Application of bee pollen phenolamine extract

Abstract

The present invention provides an application of a bee pollen phenolamine extract, which belongs to the technical field of medicine or functional food. The bee pollen phenolamine extract of the present invention can prevent and treat obesity and regulate intestinal flora and metabolites, and can be used to prepare medicines or functional foods for preventing and treating obesity and regulating intestinal flora or metabolites. The results of the test examples show that the apricot flower bee pollen phenolamine extract prepared in the embodiment of the present invention can effectively reduce the body weight of obese mice, improve the sugar metabolism and lipid metabolism disorders of obese mice; can improve the oxidative stress and inflammatory damage of obese mice; can regulate the intestinal flora disorder of obese mice, increase the richness of intestinal flora, up-regulate beneficial bacteria, and down-regulate harmful bacteria; and can also improve the metabolite changes caused by obesity in mice.

Description

Technical Field

[0001] The invention relates to the technical field of medicine or functional food, in particular to application of bee pollen phenolamine extract. Background Art

[0002] Obesity is one of the health issues that the world is paying close attention to today. The occurrence of obesity increases the risk of metabolic diseases such as type 2 diabetes, non-alcoholic fatty liver disease, atherosclerosis and cardiovascular disease. The intestinal microbiota is closely related to diet and metabolic health. Intestinal microorganisms can affect a variety of physiological functions of the body, including metabolic pathways, immune regulation, glucose and lipid metabolism, and intestinal barrier function. Intestinal flora disorders can lead to a variety of metabolic diseases such as diabetes, obesity, and non-alcoholic fatty liver disease. Many studies have shown that intestinal flora may be a potential target for regulating obesity-related metabolic diseases. At present, the main methods for preventing and treating obesity include drugs, dieting, surgery, etc., but they have disadvantages such as large side effects and difficulty in adhering to them. Therefore, there is an urgent need to find and develop new natural active ingredients to prevent and treat obesity.

[0003] Bee pollen refers to the male gametophyte of flowers collected by worker bees and stingless bees, i.e. pollen, mixed with sugars, enzymes, waxes in nectar and substances secreted in bee saliva to form irregular grain-like particles. As a dietary supplement, bee pollen has multiple biological activities and is a nutrient-rich treasure trove of natural active ingredients. Phenolamine compounds are a class of characteristic compounds rich in bee pollen. The phenolamine compounds in bee pollen have a unique structure. These compounds are composed of polyamines (putrescine, spermidine and spermine) and hydroxycinnamic acids (coumaric acid, caffeic acid, ferulic acid) combined in the form of mono- or poly-substitution through amide bonds. Phenolamine compounds have the characteristics of richness and diversity due to different arrangements and combinations of substituents, degrees of substitution and isomerism. In recent years, the discovery of phenolamine compounds' antioxidant, anticancer and antibacterial biological activities has made them a new active ingredient with broad prospects that will be the focus of future research. Therefore, as a rich source of phenolamine compounds, the structural characteristics and biological activities of phenolamine compounds in bee pollen are worthy of in-depth research and development and application. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a bee pollen phenolamine extract. The bee pollen phenolamine extract of the present invention can prevent and treat obesity and regulate intestinal flora and metabolites, and can be used to prepare medicines or functional foods for preventing and treating obesity and regulating intestinal flora or metabolites.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides an application of a bee pollen phenolamine extract in preparing functional food or medicine for preventing and treating obesity.

[0007] Preferably, the present invention provides the use of the bee pollen phenolamine extract in the preparation of functional food or medicine for reducing liver fat or epididymal fat.

[0008] Preferably, the present invention provides the use of the bee pollen phenolamine extract in the preparation of functional food or medicine for regulating blood lipids or liver function.

[0009] Preferably, the present invention provides the use of the bee pollen phenolamine extract in preparing functional food or medicine for regulating blood sugar.

[0010] Preferably, the present invention provides the use of the bee pollen phenolamine extract in the preparation of functional food or medicine for improving fasting blood sugar, glucose tolerance, insulin resistance, adiponectin level or leptin resistance.

[0011] Preferably, the present invention provides the use of the bee pollen phenolamine extract in the preparation of functional food or medicine for reducing oxidative stress damage or inflammatory damage caused by obesity.

[0012] The invention provides an application of a bee pollen phenolamine extract in preparing a functional food or medicine for regulating intestinal flora or intestinal metabolite imbalance.

[0013] Preferably, the intestinal flora induced by obesity is regulated by upregulating beneficial bacteria and downregulating harmful bacteria; the beneficial bacteria are one or more of Bacteroidetes, Muribaculaceae and Parabacteroides; the harmful bacteria are Peptostreptococcaceae and / or Romboutsia;

[0014] The metabolites include one or more of bile acid, phosphorylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine and tyrosine.

[0015] Preferably, the content of phenolamine compounds in the bee pollen phenolamine extract is 83-94%, and the phenolamine compounds in the bee pollen phenolamine extract include tricaffeoyl spermidine, dicaffeoyl coumaroyl spermidine, di-coumaroyl caffeoyl spermidine, tri-coumaroyl spermidine and isomers of tri-coumaroyl spermidine.

[0016] Preferably, the method for preparing the bee pollen phenolamine extract comprises the following steps:

[0017] The bee pollen is mixed with petroleum ether and subjected to a defatting treatment to obtain defatted bee pollen;

[0018] The defatted bee pollen is mixed with an alcohol aqueous solution for extraction, and the obtained extract is first concentrated to obtain a bee pollen alcohol extract;

[0019] The bee pollen alcohol extract is mixed with an extractant, and extracted, and the obtained extract phase is sequentially concentrated and freeze-dried to obtain a bee pollen extract; the extractant is a mixture of water and ethyl acetate;

[0020] The bee pollen extract is purified by passing it through a silica gel column using a mixture of chloroform and methanol as an eluent, and the solvent in the obtained eluent is removed to obtain a bee pollen phenolamine extract; the volume ratio of chloroform to methanol in the eluent is (80-85): (15-20).

[0021] The present invention provides the use of bee pollen phenolamine extract in the preparation of functional foods or drugs for preventing and treating obesity, and the use of bee pollen phenolamine extract in the preparation of functional foods or drugs for regulating intestinal flora or intestinal metabolite disorders. The bee pollen phenolamine extract in the present invention can prevent and treat obesity and regulate intestinal flora and metabolites, and can be used to prepare drugs or functional foods for preventing and treating obesity and regulating intestinal flora or metabolites. The results of the test examples show that the apricot flower bee pollen phenolamine extract prepared in the embodiment of the present invention can effectively reduce the body weight of obese mice, improve the sugar metabolism and lipid metabolism disorders of obese mice; can improve the oxidative stress and inflammatory damage of obese mice; can regulate the intestinal flora disorder of obese mice, increase the richness of intestinal flora, up-regulate beneficial bacteria, and down-regulate harmful bacteria; and can also improve the metabolite changes caused by obesity in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The HPLC chromatogram of the phenolamine extract of apricot bee pollen prepared in Example 1;

[0023] Figure 2 This is a graph showing the effect of the almond bee pollen phenolamine extract prepared in Example 1 on the weight gain and tissue and organ weights of obese mice;

[0024] Figure 3 This is a graph showing the effect of the almond bee pollen phenolamine extract prepared in Example 1 on blood lipids and liver function in obese mice;

[0025] Figure 4 This is a graph showing the effect of the apricot bee pollen phenolamine extract prepared in Example 1 on glucose metabolism in obese mice;

[0026] Figure 5 This is a graph showing the effect of the almond bee pollen phenolamine extract prepared in Example 1 on liver oxidative stress injury and serum inflammation in obese mice;

[0027] Figure 6 Representative H&E staining images of the liver;

[0028] Figure 7 Representative Oil Red O staining images of the liver;

[0029] Figure 8 Representative H&E staining images of epididymal adipocytes;

[0030] Fig. 9 is a bar graph of the area of ​​adipocytes in epididymal fat;

[0031] Fig.10 This is a graph showing the effect of the almond bee pollen phenolamine extract prepared in Example 1 on the intestinal flora of obese mice;

[0032] Fig.11 KEGG predicted pathway analysis diagram (NC vs HFD);

[0033] Fig.12 KEGG predicted pathway analysis diagram (HFD vs LD);

[0034] Fig.13 This is the KEGG predicted pathway analysis diagram (HFD vs HD);

[0035] Fig.14 Metabolite heat map. DETAILED DESCRIPTION

[0036] The present invention provides an application of a bee pollen phenolamine extract in the preparation of a functional food or drug for preventing and treating obesity. In the present invention, the drug preferably includes an active ingredient and a drug excipient, the active ingredient is a bee pollen phenolamine extract, the present invention has no special limitation on the drug excipient, and any drug excipient well known to those skilled in the art can be used. In the present invention, the functional food preferably includes an effective ingredient and a food excipient, the effective ingredient is a bee pollen phenolamine extract, the present invention has no special limitation on the food excipient, and any food excipient well known to those skilled in the art can be used.

[0037] As a technical solution of the present invention, the present invention provides the use of the bee pollen phenolamine extract in the preparation of functional foods or drugs for reducing liver fat or epididymal fat. The results of the test examples show that the liver index of high-fat diet mice intervened by the bee pollen phenolamine extract of the present invention is reduced, and the bee pollen phenolamine extract of the present invention can significantly reduce the epididymal fat index of obese mice.

[0038] As a technical solution of the present invention, the present invention provides the use of the bee pollen phenolamine extract in the preparation of functional food or medicine for regulating blood lipids or liver function. The results of the test example show that the bee pollen phenolamine extract of the present invention can effectively improve the blood lipid level of obese mice and has a protective effect on liver function damage caused by obesity.

[0039] As a technical solution of the present invention, the present invention provides the use of the bee pollen phenolamine extract in the preparation of functional foods or drugs for regulating blood sugar; specifically, the present invention provides the use of the bee pollen phenolamine extract in the preparation of functional foods or drugs for improving fasting blood sugar, glucose tolerance, insulin resistance, adiponectin levels or leptin resistance. The results of the test examples show that the bee pollen phenolamine extract of the present invention can regulate obesity-related glucose metabolism disorders in mice fed a high-fat diet, and improve fasting blood sugar, glucose tolerance, insulin resistance, adiponectin levels and leptin resistance.

[0040] As a technical solution of the present invention, the present invention provides the use of the bee pollen phenolamine extract in the preparation of functional food or medicine for reducing oxidative stress damage or inflammatory damage caused by obesity. The results of the test example show that the intervention of the bee pollen phenolamine extract in the present invention can reduce the oxidative stress damage caused by lipid peroxidation in obese mice, and can improve the availability of antioxidants and reduce inflammatory damage.

[0041] The present invention provides an application of a bee pollen phenolamine extract in the preparation of a functional food or medicine for regulating intestinal flora or intestinal metabolite disorders. In the present invention, the medicine preferably includes an active ingredient and a pharmaceutical excipient, and the active ingredient is a bee pollen phenolamine extract. The present invention does not specifically limit the pharmaceutical excipient, and any pharmaceutical excipient familiar to those skilled in the art may be used. In the present invention, the functional food preferably includes an effective ingredient and a food excipient, and the effective ingredient is a bee pollen phenolamine extract. The present invention does not specifically limit the food excipient, and any food excipient familiar to those skilled in the art may be used.

[0042] In the present invention, the intestinal flora caused by obesity is preferably regulated by upregulating beneficial bacteria and downregulating harmful bacteria; the beneficial bacteria are preferably one or more of Bacteroidetes, Muribacillus family and Parabacteroides; the harmful bacteria are preferably Peptostreptococcaceae and / or Romboutsia. In the present invention, the metabolites preferably include one or more of bile acid, phosphorylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine and tyrosine.

[0043] In the present invention, the content of phenolamine compounds in the bee pollen phenolamine extract is preferably 83-94%, more preferably 88-94wt%; the phenolamine compounds in the bee pollen phenolamine extract preferably include tricaffeoyl spermidine, dicaffeoyl p-coumaryl spermidine, di-p-coumaryl caffeoyl spermidine, tri-p-coumaryl spermidine and isomers of tri-p-coumaryl spermidine (such as cis-isomers, trans-isomers, etc.).

[0044] In the present invention, the preparation method of the bee pollen phenolamine extract preferably comprises the following steps:

[0045] The bee pollen is mixed with petroleum ether and subjected to a defatting treatment to obtain defatted bee pollen;

[0046] The defatted bee pollen is mixed with an alcohol aqueous solution for extraction, and the obtained extract is first concentrated to obtain a bee pollen alcohol extract;

[0047] The bee pollen alcohol extract is mixed with an extractant, and extracted, and the obtained extract phase is sequentially concentrated and freeze-dried to obtain a bee pollen extract; the extractant is a mixture of water and ethyl acetate;

[0048] The bee pollen extract is purified by passing it through a silica gel column using a mixture of chloroform and methanol as an eluent, and the solvent in the obtained eluent is removed to obtain a bee pollen phenolamine extract; the volume ratio of chloroform to methanol in the eluent is (80-85): (15-20).

[0049] The present invention mixes bee pollen with petroleum ether and performs defatting treatment to obtain defatted bee pollen. In the present invention, the bee pollen preferably includes apricot bee pollen, sunflower bee pollen or corn bee pollen, and is more preferably apricot bee pollen. In the present invention, the bee pollen is preferably crushed before mixing with petroleum ether; the present invention does not specifically limit the parameters of the crushing, and the particle size of the bee pollen can be 80 mesh. In the present invention, the boiling range of the petroleum ether is preferably 60 to 90°C. In the present invention, the degreasing treatment is preferably carried out under ultrasonic conditions, and the conditions of the degreasing treatment preferably include: the number of degreasing treatments is preferably 1 to 3 times, more preferably 2 times; the material-liquid ratio of each degreasing treatment is preferably independently 1 kg: (1.5 to 3) L, more preferably 1 kg: 2 L; the time of each degreasing treatment is preferably independently 20 to 40 min, more preferably 30 min; the temperature of the degreasing treatment is preferably 25 to 40° C., more preferably 25 to 35° C.; the power of the ultrasound is preferably 500 to 600 W, more preferably 550 to 600 W. After the degreasing treatment, the present invention preferably filters the obtained system, and then places the obtained solid material in a fume hood to dry to obtain a dry powder, which is defatted bee pollen. The present invention can remove lipids from bee pollen through degreasing treatment.

[0050] After obtaining defatted bee pollen, the present invention mixes the defatted bee pollen with an alcohol aqueous solution for extraction, and the obtained extract is first concentrated to obtain a bee pollen alcohol extract. In the present invention, the volume fraction of the alcohol aqueous solution is preferably 75-85%, more preferably 80%; the alcohol in the alcohol aqueous solution is preferably methanol or ethanol, more preferably ethanol. In the present invention, the extraction is preferably carried out under ultrasonic conditions, and the conditions for the extraction preferably include: the number of extractions is preferably 1-3 times, more preferably 2 times; the solid-liquid ratio of each extraction is preferably independently 1kg: (10-40) L, more preferably independently 1kg: (10-20) L; the time of each extraction is preferably independently 20-40min, more preferably 30min; the extraction temperature is preferably 25-40°C, more preferably 25-35°C; the ultrasonic power is preferably 500-600W, more preferably 550-600W. The invention can extract the phenolamine compounds in the defatted bee pollen to a greater extent through extraction, so as to ensure that the finally obtained bee pollen phenolamine extract has good biological activity.

[0051] After the extraction, the present invention preferably removes the residue in the obtained liquid to obtain an extract, and the obtained extract is subjected to a first concentration to obtain a bee pollen alcohol extract. In the present invention, the method for removing the residue is preferably suction filtration, and the present invention does not specifically limit the parameters of the suction filtration, and the suction filtration method well known to those skilled in the art can be used. In the present invention, the first concentration is preferably carried out under vacuum conditions, and the first concentration is preferably concentrated by evaporation in a rotary evaporator, specifically, the organic reagent in the extract is removed by the first concentration (that is, the bee pollen alcohol extract is a concentrated solution containing water).

[0052] After obtaining the bee pollen alcohol extract, the present invention mixes the bee pollen alcohol extract with an extractant to extract, and sequentially performs a second concentration and freeze-drying on the obtained extract phase to obtain a bee pollen extract. In the present invention, the extractant is a mixture of water and ethyl acetate, and the volume ratio of water to ethyl acetate in the extractant is preferably 1: (1-3), more preferably 1: (1-1.5). In the present invention, the volume ratio of the extractant to the bee pollen alcohol extract is preferably (8-4): 1, more preferably 6: 1. In the present invention, the extract phase is specifically an ethyl acetate phase. In the present invention, the second concentration is preferably evaporation concentration in a rotary evaporator. In the present invention, the freeze drying preferably includes: first pre-freezing at -50 to -60°C at normal pressure for 2h, and then vacuum freeze drying at 60 to 70Pa and -50 to -60°C for 48h; more preferably includes: first pre-freezing at -58°C at normal pressure for 2h, and then vacuum freeze drying at 66Pa and -58°C for 48h. The present invention can fully remove water-soluble impurities, such as oligosaccharides and polypeptides and other substances with relatively large polarity, through extraction, so as to ensure that the final bee pollen phenolamine extract has a higher content of phenolamine compounds.

[0053] After obtaining the bee pollen extract, the present invention uses a mixed solution of chloroform and methanol as an eluent, purifies the bee pollen extract through a silica gel column, removes the solvent in the obtained eluent, and obtains a bee pollen phenolamine extract. In the present invention, the volume ratio of chloroform and methanol in the eluent is (80-85): (15-20), preferably 85:15. The present invention directly uses only chloroform and methanol in the above volume ratio as eluents, which can effectively separate the impurity components of the crude extract, such as flavonoids, and is conducive to the enrichment of phenolamine compounds and the reduction of costs. In the present invention, the silica gel model in the silica gel column is preferably SLG12S50, and the particle size is preferably 50μm, which is specifically purchased from Japan YMC Company; in an embodiment of the present invention, the specification of the silica gel column is preferably 5cm×48cm. In the present invention, the volume of silica gel in the silica gel column is preferably 60 times the sample volume of the bee pollen extract during purification. In the present invention, the method of removing the solvent in the eluent is preferably vacuum freeze-drying.

[0054] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] Example 1

[0056] 1.5 kg of apricot bee pollen raw material was crushed into 80 meshes, and the obtained apricot bee pollen was defatted with petroleum ether under ultrasonic conditions to obtain defatted apricot bee pollen; the defatting conditions included: the number of defatting treatments was 2 times, the material-liquid ratio of each defatting treatment was 1 kg: 2 L, the time of each defatting treatment was 30 min, the temperature of the defatting treatment was 25° C., and the power of the ultrasound was 600 W;

[0057] The defatted apricot bee pollen is extracted with an ethanol aqueous solution with a volume fraction of 80% under ultrasonic conditions to obtain an extraction liquid; the extraction conditions include: the number of extractions is 2 times, the ratio of the material to the liquid for each extraction is 1kg:10L, the time for each extraction is 30min, the extraction temperature is 25°C, and the ultrasonic power is 600W; the extraction liquid is filtered to remove the residue to obtain an extraction liquid; the extraction liquid is evaporated and concentrated in a rotary evaporator to obtain an apricot bee pollen extract;

[0058] The apricot bee pollen extract and distilled water were mixed in a volume ratio of 1:2.5, and the obtained dilution was mixed with an equal volume of ethyl acetate for extraction. The upper ethyl acetate extract phase was evaporated and concentrated in a rotary evaporator, and then pre-frozen at -58°C for 2h, and then vacuum freeze-dried at 66Pa and -58°C for 48h to obtain an apricot bee pollen extract (15g);

[0059] The apricot bee pollen extract was purified by passing it through a silica gel column (the silica gel column had a specification of 5 cm×48 cm; the silica gel particle size was 50 μm, model SLG12S50, purchased from Japan YMC Company), wherein the volume of silica gel in the silica gel column was 60 times the volume of the bee pollen extract loaded during purification, and the eluent used for purification was a mixture of chloroform and methanol in a volume ratio of 85:15 (3000 mL). The solvent in the eluate obtained after purification was removed by vacuum freeze drying to obtain the apricot bee pollen phenolamine extract (9.5 g), which was stored at -20°C for future use.

[0060] The components of the almond bee pollen phenolamine extract were analyzed by high performance liquid chromatography (HPLC) and high performance liquid chromatography tandem mass spectrometry (HPLC-ESI-QTOF-MS / MS), as follows:

[0061] High performance liquid chromatography analysis: The chromatographic column is Hypersil GOLD C18 (250mm×4.6mm, 5μm), the mobile phase is 0.13% by volume formic acid aqueous solution (mobile phase A) and methanol (mobile phase B), the injection volume is 10μL, the flow rate is 0.5mL / min, and the column temperature is maintained at 30°C; the gradient elution conditions are: 0min, 50% mobile phase B; 20min, 63% mobile phase B; 22min, 50% mobile phase B; 30min, 50% mobile phase B; the detection wavelength is 280nm; and the calibration curve of p-coumaric acid standard is used for quantification.

[0062] High performance liquid chromatography tandem mass spectrometry analysis: Liquid chromatography conditions are the same as above; Mass spectrometry conditions: Ionization mode is ESI + , ionization voltage is 20-40V, scanning mass range is 100-1500, nebulizer pressure is 30-50psi, drying gas flow rate is 8-10L / min, drying gas temperature is 350℃, and capillary voltage is 3500V.

[0063] Figure 1 This is the HPLC chromatogram of the apricot bee pollen phenolamine extract prepared in Example 1. Figure 1 Peaks 1 to 3 were identified as tricaffeoyl spermidine, dicaffeoyl p-coumaryl spermidine and di-p-coumaryl caffeoyl spermidine, respectively, and peaks 4 to 7 were identified as tri-p-coumaryl spermidine and its isomers (such as cis-, trans-isomers, etc.), indicating that the components of the apricot flower bee pollen phenolamine extract prepared in Example 1 are mainly phenolamine compounds.

[0064] A p-coumaric acid standard was used to quantify the content of phenolamine compounds in the apricot bee pollen phenolamine extract prepared in Example 1 (specifically, three tests were performed). The results showed that the content of phenolamine compounds in the apricot bee pollen phenolamine extract prepared in Example 1 was 87.8±5.4wt%.

[0065] Test Example 1

[0066] This test case uses animal experiments to verify the effects of apricot bee pollen phenolamine extract on obesity suppression, improvement of intestinal flora and metabolites in high-fat diet-induced obese mice, as follows:

[0067] 1. Experimental Protocol

[0068] 44 male C57BL / 6 mice (18±2g) were housed in a constant temperature condition (22±2℃), 12h light-dark cycle, SPF environment, with free access to food and water. After one week of adaptive feeding, they were randomly divided into 2 groups, 8 mice were given a low-fat diet (10% fat) as a blank control group (NC), and 36 mice were given a high-fat diet (60% fat) as a model group. The amount of food given, the amount of food scattered, and the amount of food left were recorded every week, and the body weight was weighed once. After 2 weeks of feeding, the mice given a high-fat diet were sorted according to weight gain, and the 1 / 3 obesity-resistant mice with lower weight gain were eliminated. The remaining 2 / 3 (24) mice were randomly divided into 3 groups, namely a high-fat diet group (HFD), a low-dose group (LD), and a high-dose group (HD), and were given a high-fat diet (60% fat). The drug was administered by gavage every day until 12 weeks. The specific dosing conditions of each group of animals are as follows:

[0069] Blank control group (NC): 0.1% CMC-Na (prepared from sodium carboxymethylcellulose [CMC-Na]: water w / v);

[0070] High-fat diet group (HFD): 0.1% CMC-Na;

[0071] Low dose group (LD): 50 mg / kg BW of almond bee pollen phenolamine extract prepared with 0.1% CMC-Na;

[0072] High-dose group (HD): 200 mg / kg BW of almond bee pollen phenolamine extract prepared with 0.1% CMC-Na.

[0073] Body weight and food intake were measured every day. Three days before the mice were sacrificed, fresh fecal pellets of each mouse were collected in sterile EP tubes and stored at -80°C for further analysis. Before sacrifice, the mice were fasted overnight and had free access to water. All mice were anesthetized to draw eyeball blood, and then centrifuged at 4°C, 3500rpm for 10min to collect serum. Epididymal fat and liver were dissected and weighed. Organ index (epididymal fat and liver) is defined as the ratio of organ tissue weight to body weight. The liver was quickly frozen in liquid nitrogen and stored at -80°C for further analysis. In addition, part of the liver and epididymal adipose tissue was fixed in 4% paraformaldehyde for histological analysis.

[0074] Figure 2 This is a graph showing the effect of the almond bee pollen phenolamine extract prepared in Example 1 on the weight gain and tissue and organ weight of obese mice. Figure 2 A corresponds to body weight, B corresponds to daily food intake, C corresponds to liver index, and D corresponds to epididymal fat index. Figure 2 As shown in A, at week 0, the body weights of mice in the HFD, LD, and HD groups were all higher than those in the NC group, and there was no difference among the three groups. However, at week 12, the body weights of mice in the LD and HD groups were significantly lower than those in the HFD group (p < 0.01). Figure 2 From B, we can see that there is no significant difference in the food intake of the four groups of mice, which means that there is no significant difference in the energy intake of the three groups of mice on a high-fat diet. This shows that the intervention of the almond bee pollen phenolamine extract did not affect the appetite of mice, and its effect on body weight was not due to a reduction in energy intake. Figure 2 As shown in C and D, the liver index of mice in the HFD group increased, while that of mice on a high-fat diet intervened with the phenolamine extract of apricot blossom and bee pollen decreased. Epididymal fat showed more obvious changes, and the epididymal fat index of mice in the HFD group was significantly higher than that of the other three groups, while the epididymal fat index of mice in the LD and HD groups intervened with the phenolamine extract of apricot blossom and bee pollen could significantly reduce the epididymal fat index of obese mice (p < 0.05).

[0075] 2. Detection plan

[0076] 2.1 Serum biochemical analysis

[0077] The levels of serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and alanine aminotransferase (ALT) were measured by colorimetric assay kits.

[0078] Figure 3 This is a graph showing the effect of the almond bee pollen phenolamine extract prepared in Example 1 on blood lipids and liver function in obese mice. Figure 3 A corresponds to TC, B corresponds to LDL-C, C corresponds to HDL-C, and D corresponds to ALT. Figure 3 It can be seen that the levels of TC (p<0.05) and LDL-C (p<0.05) in the serum of mice in the HFD group increased, and the level of HDL-C (p<0.05) decreased. However, the intervention of apricot bee pollen phenolamine extract can alleviate the abnormal lipid metabolism of obese mice. After oral administration of apricot bee pollen phenolamine extract, the TC content of mice in the LD and HD groups decreased (p<0.05). In addition, the intervention of apricot bee pollen phenolamine extract had the greatest effect on the levels of HDL-C and LDL-C. The LDL-C content of mice in the LD and HD groups decreased by about 18% and 24% respectively compared with the HFD group; the HDL-C content increased by about 8% and 52% respectively compared with the HFD group, and it was dose-dependent. Therefore, apricot bee pollen phenolamine extract can effectively improve the blood lipid level of obese mice. At the same time, the ALT activity of mice in the HFD group increased significantly, and the ALT activity of mice in the LD and HD groups decreased significantly (p<0.05), and there was no statistical difference with the mice in the NC group ( Figure 3D). Therefore, the almond bee pollen phenolamine extract has a protective effect on obesity-induced liver damage.

[0079] 2.2 OGTT and insulin, adiponectin and leptin

[0080] At week 11, oral glucose tolerance test (OGTT) was performed on mice fasted for 12 h. Fasted mice were given glucose at 2 g / kg BW, and blood was collected from the tail vein at 0 min before, 15 min, 30 min, 60 min, and 120 min after glucose administration, and blood glucose levels were measured using a reagent strip with a glucometer. The area under the curve (AUC) was calculated using the trapezoidal rule method. Serum insulin, adiponectin, and leptin were measured using ELISA kits. HOMA-IR was calculated using the formula: HOMA-IR = fasting insulin (mU / L) × fasting glucose (mM) / 22.5.

[0081] Figure 4 This is a graph showing the effect of the almond bee pollen phenolamine extract prepared in Example 1 on glucose metabolism in obese mice. Figure 4 A in the figure corresponds to OGTT and the area under the curve (AUC), B corresponds to fasting glucose, C corresponds to insulin, D corresponds to HOMA-IR, E corresponds to adiponectin, and F corresponds to leptin. Figure 4 From A in the figure, we can see that the AUC of obese mice in the HFD group was significantly higher than that in the NC group, indicating that obese mice had impaired glucose tolerance. The AUC of mice after intervention with a high dose of apricot bee pollen phenolamine extract was significantly lower than that in the HFD group (p < 0.05), and the blood glucose levels of mice in the HD group at 15min, 30min, 60min and 120min were all lower than those in the HFD group. This shows that the apricot bee pollen phenolamine extract can improve the glucose tolerance of obese mice. The fasting blood glucose, insulin content and insulin resistance index of obese mice increased significantly compared with those of mice in the NC group (p < 0.05), indicating that mice in the HFD group developed insulin resistance ( Figure 4 B~D in the figure), the fasting blood glucose, insulin content and insulin resistance index of mice in the LD and HD groups were significantly lower than those in the HFD group (p<0.05). This shows that the mice intervened with the phenolamine extract of apricot bee pollen reduced the insulin resistance of mice on a high-fat diet, and it was dose-dependent. Adiponectin and leptin are the main adipose cell factors, which are closely related to insulin resistance. Their circulating concentrations (low adiponectin and high leptin) can be used to predict insulin resistance caused by obesity and metabolic syndrome. The level of serum adiponectin decreases with obesity. The adiponectin level of mice in the HFD group was significantly reduced (p<0.05), while the adiponectin level of mice in the LD and HD groups was significantly increased (p<0.05) and there was no statistical difference with the NC group ( Figure 4 E in the figure). Serum leptin levels are related to energy storage. Leptin has the function of suppressing appetite and increasing heat production. However, when the human body develops "leptin resistance", even if leptin levels increase, it cannot prevent obese people from gaining weight. Figure 4 F in the figure shows that the leptin level in the HFD group was significantly higher than that in the NC group (p < 0.05), indicating that the high-fat diet caused leptin resistance in obese mice, while the serum leptin level in the LD group was significantly lower than that in the HFD group (p < 0.01), and the leptin level in the HD group also decreased. This shows that the phenolamine extract of apricot flower bee pollen can regulate the glucose metabolism disorder associated with obesity in mice fed a high-fat diet, and improve glucose tolerance, insulin resistance, adiponectin levels and leptin resistance.

[0082] 2.3 Oxidative stress and inflammation

[0083] The levels of liver malondialdehyde (MDA) and reduced glutathione (GSH) were determined by colorimetric assay kits. The levels of serum interleukin-6 (IL-6) and interleukin-10 (IL-10) were detected according to the instructions of the ELISA kits.

[0084] Figure 5 This is a graph showing the effect of the almond bee pollen phenolamine extract prepared in Example 1 on liver oxidative stress injury and serum inflammation in obese mice. Figure 5 A corresponds to MDA, B corresponds to GSH, C corresponds to IL-6, and D corresponds to IL-10. Figure 5 It can be seen that the content of MDA in the liver of mice in the HFD group increased significantly (p < 0.05), and the content of GSH decreased significantly (p < 0.05). The content of MDA in the liver of mice in the LD and HD groups with high-fat diets decreased significantly compared with the HFD group, and the content of GSH increased significantly (p < 0.05). The serum IL-6 content of obese mice fed with a high-fat diet increased significantly (p < 0.05), and the serum IL-6 of mice in the LD and HD groups decreased significantly (p < 0.05). In addition, after intervention with the phenolamine extract of apricot bee pollen, IL-10, an anti-inflammatory factor, increased significantly in the serum of mice in the LD group (p < 0.05). Obese mice caused by long-term high-fat diet may be more prone to lipid peroxidation and produce a pro-inflammatory environment. Intervention with the phenolamine extract of apricot bee pollen can reduce the oxidative stress damage caused by lipid peroxidation in obese mice, and can increase the availability of antioxidants and reduce inflammatory damage.

[0085] 2.4 Histopathological sections

[0086] Figures 6 to 9 This is a graph showing the effect of the almond bee pollen phenolamine extract prepared in Example 1 on fat accumulation in tissues and organs of obese mice, wherein: Figure 6Representative H&E staining images of the liver (200×), scale bar: 100 μm; Figure 7 Representative Oil Red O staining images of the liver (200×), scale bar: 100 μm; Figure 8 Representative H&E staining images of epididymal adipocytes (200×), scale bar: 100 μm; Fig. 9 The figure is a bar graph of the area of ​​adipocytes in epididymal fat. Obese mice will develop fatty degeneration and adipocyte hypertrophy in the liver. Through liver H&E staining, it can be seen that compared with the NC group, the liver of the HFD group mice has many vacuoles, which are fat in the liver, that is, there is abnormal lipid accumulation in the liver of the HFD group mice. However, compared with the HFD group, the liver cells of the LD and HD groups are closely arranged and there are no large vacuoles ( Figure 6 Oil red O staining of the liver showed the accumulation of lipid droplets in the liver tissue of the HFD group mice. The red lipid droplets in the LD group were significantly reduced compared with the HFD group, and the red lipid droplets in the HD group were even less than those in the LD group ( Figure 7 ). According to H&E staining of epididymal adipose tissue, it can be seen that the epididymal adipose adipocytes in the HFD group were significantly larger than those in the NC group, and were unevenly arranged ( Figure 8 ), the adipocytes in the LD and HD groups were significantly reduced compared with those in the HFD group in a dose-dependent manner (p<0.05) ( Fig. 9 ). It can be seen that the almond bee pollen phenolamine extract can improve the liver lipid accumulation and adipocyte hypertrophy in obese mice induced by a high-fat diet.

[0087] 2.5 Analysis of intestinal flora

[0088] Fig.10 This is a graph showing the effect of the almond bee pollen phenolamine extract prepared in Example 1 on the intestinal flora of obese mice. Fig.10 A corresponds to Chao1 index, B corresponds to PCoA, C corresponds to the relative content of key bacterial species at the phylum level, D corresponds to the relative content of key bacterial species at the family level, and E corresponds to the relative content of key bacterial species at the genus level.

[0089] Chao1 index was used to evaluate the richness of intestinal flora. Fig.10 As shown in A, the Chao1 index of the HFD group was significantly lower than that of the NC group (p < 0.05), and the Chao1 index of the LD and HD groups increased compared with the HFD group, and the HD group increased significantly (p < 0.01). This shows that a high-fat diet can reduce the richness of the intestinal flora of obese mice, while intervention with almond bee pollen phenolamine extract can increase the richness of the intestinal flora of obese mice on a high-fat diet. PCoA analysis based on UnweightedUniFrac distance ( Fig.10In B), it can be seen that the explanatory value of PCoA1 is 16.66%, which can well explain the effect of feed on the differences in intestinal flora of mice in the NC group and the HFD, LD and HD groups. The explanatory value of PCoA2 is 8.71%, which shows that the intervention of apricot bee pollen phenolamine extract can improve the composition of mouse intestinal flora.

[0090] In obese humans and animals, the intestinal flora is characterized by reduced levels of Bacteroidetes and increased Firmicutes. Phenolamine extract from almond bee pollen significantly increased the relative abundance of Bacteroidetes (p < 0.05) and significantly reduced the Firmicutes / Bacteroidetes (F / B) ratio (p < 0.05) ( Fig.10 C).

[0091] Peptostreptococcaceae was enriched in both proctitis and obese mice. Fig.10 As shown in D, the relative abundance of Peptostreptococcaceae increased in the HFD group, while after treatment with almond bee pollen phenolamine extract, the relative abundance of Peptostreptococcaceae in the LD and HD groups was significantly downregulated in a dose-dependent manner (p < 0.05, p < 0.01). In addition, Muribaculaceae is a butyrate-producing bacterium that also produces propionate as a fermentation end product. The relative abundance of Muribaculaceae was significantly reduced in the HFD group (p < 0.01), and the relative abundance of Muribaculaceae was significantly upregulated in the LD and HD groups (p < 0.05).

[0092] Studies have shown that Parabacteroides is a bacteria associated with obesity prevention, specifically related to anti-obesity and anti-inflammatory activity. Romboutsia is a harmful genus. Studies have shown that the expression level of pro-inflammatory central genes is positively correlated with the abundance of Romboutsia, and a high-fat and high-sugar diet will increase the abundance of this genus in the intestine. Fig.10 As shown in E, the relative abundance of Parabacteroide in the intestine of obese mice was downregulated and the relative abundance of Romboutsia was upregulated. The HD group could significantly increase the relative abundance of Parabacteroides (p < 0.01), and the LD and HD groups could significantly downregulate the relative abundance of Romboutsia in a dose-dependent manner (p < 0.05, p < 0.01).

[0093] The above results indicate that the phenolamine extract of apricot bee pollen reshapes the structure of the intestinal microbiota in obese mice induced by a high-fat diet, alleviates the changes in the microbiota of obese mice caused by a high-fat diet, downregulates harmful bacteria, upregulates beneficial bacteria, and alleviates the imbalance of the microbiota in mice.

[0094] 2.6 Fecal metabolomics

[0095] Eight mouse fecal samples were randomly selected from each group for non-targeted metabolomics analysis. 100 mg of feces was taken, 200 mg of glass beads were added, 1 mL of pre-cooled 50% methanol aqueous solution was added, high-speed vortexed for 30 min, centrifuged at 10000g and 4°C for 10 min, and the supernatant was aspirated and filtered through a 0.45 μm Millipore filter for analysis.

[0096] Metabolites in fecal samples were analyzed by Q Exactive Orbitrap LC-MS / MS. The liquid phase system used was Thermo Scientific 3000UHPLC. A Thermo Hypersil GOLD C18 (100×2.1mm, 1.9μm) chromatographic column was used, and the column temperature was 40°C. The mobile phase was a 0.1% volume fraction of formic acid in water (mobile phase A) and acetonitrile (mobile phase B), and the gradient elution conditions were: 0 min, 5% mobile phase B; 2 min, 5% mobile phase B; 10 min, 30% mobile phase B; 18 min, 50% mobile phase B; 20 min, 80% mobile phase B; 21 min, 95% mobile phase B; 22 min, 5% mobile phase B; 25 min, 5% mobile phase B. The flow rate was set to 0.3 mL / min. The sample injection volume was 2 μL. The mass spectrometry conditions were as follows: electrospray ionization (ESI + ) ionization mode, positive ion mode, mass spectrometry acquisition mass-to-charge ratio (m / z) range of 100-1500.

[0097] The same type of samples were randomly divided into groups and injected once per sample. 10 μL of each sample of the same type was mixed to make quality control samples (QC). Pure methanol was used as a blank sample. After every 8 injections of samples, 1 injection of QC sample and 1 injection of blank sample were performed for quality control and correction of peak drift.

[0098] Figures 11 to 14 This is a graph showing the effect of the almond bee pollen phenolamine extract prepared in Example 1 on the intestinal metabolite levels of obese mice, wherein: Fig.11 This is the KEGG predicted pathway analysis diagram (NC vs HFD). Fig.12This is the KEGG predicted pathway analysis diagram (HFD vs LD). Fig.13 This is the KEGG predicted pathway analysis diagram (HFD vs HD). Fig.14 is the metabolite heat map, Fig.14 The numbers (1, 2) after the compound indicate the isomers of the compound.

[0099] KEGG was used to analyze the enrichment of differential metabolite pathways to further explain the effect of apricot bee pollen phenolamine extract on high-fat diet-induced obese mice ( Figures 11 to 13 ). The results showed that the main metabolic pathways affected by the apricot bee pollen phenolamine extract were linoleic acid metabolism, glycerophospholipid metabolism, α-linolenic acid metabolism and primary bile acid biosynthesis. The main key host metabolites were bile acid, phosphorylcholine (PC), lysophosphatidylcholines (lysoPC), lysophosphatidylethanolamine (lysoPE) and tyrosine. A heat map analysis of these key compounds and possible metabolites produced by the apricot bee pollen phenolamine extract was performed ( Fig.14In addition, the levels of 27-norcholestanehexol, 8Z,11Z,14Z-eicosatrienoyl-CoA, lysoPC (18:2(9Z,12Z)) and lysoPE were down-regulated in the HFD group, while PC, lysoPC (20:3(5Z,11Z,8Z)), N-(1-deoxy-1-fructosyl)tyrosine, bile acid (7alpha-hydroxy-3-oxochol-4-en-24-oic acid and 7-ketodeoxycholic acid) were up-regulated. The changes of these metabolites in obese mice were reversed after intervention with the phenolamine extract of almond bee pollen. Tyrosine is a biomarker of the phenylalanine metabolic pathway, and metabolic abnormalities are associated with hepatic steatosis. Bile acid is the final metabolite of cholesterol, and reducing intestinal bile acid levels can reduce hyperlipidemia and lipid absorption. 27-norcholestanehexol is a bile alcohol. Cholesterol is the final product of cholesterol elimination and is also a type of bile salt. After ingestion of lipids, bile salts are released into the duodenum as emulsifiers to dissolve lipids. Phenolamine extract of apricot bee pollen can reduce the bile acid content in the feces of obese mice and regulate the bile acid metabolic cycle. PC is the main phospholipid in mammalian cell membranes, and the increase in fecal PC may be due to intestinal damage caused by a high-fat diet. LysoPC is a potential biomarker of the glycerophospholipid metabolic pathway. Metabolic imbalance between PC and lysoPC can lead to dysregulation of glycerophospholipid metabolism, thereby affecting the occurrence of atherosclerosis, diabetes, and hyperlipidemia. LysoPE is a product of the hydrolysis of phosphatidylethanolamine (PE), which can induce lipid accumulation in the liver and affect liver lipid metabolism. After intervention with almond bee pollen phenolamine extract, the abundance of lysoPE in obese mice was upregulated, indicating that almond bee pollen phenolamine extract may regulate lipid metabolism by promoting lipid excretion.

[0100] It can be seen from the above embodiments and test examples that the present invention studies the effect of apricot bee pollen phenolamine extract on obese mice, and the role of key intestinal microorganisms and related metabolites in regulating high-fat diet-induced obesity in mice by apricot bee pollen phenolamine extract. At the same time, the structural characteristics of phenolamine compounds in apricot bee pollen and its regulatory effect on the glucose and lipid metabolism of obese mice, as well as its effect on the intestinal microbiome and metabolites of obese mice are studied, which is conducive to promoting the application of bee pollen in the field of medicine and functional food.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A use of a bee pollen phenolamine extract in the preparation of a drug for preventing and treating obesity, wherein the preparation method of the bee pollen phenolamine extract comprises the following steps: The almond bee pollen is mixed with petroleum ether and subjected to a defatting treatment to obtain defatted bee pollen; The degreasing treatment is carried out under ultrasonic conditions, and the conditions of the degreasing treatment include: the number of degreasing treatments is 2 to 3 times; the material-liquid ratio of each degreasing treatment is 1 kg: (2 to 3) L; the time of each degreasing treatment is 30 to 40 minutes; the temperature of the degreasing treatment is 25 to 35° C.; the power of the ultrasound is 550 to 600 W; The defatted bee pollen is mixed with an ethanol aqueous solution with a volume fraction of 80-85%, and extracted, and the obtained extract is first concentrated to obtain a bee pollen alcohol extract; the extraction is carried out under ultrasonic conditions, and the extraction conditions include: the number of extractions is 2-3 times; the material-liquid ratio of each extraction is 1kg: (10-20) L; the time of each extraction is 30-40 minutes; the extraction temperature is 25-35°C; the ultrasonic power is 550-600W; The bee pollen alcohol extract is mixed with an extractant, extracted, and the obtained extract phase is sequentially concentrated and freeze-dried to obtain a bee pollen extract; the extractant is a mixture of water and ethyl acetate, and the volume ratio of water to ethyl acetate in the extractant is 1: (1-1.5); the volume ratio of the extractant to the bee pollen alcohol extract is (6-8): 1; The bee pollen extract is purified by passing through a silica gel column using a mixed solution of chloroform and methanol as an eluent, and vacuum freeze-dried to obtain a bee pollen phenolamine extract; the volume ratio of chloroform to methanol in the eluent is (80-85): (15-20); the silica gel in the silica gel column is SLG12S50, the particle size is 50 μm, and the specification of the silica gel column is 5 cm×48 cm; the volume of the silica gel in the silica gel column is 60 times the sample volume of the bee pollen extract during purification.

2. The use according to claim 1, characterized in that: The bee pollen phenolamine extract can reduce liver fat or epididymal fat.

3. The use according to claim 1, characterized in that: The bee pollen phenolamine extract can regulate blood lipids or liver function.

4. The use according to claim 1, characterized in that: The bee pollen phenolamine extract can regulate blood sugar.

5. The use according to claim 1, characterized in that: The bee pollen phenolamine extract can improve fasting blood sugar, glucose tolerance, insulin resistance, adiponectin level or leptin resistance.

6. The use according to claim 1, characterized in that: The bee pollen phenolamine extract can reduce oxidative stress damage or inflammatory damage caused by obesity.

7. Use of a bee pollen phenolamine extract in the preparation of a drug for regulating intestinal flora or intestinal metabolite imbalance caused by obesity, the preparation method of the bee pollen phenolamine extract comprising the following steps: The almond bee pollen is mixed with petroleum ether and subjected to a defatting treatment to obtain defatted bee pollen; The degreasing treatment is carried out under ultrasonic conditions, and the conditions of the degreasing treatment include: the number of degreasing treatments is 2 to 3 times; the material-liquid ratio of each degreasing treatment is 1 kg: (2 to 3) L; the time of each degreasing treatment is 30 to 40 minutes; the temperature of the degreasing treatment is 25 to 35° C.; the power of the ultrasound is 550 to 600 W; The defatted bee pollen is mixed with an ethanol aqueous solution with a volume fraction of 80-85%, and extracted, and the obtained extract is first concentrated to obtain a bee pollen alcohol extract; the extraction is carried out under ultrasonic conditions, and the extraction conditions include: the number of extractions is 2-3 times; the material-liquid ratio of each extraction is 1kg: (10-20) L; the time of each extraction is 30-40 minutes; the extraction temperature is 25-35°C; the ultrasonic power is 550-600W; The bee pollen alcohol extract is mixed with an extractant, extracted, and the obtained extract phase is sequentially concentrated and freeze-dried to obtain a bee pollen extract; the extractant is a mixture of water and ethyl acetate, and the volume ratio of water to ethyl acetate in the extractant is 1: (1-1.5); the volume ratio of the extractant to the bee pollen alcohol extract is (6-8): 1; The bee pollen extract is purified by passing through a silica gel column using a mixed solution of chloroform and methanol as an eluent, and vacuum freeze-dried to obtain a bee pollen phenolamine extract; the volume ratio of chloroform to methanol in the eluent is (80-85): (15-20); the silica gel in the silica gel column is SLG12S50, the particle size is 50 μm, and the specification of the silica gel column is 5 cm×48 cm; the volume of the silica gel in the silica gel column is 60 times the sample volume of the bee pollen extract during purification.

8. The use according to claim 7, characterized in that: The intestinal flora caused by obesity is regulated by upregulating beneficial bacteria and downregulating harmful bacteria; the beneficial bacteria are Bacteroidetes, Muribaculaceae and Parabacteroides One or more of the following; the harmful bacteria are Peptostreptococcaceae and / or Romboutsia ; The metabolites include one or more of bile acid, phosphorylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine and tyrosine.

9. The use according to any one of claims 1 to 8, characterized in that: The content of phenolamine compounds in the bee pollen phenolamine extract is 83-94%, and the phenolamine compounds in the bee pollen phenolamine extract include tricaffeoyl spermidine, dicaffeoyl p-coumaryl spermidine, di-p-coumaryl caffeoyl spermidine, tri-p-coumaryl spermidine and isomers of tri-p-coumaryl spermidine.

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