract in the preparation of medicaments, foods and / or health products for the prevention and / or treatment of obesity is provided.

[0088] In a preferred embodiment, the obesity is caused by dietary habits, genetics, metabolism, and / or fat cell abnormalities.

[0089] In a preferred embodiment, the abnormality is an increase in the number of adipocytes and / or adipocyte hypertrophy.

[0090] In a preferred embodiment, the first and / or second honeysuckle extracts prevent and / or treat obesity through one or more of the following mechanisms: reducing lipid accumulation in cells, reducing adipocyte diameter, reducing body weight, reducing the relative weight of peripheral adipose tissue, and increasing energy metabolism levels.

[0091] In a preferred embodiment, the adipocytes are selected from one or more of the following: inguinal white adipose tissue cells, epididymal white adipose tissue cells, and brown adipose tissue cells.

[0092] In a preferred embodiment, the peripheral adipose tissue includes inguinal white adipose tissue and / or epididymal white adipose tissue.

[0093] In a preferred embodiment, the increased energy metabolism level is achieved through one or more of the following methods: increased oxygen consumption, increased carbon dioxide excretion, and increased heat production by the body.

[0094] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0096] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0097] Example 1: Evaluation of the therapeutic effect of honeysuckle water extract on obesity using a high-fat diet (HFD) induced obesity animal model.

[0098] 1.1 Experimental Materials

[0099] 1.1.1 Laboratory Animals

[0100] Male C57BL / 6J mice, 4 weeks old, weighing 20±2 g, were purchased from Shanghai Slake Experimental Animal Co., Ltd. [Certificate number: SCXK (Shanghai) 2017-0005] and raised in the SPF-grade experimental animal center of Shanghai University of Traditional Chinese Medicine. The room temperature was 25°C, the humidity was 60%-70%, and the day-night cycle was 12 hours. The mice had free access to food and water.

[0101] 1.1.2. Test drug and its preparation

[0102] Preparation of aqueous extract of Lonicerae Japonicae Thunb. Flos: Referring to the preparation method of Nanjing Jinling Mailuoning Oral Liquid, the specific implementation is as follows: Take the Lonicerae Japonicae Thunb. Flos medicinal materials, add 6 times the amount of water and soak for 2 hours. Decoct twice, each time for 1.5 hours, filter, combine the decoction liquids, concentrate the filtrate to a relative density of 1.16 (80°C), add ethanol to an ethanol content of 66%, let it stand, take the supernatant, recover the ethanol and concentrate to a relative density of 1.36 (80°C). Add ethanol to an ethanol content of 80%, let it stand, take the supernatant, recover all the ethanol, dissolve in water, and refrigerate and filter.

[0103] The administration doses were: low dose was 5.97 g / kg; high dose was 10.61 g / kg.

[0104] 1.2. Experimental methods

[0105] 1.2.1. Animal experiment protocol

[0106] After one week of adaptive feeding of the mice, they were randomly divided into a normal control group and a model group. The normal control group (NCD, n = 6) was fed with normal feed, and the mice in the model group were fed with high-fat feed. The high-fat feed was continuously fed for 12 weeks. After 8 weeks of feed feeding, the mice were grouped according to their body weights as follows: high-fat model group (HFD, n = 7), low-dose Lonicerae Japonicae Thunb. Flos administration group (HFD+LW(5.97 g / kg), n = 7), high-dose Lonicerae Japonicae Thunb. Flos administration group (HFD+LW(10.61 g / kg), n = 7). Drug intervention was given in the last four weeks. The normal control group and the high-fat model group were given the same volume of normal saline.

[0107] 1.2.2. Measurement of mouse energy metabolism

[0108] After 4 weeks of drug administration, the mice were placed in an energy metabolism cage (single-cage housing). Each mouse was given sufficient feed and water. After the mice adapted to the metabolism cage for 24 hours, the detection instrument of the metabolism cage was turned on. After the machine was stable, the metabolic indexes of each mouse, such as oxygen consumption, carbon dioxide exhalation, and heat production, within 24 hours were detected.

[0109] 1.2.3. H&E staining of adipose tissue

[0110] White adipose tissue, including inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), and brown adipose tissue (BAT), was collected separately, fixed with 4% paraformaldehyde solution, embedded in paraffin, sectioned, and stained with H&E.

[0111] 1.2.4 Data Statistics

[0112] Experimental data are expressed as mean ± standard error (Mean ± SEM). Statistical analysis was performed using SPSS 16.0 software. One-way ANOVA was used for variance analysis between groups. Compared with the control group, * P<0.05, ** P<0.01, *** P<0.001; compared with the model group, # P<0.05, ## P<0.01, ### P<0.001.

[0113] 1.3 Experimental Results

[0114] 1.3.1. Honeysuckle water extract (LW) reduced body weight and peripheral adipose tissue weight in obese mice.

[0115] Table 1 shows that, compared with the NCD group, the final body weight of mice in the HFD group was significantly increased, and the relative weight of eWAT and iWAT was significantly increased. However, after administering high and low doses (5.97 and 10.61 g / kg) of honeysuckle water extract, the body weight and the relative weight of eWAT and iWAT of obese mice were significantly reduced. Figure 2 H&E staining results of adipose tissue showed that administration of honeysuckle water extract significantly reduced the diameter of adipocytes in white adipose tissue (iWAT, eWAT) and brown adipose tissue of obese mice.

[0116] Table 1. Honeysuckle water extract reduced the final body weight and peripheral adipose tissue weight in obese mice.

[0117]

[0118] Note: Compared with the control group, ***p<0.001; compared with the model group, # p<0.05; data = mean ± SEM, n=6~7.

[0119] 1.3.2. Honeysuckle water extract (LW) improves energy metabolism in obese mice.

[0120] The energy metabolism levels of mice in each group were measured using an energy metabolism analyzer. Results are shown in Table 2. Figure 1 As shown, compared with the NCD group, the oxygen consumption of mice in the HFD group was significantly lower. Figure 1 -a) Exhaled carbon dioxide (CO2) Figure 1 -b) and the heat generated during the day and night (Table 2, Figure 1 Both -c) were significantly reduced, and the energy metabolism level of obese mice was significantly reduced. After intervention with high and low doses (5.97, 10.61 g / kg) of honeysuckle water extract, the oxygen consumption of obese mice ( Figure 1 -a) Exhaled carbon dioxide (CO2) Figure 1 -b) and the heat generated (Table 2, Figure 1 Both -c) were significantly increased, suggesting that honeysuckle water extract can significantly improve the energy metabolism level of obese mice.

[0121] Table 2. Honeysuckle water extract improves energy metabolism in obese mice.

[0122]

[0123] Note: Values ​​= Average ± SEM, n = 3.

[0124] Example 2: Evaluation of the lipid-reducing effect of extracts from honeysuckle processed using different methods in a 3T3-L1 cell-induced lipid-accumulating adipocyte model.

[0125] 2.1 Experimental Materials

[0126] Mouse embryonic fibroblasts (3T3-L1) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.; 3-isobutyl-1-methylxanthine (IBMX) and dexamethasone were purchased from Sigma; insulin and Oil Red O staining kit were purchased from Beyotime Biotechnology Co., Ltd.; honeysuckle was provided by Jinling Pharmaceutical Co., Ltd.

[0127] BP211D electronic analytical balance (Mettler Toledo, Switzerland); rotary evaporator, SF-TGL-16M centrifuge (Shanghai Fichar Analytical Instruments Co., Ltd.); KQ-250DB ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).

[0128] 2.2 Experimental Methods

[0129] 2.2.1 Preparation of extracts from honeysuckle using different processing methods

[0130] 75% ethanol extract of honeysuckle (Sample A): Take 5g of honeysuckle, add 50 times the amount of 75% ethanol, heat under reflux for 2 hours, filter, concentrate the filtrate under reduced pressure until there is no alcohol taste, add water to prepare a certain concentration for later use, concentrate the filtrate to 30mL, filter through a 0.22μm filter membrane, and store at -20℃.

[0131] 95% ethanol extract of honeysuckle (Sample B): Weigh 5 grams of honeysuckle, add 50 times the amount of 95% ethanol, heat under reflux for 2 hours, filter, concentrate the filtrate under reduced pressure until there is no alcohol taste, add water to prepare a certain concentration for later use.

[0132] Aqueous extract of honeysuckle (Sample C): Weigh 5 grams of honeysuckle, add 6 times the amount of water and soak for 2 hours, simmer twice for 1.5 hours each time, filter, concentrate the filtrate under reduced pressure, and prepare a certain concentration for later use.

[0133] Honeysuckle water extract (66% ethanol removed polysaccharide) (Sample D): Weigh 10g of honeysuckle, add 6 times the amount of water and soak for 2 hours, simmer twice for 1.5 hours each time, filter, concentrate the filtrate under reduced pressure until there is no alcohol odor, add an appropriate amount of anhydrous ethanol to 66% alcohol content, let stand, filter, precipitate and freeze dry to obtain 66% ethanol precipitated polysaccharide, add water to prepare a certain concentration, prepare 66% ethanol precipitated polysaccharide; take the supernatant, recover the ethanol and concentrate under reduced pressure until there is no alcohol odor, add an appropriate amount of anhydrous ethanol to 80% alcohol content, let stand, filter, take the supernatant, concentrate under reduced pressure until there is no alcohol odor, add water to prepare a certain concentration for later use;

[0134] Honeysuckle water extract (without 50% ethanol polysaccharide) (Sample E): Weigh 10g of honeysuckle medicinal material, add 6 times the amount of water and soak for 2 hours, decoct twice, 1.5 hours each time, filter, concentrate the filtrate until there is no alcohol taste, add an appropriate amount of anhydrous ethanol to 50% alcohol content, let stand, take the supernatant, recover the ethanol and concentrate under reduced pressure to a concentrated extract, add an appropriate amount of anhydrous ethanol to 70% alcohol content, let stand, take the supernatant, concentrate until there is no alcohol taste, add water to prepare a certain concentration for later use;

[0135] Honeysuckle water extract (40% ethanol removed polysaccharide) (Sample F): Weigh 10g of honeysuckle, add 6 times the amount of water and soak for 2 hours, decoct twice, 1.5 hours each time, filter, concentrate the filtrate under reduced pressure until there is no alcohol taste, add an appropriate amount of anhydrous ethanol to the alcohol content of 40%, let stand, take the supernatant, recover the ethanol under reduced pressure and concentrate to a concentrated extract, add an appropriate amount of anhydrous ethanol to the alcohol content of 75%, let stand, take the supernatant, concentrate under reduced pressure until there is no alcohol taste, add water to prepare a certain concentration for later use.

[0136] Honeysuckle water extract (without 20% ethanol polysaccharide): (Sample G) Weigh 10g of honeysuckle, add 6 times the amount of water and soak for 2 hours, decoct twice, 1.5 hours each time, filter, concentrate the filtrate under reduced pressure until there is no alcohol taste, add an appropriate amount of anhydrous ethanol to the alcohol content to 20%, let stand, take the supernatant, recover the ethanol under reduced pressure and concentrate to a concentrated extract, add an appropriate amount of anhydrous ethanol to the alcohol content to 60%, let stand, take the supernatant, concentrate under reduced pressure until there is no alcohol taste, add water to prepare a certain concentration for later use.

[0137] Honeysuckle extracts were prepared using different processes according to the above conditions, and the corresponding yields were calculated.

[0138] 2.2.2 3T3-L1 cell culture

[0139] 3T3-L1 cells were cultured at 37°C in a cell culture incubator containing 5% CO2. The culture medium was DMEM containing 10% fetal bovine serum and antibiotics. Cells were passaged when they reached 80% confluence.

[0140] 2.2.3, 3T3-L1 adipogenesis induction

[0141] Two days after contact inhibition, 3T3-L1 precursor adipocytes were induced to differentiate. Differentiation induction medium ① (DMEM + 10% FBS + 1% penicillin + 0.5 mM IBMX + 1 μm dexamethasone + 10 μm insulin) was added for 4 days. Then, the induction medium ② (DMEM + 10% FBS + 1% penicillin + 10 μm insulin) was added and the cells were cultured for another 4 days. After that, the medium was changed to ordinary medium (DMEM + 10% FBS + 1% penicillin) and the cells were incubated with the drug. Two days later, the cells were harvested and stained with Oil Red O.

[0142] 2.2.4 Oil Red O staining of 3T3-L1 cells

[0143] The culture medium was aspirated, and 4% paraformaldehyde fixative was added for fixation for 10 minutes. The cells were washed twice with PBS, and then an appropriate amount of staining and washing buffer was added to cover the cells for 20 seconds. The staining and washing buffer was aspirated, and an appropriate amount of Oil Red O staining working solution was added for staining for 30 minutes. The Oil Red O staining working solution was removed, and an appropriate amount of staining and washing buffer was added. The cells were allowed to stand for 30 seconds, and then the staining and washing buffer was removed. The cells were observed and photographed under a microscope, and the lipid droplets were quantitatively analyzed using ImageJ software.

[0144] 2.3 Experimental Results

[0145] 2.3.1 Extraction yield of different samples of honeysuckle

[0146] Extracts of honeysuckle were prepared using different processes according to step “2.2.1”, and the corresponding yields were calculated. The results are shown in Table 3. The extraction yield of sample A was 50.20%, sample B was 25.05%, sample C was 39.37%, sample D was 12.53%, sample E was 29.85%, sample F was 34.73%, and sample H was 29.82%.

[0147] Table 3. Yield of Honeysuckle Flos by Different Extraction Processes

[0148]

[0149] 2.3.2 Screening of lipid-lowering activity of honeysuckle extract samples obtained by different processes

[0150] The results are as follows Figure 3 As shown, compared with the blank group, the intracellular lipid droplet content in the model group was significantly increased. After administering samples A, B, C, D, E, F, and G at a concentration of 1 g / mL, it was found that, compared with the model group, the lipid content in 3T3-L1 cells was significantly reduced after administering samples C, D, and F, respectively. This indicates that the samples prepared by the different methods significantly reduced lipid accumulation in 3T3-L1 cells, showing lipid-lowering activity. However, after administering samples A, B, E, and G, there was no change in lipid accumulation in 3T3-L1 cells.

[0151] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing honeysuckle extract, characterized in that, The preparation method includes the following steps: (1) Weigh an appropriate amount of honeysuckle medicinal material, soak it in water, decoct and extract, filter, concentrate, and obtain the first honeysuckle extract; and Optionally (2) ethanol is added to the first honeysuckle extract to remove polysaccharides, thereby obtaining a second honeysuckle extract.

2. The preparation method according to claim 1, characterized in that, The method for removing polysaccharides includes the following steps: (a) Add ethanol to the first honeysuckle extract to obtain the first alcohol-containing solution, let stand, and obtain the first supernatant; (b) The first supernatant is recovered from ethanol and concentrated to obtain a concentrated solution; (c) Add ethanol to the concentrated solution to obtain a second alcohol-containing solution, allow it to stand, and obtain a second supernatant; and (d) The second supernatant was recovered with ethanol and concentrated to obtain the second honeysuckle extract.

3. The preparation method according to claim 1, characterized in that, The weight of the honeysuckle medicinal material is 1-20g, for example, about 5g or about 10g; Preferably, the volume / mass (ml / g) ratio of the water to the honeysuckle medicinal material is 1 to 10, for example, about 6; Preferably, the soaking time is 1 to 3 hours, for example, about 2 hours; More preferably, the simmering is done over a low flame; More preferably, the decoction is performed 1 to 3 times, for example, 2 times; More preferably, the decocting time is 60-120 minutes per cycle, for example, about 90 minutes per cycle; Preferably, the concentration is vacuum concentration; Preferably, the concentration under reduced pressure is carried out until no alcohol odor is detected; More preferably, the first honeysuckle extract has a relative density of 1.00 to 1.30, for example, about 1.16, at 80°C.

4. The preparation method according to claim 2, characterized in that, In step (a), the ethanol is anhydrous ethanol; Preferably, in step (a), the concentration of ethanol in the first alcohol-containing solution is 10% to 30%, for example, about 20%; More preferably, in step (b), the recovery is reduced pressure recovery; More preferably, in step (b), the concentration is reduced pressure concentration; More preferably, in step (b), the reduced pressure concentration is carried out until there is no alcohol odor; More preferably, in step (b), the concentrate is a concentrated extract; Preferably, in step (c), the ethanol is anhydrous ethanol; Preferably, in step (c), the concentration of ethanol in the second alcohol-containing solution is 50% to 70%, for example, about 60%. Preferably, in step (d), the concentration is reduced pressure concentration; Preferably, in step (d), the reduced pressure concentration is carried out until there is no alcohol odor.

5. A first honeysuckle extract and / or a second honeysuckle extract prepared by the preparation method according to any one of claims 1 to 4.

6. The use of the first and / or second honeysuckle extracts according to claim 5 in the preparation of medicaments, foods and / or health products for the prevention and / or treatment of obesity.

7. The use according to claim 6, characterized in that, The obesity mentioned refers to obesity caused by dietary habits, genetics, metabolism and / or fat cell abnormalities; Preferably, the abnormality is an increase in the number of adipocytes and / or adipocyte hypertrophy.

8. The use according to claim 6, characterized in that, The effects of the first and / or second honeysuckle extracts in preventing and / or treating obesity are achieved through one or more of the following mechanisms: reducing lipid accumulation in cells, reducing adipocyte diameter, reducing body weight, reducing the relative weight of peripheral adipose tissue, and increasing energy metabolism levels.

9. The use according to claim 8, characterized in that, The adipocytes are selected from one or more of the following: inguinal white adipose tissue cells, epididymal white adipose tissue cells, and brown adipose tissue cells.

10. The use according to claim 8, characterized in that, The peripheral adipose tissue includes inguinal white adipose tissue and / or epididymal white adipose tissue; Preferably, the increase in energy metabolism is achieved through one or more of the following methods: increased oxygen consumption, increased carbon dioxide excretion, and increased heat production by the body.

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