Application of hippophae rhamnoides leaf extract in preparation of medicine for reducing blood sugar and blood lipid

Abstract

The invention belongs to the technical field of hippophae rhamnoides leaf utilization, and particularly relates to application of a hippophae rhamnoides leaf extract in preparation of a medicine for reducing blood sugar and blood lipid. Experiments find that 33 compounds, mainly including fatty acid compounds, flavonoid compounds, phenolic acid compounds and tannin compounds, are identified in the hippophae rhamnoides leaf extract obtained through double-solvent extraction. Network pharmacological analysis results show that core components for reducing blood sugar and lipid in the SLE mainly comprise crowndaisy chrysanthemum acid, malvacic acid, aspartic acid and 6-octadecenoic acid, the number of acting core targets is 43, and main involved channels comprise lipid and atherosclerosis, a PI3K-Akt signal channel, an AGE-RAGE signal channel in diabetic complications and an HIF-1 signal channel.

Description

Technical Field

[0001] The invention belongs to the technical field of seabuckthorn leaf utilization, and particularly relates to application of seabuckthorn leaf extract in preparing blood sugar and lipid lowering medicines. Background Art

[0002] Sea buckthorn berries are high in vitamins, earning them the nickname "King of Vitamin C." They are also rich in over 200 bioactive substances beneficial to the human body, making them a promising resource for applications in medicine and healthcare. Sea buckthorn leaves are narrowly lanceolate or oblong-lanceolate, 30 to 80 mm long and 4 to 10 mm wide. They are dark green on the surface and silvery-gray on the underside. They are the primary byproduct of harvesting sea buckthorn berries. Compared to sea buckthorn berries, sea buckthorn leaves offer advantages such as greater biomass, a longer growth cycle, and ease of harvesting and storage.

[0003] Currently, the main uses of seabuckthorn leaves include making seabuckthorn leaf tea, adding it to feed, and extracting total flavonoids. Extraction of beneficial components is a major research focus. Currently, research on the active ingredients in seabuckthorn leaves primarily focuses on flavonoids, with the extracted flavonoids typically used for antioxidant and anti-tumor benefits. Flavonoids generally possess antioxidant and anti-tumor properties. However, seabuckthorn leaves contain numerous other active ingredients besides flavonoids, and different extraction methods yield different active ingredients. Therefore, further research on seabuckthorn leaf extracts is needed to explore new applications for these extracts. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides the use of seabuckthorn leaf extract in the preparation of a drug for lowering blood sugar and lipids.

[0005] The first aspect of the present invention provides the use of a seabuckthorn leaf extract in the preparation of a drug for lowering blood sugar and lipids, wherein the seabuckthorn leaf extract is obtained by ultrasonic extraction of a mixed reagent obtained by mixing methanol and acetone in a volume ratio of 1:1 to 5;

[0006] The lipid-lowering refers to lowering cholesterol and triglycerides, and the sugar-lowering refers to lowering blood sugar.

[0007] The present invention experimentally discovered that 33 compounds were identified in the seabuckthorn leaf extract obtained by extracting seabuckthorn leaves using a dual solvent consisting of methanol and acetone in a volume ratio of 1:1 to 5. These compounds primarily included fatty acids, flavonoids, phenolic acids, and tannins. Further network pharmacology analysis revealed that the core components of the seabuckthorn leaf extract that lower blood sugar and lipids are chrysanthemum acid, malvaceaic acid, aspartic acid, and 6-octadecenoic acid. The extract also targets 43 pathways, primarily involved in lipid metabolism and atherosclerosis, the PI3K-Akt signaling pathway, the AGE-RAGE signaling pathway in diabetic complications, and the HIF-1 signaling pathway.

[0008] In another preferred embodiment, the effective ingredients in the seabuckthorn leaf extract are chrysanthemum acid, malvaceae acid, aspartic acid and 6-octadecenoic acid.

[0009] In another preferred embodiment, the specific extraction process of the seabuckthorn leaf extract is as follows:

[0010] After the sea buckthorn leaves are crushed, sea buckthorn leaf powder is obtained;

[0011] Methanol and acetone are mixed in a volume ratio of 1:1-5 to obtain a mixed reagent, sea buckthorn leaf powder and the mixed reagent are mixed in a material-liquid ratio of 1:10-30, ultrasonic extraction is performed at 30°C-70°C and 120W-360W for 15min-75min, the mixture is centrifuged, the supernatant is collected, concentrated, and dried to obtain the sea buckthorn leaf extract.

[0012] In another preferred embodiment, the drug is any one of an oral preparation, an injection and an external preparation.

[0013] In another preferred embodiment, the oral preparation is any one of tablets, capsules, pills, powders, granules and syrups.

[0014] In another preferred embodiment, the injection is a solution or emulsion.

[0015] The second aspect of the present invention provides a drug for alleviating hyperlipidemia, which contains the seabuckthorn leaf extract as the only active ingredient.

[0016] The third aspect of the present invention provides a drug for alleviating diabetes, which contains the seabuckthorn leaf extract as the only active ingredient.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention experimentally discovered that 33 compounds were identified in seabuckthorn leaf extract obtained by dual-solvent extraction using a mixture of methanol and acetone in a volume ratio of 1:1 to 5, primarily including fatty acids, flavonoids, phenolic acids, and tannins. Network pharmacology analysis revealed that the core components of SLE that lower blood sugar and lipids are primarily chrysanthemum acid, malvaceaic acid, aspartic acid, and 6-octadecenoic acid. There are 43 core targets, primarily implicated in pathways such as lipids and atherosclerosis, the PI3K-Akt signaling pathway, the AGE-RAGE signaling pathway in diabetic complications, and the HIF-1 signaling pathway. Molecular docking results indicate that luteolin has a strong affinity for key targets. By selecting specific extraction reagents, the present invention enables the targeted extraction of active ingredients from seabuckthorn leaves that have a positive effect on lowering blood sugar and lipids, thereby increasing the utilization rate of seabuckthorn leaves and providing a new application for seabuckthorn leaf extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of the effects of different solvent extracts on enzyme activity are shown in the figure; among them, (a) is the inhibition result of α-amylase, (b) is the inhibition result of α-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 figure, α-Amy represents α-amylase, α-Glu represents glucosidase, PL represents pancreatic lipase, CE represents cholesterol esterase, and XO represents xanthine oxidase; 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 the ethyl acetate, n-BuoH represents the n-butanol extraction group, and Acarbose represents the acarbose extraction group.

[0020] Figure 2 The results of different extraction solvent ratios are shown in Figure 2.

[0021] Figure 3 The results of different material-liquid ratios are shown in the figure.

[0022] Figure 4 The results of different ultrasonic powers are shown in Figure 2.

[0023] Figure 5 The results of different extraction times are shown in Figure 2.

[0024] Figure 6 The results of different extraction temperatures are shown in Figure 2.

[0025] Figure 7are three-dimensional response surface plots and corresponding contour plots; (a) is a three-dimensional response surface plot of solvent ratio and ultrasonic power, (b) is a three-dimensional response surface plot of solvent ratio and extraction time, (c) is a three-dimensional response surface plot of extraction time and three-dimensional response surface plot, (d) is the contour plot of (a), (e) is the contour plot of (b), and (f) is the contour plot of (c).

[0026] Figure 8 This is a Venn diagram of diabetes, hyperlipidemia and seabuckthorn leaf extract, where DM represents diabetes, HLP represents hyperlipidemia, and SLE represents seabuckthorn leaf extract.

[0027] Figure 9 A protein interaction network diagram.

[0028] Figure 10 These are the enrichment analysis diagrams of GO and KEGG; among them, (a) is the bubble diagram of GO analysis, and (b) is the KEGG pathway analysis diagram.

[0029] Figure 11 Molecular docking diagrams of active ingredients and targets; among them, (a) is the molecular docking diagram of luteolin-IL6, (b) is the molecular docking diagram of luteolin-AKT1, (c) is the molecular docking diagram of luteolin-TNF, and (d) is the molecular docking diagram of luteolin-PPARG. DETAILED DESCRIPTION

[0030] The technical solutions of the present invention will be clearly and completely described in conjunction with the specific embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0031] Unless otherwise specified, the methods described in the embodiments of the present invention are conventional methods. The materials and reagents used are all commercially available unless otherwise specified.

[0032] 1. Experimental Materials

[0033] Sea buckthorn was purchased from Aheqi County, Kizilsu Kirgiz Autonomous Prefecture, Xinjiang. The sea buckthorn leaf powder was obtained by drying and crushing the sea buckthorn leaves, and then passing them through a 60-mesh sieve. The obtained sea buckthorn leaf powder was sealed and stored at -20°C for later use.

[0034] Anhydrous ethanol, ethyl acetate, acetone, n-butanol, and methanol were all of analytical grade, and acetonitrile was of HPLC grade and purchased from Sinopharm Chemical Reagents. Acarbose and cholesterol esterase were purchased from Beijing Solaibao Technology Co., Ltd. α-amylase (porcine pancreas), α-glucosidase, xanthine oxidase, DNS reagent, 4-nitrophenyl butyrate (p-NPB), p-nitrophenyl-α-D-glucopyranoside (p-NPG), allopurinol, metformin hydrochloride (Met), 4% paraformaldehyde, D(+)-anhydrous glucose, and normal saline were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Orlistat was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Porcine pancreatic lipase was purchased from Beijing Bailingwei Technology Co., Ltd. IL-6, IL-1β, TNF-α, and FINS ELISA kits were purchased from Thermo Fisher Scientific.

[0035] 2. Preparation of Seabuckthorn Leaf Extract

[0036] Ten organic solvents of varying polarity were used in conjunction with ultrasound-assisted extraction of active substances from seabuckthorn leaves. The ten organic solvents were as follows: water (H2O), 25% (v / v) ethanol, 50% (v / v) methanol, 50% (v / v) ethanol, 75% (v / v) ethanol, methanol, ethanol, acetone, ethyl acetate, and n-butanol (in descending order of polarity). Accurately weighing 10 g of seabuckthorn leaves at each time, the solvents were mixed with each of the ten solvents at a solid-liquid ratio of 1:20. Ultrasonic extraction was performed at 50°C for 45 minutes at an ultrasonic power of 300w. The extract was then centrifuged at 4000 rpm for 20 minutes. The supernatant was filtered, the filtrate was concentrated under reduced pressure, and then freeze-dried in a freeze dryer under low vacuum for 3 days to obtain a solid seabuckthorn leaf extract, designated SLE, which was stored in a -20°C refrigerator.

[0037] 3. Evaluation of enzyme activity inhibition ability

[0038] α-Amy and α-Glu are the main enzymes that hydrolyze ingested carbohydrates into glucose. Therefore, inhibiting the activity of starch-digesting enzymes in the gastrointestinal tract can significantly reduce the hydrolysis and absorption of carbohydrates, thereby lowering postprandial blood glucose levels. Pancreatic lipase (PL) is a key enzyme in the digestion of hypertriglyceridemia, catalyzing the decomposition of hypertriglyceridemia into monoacylglycerols and free fatty acids, promoting their absorption. Cholesterol esterase (CE) is a key enzyme in cholesterol absorption, responsible for hydrolyzing cholesterol esters into free cholesterol, enabling intestinal absorption. Xanthine oxidase (XO) is a purine catabolism enzyme associated with hyperuricemia and gout. XO inhibition is an important means of evaluating the anti-hyperuricemic effects of natural products. Therefore, by separately inhibiting α-Amy, α-Glu, PL, CE, and XO, a seabuckthorn leaf extract with excellent lipid-lowering and blood glucose-lowering effects was obtained.

[0039] 1) Inhibitory ability against α-Amy

[0040] In a 5 ml test tube, add 100 μL of samples with different gradient mass concentrations, 100 μL of 0.1 mol / L phosphate buffer (PBS, pH = 7), and the newly prepared 100 μL 4 U / mL α-Amy solution, mix well, incubate at 37 ° C for 10 minutes, add 200 μL of 1% soluble starch solution, incubate at 37 ° C for 10 minutes, add 0.5 mL of DNS reagent, place the test tube in a boiling water bath for 5 minutes, cool in an ice water bath for 5 minutes, and finally add 1 mL of deionized water for dilution. After the experiment, take 200 μL of the reaction solution from each test tube and place it in a 96-well plate to measure the absorbance at a wavelength of 540 nm on a microplate reader. Acarbose (Acarbose) was used as a positive control. The α-Amy inhibition rate was calculated according to formula (1):

[0041]

[0042] Wherein, A represents the absorbance value of the group with enzyme and sample; B represents the absorbance value of the group with sample without enzyme; C represents the absorbance value of the group with enzyme but no sample; D represents the absorbance value of the group with no enzyme and no sample.

[0043] 2) Inhibitory ability against α-Glu

[0044] Add 50 μL of samples with different mass concentration gradients, 50 μL of 0.05 mol / L phosphate buffer (PBS, pH = 6.8) and 50 μL of 2 U / mL α-Glu solution into a 96-well plate, mix well and incubate at 37°C for 10 min, add 50 μL of 0.377 mol / L p-NPG (both p-NPG and enzyme solution are prepared with PBS solution), incubate at 37°C for 20 min, and finally measure the absorbance at a wavelength of 405 nm. Acarbose is used as a positive control.

[0045] 3) PL inhibition ability

[0046] 50 μL of samples with different mass concentration gradients, 50 μL of 0.1 mol / L Tris-HCl buffer solution (pH = 8.2) and 50 μL of 100 U / mL PL solution were added to a 96-well plate and incubated at 37°C for 5 min. 50 μL of 0.5 mg / mL P-npb solution (prepared with 5 wt% DMSO solution) was added and incubated at 37°C for 20 min. Finally, the absorbance at 405 nm was measured, and orlistat was used as a positive control.

[0047] 4) CE inhibition ability

[0048] A 4 mmol / L p-NPB solution was prepared in acetonitrile, followed by a buffer solution containing sodium taurocholate at a final concentration of 5.16 mmol / L and sodium hydroxide at a final concentration of 0.1 mol / L. To a 96-well plate, 50 μL of sample with varying concentrations, 150 μL of buffer, and 50 μL of 5 U / mL CE solution were added and mixed thoroughly. The plates were incubated at 25°C for 5 minutes. Then, 20 μL of p-NPB solution was added and incubated at 25°C for 25 minutes. The absorbance at 405 nm was measured. Orlistat was used as a positive control.

[0049] 5) XO's inhibitory ability

[0050] A 0.48 mmol / L xanthine solution was prepared using PBS (0.2 M, pH = 7.2). A small amount of 1 mol / L NaOH solution was added to aid dissolution. 50 μL of samples with varying concentrations, 50 μL of 0.02 U / mL XO solution, and 50 μL of PBS solution were added to a 96-well plate. The plates were incubated at 25°C for 5 minutes. After adding 100 μL of xanthine solution and incubating at 25°C for 25 minutes, the absorbance at 290 nm was measured. Allopurinol was used as a positive control, and the XO inhibition rate was calculated according to formula (1).

[0051] 4. Single-factor experiment

[0052] A mixed solvent of methanol and acetone was used to extract the active substances from seabuckthorn leaves, with α-Amy and CE inhibition rates used as evaluation indicators. The factors investigated in the single-factor seabuckthorn leaf extraction experiment included solvent ratio (1:5-5:1), solid-liquid ratio (1:10 g / mL-1:30 g / mL), extraction time (15-75 min), extraction temperature (30-70°C), and ultrasonic power (120-360 W).

[0053] 5. Entropy weight assignment

[0054] 1) Standardization

[0055] The raw data of each measured indicator is standardized. Assuming there are m evaluation indicators and n evaluation objects, they form a matrix A = (Aij)mn. After standardization using matrix formula 2-2, the resulting indicators are B = (Bij)mn. In this experiment, m = 17 and n = 2.

[0056]

[0057] Where Aij represents the jth evaluation index in the i-th experiment, where i = 1, 2, 3…17, j = 1, 2.

[0058] 2) Probability calculation

[0059] Use formula (2) to find the original number matrix A=(A ij )The probability matrix P of each data in mn ij , where P represents the probability of the jth evaluation indicator in the i-th experiment, i = 1, 2, 3, …, 17; j = 1, 2; and P satisfies 0≤P≤1.

[0060]

[0061] 3) Information entropy algorithm

[0062] According to formula (3), the information entropy (H j ).

[0063] Where m = 17;

[0064] 4) Weight coefficient algorithm

[0065] The weight coefficients of each indicator (W j ).

[0066]

[0067] From formula (3), we can see that when the information entropy (H j ) is smaller, the weight coefficient (W j ) is larger, when A ij When the numerical values ​​differ greatly, it means that the sub-indicator conveys a large amount of information and has a large weight coefficient.

[0068] 5) Box-Behnken design

[0069] Based on single-factor experiments, a three-factor, three-level response surface experiment was designed using Design-Expert 13 software to optimize the seabuckthorn leaf extraction process, using the combined score of α-amylase and cholesterol esterase inhibition rates as the response value. The experimental factors and levels are shown in Table 1. The optimal process conditions were determined by analyzing the experimental data using BBD-RSM. Three validation experiments were conducted under these conditions, and the data were compared with the predictions of the relevant model.

[0070] Table 1 Factors and levels of Box-Behnken experimental design

[0071]

[0072] 6) UPLC QTOF / MS

[0073] UPLC-Q-TOF / MS was used to analyze the components of the optimized seabuckthorn leaf extract. The extract was dissolved in acetonitrile and diluted to the appropriate concentration. The extract was passed through a 0.25 μm pore size filter and placed in a 1.5 mL liquid phase vial. An XBridge BEHC18 column (150 mm × 2.1 mm, 1.7 μm) was used, with a column temperature of 45°C and a mobile phase flow rate of 0.3 mL / min. Mobile phase A consisted of acetonitrile and mobile phase B consisted of 125 mmol formic acid. The gradient elution conditions were: 100% B from 0 min to 10 min; 20% A + 80% B from 10 min to 18 min; 100% A from 18 min to 21 min; and 100% B from 21 min to 24 min. The PDA detector wavelength was set to 800 nm. Mass spectrometry conditions: mass spectrometry mode ESI- and ESI+, ion source temperature 110°C, desolvation temperature 450°C, desolvation gas flow rate 800 L / h, cone gas flow rate 50 L / h, collision energy 4 eV, detector voltage 2000 V, scanning m / z range 50 Da to 1200 Da.

[0074] 7) Network pharmacology analysis

[0075] Screening of active ingredients in SLE, diabetes and hyperlipidemia-related targets

[0076] Active ingredients and related targets of SLE were searched using databases such as TCMSP (https: / / old.tcmsp-e.com / tcmsp.php) and Swiss Target Prediction (http: / / swisstargetprediction.ch / ). Gene targets for diabetes and hyperlipidemia were obtained and screened using databases such as GeneCards, OMIM, and TTD. The resulting targets were converted to standard gene names using the UniProt database (https: / / www.uniprot.org / ). Subsequently, a Venn diagram was constructed to depict the SLE targets associated with diabetes and hyperlipidemia, respectively. Cross-talking targets were considered potential targets for the simultaneous treatment of diabetes and hyperlipidemia.

[0077] Screening of core active ingredients in SLE

[0078] The complex relationships between the active ingredients and cross-targets of SLE were imported into Cytoscape 3.10.2 software. Using the "Network Analyzer" add-on feature within Cytoscape, the degree importance of the ingredients and targets was evaluated. A larger degree value indicates a more important role played by the node in the network. Core ingredients were selected based on the degree values ​​as ligands for molecular docking.

[0079] Constructing protein-protein interaction (PPI) networks

[0080] Potential anti-diabetic and hyperlipidemia target genes for SLE were entered into the STRING database (https: / / string-db.org / ) to obtain protein interaction relationships. These relationships were then imported into Cytoscape 3.10.2 software to construct a PPI network. The CytoNCA plug-in was then used to analyze three topological parameters: degree, betweenness, and proximity, to identify important nodes and screen for core targets.

[0081] GO and KEGG enrichment analysis

[0082] The core targets of SLE and SPE were analyzed for GO functional and KEGG pathway enrichment using the Metascape database (https: / / metascape.org / ). GO functional enrichment analysis was categorized into three categories: cellular component (CC), molecular function (MF), and biological process (BP). Finally, GO and KEGG enrichment visualization was performed using the Microbiology Platform (https: / / www.bioinformatics.com.cn / ).

[0083] Molecular docking

[0084] Molecular docking was performed between the core components of SLE, which are key to preventing diabetes and hyperlipidemia, and the core targets. The 3D structures of the active components of SLE were obtained from the TCMSP and PubChem databases, and the 3D structures of the core targets were obtained from the PDB database. The macromolecules and small molecules were pre-processed using AutoDock Tool 1.5.7, including dehydration and hydrogenation, and then molecular docking was performed. The key targets were evaluated using the binding energy of the optimal conformation of the target and the component. The docking results were visualized using PyMOL software.

[0085] 6. Results and Discussion

[0086] 1) Evaluation of metabolic enzyme inhibition ability

[0087] Evaluation of the metabolic enzyme inhibitory ability of seabuckthorn leaf extract

[0088] Figure 1 The inhibitory ability of SLE in 10 solvents of different polarity on 5 metabolic enzymes is shown, and the IC50 value on the vertical axis is used to judge the inhibitory ability of the extract on enzyme activity. Among them, α-Glu and α-Amy are metabolic enzymes closely related to sugar metabolism. By inhibiting the activity of α-Glu and α-Amy, blood sugar levels can be effectively regulated. Figure 1 a and Figure 1 As shown in Figure 2b, the inhibitory abilities of methanol, ethanol and acetone extracts on α-Amy were strong and significantly higher than those of the positive control acarbose (IC50 = 19.68 ± 0.20 mg / mL). The methanol extract (IC50 = 1.59 ± 0.16 mg / mL) had the best inhibitory effect. It is speculated that methanol solvent can effectively extract substances that inhibit the activity of α-Amy from sea buckthorn leaves, while the 10 SLEs had weak inhibitory abilities on α-Glu, and generally did not have as good an inhibitory effect as acarbose.

[0089] PL and CE are metabolic enzymes closely related to hyperlipidemia. Inhibiting the activity of PL and CE can effectively regulate lipid metabolism. Figure 1 c and Figure 1 As shown in Figure d, the inhibitory effects of the 10 SLEs on PL were weak, significantly lower than that of the positive control orlistat, and there was no obvious pattern in the inhibitory effect of solvent polarity on PL. In the CE inhibition ability evaluation, acetone (IC50 = 8.14 ± 0.43 mg / mL) and n-butanol (IC50 = 5.15 ± 0.10 mg / mL) showed strong inhibitory effects, and their IC50 values ​​were close to that of orlistat (IC50 = 4.86 ± 0.09 mg / mL).

[0090] XO is a purine catabolism enzyme associated with hyperuricemia and gout. XO inhibition ability is an important means to evaluate the anti-hyperuricemia effect of natural products. Figure 1 e shows the inhibitory effects of 10 solvents SLE with different polarity and the positive control allopurinol on XO. The solvent extracts with stronger polarity have better inhibitory effects on XO, but the inhibitory activities of the 10 SLEs on XO are generally lower than that of allopurinol.

[0091] In summary, the methanol extract has strong α-Amy inhibitory activity, and the acetone and n-butanol extracts have strong CE inhibitory activity. Considering the poor removal effect of n-butanol solvent, methanol and acetone mixed solvent will be used to extract sea buckthorn leaves to prepare SLE that simultaneously inhibits α-Amy and CE activity.

[0092] 2) Analysis of single-factor experimental results

[0093] Effect of solvent ratio on the inhibitory ability of SLE enzyme activity

[0094] In order to explore the effect of SLE solvent ratio on the inhibition rate of α-Amy and CE ( Figure 2 ), experiments were conducted in the ranges of 1:5 to 5:1 and 1:3 to 3:1, while keeping other parameters constant. Changing the solvent ratio significantly affects solvent polarity and solubility, which in turn affects the dissolution and extraction efficiency of the target component, as well as the amount of impurities extracted. Figure 2 The effect of the methanol / acetone solvent ratio in SLE on the inhibition rates of α-Amy and CE is shown in the figure. As shown, when the solvent ratio was 1:3, SLE had the highest inhibition rate against α-Amy, reaching (53.60±1.71)%. When the solvent ratio was 1:1, SLE had the highest inhibition rate against CE, reaching (57.78±1.69)%. When extracting seabuckthorn leaves, the methanol solvent extract exhibited strong α-Amy inhibitory activity and some CE inhibitory activity, while the acetone solvent extract exhibited strong CE inhibitory activity and some α-Amy inhibitory activity. As the solvent ratio of methanol to acetone gradually increased, the inhibition rates of α-Amy and CE both increased and then decreased. However, when the solvent ratio decreased from 1:3 to 1:1, i.e., when the amount of methanol decreased and the amount of acetone increased, the inhibition rate of α-Amy decreased, while the inhibition rate of CE increased. This may be because the effects of methanol and acetone differed at different solvent ratios. Taking all factors into consideration, a solvent ratio of methanol to acetone of 1:3 was considered to be the optimal solvent ratio for SLE with strong α-Amy and CE inhibitory activities.

[0095] Effect of solid-liquid ratio on the inhibitory ability of SLE enzyme activity

[0096] In order to explore the effect of the solid-liquid ratio of SLE on the inhibition rate of α-Amy and CE ( Figure 3), experiments were conducted in the range of 1:10 to 1:30, and other parameters were kept unchanged. Figure 3 The effect of the solid-liquid ratio of SLE on the inhibition rates of α-Amy and CE was shown. From a solid-liquid ratio of 1:10 to 1:20, the inhibition rates of α-Amy and CE gradually increased, reaching their maximum values ​​at a solid-liquid ratio of 1:20. At this point, the inhibition rate of α-Amy was (47.89±0.42)%, and the inhibition rate of CE was (58.60±0.16)%. At lower solid-liquid ratios, increasing the solid-liquid ratio increased the contact area between the solid and solvent, promoting solute diffusion from the solid to the solvent. Furthermore, the cavitation effect and mechanical vibration of ultrasound accelerated solvent penetration through the cell wall, promoting solute release. When the solid-liquid ratio exceeded 1:20, the inhibition rates of α-Amy and CE slowly decreased. This may be because an excessively high solid-liquid ratio leads to the precipitation of certain impurities and the physical effects of ultrasound may be diluted by the excess solvent, resulting in a weakened cavitation effect. Taking all these factors into consideration, a solid-liquid ratio of 1:20 is considered the optimal solid-liquid ratio for SLE with strong inhibitory activity for both α-Amy and CE.

[0097] Effect of ultrasound power on the inhibitory ability of SLE enzyme activity

[0098] In order to explore the effect of ultrasound power on the inhibition rate of α-Amy and CE in SLE ( Figure 4 ), experiments were conducted in the range of 120W to 360W, and other parameters were kept unchanged. Figure 4 The effect of ultrasound power on the inhibition of α-Amy and CE in SLE was shown. Within the experimental range, the inhibition rates of both α-Amy and CE showed an initial upward and then downward trend. The α-Amy inhibition rate reached a maximum of (46.00±0.88)% at an ultrasound power of 300W, with no significant difference from the inhibition rate at 240W (43.58±1.90)%. The CE inhibition rate reached a maximum of (66.61±1.15)% at an ultrasound power of 240W. This is attributed to the enhanced cavitation and mechanical shear forces that facilitated the cell wall disruption of seabuckthorn leaf cells, promoting the diffusion and dissolution of active ingredients within the cells. Excessive ultrasound power can lead to localized high temperatures and excessive mechanical shear, disrupting the structure of certain active ingredients (flavonoids and dietary fiber), thereby reducing the inhibitory activity of α-Amy and CE. Taking all these factors into consideration, 240W is considered the optimal ultrasound power for SLE extraction, achieving both strong α-Amy and CE inhibition.

[0099] Effect of extraction time on the inhibitory ability of SLE enzyme activity

[0100] In order to explore the effect of SLE extraction time on the inhibition rate of α-Amy and CE ( Figure 5 ), experiments were conducted in the range of 15 min to 75 min, and other parameters were kept unchanged. Figure 5The effect of SLE extraction time on the inhibition rates of α-Amy and CE was shown. As extraction time increased from 15 to 45 minutes, the inhibition rates of α-Amy and CE increased significantly, reaching a maximum at 45 minutes, with an α-Amy inhibition rate of (64.48±0.93)% and a CE inhibition rate of (55.32±0.95)%. With increasing extraction time, the inhibition rates of α-Amy and CE began to decrease, with the CE inhibition rate showing a more pronounced downward trend. The inhibition rates of α-Amy and CE showed an initial upward and then downward trend with extraction time, with the CE inhibition rate showing a more pronounced trend, indicating that the SLE extraction time had a more significant effect on the CE inhibition rate. Within a short period of time, the microjets generated by the collapse of ultrasonic cavitation bubbles can enhance cell wall perforation efficiency, disrupting the plant cell wall and facilitating the extraction of intracellular components. However, prolonged ultrasonic treatment can increase the dissolution of certain macromolecular impurities and the decomposition of heat-sensitive substances. Taking all these factors into consideration, a 45-minute extraction time was considered the optimal extraction time for SLE to exhibit both strong α-Amy and CE inhibitory activity.

[0101] Effect of extraction temperature on the inhibitory ability of SLE enzyme activity

[0102] In order to explore the effect of SLE extraction temperature on the inhibition rate of α-Amy and CE ( Figure 6 ), experiments were conducted in the range of 30℃~70℃, and other parameters were kept unchanged. Figure 6 The effect of SLE extraction temperature on the inhibition rates of α-Amy and CE was shown. Within the extraction temperature range of 30°C to 70°C, the inhibition rates of α-Amy and CE increased with increasing extraction temperature, reaching their maximum values ​​at 50°C, where the α-Amy inhibition rate was (48.25±1.47)% and the CE inhibition rate was (54.23±1.53)%. This may be because increasing temperature promotes molecular diffusion and permeation, making the intracellular active components more easily dissolvable. However, with continued temperature increases, the inhibition rates of α-Amy and CE gradually decreased. Excessively high temperatures increase solvent viscosity, reducing extraction efficiency. Furthermore, compounds such as flavonoids, phenolic acids, and anthocyanins are susceptible to decomposition at high temperatures. Taking all these factors into consideration, 50°C is considered the optimal extraction temperature for SLE to exhibit both strong α-Amy and CE inhibitory activity.

[0103] 3) Entropy weight assignment

[0104] The calculation results of the information entropy value (Hj) and weight coefficient (Wj) of each evaluation index are shown in Table 2. The evaluation indicators of this experiment are the inhibition rate of α-Amy and CE. The comprehensive score (Y) of these two indicators is calculated using the entropy weight method. The calculation formula for the comprehensive score is as follows: Y (%) = α-Amy inhibition rate × 0.53 + CE inhibition rate × 0.47.

[0105] Table 2 Information entropy values ​​and weight coefficients of each evaluation index

[0106]

[0107] 4) Box-Behnken design results analysis

[0108] The results of the BBD experiment, with the comprehensive score of α-Amy and CE inhibition rate as the response value, are listed in Table 3. The data were subjected to multivariate quadratic regression and variance analysis (Table 4) using the software Design-Expert 13, and the multivariate quadratic regression equation was obtained as follows:

[0109] Overall score = 64.28-3.14A-0.1411B-0.3592C-1.95AB-1.15AC+3.29BC-3.93A2-9.22B2-5.76C2

[0110] In the quadratic polynomial regression model, P < 0.0001 indicated a significant model and good regression performance. The linear coefficient A, quadratic coefficients A², B², and C², and cross-coefficients AB, AC, and BC were all significant (P < 0.05). The lack-of-fit value (P = 0.3757) was not significant, indicating a good model fit. The model R² = 0.9926, indicating good correlation and ability to predict the actual values ​​of the independent variables within the experimental range. The Adj R² = 0.9832 and Pre R² = 0.9349, with a difference of less than 0.2, indicate that the quadratic model fits well and fully explains the actual production process. The CV value was 1.62, indicating that the quadratic model is accurate and reliable. The model signal-to-noise ratio was 26.8197 (>4), which is also within a reasonable range. These analyses demonstrate that the established quadratic model is statistically sound and reliable. F-score analysis indicates that the influence of each factor on the comprehensive score is ranked as follows: A > C > B.

[0111] The interaction between the factors was analyzed by the software Design-Expert 13. Figure 7 The steeper the curvature of the response surface, the greater the interaction between the two factors. The denser the contour lines are and the more elliptical they are, the stronger the interaction between the two factors is. The sparser the contour lines are and the more circular they are, the weaker the interaction between the two factors is. Figure 7 c It can be seen that the surfaces along the B axis and the C axis are the steepest, indicating that the interaction between factors B (extraction time) and C (extraction temperature) is the strongest, which is consistent with the results of variance analysis.

[0112] Table 3 SLE-BBD experiment plan and results

[0113]

[0114] Table 4 Analysis of variance of SLE-BBD experiment

[0115]

[0116]

[0117] Note: “-” indicates that the item is not included, the same below; **** indicates p<0.0001, *** indicates p<0.001, ** indicates p<0.01, and * indicates p<0.05.

[0118] The optimal extraction conditions for the SLE process, selected by fitting with Design-Expert 13 software, were a solvent ratio of 1:4.473, an extraction time of 43.361 minutes, and an extraction temperature of 48.639°C. Based on actual production conditions, the fitting conditions were modified to a solvent ratio of 1:4, an extraction time of 40 minutes, and an extraction temperature of 48°C. The remaining parameters included a solid-liquid ratio of 1:20 and an ultrasonic power of 240 W. Under these conditions, the predicted value of the comprehensive score was 60.00%, while the experimental value was (58.85±0.65)%. The actual value closely matched the prediction of the quadratic model, with a relative error of 1.92%, demonstrating the reliability of the model.

[0119] 5)UPLC-Q-TOF / MS analysis

[0120] Preliminary identification of active substances. UPLC-Q-TOF / MS and UNIFI software were used to further analyze the components of SLE. Mass spectrometry data were analyzed using MassLynx and compared with data recorded in the Massbank database. Thirty-three compounds were preliminarily identified, which can be roughly divided into fatty acids, phenolic acids, flavonoids, tannins, and other organic compounds, as shown in Table 5.

[0121] Fifteen fatty acids and their derivatives have been preliminarily identified. Saturated fatty acids include behenic acid, heptadecanoic acid, palmitic acid, stearic acid, tricosanoic acid, and tetracosanoic acid, which are long-chain saturated fatty acids with high fat solubility. Unsaturated fatty acids include α-linolenic acid, linoleic acid, 6-octadecenoic acid, chrysanthemum acid, ricinoleic acid, and malvaceae. α-linolenic acid and linoleic acid are ω-3 and ω-6 polyunsaturated fatty acids, respectively, essential fatty acids for humans and possess hypoglycemic and lipid-lowering properties. Chang Songlin et al. fed diabetic mice with varying doses of α-linolenic acid for 42 days and found that α-linolenic acid modulated glucose and lipid metabolism by improving insulin resistance and reducing serum TC and TG levels. Linoleic acid has also been shown to lower cholesterol levels in hypercholesterolemic rats and may have potential in preventing atherosclerosis and obesity. Chrysanthemum acid is a naturally occurring epoxidized unsaturated fatty acid, primarily found in chrysanthemum seed oil, and possesses antioxidant and antimicrobial properties. (E,E)-9-keto-10,12-octadecadienoic acid, α-hydroxytetracosanoid, and 2-palmitoylmonoglyceride are fatty acid derivatives. (E,E)-9-keto-10,12-octadecadienoic acid has two double bonds (at carbon atoms 10 and 12) and a keto group (at carbon atom 9) within its 18-carbon chain, with the double bonds in a trans (E) configuration. α-hydroxytetracosanoid is characterized by a hydroxyl group (-OH) at the α position (i.e., the carbon atom adjacent to the carboxyl group) within its 24-carbon chain. This structure gives α-hydroxytetracosanoid unique chemical and biological activities. 2-palmitoylmonoglyceride is an esterification product of glycerol and palmitic acid. Sea buckthorn leaves contain a small amount of fat-soluble components, which can be effectively dissolved and enriched by organic solvents. Despite the low content, efficient separation can be achieved through optimized ultrasound-assisted extraction.

[0122] Six phenolic acids and their derivatives have been preliminarily identified: 2-hydroxy-5-butoxyphenylacetic acid, acetoeugenol, ellagic acid, p-hydroxyphenylpropionic acid, cinnamic acid, and isobutyl cinnamate. 2-Hydroxy-5-butoxyphenylacetic acid is a phenylacetic acid derivative containing a phenolic hydroxyl group and a butoxy group. Acetoeugenol is an acetylated derivative of eugenol, possessing properties of both phenolic and ester compounds. Ellagic acid is a common phenolic acid found in medicinal plants and a dimeric derivative of gallic acid. It exists not only in free form but also, more commonly, in condensed forms (such as ellagitannins and glycosides). Ellagic acid has multiple pharmacological activities and is beneficial for the treatment of metabolic syndrome and diabetes. p-Hydroxyphenylpropionic acid is a phenylpropionic acid derivative whose chemical structure contains a benzene ring, a hydroxyl group (-OH), and a propionic acid group (-CH-CH-COOH). Cinnamic acid is another important natural phenolic acid that can enhance lipid metabolism by improving mitochondrial function, reducing serotonin levels, and promoting autophagy-mediated lipid clearance. Isobutyl cinnamate is a derivative of cinnamic acid and belongs to lipid compounds.

[0123] Five flavonoids and their derivatives have been preliminarily identified, including cyanidin, luteolin, delphinidin, glabripenol, and cynaroside. The structure of this type of compound is based on benzopyrone as the core skeleton. Cyanadin, delphinidin, and luteolin are all natural anthocyanidin compounds, which are common pigments in plants. In animal studies, cyanidin has been shown to regulate lipid metabolism. Glabripenol is an important isoflavone compound, mainly extracted from Glycyrrhiza glabra. Studies have shown that it can inhibit the activity of cholesterol acyltransferase (ACAT) and diacylglycerol acyltransferase (DGAT), and has potential in improving hyperlipidemia and diabetes. Cynaroside is an active flavonoid glycoside with multiple biological activities, including antihypertensive, myocardial protection, anti-diabetic, lipid-lowering, anti-inflammatory and antioxidant effects.

[0124] Tannins can be divided into hydrolyzable tannins and condensed tannins. Proanthocyanidin A2 is a condensed tannin characterized by an additional ether bond. Its structure is more complex than that of proanthocyanidin B and is bioavailable both in vivo and in vitro. Proanthocyanidins exhibit significant antioxidant activity and can affect lipid metabolism and blood glucose levels by regulating intestinal microbiota, promoting insulin release, and modulating endoplasmic reticulum stress. Terminalia flavonoids A is a hydrolyzable tannin belonging to the polyphenol class. It is primarily found in certain plants, particularly the bark and fruit of the Terminalia catappa tree. In addition, several other compounds have been identified, including malonic acid, azelaic acid, taurine, punicalagin, and punicalagin B. Punicalagin is a polyphenol formed by ester linkages of multiple ellagic acid molecules with antioxidant, anti-inflammatory, antibacterial, antiviral, and anticancer properties. Punicalagin B is a naturally occurring triterpenoid compound primarily isolated from Agrimonia.

[0125] Table 5 UPLC-Q-TOF / MS identification of SLE components

[0126]

[0127]

[0128] 6) Network pharmacology results

[0129] Target prediction results: 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. 33 active substances in SLE and 713 of their targets were found through the TCMSP and SwissTarget Prediction databases. The targets of SLE active substances were intersected with those related to diabetes and hyperlipidemia, and a Venn diagram was drawn ( Figure 9 ). 106 intersection targets were obtained for SLE as potential targets for the treatment of diabetes mellitus with hyperlipidemia.

[0130] Core ingredient screening: The top four active ingredients in SLE with the highest degree values ​​were selected as ligands for molecular docking (Table 6). Among them, malvaccin and malvaccin are the core active ingredients of SLE. These active ingredients with higher degree values ​​may be the main active ingredients in SLE for preventing and treating diabetes and hyperlipidemia.

[0131] Table 6 Core active ingredients of SLE

[0132]

[0133] PPI Network Analysis

[0134] Potential targets were entered into the STRING database, and the PPI network diagram of potential targets was drawn using Cytoscape 3.10.2 ( Figure 10 The darker the target color, the larger the connecting node, and the more connections between targets, the stronger the synergy in the network and the more likely it is to be a core target for SLE or SPE. The average of the three topological parameters of SLE calculated using the CytoNCA plug-in is BC (25.58), CC (0.58), and DC (31). As a result, SLE has 43 core targets. It can be seen that the top five core targets of SLE are ALB, IL6, AKT1, TNF, and PPARG.

[0135] GO and KEGG enrichment analysis

[0136] In order to explore the biological pathways and mechanisms of SLE in treating diabetes and hyperlipidemia, the core targets were analyzed by GO and KEGG, ranked by p-value and visualized using the Microbiome Information Platform. Figure 10 In the GO enrichment results, the biological processes of SLE treatment of diabetes and hyperlipidemia mainly include cellular responses to lipids, cellular responses to nitrogen compounds, and responses to steroid hormones. Cellular components and molecular functions involve membrane rafts, membrane microdomains and transcription factor binding, protein kinase binding, etc. The main pathways involved in KEGG pathway analysis include lipids and atherosclerosis, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and HIF-1 signaling pathway.

[0137] Molecular docking analysis

[0138] When a ligand and receptor interact, it is generally believed that a binding energy less than -5kcal / mol indicates good binding activity. The results are shown in Table 7. The docking binding energy between the core component luteolin and the target is less than -5kcal / mol, indicating that the affinity between luteolin and the core target is strong. The docking results were further visualized using PyMol software. Figure 11 As shown, core components such as luteolin mainly interact with different sites of each target through hydrogen bonds. This indicates that these core components can bind to different amino acid residues of key disease targets through hydrogen bonds, thereby playing a role in treating hyperlipidemia and diabetes.

[0139] Table 7 Molecular docking binding energy of SLE core active ingredients and core targets (kcal / mol)

[0140] Active ingredient IL6 AKT1 TNF PPARG Tianshi acid -3.1 -2.85 -4.28 -3.01 Malvaceae -3.83 -3.53 -4.33 -4.38 6-Octadecenoic acid -2.1 -3.98 -4.78 -4.02 α-linolenic acid -3.58 -4.86 -4.24 -4.67

[0141] The embodiment of the present invention uses 10 organic solvents of different polarities combined with ultrasound-assisted extraction of active substances in sea buckthorn leaves to prepare sea buckthorn leaf extracts. The inhibitory effects of SLE extracted with different solvents on α-glucosidase, α-amylase, pancreatic lipase, cholesterol esterase and xanthine oxidase were determined. The methanol extract has strong α-Amy inhibitory activity, and the acetone extract has strong CE inhibitory activity. The SLE extraction process was optimized using two solvents, and the optimal process conditions for SLE were obtained as follows: a methanol and acetone solvent ratio of 1:4, an extraction time of 40 min, an extraction temperature of 48°C, a solid-liquid ratio of 1:20, and an ultrasonic power of 240W.

[0142] UPLC-Q-TOF / MS and UNIFI software were used to analyze the components of seabuckthorn leaf extract, revealing the main compounds, including fatty acids, flavonoids, phenolic acids, and tannins. Network pharmacology was then used to analyze the key components, core targets, and pathways involved in hypoglycemic and lipid-lowering effects in SLE. The results showed that the key components in SLE for hypoglycemic and lipid-lowering effects include chrysanthemum acid, malvaceaic acid, aspartic acid, and 6-octadecenoic acid, with 43 core targets, primarily implicated in pathways such as lipid metabolism and atherosclerosis, the PI3K-Akt signaling pathway, the AGE-RAGE signaling pathway in diabetic complications, and the HIF-1 signaling pathway.

[0143] The sea buckthorn leaf extract obtained under the above optimal extraction conditions was used as an example to prepare a syrup, which was obtained by mixing the syrup with excipients and a solvent. The excipients were commonly used excipients for oral preparations, such as sorbitol, high fructose syrup, and glycerin, and the solvent was water.

[0144] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of seabuckthorn leaf extract in the preparation of a drug for lowering blood sugar and lipids, characterized in that: The seabuckthorn leaf extract is obtained by ultrasonic extraction of a mixed reagent obtained by mixing methanol and acetone in a volume ratio of 1:1-5; the lipid-lowering method refers to lowering cholesterol and triglycerides, and the sugar-lowering method lowers blood sugar.

2. The use of the seabuckthorn leaf extract according to claim 1 in the preparation of a drug for lowering blood sugar and lipids, characterized in that: The effective ingredients in the seabuckthorn leaf extract are chrysanthemum acid, malvaceae acid, aspartic acid and 6-octadecenoic acid.

3. The use of the seabuckthorn leaf extract according to claim 1 in the preparation of a drug for lowering blood sugar and lipids, characterized in that: The specific extraction process of the seabuckthorn leaf extract is as follows: After the sea buckthorn leaves are crushed, sea buckthorn leaf powder is obtained; Methanol and acetone are mixed in a volume ratio of 1:1-5 to obtain a mixed reagent, sea buckthorn leaf powder and the mixed reagent are mixed in a material-liquid ratio of 1:10-30, ultrasonic extraction is performed at 30°C-70°C and 120W-360W for 15min-75min, the mixture is centrifuged, the supernatant is collected, concentrated, and dried to obtain the sea buckthorn leaf extract.

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

5. The use according to claim 4, characterized in that The oral preparation is any one of tablets, capsules, pills, powders, granules and syrups.

6. The use according to claim 5, characterized in that The injection is a solution or emulsion.

7. A drug for alleviating hyperlipidemia, characterized in that: The seabuckthorn leaf extract according to claim 1 is the only active ingredient.

8. A drug for alleviating diabetes, characterized in that: The seabuckthorn leaf extract according to claim 1 is the only active ingredient.

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