Application of hippophae rhamnoides fruit residue extract in preparation of medicine for treating diabetic complications

Abstract

The invention belongs to the technical field of hippophae rhamnoides fruit residue utilization, and particularly relates to application of hippophae rhamnoides fruit residue extract in preparation of a medicine for treating diabetic complications. Experiments find that the sea buckthorn pomace extract has a high inhibition rate on cholesterol esterase and alpha-amylase, and the sea buckthorn pomace extract mainly contains malvacic acid, aspartic acid, luteolin and vernonic acid, and key targets mainly include IL6, AKT1, TNF and PPARG, and the sea buckthorn pomace extract has a high inhibition rate on cholesterol esterase and alpha-amylase. The fructus hippophae pomace extract plays a role in regulating blood fat regulation, atherosclerosis, HIF-1 signal transduction, AGE-RAGE signal transduction and other pathways in diabetic complications.

Description

Technical Field

[0001] The invention belongs to the technical field of seabuckthorn pomace utilization, and particularly relates to application of seabuckthorn pomace extract in preparing medicine for treating diabetic complications. Background Art

[0002] Sea buckthorn ( Hippophae rhamnoides L Seabuckthorn is a perennial deciduous shrub or tree of the genus Hippophae rhamnoides in the family Hippophaeaceae. Seabuckthorn is a valuable plant that is both medicinal and edible. Its berries are rich in nutrients such as protein, amino acids, vitamins, trace elements, and polysaccharides, as well as bioactive substances such as flavonoids, steroids, proanthocyanidins, triterpenes, tannins, and serotonin.

[0003] Seabuckthorn is reported to have a variety of pharmacological effects, including antioxidant and immunomodulatory properties, cardioprotection and anti-atherosclerosis, antibacterial and antiviral effects, acute and chronic wound healing, and anti-radiation, anti-inflammatory, anti-cancer, and anti-dermatological properties. This has led to its widespread attention in the research and development of health supplements and foods. Seabuckthorn berries can be eaten directly or used to make juice. Seabuckthorn pomace is a byproduct of juicing.

[0004] With the continuous expansion of the seabuckthorn industry, a large amount of seabuckthorn pomace is discarded as waste, and only a small amount is added to animal feed, resulting in a huge waste of resources. Seabuckthorn pomace also contains a large number of bioactive substances, so further research on seabuckthorn pomace is needed to provide it with more application paths. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a use of a seabuckthorn pomace extract in the preparation of a medicine for treating diabetic complications.

[0006] The first aspect of the present invention provides the use of a seabuckthorn pomace extract in preparing a drug for treating diabetic complications, wherein the diabetic complications are at least one of elevated blood lipids and atherosclerosis; the seabuckthorn pomace extract is obtained by extracting seabuckthorn pomace with an organic solvent.

[0007] The present invention discovered through experiments that seabuckthorn pomace extract has a high inhibitory effect on both cholesterol esterase and α-amylase. Using network pharmacology analysis, it was found that the key components of seabuckthorn pomace extract that are anti-diabetic and anti-hyperlipidemia are malvaceae, aspartic acid, luteolin, and vernolic acid, and that key targets include IL6, AKT1, TNF, and PPARG. The seabuckthorn pomace extract exerts its effects by regulating multiple pathways involved in diabetic complications, including lipid regulation, atherosclerosis, HIF-1 signaling, and AGE-RAGE signaling. Molecular docking results indicate that luteolin has a strong affinity for the key targets.

[0008] In another preferred embodiment, the main active ingredients in the Hippophae rhamnoides L. pomace extract are malvalic acid, aspartic acid, chrysosplenetin and heleninic acid.

[0009] In another preferred embodiment, the Hippophae rhamnoides L. pomace extract is used to inhibit cholesteryl esterase and α-amylase.

[0010] In another preferred embodiment, the organic solvent is obtained by mixing ethyl acetate and ethanol solution at a volume ratio of 1:1-2. The ethanol solution can make the extract have strong α-amylase inhibitory activity, and the ethyl acetate can make the extract have strong cholesteryl esterase inhibitory activity.

[0011] In another preferred embodiment, the mass percentage concentration of the ethanol solution is 25%-75%.

[0012] In another preferred embodiment, the specific extraction process of the Hippophae rhamnoides L. pomace extract is as follows: After the Hippophae rhamnoides L. pomace is dried, it is crushed to obtain Hippophae rhamnoides L. pomace powder; The Hippophae rhamnoides L. pomace powder is mixed with an organic solvent at a solid-liquid ratio of 1:10-30, ultrasonic extraction, centrifugation, taking the supernatant, concentration, drying to obtain the Hippophae rhamnoides L. pomace extract; The organic solvent is obtained by mixing ethyl acetate and ethanol solution at a volume ratio of 1:1-2; The mass percentage concentration of the ethanol solution is 25%-75%.

[0013] In another preferred embodiment, the ultrasonic temperature is 30°C-70°C, the time is 15 min-75 min, and the power is 120w-360w.

[0014] In another preferred embodiment, the medicine is any one of oral preparations, injections and external preparations.

[0015] In another preferred embodiment, the oral preparation is any one of tablets, capsules, pills, powders, granules and syrup.

[0016] In another preferred embodiment, the injection is a solution or an emulsion.

[0017] Compared with the prior art, the present application has the following beneficial effects: The present application finds through experiments that the inhibition rates of seabuckthorn pomace extract on cholesterol esterase and alpha-amylase are 70.17%±1.05% and 52.06%±0.67% respectively, and through network pharmacology analysis, it is found that the key components of seabuckthorn pomace extract for resisting diabetes and hyperlipidemia mainly include malvaicine, aspartic acid, jacobine and helenalin, and the key targets mainly include IL6, AKT1, TNF and PPARG, and the seabuckthorn pomace extract plays a role by regulating multiple pathways such as blood lipid regulation, atherosclerosis, HIF-1 signal transduction and AGE-RAGE signal transduction in diabetic complications. The molecular docking result shows that jacobine has strong affinity with the key targets. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is the result graph of the influence of different solvent extracts on enzyme activity; wherein (a) is the inhibition result of alpha-amylase, (b) is the inhibition result of alpha-glucosidase, (c) is the inhibition result of pancreatic lipase, (d) is the inhibition result of cholesterol esterase, and (e) is the inhibition result of xanthine oxidase. In the graph, H2O represents the water extraction group, 25EtOH represents the 25wt% ethanol extraction group, 50MeOH represents the 50wt% methanol extraction group, 50EtOH represents the 50wt% ethanol extraction group, 75EtOH represents the 75wt% ethanol extraction group, MeOH represents the methanol extraction group, EtOH represents the ethanol extraction group, AC represents the acetone extraction group, EA represents ethyl acetate, n-BuoH represents the n-butanol extraction group, and Acarbose represents the acarbose extraction group.

[0019] Figure 2 It is the result graph of single factor test; wherein (a) is the result graph of solvent ratio, (b) is the result graph of solid-liquid ratio, (c) is the result graph of ultrasonic power, (d) is the result of extraction time, and (e) is the result graph of extraction temperature; in the graph, CE represents cholesterol esterase, alpha-Amy represents alpha-amylase, and SPE represents seabuckthorn pomace extract.

[0020] Figure 3 It is the three-dimensional response surface graph and the corresponding contour graph; wherein (a) is the three-dimensional response surface graph of solvent ratio and ultrasonic power, (b) is the three-dimensional response surface graph of solvent ratio and extraction time, (c) is the three-dimensional response surface graph of extraction time and three-dimensional response surface graph, (d) is the contour graph of (a), (e) is the contour graph of (b), and (f) is the contour graph of (c).

[0021] Figure 4 It is the Wayne graph of diabetes, hyperlipidemia and seabuckthorn pomace extract, wherein DM represents diabetes, HLP represents hyperlipidemia, and SPE represents seabuckthorn pomace extract.

[0022] Figure 5 Figure 8 is a protein interaction network diagram.

[0023] Figure 6 Figure 9 is a GO and KEGG enrichment analysis diagram; wherein (a) is a bubble diagram of GO analysis, and (b) is a KEGG pathway analysis diagram.

[0024] Figure 7 Figure 10 is a molecular docking diagram of active ingredients and targets; wherein (a) is a molecular docking diagram of syringaldehyde-IL6, (b) is a molecular docking diagram of syringaldehyde-AKT1, (c) is a molecular docking diagram of syringaldehyde-TNF, and (d) is a molecular docking diagram of syringaldehyde-PPARG. DETAILED DESCRIPTION

[0025] The technical solutions in the present application will be clearly and completely described in combination with the specific embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0026] The methods described in the embodiments of the present application are all conventional methods unless otherwise specified. The materials, reagents, etc. used are all commercially available unless otherwise specified.

[0027] 1. Experimental materials Hippophae rhamnoides L. was purchased from Aheqi County, Kizilsu Kyrgyz Autonomous Prefecture, Xinjiang. Hippophae rhamnoides L. pomace powder was obtained by drying the by-product after juicing, crushing, and passing through a 60-mesh sieve. The obtained Hippophae rhamnoides L. pomace powder was sealed and stored at -20℃ for standby.

[0028] Anhydrous ethanol, ethyl acetate, acetone, n-butanol, and methanol were all analytical pure, and acetonitrile was HPLC grade, which were purchased from National Pharmaceutical Group Chemical Reagent; Acarbose and cholesterol esterase were purchased from Beijing Solabio Science and Technology Co., Ltd.; α-amylase (pig pancreas), α-glucosidase, xanthine oxidase, DNS reagent, 4-nitrophenyl butyrate (p-NPB), p-nitrophenyl-α-D-glucopyranoside (p-NPG), and allopurinol were purchased from Shanghai Yuanye Biological Technology Co., Ltd.; Orlistat was purchased from Shanghai Aladdin Biochem Technology Co., Ltd.; Porcine pancreatic lipase was purchased from Beijing Bailingwei Technology Co., Ltd.

[0029] 2. Preparation of Hippophae rhamnoides L. pomace extract Ten different polar organic solvents were used to extract the active substances from seabuckthorn pomace by ultrasonic-assisted extraction. The ten organic solvents were as follows: water, 25wt% ethanol, 50wt% methanol, 50wt% ethanol, 75wt% ethanol, methanol, ethanol, acetone, ethyl acetate, and n-butanol. 10 g of seabuckthorn pomace powder was accurately weighed each time, mixed with the above-mentioned 10 solvents at a liquid-solid ratio of 20:1, and ultrasonically extracted at 50°C and 300w for 45 min. The supernatant was centrifuged at 4000r / min for 20 min, the filtrate was filtered, and the filtrate was concentrated under reduced pressure. Then, the filtrate was freeze-dried for 3 days using a freeze-drier under low vacuum to obtain 10 kinds of seabuckthorn pomace extracts extracted by different solvents, which were stored in a-20°C refrigerator.

[0030] 3. Evaluation of enzyme activity inhibition ability α-amylase inhibition rate determination: 100 μL of seabuckthorn pomace extract extracted by the above-mentioned 10 different solvents, 100 μL of 0.1 mol / L, pH 7 phosphate buffer, and 100 μL of 4 U / mL α-amylase solution were added to a 5 mL test tube, mixed well, incubated at 37°C for 10 min, added with 200 μL of 1wt% soluble starch solution, incubated at 37°C for 10 min, added with 0.5 mL of DNS reagent, and placed in a boiling water bath for 5 min. The test tube was cooled in an ice water bath for 5 min, and finally diluted with 1 mL of deionized water. After the experiment, 200 μL of the reaction solution was taken from each test tube and placed in a 96-well plate to measure the absorbance at 540 nm wavelength on an enzyme marker. Acarbose was used as a positive control, and the α-amylase inhibition rate was calculated.

[0031] α-glucosidase inhibition rate determination: 50 μL of seabuckthorn pomace extract extracted by the above-mentioned 10 different solvents, 50 μL of 0.05 mol / L, pH 6.8 phosphate buffer, and 50 μL of 2 U / mL α-glucosidase solution were added to a 96-well plate, mixed well, and incubated at 37°C for 10 min. 50 μL of 0.377 mol / L p-NPB was added, incubated at 37°C for 20 min, and finally the absorbance was measured at 405 nm wavelength. Acarbose was used as a positive control, and the α-glucosidase inhibition rate was calculated.

[0032] Determination of pancreatic lipase inhibition rate: 50 μL of seabuckthorn pomace extract extracted by the above 10 different solvents, 50 μL of 0.1 mol / L Tris-Hcl buffer solution, 50 μL of 100 U / mL porcine pancreatic lipase solution were added into a 96-well plate, incubated at 37℃ for 5 min, 50 μL of 0.5 mg / mL 4-nitrophenyl butyrate solution (5% DMSO solution) was added, incubated at 37℃ for 20 min, and finally the absorbance at 405 nm was measured. Olibutin was used as a positive control, and the pancreatic lipase inhibition rate was calculated.

[0033] Determination of cholesterol esterase inhibition rate: p-NPB was diluted with acetonitrile to a concentration of 4 mmol / L, 5.16 mmol / L sodium taurocholate and 0.1 mol / L sodium hydroxide PB buffer. 50 μL of seabuckthorn pomace extract extracted by the above 10 different solvents, 150 μL of PB buffer, 50 μL of 5 U / mL cholesterol esterase solution were added into a 96-well plate, mixed well, incubated at 25℃ for 5 min, added 20 μL PNPB solution, incubated at 25℃ for 25 min, and the absorbance at 405 nm was measured. Olibutin was used as a positive control, and the cholesterol esterase inhibition activity was calculated.

[0034] Determination of xanthine oxidase inhibition rate: 0.48 mmol / L xanthine solution was prepared using 0.2 mol / L PBS solution, pH 7.2, and 1 mol / L NaOH solution was added to aid dissolution. 50 μL of seabuckthorn pomace extract extracted by the above different solvents, 50 μL of 0.02 U / mL xanthine oxidase solution, 50 μL of PBS solution were added into a 96-well plate, incubated at 25℃ for 5 min, added 100 μL xanthine solution, incubated at 25℃ for 25 min, and the absorbance at 290 nm was measured. Allopurinol was used as a positive control, and the xanthine oxidase inhibition rate was calculated.

[0035] 4. Single factor experiment According to the screening, 50wt% ethanol extract has equivalent α-amylase activity inhibition ability to the positive control acarbose, and the ethyl acetate extract has stronger cholesterol esterase activity inhibition ability than the positive control olibutin. Therefore, 50wt% ethanol and ethyl acetate mixed solvent was used to extract active substances from seabuckthorn pomace, and the α-amylase and cholesterol esterase inhibition rate comprehensive score was used as an index. In the experiment, the factors investigated included solvent ratio 1~3:1~3, solid-liquid ratio 1g:10mL~30mL, extraction time 15min~75min, extraction temperature 30℃~70℃, and ultrasonic power 120℃~360w.

[0036] 5. Entropy weight assignment

[0037] 1) Standardization

[0038] The measured raw data of each index was standardized. Assuming there are m evaluation indexes and n evaluation objects, they will form a matrix A=(A ij )mn, which is standardized by matrix formula (1) to obtain B=(B ij )mn indexes. In this experiment, m=17 and n=2.

[0039]

[0040] In the formula, A ij represents the jth evaluation index in the ith experiment, where i=1, 2, 3, …, 17; j=1, 2;

[0041] B ij represents the jth index obtained after standardization in the ith experiment, where i=1, 2, 3, …, 17; j=1, 2.

[0042] 2) Probability calculation

[0043] The probability matrix P ij of each data in the raw number matrix A=(A ij )mn is calculated by formula (2), where P represents the probability of the jth evaluation index in the ith experiment, i=1, 2, 3, …, 17; j=1, 2; and P satisfies 0≤P≤1. 3) Information entropy algorithm The information entropy (H j ) of each evaluation index is calculated according to formula (3). In the formula, m=17; 6) Weight coefficient algorithm The weight coefficient (W j ) of each index is calculated by formula (4). According to formula (3), when the information entropy (H j ) is smaller, the weight coefficient (W j ) is larger. When the A ij values differ greatly, it means that the information amount of the index is large, and the weight coefficient is large. 1) Box-Behnken design On the basis of single factor experiment, the extraction process of seabuckthorn pomace was optimized by Design-Expert 13 software based on response surface methodology (RSM) with the comprehensive score of α-amylase and cholesterol esterase inhibitory rate as response value. The experimental factors and levels are shown in Table 1. Through the analysis of BBD-RSM experimental data, the optimal process conditions were obtained. Under these conditions, three validation experiments were carried out, and the data were compared with the predicted values of the related model.

[0044] Table 1 Factors and levels of Box-Behnken experimental design

[0045] 2) UPLC-Q-TOF / MS Analysis The optimal process extract of seabuckthorn pomace was analyzed by UPLC-Q-TOF / MS. The extract was dissolved and diluted to the appropriate concentration with acetonitrile, filtered through a filter membrane with a pore size of 0.25 μm, and placed in a 1.5 mL liquid phase bottle. An XBridge BEHC18 column (150 mm x 2.1 mm, 1.7 μm) was used with a column temperature of 45°C and a flow rate of 0.3 mL / min. The mobile phase A was acetonitrile, and the mobile phase B was 125 mmol formic acid. The gradient elution conditions were as follows: 0 min-10 min, mobile phase B; 10 min-18 min, mobile phase A to mobile phase B at a volume ratio of 2:8; 18 min-21 min, mobile phase A; 21 min-24 min, mobile phase B. The wavelength of the PDA detector was set to 800 nm. The mass spectrometry conditions were as follows: mass spectrometry mode ESI- and ESI+, ion source temperature 110°C, desolvation temperature 450°C, desolvation gas flow rate 800 L / h, cone hole gas flow rate 50 L / h, collision energy 4 eV, detector voltage 2000 V, and scanning m / z range 50 Da-1200 Da. 3) Network pharmacology analysis Screening of SPE active ingredients, diabetes and hyperlipidemia related targets The active ingredients and related targets were queried through TCMSP (https: / / old.tcmsp-e.com / tcmsp.php) and Swiss Target Prediction (http: / / swisstargetprediction.ch / ) databases. The gene targets of diabetes and hyperlipidemia were obtained and screened through GeneCards, OMIM, TTD and other databases. The UniProt (https: / / www.uniprot.org / ) database was used to convert the obtained targets into standard gene names. Subsequently, a Venn diagram was constructed to describe the targets related to SPE, diabetes and hyperlipidemia. The cross-targets were considered as potential targets for simultaneously treating diabetes and hyperlipidemia.

[0046] PPI protein network construction The potential target genes were input into the STRING database (https: / / string-db.org / ), and the PPI network was generated and drawn by Cytoscape 3.10.2. Then the CytoNCA plug-in was used to analyze three topological parameters, including degree, betweenness and closeness, to find important nodes.

[0047] GO function and KEGG enrichment analysis The metascape database was used to analyze the GO function and KEGG pathway enrichment of the core target points. GO function enrichment analysis was divided into three categories: cellular components (CC), molecular functions (MF) and biological processes (BP), and finally the enrichment bubble chart was constructed by microbioinformatics platform.

[0048] Molecular docking The top four key active ingredients of SPE were docked with the core target points IL6, AKT1, TNF and PPARG. The 3D structure of SPE active ingredients was obtained from the TCMSP database, and the 3D structure of the core target points was obtained from the PDB database. AutoDock Tool 1.5.7 was used for pretreatment of macromolecules and small molecules such as dehydration and hydrogenation, and molecular docking was performed. The binding energy of the optimal conformation of the target and components was used to evaluate the key target, and the docking results were visualized using PyMOL software.

[0049] 7、Results and discussion 1) Metabolic enzyme inhibition ability evaluation Alpha-glucosidase and alpha-amylase are closely related to diabetes mellitus, and the activity of alpha-glucosidase and alpha-amylase can be effectively regulated by inhibiting the activity of alpha-glucosidase and alpha-amylase. Figure 1 The a and b in Table 1 show the inhibition levels of the 10 different polar solvent seabuckthorn pomace extracts and the positive control acarbose on alpha-glucosidase and alpha-amylase, respectively. The vertical coordinate IC 50 value determines the inhibition ability of the extract on enzyme activity. It can be seen that the inhibition ability of the extract of strong polar solvent on alpha-glucosidase and alpha-amylase is significantly higher than that of weak polar solvent, indicating that strong polarity is helpful for the dissolution of substances in the pomace that inhibit the activity of alpha-glucosidase and alpha-amylase. The inhibition ability of the 10 solvent extracts on alpha-glucosidase is generally lower than that of acarbose, while the inhibition ability of the 50% ethanol solvent extract on alpha-amylase is slightly higher than that of acarbose. The IC 50 value of the 50% ethanol extract on alpha-amylase is 15.49±0.032, and the IC50 The value is 19.68±0.099. It is speculated that 50% ethanol solvent can effectively extract the substances in seabuckthorn pomace that inhibit the activity of α-amylase, and can be used for the development of related foods for regulating diabetes.

[0050] Pancreatic lipase and cholesterol esterase are metabolic enzymes closely related to hyperlipidemia, and the activity of pancreatic lipase and cholesterol esterase can be effectively regulated by inhibiting the activity of pancreatic lipase and cholesterol esterase. Figure 1 c and 1d respectively show the inhibition levels of 10 different polar solvent seabuckthorn pomace extracts and positive control orlistat on pancreatic lipase and cholesterol esterase, with the vertical coordinate IC 50 The value determines the inhibition ability of the extract on enzyme activity. It can be seen that the inhibition ability of the weak polar solvent extract on pancreatic lipase and cholesterol esterase is significantly higher than that of the strong polar solvent extract, indicating that weaker polarity is helpful for the dissolution of substances in the pomace that inhibit the activity of pancreatic lipase and cholesterol esterase. In pancreatic lipase, 10 kinds of solvent extracts have certain inhibitory effect, but generally lower than that of orlistat. In cholesterol esterase, the inhibition effect of weak polar solvent extract, especially ethyl acetate extract, is very obvious, and better than that of orlistat. The IC 50 value of ethyl acetate extract on cholesterol esterase is 1.08±0.052, and the IC 50 value of orlistat is 4.86±0.045. It is speculated that ethyl acetate solvent can effectively extract the substances in seabuckthorn pomace that inhibit the activity of cholesterol esterase, and can be used for the development of related foods for regulating hyperlipidemia.

[0051] Figure 1 e shows the inhibition effect of 10 different polar solvent seabuckthorn pomace extracts and positive control allopurinol on xanthine oxidase, which is a purine catabolism enzyme related to hyperuricemia and gout. It can be seen that the inhibition ability of strong polar solvent extract on xanthine oxidase is significantly higher than that of weak polar solvent extract, indicating that strong polarity is helpful for the dissolution of substances in the pomace that inhibit the activity of xanthine oxidase.

[0052] 2) Single factor experiment results In order to explore the effect of 50% ethanol and ethyl acetate solvent ratio on enzyme inhibition, experiments were carried out in the solvent ratio range of 1:3-3:1, and other parameters were kept unchanged. With the increase of solvent ratio, the inhibition rate of seabuckthorn pomace extract on α-amylase and cholesterol esterase showed a trend of first increasing and then decreasing, as shown in Figure 2The results show that the higher polarity solvent extract, such as 50% ethanol, has better α-amylase inhibitory capacity, while the lower polarity solvent extract, such as ethyl acetate, has better cholesterase inhibitory capacity. The cholesterase inhibitory rate reaches the maximum when the solvent ratio is 1:1. On the one hand, ethyl acetate extracts the main active substances, and on the other hand, 50% ethanol also extracts some substances that can inhibit the activity of cholesterase. The highest cholesterase inhibitory rate is obtained when the solvent ratio is 1:1. The α-amylase inhibitory rate is the maximum when the solvent ratio is 2:1 for the same reason. Considering the inhibitory effect of α-amylase and cholesterase, the solvent ratio of 2:1 is selected as the best single factor result.

[0053] In order to explore the effect of liquid-solid ratio on enzyme inhibition, experiments were conducted in the range of 10 mL / g~30 mL / g liquid-solid ratio, and other parameters were kept constant. With the increase of liquid-solid ratio, the inhibition rate of seabuckthorn pomace extract on α-amylase and cholesterase gradually increased and then decreased, as shown in Fig. 2b. Figure 2 The greater the liquid-solid ratio, the greater the contact area between the solvent and the solid sample, and the stronger the ultrasonic effect, which is beneficial to the dissolution of active substances. The α-amylase inhibitory rate reaches the maximum value when the liquid-solid ratio is 20 mL / g, and the cholesterase inhibitory rate reaches the maximum value when the liquid-solid ratio is 25 mL / g. With the continuous increase of liquid-solid ratio, the enzyme inhibition rate shows a downward trend, which may be due to the increase of cavitation effect, leading to the dissolution of more impurities. Considering the inhibitory effect of the two enzymes, 25 mL / g is selected as the best liquid-solid ratio.

[0054] In order to explore the effect of ultrasonic power on enzyme inhibition, experiments were conducted in the range of 120W-360W, and other parameters were kept constant. When the ultrasonic power changes from 120W to 180W, the inhibition rate of α-amylase and cholesterase begins to rise, reaches the maximum inhibition rate value at 180W, and then shows a downward trend Figure 2 as shown in Fig. 2c. The reason may be that the greater the power, the stronger the cavitation effect, the faster the active substance leaching rate, and the higher the enzyme inhibition rate. When the ultrasonic power increases to 240W, the power may be too high to destroy the molecular structure of the active substance, causing loss, resulting in a decrease in enzyme inhibition rate. Considering the inhibitory effect of the two enzymes, 180W is selected as the best ultrasonic power for extraction.

[0055] In order to explore the effect of extraction time on enzyme inhibition, experiments were conducted in the range of 15min~75min, and other parameters were kept constant. When the extraction time changes from 15min to 30min, the inhibition rate of α-amylase and cholesterase begins to rise, reaches the maximum inhibition rate value at 30min, as shown in Fig. 2d. Figure 2The inhibitory rate of cholesterol esterase decreased significantly after 45 min, and the inhibitory rate of α-amylase showed a slight downward trend but the change was not obvious. Ultrasonic time is an important factor affecting the extraction process. The cavitation effect generated by ultrasonic waves can break the cell wall of plant materials and accelerate the extraction of intracellular components. Compared with traditional solvent extraction, the use of ultrasonic-assisted extraction can reduce the extraction time and improve the recovery rate of compounds. In addition, long-term ultrasonic treatment can produce transient high temperature and high pressure, which can cause the decomposition of some compounds, which may be the reason for the decrease in enzyme inhibition rate. Therefore, 30 min is the optimal extraction time obtained from single-factor experiments.

[0056] To explore the effect of extraction temperature on enzyme inhibition, experiments were conducted at 30-70℃, and other parameters were kept constant. When the extraction temperature changed from 30℃ to 40℃, the inhibitory rates of α-amylase and cholesterol esterase began to rise, reaching the maximum inhibitory rate at 40℃, and then gradually decreasing, as shown in Fig. 2e. Increasing the extraction temperature reduces the viscosity of the solvent, increases molecular motion, and accelerates the mass transfer of intracellular substances. However, excessive temperature and strong cavitation effect can also cause the decomposition of some active substances, leading to a decrease in inhibition rate. Therefore, 40℃ is chosen as the optimal extraction temperature. Figure 2

[0057] 3)Entropy weight assignment The information entropy values and weight coefficients of each evaluation index are shown in Table 2.

[0058] Table 2 Calculation results of information entropy values and weight coefficients of each evaluation index 4)BBD RSM analysis The BBD experimental results of the comprehensive score of α-amylase and cholesterol esterase inhibition rate as the response value are listed in Table 3. Through the software Design-Expert 13, the data is subjected to multivariate quadratic regression and variance analysis, as shown in Table 4, and the multivariate quadratic regression equation is obtained as follows: comprehensive score TPC=62.66-2.71A-1.37B+2.27C+3.62AB-0.648AC+2.35BC-3.85A 2 -5.1B 2 -5.61C 2 In the quadratic polynomial regression model, P<0.0001, indicating that the model is significant and the regression effect is good. The linear coefficient, quadratic coefficient and cross coefficient AB, BC are significant (P<0.05), and only the interaction coefficient AC is not significant (P>0.05). The lack of fit value P=0.8428 is not significant, indicating that the model fitting effect is good. The R 2 =0.9907 of the model indicates that the model has good correlation and can predict the actual value of the independent variable within the experimental range. Adj R​2 =0.9788, Pre R 2 =0.9628, the difference between the two is less than 0.2, indicating that the quadratic model fits well and can completely explain the actual production process. The C.V. value is 1.51, indicating that the quadratic model is accurate and reliable. The signal-to-noise ratio of the model is 25.8736 (>4), which is also within a reasonable range. The above analysis shows that the established quadratic model is reasonable and reliable in statistics. According to the analysis of variance, the degree of influence of each factor on the comprehensive score is as follows: A > C > B.

[0059] Table 3 Results of Box-Behnken experimental design Table 4 Analysis of variance of Box-Behnken experimental design Note: "-" means not containing this item, the same below.

[0060] The interaction between each factor is analyzed by software Design-Expert 13 Figure 3 The greater the curvature of the response surface and the greater the slope, the greater the effect between the factor and the response value. As can be seen from the figure, the curve along the A axis and the curve along the C axis are steep, and both are steeper than the curve along the C axis, indicating that factors A and B have a greater effect on the comprehensive score, which is consistent with the results of the analysis of variance. The more the contour lines tend to be elliptical, the stronger the interaction between each factor, and the more the contour lines tend to be circular, the weaker the interaction between each factor.

[0061] The optimal extraction conditions fitted by Design-Expert 13 software are as follows: solvent ratio 1.507:1, extraction time 25.963 min, and ultrasonic power 190.46 W. According to the actual production conditions, the fitting conditions are modified as follows: solvent ratio 1.5:1, extraction time 26 min, and ultrasonic power 190 W, and the remaining parameters are as follows: liquid-solid ratio 25:1, and extraction temperature 40°C. Under these conditions, the predicted value of the comprehensive score is 63.71%, and the experimental value is (60.21±1.32)%. The actual value is very close to the predicted value of the quadratic model, with a relative error of 5.49%, which proves that the model is good in reliability.

[0062] 5) UPLC-Q-TOF / MS analysis The active substances identified initially are shown in Table 5.

[0063] Table 5 Active substances From Table 5, it can be seen that it can also be roughly divided into fatty acids, phenolic acids, flavonoids, tannins and other organic compounds. Fourteen kinds of fatty acids and their derivatives were identified, including saturated fatty acids (wax acid, heptadecanoic acid, palmitic acid, stearic acid and senecio acid) and unsaturated fatty acids (6-octadecenoic acid, malvalic acid, azelaic acid, ricinoleic acid, trichosanic acid, 8,11-octadecadienoic acid and γ-linolenic acid). Senecio acid is a monounsaturated fatty acid with a special epoxy structure, mainly exists in plants of the genus Senecio. Sun Meili

[110] found that the content of senecio acid in the extract of Senecio fruit was large, and proved by mouse experiment that the extract of 30% ethanol group could affect the insulin signaling pathway and had certain hypoglycemic effect. Azelaic acid is a naturally occurring saturated linear dicarboxylic acid, widely exists in cereals such as wheat, rye and barley, and is also an important chemical raw material and pharmaceutical ingredient. Trichosanic acid is a polyunsaturated fatty acid, mainly extracted from the seeds of Trichosanthes. Trichosanthes is a traditional Chinese medicinal material, widely used for treating respiratory diseases and inflammation, etc. Trichosanic acid is an important active ingredient in Trichosanthes, with multiple biological activities. γ-linolenic acid (GLA) is a polyunsaturated fatty acid belonging to the ω-6 fatty acid family, one of the essential fatty acids for human body, with important physiological functions and health benefits. GLA is famous for its multiple biological activities, including relieving diabetes and its complications, reducing blood lipids and preventing cardiovascular diseases. (E,E)-9-keto-10,12-octadecadienoic acid and α-hydroxy tetracosanoic acid belong to fatty acid derivatives. Six kinds of phenolic acids and their derivatives were preliminarily identified, including 2-hydroxy-5-butoxyphenylacetic acid, acetyl eugenol, ellagic acid, p-hydroxyphenylpropionic acid, cinnamic acid and cinnamic acid isobutyl ester.

[0064] Five kinds of flavonoids and their derivatives were preliminarily identified, including cyanidin, glabrol, kaempferol-3-glucoside-2''-p-coumaroyl ester, jacobinin and esculin-3-O-β-D-glucoside. Kaempferol-3-glucoside-2''-p-coumaroyl ester is a naturally occurring flavonoid compound, which is a complex compound formed by the connection of kaempferol (a flavonol), glucose and p-coumaric acid through glycosidic bond and ester bond. Esculin-3-O-β-D-glucoside is also a flavonoid compound, which is a glycoside compound formed by the connection of esculin and glucose through glycosidic bond, widely exists in certain plants, with multiple biological activities.

[0065] Tannins include procyanidin A2, corilagin A and chebulagic acid. A kind of chebulagic acid tannin was identified in SPE. Chebulagic acid is a hydrolysable ellagitannin belonging to polyphenols, mainly exists in Terminalia chebula plants. Chebulagic acid is composed of multiple gallic acid and six hydroxydiphenylbenzoyl units connected by ester bond, contains multiple phenolic hydroxyl groups, and has strong antioxidant activity. In addition, scirpusin B, malonic acid and tianshao acid substances were also identified.

[0066] 6) Network pharmacology results Target prediction results The TCMSP and Swiss Target Prediction databases were used to query 31 active substances in SPE and their 725 targets. Targets related to diabetes and hyperlipidemia were obtained through GeneCards, OMIM, and TTD databases. After screening and deduplication, 1380 diabetes-related targets and 1128 hyperlipidemia-related targets were obtained. The intersection of the 725 SPE active substance targets with the 1380 diabetes-related targets and the 1128 hyperlipidemia-related targets was taken to draw a Venn diagram ( Figure 4 ), 104 intersection targets were obtained as potential targets for SPE treatment of diabetes mellitus combined with hyperlipidemia.

[0067] Core ingredient screening The top four active ingredients with the highest degree values ​​in SPE were selected as ligands for molecular docking, as shown in Table 6. Among them, malvaccin and malvaccin are the core active ingredients of SPE. These active ingredients with higher degree values ​​may be the main active ingredients of SPE for preventing and treating diabetes and hyperlipidemia.

[0068] Table 6 Core active ingredients of SPE Potential targets were entered into the STRING database, and the PPI network diagram of potential targets was drawn using Cytoscape 3.10.2 ( Figure 5 The darker the target color, the larger the connecting node, and the more connections between targets, the stronger the synergy within the network and the more likely it is to be a core target for SPE. The average values ​​of the three topological parameters calculated using the CytoNCA plug-in were BC (69.87), CC (0.61), and DC (34.4). Parameter values ​​greater than the average were considered core targets, resulting in 24 core targets.

[0069] GO and KEGG enrichment analysis The Metascape database was used to perform GO function and KEGG pathway enrichment analysis on the screened core targets. GO analysis results showed that 872 potential target gene pathways were enriched, including 810 BPs, 20 CCs, and 42 MFs. The top 10 entries were plotted in a bubble chart as shown in the figure below. Figure 6KEGG pathway analysis showed that SPE could regulate 119 signaling pathways in the treatment of diabetes combined with hyperlipidemia. The results showed that SPE could play a role by regulating blood lipid regulation in diabetic complications, atherosclerosis, HIF-1 signaling and AGE-RAGE signaling pathways, etc. The top 20 pathways were taken to draw a bubble chart, as shown in Figure 6 . .

[0070] Molecular docking verification When the ligand and the receptor interact, it is generally believed that the binding energy less than-5 kcal / mol indicates good binding activity. The results are shown in Table 7. The docking binding energy between the key ingredient osimander and the target is less than-5 kcal / mol, indicating that the affinity between osimander and the key target is strong. Further, the docking results were visualized using PyMol software. As shown in Figure 7 , the key ingredients such as osimander mainly interact with different sites of each target to form hydrogen bonds. This indicates that these key ingredients can bind to different amino acid residues of the key target of the disease through hydrogen bonds, thereby playing a role in the treatment of hyperlipidemia and diabetes.

[0071] Table 7 Molecular docking binding energy (kcal / mol) of core active ingredients in SPE and core targets Ten seabuckthorn pomace extracts were prepared using organic solvents obtained by mixing ethyl acetate and ethanol in a volume ratio of 1:1-2 with ultrasonic assistance. Enzyme activity inhibition showed that the 50% ethanol extract had strong α-amylase inhibitory activity, and the ethyl acetate extract had strong cholesterol esterase inhibitory activity. Further optimization of the extraction process was carried out, and the optimal process conditions were as follows: the volume ratio of 50% ethanol and ethyl acetate was 1.5:1, the extraction time was 26 min, the ultrasonic power was 190 W, the liquid-solid ratio was 25:1, and the extraction temperature was 40°C. The UNIFI software identified the composition analysis results of the seabuckthorn pomace extract obtained under the optimal process, and SPE identified 31 compounds, mainly including fatty acids, flavonoids, phenolic acids and tannins. Network pharmacology analysis results showed that the core ingredients for reducing blood sugar and blood fat in SPE mainly included malvaicine, aspartic acid, osimander and senecioic acid, etc. The core target points had 24, mainly involving the pathways of lipid and atherosclerosis, insulin resistance, cancer pathogenesis and HIF-1 signaling pathway, etc. This provides relevant references for further studying the mechanism of SBPE in the treatment of hyperglycemia and hyperlipidemia.

[0072] The seabuckthorn pomace extract obtained under the optimal extraction conditions was used as an example to prepare a syrup. The syrup was obtained by mixing the syrup with excipients and solvents, wherein the excipients were common oral excipients such as sorbitol, high fructose syrup and glycerol, and the solvent was water.

[0073] The above-described embodiments are merely intended to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements of the present application made by those skilled in the art based on the above-described embodiments should fall within the scope of the present application defined by the claims.

Claims

1. Use of seabuckthorn pomace extract in preparing a medicament for treating diabetic complications, characterized in that: The diabetic complication is at least one of elevated blood lipids and atherosclerosis; The seabuckthorn pomace extract is obtained by extracting the seabuckthorn pomace with an organic solvent.

2. The use according to claim 1, characterized in that The main active ingredients in the seabuckthorn pomace extract are malvalic acid, aspartic acid, luteolin and vernolic acid.

3. The use according to claim 1, characterized in that The seabuckthorn pomace extract is used for inhibiting the activities of cholesterol esterase and alpha-amylase.

4. The use according to claim 1, characterized in that The organic solvent is obtained by mixing ethyl acetate and ethanol solution in a volume ratio of 1:1-2.

5. The use according to claim 4, characterized in that The mass percentage concentration of the ethanol solution is 25% to 75%.

6. The use according to claim 1, characterized in that The specific extraction process of the seabuckthorn pomace extract is as follows: drying the seabuckthorn pomace and then crushing it to obtain seabuckthorn pomace powder; The seabuckthorn pomace powder is mixed with an organic solvent at a solid-liquid ratio of 1:10-30, ultrasonically extracted, centrifuged, the supernatant is collected, concentrated, and dried to obtain the seabuckthorn pomace extract; The organic solvent is obtained by mixing ethyl acetate and ethanol solution in a volume ratio of 1:1-2; The mass percentage concentration of the ethanol solution is 25% to 75%.

7. The use according to claim 6, characterized in that The temperature of the ultrasound is 30° C. to 70° C., the time is 15 min to 75 min, and the power is 120 W to 360 W.

8. The use according to claim 1, characterized in that The medicine is any one of an oral preparation, an injection and an external preparation.

9. The use according to claim 8, characterized in that The oral preparation is any one of tablets, capsules, pills, powders, granules and syrups.

10. The use according to claim 9, characterized in that The injection is a solution or emulsion.

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