Preparation of active components of suaeda salsa and isoquercitrin and use thereof in resisting obesity and regulating glycolipid metabolism

Abstract

The application relates to the technical field of medicines, and particularly discloses preparation of active components of Suaeda salsa and isoquercitrin and anti-obesity and sugar and lipid metabolism regulating purposes, wherein the active components are 30% ethanol elution parts of Suaeda salsa extracts obtained through macroporous adsorption resin column chromatography, and the isoquercitrin is prepared by taking SSEE-30% D as a starting material and then being separated through a silica gel column chromatography. The SSEE-30% D effective parts of Suaeda salsa and the monomer component isoquercitrin provided by the application both show remarkable effects in anti-obesity, sugar and lipid metabolism regulation, insulin resistance improvement, anti-inflammation, liver and kidney injury protection and the like, the mechanism of action involves mitochondrial respiratory chain regulation and mitochondrial autophagy activation, and the application has broad medical application prospects and important social and economic values.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the preparation of active components of Suaeda salsa and isoquercitrin, and their uses in anti-obesity and regulation of glucose and lipid metabolism. Background Technology

[0002] Obesity is a chronic metabolic disease caused by a combination of factors, including genetics, environment, and lifestyle, and has become a global public health problem. Data from the World Health Organization shows that the global obese population continues to rise. Obesity not only seriously affects patients' quality of life but is also a significant risk factor for type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease, hypertension, and various cancers. Currently used anti-obesity drugs, such as orlistat, have limitations such as gastrointestinal adverse reactions. Anti-obesity active substances derived from natural products are increasingly attracting researchers' attention due to their multi-target and low-toxicity characteristics.

[0003] Suaeda salsa, also known as yellow-bearded vegetable or winged suaeda, is an annual herbaceous plant belonging to the genus Suaeda in the family Amaranthaceae. It mainly grows in saline-alkali soils, beaches, and salt lake edges, and is widely distributed in both inland and coastal areas of my country. Suaeda salsa has high nutritional value, containing abundant protein, fatty acids, polysaccharides, dietary fiber, and other chemical components. Its tender branches and leaves can be eaten as vegetables. Modern pharmacological studies have shown that Suaeda salsa possesses various biological activities, including anti-inflammatory, antioxidant, anti-tumor, hypoglycemic, and lipid-lowering effects. Chemically, Suaeda salsa is rich in flavonoids, polyphenols, polysaccharides, steroids, terpenoids, and various alkaline mineral elements. Currently, the main extraction methods for the effective components of Suaeda salsa include solvent extraction, ultrasonic or microwave-assisted extraction, and supercritical CO2 extraction. Researchers have used ultraviolet spectrophotometry to determine the total flavonoid content of Suaeda salsa and developed an HPLC fingerprint of Suaeda salsa using high-performance liquid chromatography. However, existing studies have mostly focused on the in vitro activity or total flavonoid content determination of crude extracts of Suaeda salsa, and the in vivo pharmacodynamic evaluation and mechanism of action of specific effective fractions obtained through systematic separation and purification are still insufficient.

[0004] Isoquercitrin, also known as quercetin-3-O-β-D-glucoside, is a natural flavonoid compound widely found in various edible plants. Existing research indicates that isoquercitrin possesses diverse pharmacological activities. Regarding anti-obesity and glucose metabolism regulation, literature reports that isoquercitrin can prevent high-fat diet-induced obesity and hyperglycemia by activating the AMPKα pathway; it can exert anti-obesity effects by targeting targets such as C-1-tetrahydrofolate synthase to regulate the AMPK / SREBP-1c / FAS / CD36 signaling pathway; it can improve fatty acid metabolism disorders in the liver of db / db mice, the mechanism of which may be related to downregulating the expression of hepatic SREBP1c and FAS mRNA and upregulating the expression of hepatic PGC1α and PPARα; it can also regulate the secretion of obesity-induced pro-inflammatory factors and affect adipokines expression. Furthermore, isoquercitrin and quercetin-3-O-gentiobiglycoside in okra extract can reduce blood glucose and serum insulin levels in obese mice and improve glucose tolerance. Regarding patents, there are already public reports on the application of isoquercitrin in the preparation of drugs to improve insulin resistance and diabetes-related liver disease.

[0005] Although the pharmacological activity of isoquercitrin has been studied to some extent, the existing technology still has the following shortcomings: (1) At present, isoquercitrin is mostly obtained from plants such as okra, buckwheat, and sophora japonica. There are no reports on the systematic isolation and preparation of isoquercitrin from Suaeda salsa and the systematic in vivo anti-obesity pharmacodynamic evaluation of it; (2) Existing studies focus on the single aspect of isoquercitrin's hypoglycemic or lipid-lowering effects, and lack a systematic evaluation of its overall anti-obesity effect, including a comprehensive evaluation of multiple dimensions such as body weight, adipose tissue morphology, glucose and lipid metabolism, inflammatory response, and liver and kidney damage; (3) The study of the anti-obesity mechanism of isoquercitrin is not in-depth, especially its regulatory effect on mitochondrial respiratory chain function and mitochondrial autophagy has not been reported; (4) As a natural source of isoquercitrin, the effective part of Suaeda salsa, SSEE-30%D, has a blank in terms of its anti-obesity activity and mechanism of action. Summary of the Invention

[0006] The purpose of this invention is to provide the preparation of active components of *Suaeda salsa* and isoquercitrin, and their applications in anti-obesity and regulation of glucose and lipid metabolism. This addresses the shortcomings of existing technologies, such as insufficient research on the active components of *Suaeda salsa*, the lack of effective fractions with clear quality control standards, and the lack of systematic anti-obesity pharmacodynamic evaluation. It also addresses the incomplete understanding of the mechanism of isoquercitrin's role in anti-obesity and regulation of glucose and lipid metabolism.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] An active component of Suaeda salsa, wherein the active component is the 30% ethanol elution fraction (SSEE-30%D) obtained by macroporous adsorption resin column chromatography separation of Suaeda salsa extract.

[0009] A method for preparing isoquercitrin includes the following preparation steps;

[0010] S1. First silica gel column chromatography: SSEE-30%D was loaded onto a silica gel column and eluted with a gradient of dichloromethane-methanol mixed solvent. Each fraction was detected by thin-layer chromatography (TLC). Fractions with consistent spot characteristics were combined to obtain component II.

[0011] S2. Second silica gel column chromatography: Component II was loaded onto a silica gel column and eluted with a gradient of dichloromethane-methanol mixed solvent. The fraction that showed a single spot on TLC and a single peak on HPLC was collected and dried to obtain isoquercitrin monomer.

[0012] Preferably, in S1, the volume ratio of dichloromethane to methanol in the gradient elution is 20:1, 15:1, 10:1, 5:1, 3:1, 1:1, and 0:1, respectively; the component II is obtained by combining fractions with an Rf value of 0.15-0.25 and similar target spots when using a dichloromethane to methanol eluent with a volume ratio of 20:1 for TLC detection.

[0013] Preferably, in step S2, the volume ratios of dichloromethane and methanol in the gradient elution are 12:1, 10:1, and 0:1, respectively; the resulting isoquercitrin monomer has the molecular formula C2. 21 H 20 O 12 Its molecular weight is 464.38.

[0014] Application of active components of Suaeda salsa in the preparation of anti-obesity drugs or health products.

[0015] Application of active components of Suaeda salsa in the preparation of drugs or health products that regulate glucose and lipid metabolism disorders.

[0016] Application of active components of Suaeda salsa in the preparation of drugs or health products that improve insulin resistance.

[0017] Application of active components of Suaeda salsa in the preparation of drugs or health products for improving non-alcoholic fatty liver disease.

[0018] Applications of isoquercitrin in the preparation of drugs or health products for anti-obesity, regulation of glucose and lipid metabolism disorders, improvement of insulin resistance and improvement of non-alcoholic fatty liver disease.

[0019] The present invention also provides a pharmaceutical composition for the prevention and treatment of obesity and disorders of glucose and lipid metabolism. The pharmaceutical composition comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is SSEE-30%D and isoquercitrin.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] This invention clarifies the quality control standard for the effective fraction SSEE-30%D from *Suaeda salsa*. The SSEE-30%D fraction, eluted with 30% ethanol, was obtained from *Suaeda salsa* via macroporous adsorption resin column chromatography, and a quality control method for its HPLC fingerprint was established. This method identified four common peaks, with the RSD values ​​of the relative peak areas of each common peak being less than 3%. The similarity between the fingerprint spectra of eight batches of samples and the control was all above 0.9, indicating that the quality of this effective fraction is highly stable and controllable across batches. This solves the technical problem of uncontrollable quality and lack of standardized quality control methods for *Suaeda salsa* extract in existing technologies.

[0022] This invention also provides a method for preparing high-purity isoquercitrin from Suaeda salsa. The invention utilizes a two-step silica gel column chromatography separation and purification process to successfully prepare high-purity isoquercitrin monomers from SSEE-30%D, and the structure was confirmed by NMR and MS. This method is simple to operate, has good reproducibility, and can obtain high-purity target compounds.

[0023] Simultaneously, SSEE-30%D and isoquercitrin were found to have significant anti-obesity effects. After 8 weeks of treatment, SSEE-30%D (high dose) reduced the average body weight of DIO mice by approximately 7.96g, and isoquercitrin (high dose) reduced the average body weight by approximately 7.30g. SSEE-30%D also significantly reduced the weight of epididymal fat (eWAT), inguinal fat (iWAT), and perirenal white fat (pWAT), and significantly reduced the volume of adipocytes, improving adipose tissue hypertrophy and vacuolation, indicating that both can effectively inhibit high-fat diet-induced weight gain and fat accumulation.

[0024] This study is the first to discover that SSEE-30%D and isoquercitrin can significantly improve glucose metabolism disorders and insulin resistance. Both significantly reduced fasting blood glucose (FBG) levels in DIO mice (P < 0.001), improved oral glucose tolerance (OGTT) and insulin tolerance (ITT), reduced fasting insulin (FINS) levels (P < 0.01), and improved HOMA-IR and HOMA-IS indices (P < 0.001), indicating that they can effectively restore insulin sensitivity and have potential therapeutic value for type 2 diabetes and related metabolic diseases.

[0025] SSEE-30%D and isoquercitrin were found to significantly improve lipid metabolism disorders. Both significantly reduced serum TG, TC, NEFA, GL and LDL-C levels in DIO mice (P < 0.01), effectively improving lipid metabolism abnormalities induced by a high-fat diet.

[0026] SSEE-30%D and isoquercitrin were found to have significant anti-inflammatory effects. Both significantly reduced serum levels of pro-inflammatory factors TNF-α, IL-1β, MCP-1, and LEP (P < 0.01) and increased levels of anti-inflammatory factors IL-4 and ADPN (P < 0.05). The anti-inflammatory effect of high-dose isoquercitrin was particularly prominent (P < 0.001), indicating that both can improve obesity-related chronic low-grade inflammation by modulating the inflammatory response.

[0027] SSEE-30%D and isoquercitrin were found to improve liver and kidney damage. Both significantly reduced serum ALT and AST levels in DIO mice (P < 0.05), decreased BUN and Cr levels, improved liver and kidney indices, and alleviated hepatic steatosis and microvesicular steatosis, indicating that they have a significant protective effect against high-fat diet-induced liver and kidney damage, especially showing potential for improving non-alcoholic fatty liver disease.

[0028] This invention also reveals the mechanism by which SSEE-30%D and isoquercitrin improve energy metabolism by regulating the mitochondrial respiratory chain. Both can significantly upregulate the enzyme activity of hepatic mitochondrial respiratory chain complexes I, II, III, IV, and V, improve mitochondrial dysfunction, and enhance ATP production and energy metabolism levels, revealing their molecular basis for anti-obesity from the perspective of energy metabolism.

[0029] This study reveals a novel anti-obesity mechanism by which SSEE-30%D and isoquercitrin promote brown adipose tissue thermogenesis through activation of mitophagy. Both upregulated the expression levels of PPAR-α, PPAR-γ, and PPAR-δ proteins (P < 0.05), significantly upregulated the expression levels of UCP-1, LC3-II / LC3-I, Beclin-1, and Bax proteins (P < 0.05 or P < 0.01), and downregulated the expression levels of Bcl-2 and p62 proteins (P < 0.01). This mechanism is complete and has clearly defined targets, providing new intervention targets and theoretical basis for the development of anti-obesity drugs.

[0030] In summary, the effective fraction SSEE-30%D of Suaeda salsa and its monomeric component isoquercitrin provided by this invention have shown significant effects in anti-obesity, regulation of glucose and lipid metabolism, improvement of insulin resistance, anti-inflammation, and protection against liver and kidney damage. Its mechanism of action involves mitochondrial respiratory chain regulation and mitochondrial autophagy activation, and it has broad prospects for medical applications and important socio-economic value. Attached Figure Description

[0031] Figure 1 This is the HPLC fingerprint of the active component SSEE-30%D of Suaeda salsa in this invention;

[0032] Figure 2This is a graph showing the detection results of the first silica gel column chromatography separation of SSEE-30%D according to the present invention. Figure 2 'a' represents the TLC unfolding system screening results. Figure 2 b represents the TLC detection results of all fractions collected in the first column chromatography analysis. Figure 2 c to g are TLC re-examination results after merging fractions with similar spot characteristics;

[0033] Figure 3 The images shown are the detection chromatograms of fraction II purified by silica gel column chromatography in this invention, where (a) is the elution solvent screening chromatogram, (b) is the TLC chromatogram corresponding to elution with a dichloromethane-methanol volume ratio of 12:1, (c) is the TLC chromatogram corresponding to elution with a dichloromethane-methanol volume ratio of 10:1, and (d) is the analytical HPLC chromatogram of multiple target fractions.

[0034] Figure 4 The chemical structural formula of the isoquercitrin monomer isolated in this invention is shown below.

[0035] Figure 5 The ¹H-NMR hydrogen spectrum of the isoquercitrin monomer of this invention is shown below.

[0036] Figure 6 The ¹³C-NMR carbon spectrum of the isoquercitrin monomer of this invention is shown below.

[0037] Figure 7 This is the ESI-MS mass spectrum of isoquercitrin in anion mode according to the present invention;

[0038] Figure 8 This is the ESI-MS mass spectrum of isoquercitrin in positive ion mode according to the present invention;

[0039] Figure 9 The HPLC chromatograms of the crude extract of SSEE-30%D and isoquercitrin monomer of this invention are shown below.

[0040] Figure 10 The graph shows the changes in body weight, food intake, water intake, and energy metabolism in high-fat obese mice after intervention with orlistat and SSEE-30%D according to this invention.

[0041] Figure 11 This is a graph showing the changes in body weight, food intake, water intake, and energy metabolism in high-fat obese mice after intervention with isoquercitrin according to the present invention.

[0042] Figure 12 The images show the appearance of mice in the NFD and HFD groups after 8 weeks of drug administration, as well as bar charts showing the final weight of mice in each group.

[0043] Figure 13 This is a graph showing the detection results of glucose metabolism and insulin sensitivity-related indicators in hyperlipidemic obese mice using SSEE-30%D of the present invention;

[0044] Figure 14 The figure shows the detection results of isoquercitrin on glucose metabolism and insulin sensitivity in hyperlipidemic obese mice according to the present invention.

[0045] Figure 15 This is a bar chart showing the detection of serum lipid metabolism indicators in hyperlipidemic obese mice using SSEE-30%D according to the present invention.

[0046] Figure 16 This is a bar chart illustrating the effect of isoquercitrin on serum lipid metabolism indicators in hyperlipidemic obese mice according to the present invention.

[0047] Figure 17 This is a bar chart showing the detection of serum inflammatory factor levels in hyperlipidemic obese mice using SSEE-30%D according to the present invention.

[0048] Figure 18 This is a bar chart showing the detection of serum inflammatory factor levels in high-fat obese mice by isoquercitrin according to the present invention.

[0049] Figure 19 The figure shows the results of the SSEE-30%D of the present invention regulating the mitochondrial respiratory chain to improve liver injury in hyperlipidemic mice. Among them, (ae) is the detection result of enzyme activity of liver mitochondrial respiratory chain complex I~V, (f) is the actual anatomical diagram of the liver of each group of mice, and (g) is the H&E pathological staining diagram of liver tissue.

[0050] Figure 20 The figure shows the results of isoquercitrin regulating mitochondrial respiratory chain to improve liver injury in hyperlipidemic mice. (ae) shows the detection results of enzyme activity of liver mitochondrial respiratory chain complex I-V, (f) shows the actual anatomical diagram of the liver of each group of mice, and (g) shows the H&E pathological staining diagram of liver tissue.

[0051] Figure 21 This is a schematic diagram illustrating the effects of SSEE-30%D and isoquercitrin of the present invention on the improvement of adipose tissue weight and tissue morphology in hyperlipidemic obese mice.

[0052] Figure 22 This is a bar chart showing the detection of organ indices (heart, liver, spleen, lung, kidney, and pancreas) in hyperfat obese mice by SSEE-30%D according to the present invention.

[0053] Figure 23 This is a bar chart showing the effects of isoquercitrin of the present invention on the organ indices of the heart, liver, spleen, lung, kidney, and pancreas in hyperlipidemic obese mice.

[0054] Figure 24 The bar chart shows the biochemical indicators of SSEE-30%D and isoquercitrin in improving liver and kidney damage in hyperlipidemic mice according to the present invention.

[0055] Figure 25This is a Western blot result of the SSEE-30%D assay on the expression of autophagy-related proteins in brown adipose tissue of high-fat mice.

[0056] Figure 26 This is a Western blot result of isoquercitrin's effect on the expression of autophagy-related proteins in brown adipose tissue mitochondria of high-fat mice. Detailed Implementation

[0057] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] Example 1: Preparation of SSEE-30%D, an effective component of Suaeda salsa.

[0059] Take dried whole plant of Suaeda salsa, crush it and pass it through a 40-mesh sieve. Add 8 times (V / W) of 70% ethanol and heat and reflux in a 60℃ water bath twice for 2 hours each time. Combine the extracts, filter, and concentrate under reduced pressure until there is no alcohol taste to obtain total extract of Suaeda salsa.

[0060] The above extract was dispersed in an appropriate amount of distilled water and loaded onto a pre-treated D101 macroporous adsorption resin column (the weight ratio of resin to sample was 10:1). The column was eluted sequentially with distilled water, 30% ethanol, 50% ethanol, 70% ethanol and 95% ethanol, with each gradient elution consisting of 3 column volumes. The 30% ethanol eluent was collected, concentrated under reduced pressure, and freeze-dried to obtain the effective fraction of Suaeda salsa, SSEE-30%D.

[0061] Following the above method, a total of 8 batches of SSEE-30%D samples were prepared for later use.

[0062] Example 2: Establishment of HPLC fingerprint of SSEE-30%D

[0063] (1) Preparation of test solution: Accurately weigh an appropriate amount of SSEE-30%D sample prepared in Example 1, dissolve it in methanol and make up to volume to prepare a solution with a concentration of 1.0 mg / mL. Filter the solution through a 0.45 μm microporous membrane and take the filtrate.

[0064] (2) Preparation of reference solution: Accurately weigh an appropriate amount of isoquercitrin reference standard, dissolve it in methanol and make up to volume to prepare a solution with a concentration of 0.5 mg / mL. Filter the solution through a 0.45 μm microporous membrane and collect the filtrate.

[0065] (3) Chromatographic conditions: Agilent ZORBAX SB-C 18 The chromatographic column was 250 mm × 4.6 mm, 5 μm; mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile; the gradient elution program was as follows: 0–10 min, 5%–15% B; 10–30 min, 15%–25% B; 30–45 min, 25%–40% B; 45–50 min, 40%–5% B; the flow rate was 1.0 mL / min; the column temperature was 30 °C; the detection wavelength was 254 nm; and the injection volume was 10 μL.

[0066] (4) Establishment of fingerprint chromatograms: Eight batches of SSEE-30%D test solution were precisely pipetted and analyzed under the chromatographic conditions described above, and the chromatograms were recorded. The chromatograms of the eight batches of samples were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for peak matching analysis to generate a reference fingerprint chromatogram. The results are as follows: Figure 1 As shown, all 8 batches of samples had 4 common peaks, and the RSD value of the relative peak area of ​​each common peak was less than 3%. The similarity between all samples and the control fingerprint spectrum was above 0.9.

[0067] (5) Methodological validation:

[0068] Precision test: The same batch of SSEE-30%D test solution was injected six times consecutively, and the relative peak areas of each common peak were recorded. The RSD value was calculated. The results showed that the RSD values ​​of the relative peak areas of each common peak were all less than 3%, indicating that the instrument precision was good.

[0069] Repeatability test: Six samples of the same batch of SSEE-30%D were taken, and test solutions were prepared separately. The samples were injected and analyzed under the chromatographic conditions described above. The relative peak areas of each common peak were recorded, and the RSD values ​​were calculated. The results showed that the RSD values ​​of the relative peak areas of each common peak were all less than 3%, indicating that the method has good repeatability.

[0070] Stability test: The same batch of SSEE-30%D test solution was injected and analyzed at 0, 2, 4, 6, 8, 12, and 24 hours. The relative peak areas of each common peak were recorded, and the RSD values ​​were calculated. The results showed that the RSD values ​​of the relative peak areas of each common peak were all less than 3%, indicating that the test solution had good stability within 24 hours.

[0071] Example 3: Isolation and purification of isoquercitrin

[0072] (1) First silica gel column chromatography separation:

[0073] Take 0.98g of the SSEE-30%D sample prepared in Example 1, dissolve it in an appropriate amount of methanol, mix it with 2.0g of silica gel (100-200 mesh), evaporate the solvent, and set aside.

[0074] Column packing: Weigh 30g of silica gel (200-300 mesh) and use dichloromethane as a homogenizing solvent to pack the column using a wet method (column size: 2.5cm×40cm). After compaction, spread the mixed silica gel evenly on the top of the column.

[0075] Elution system screening: First, mixed solvents with dichloromethane to methanol volume ratios of 40:1, 30:1, 20:1, 15:1, 10:1, 5:1, and 1:1 were selected as the developing solvent for TLC analysis of SSEE-30%D samples. Results are as follows... Figure 2 As shown in Figure a, when the volume ratio of dichloromethane to methanol is 20:1, the Rf value of the target spot is approximately 0.2, indicating a good separation effect. Therefore, a mixed solvent with a volume ratio of dichloromethane to methanol of 20:1 was selected as the initial elution solvent for silica gel column chromatography.

[0076] Column chromatography elution: Gradient elution was performed sequentially using mixed solvents of dichloromethane and methanol at volume ratios of 20:1, 15:1, 10:1, 5:1, 3:1, 1:1, and 0:1, with two column volumes eluted for each gradient. Approximately 10 mL was collected per tube. TLC was used to detect each fraction, with a 20:1 volume ratio of dichloromethane and methanol as the developing solvent and 10% sulfuric acid in ethanol as the colorimetric reagent. The mixture was heated to 105°C for color development. Fractions with similar spot characteristics were initially combined, and then the combined fractions were re-examined by TLC. Further merging was performed based on the re-examination results. The results are shown below. Figure 2 As shown in b to g.

[0077] Based on the TLC analysis results ( Figure 2 The collected samples were combined into six fractions: samples 1-2 were combined into fraction I (0.0231 g, yield 2.36%), samples 3-6 were separated, samples 7-10 were combined into fraction II (0.1262 g, yield 12.88%), samples 11-13 were combined into fraction III (0.0867 g, yield 8.85%), samples 14-17 were combined into fraction IV (0.0709 g, yield 7.23%), samples 18-19 were combined into fraction V (0.0385 g, yield 3.93%), and samples 20-23 were combined into fraction VI (0.1427 g, yield 14.56%). Fraction II showed a more concentrated TLC target spot and relatively fewer impurity spots; therefore, fraction II was selected for further separation.

[0078] (2) Second silica gel column chromatography separation:

[0079] Take 0.1262 g of the above component II, dissolve it in an appropriate amount of methanol, mix it with 0.3 g of silica gel (100-200 mesh), evaporate the solvent, and set aside for later use.

[0080] Column packing: Weigh 10g of silica gel (200-300 mesh) and use dichloromethane as a homogenizing solvent to pack the column using a wet method (column size: 1.5cm×30cm). After compaction, spread the mixed silica gel evenly on the top of the column.

[0081] Elution: Based on the TLC results (Rf value < 0.2), gradient elution was performed sequentially using a mixed solvent of dichloromethane:methanol = 12:1, 10:1, and 0:1 (V / V). Figure 3 a) Elute 3 column volumes per gradient, collecting approximately 5 mL per tube. Detect by TLC (developing solvent: dichloromethane:methanol = 10:1, colorimetric reagent: 10% sulfuric acid in ethanol, heated to 105℃), and combine fractions with the same spot.

[0082] like Figure 3 As shown in b, when eluted with dichloromethane:methanol = 12:1, the thin-layer chromatographic spots of samples 8 and 9 are similar, and the spots of samples 12-15 also show similar patterns. Figure 3 As shown in c, when eluted with dichloromethane:methanol = 10:1, samples 6-31 all showed a single spot, suggesting they may be the same compound. Further verification was performed by HPLC. Figure 3 d) Except for the solvent peak, all samples showed single peaks, further confirming the results of the TLC analysis.

[0083] Samples 6-31 were combined and concentrated under reduced pressure to obtain a pale yellow powdery solid, which is isoquercitrin (IQ).

[0084] Example 4: Structural identification of isoquercitrin

[0085] The isoquercitrin sample prepared in Example 3 was analyzed by NMR and MS.

[0086] Appearance: Pale yellow powder, soluble in methanol.

[0087] The molecular formula is C21H20O12, and the molecular weight (MW) is 464.38. Analysis was performed using electrospray ionization (ESI-MS) mass spectrometry. Figure 7 and Figure 8 In the mass spectrometry results, the molecular ion peak of isoquercitrin is 487.08411 [M+Na]+, which is the sodium ion binding peak; and 463.08795 [MH]-, which is the dehydrogenation ion peak. These mass spectrometry peaks are consistent with the molecular weight and structural characteristics of isoquercitrin, further confirming its molecular composition.

[0088] 1H-NMR (600MHz, CD3SOCD3) spectrum ( Figure 5The results showed: 1 hydroxyl proton signal: δ12.64 (1H, s, 5-OH); 5 aromatic proton signals: δ7.59 (1H, dd, H-2'), 7.58 (1H, d, H-6'), 6.85 (1H, d, H-5'), 6.40 (1H, d, H-8), 6.19 (1H, d, H-6); and 1 set of proton signals on glucose: 5.49 (1H, d, H-1''), 3.17-3.36 (4H, m, H-2", 3", 4", 5"), 3.59 (2H, d, H-6").

[0089] like Figure 6 As shown, in the 13C-NMR (600Hz, CD3SOCD3) spectrum, the signals for one carbonyl carbon on the C ring are: δ177.41 (C-4); the signals for two olefin carbons are δ156.37 (C-2) and 133.30 (C-3); the signals for ten carbons on the A and B rings are: δ161.25 (C-5), 156.37 (C-9), 148.56 (C-4'), 144.85 (C-3'), 121.61 (C-6'), 116.17 (C-5'), 115.22 (C-2'), 103.88 (C-10), 9 8.77 (C-6), 93.57 (C-8); the six carbon signals on glucose are δ100.88 (C-1''), 77.59 (C-5''), 76.52 (C-3''), 74.11 (C-2''), 69.94 (C-4"), and 60.98 (C-6''). Comprehensive analysis of the mass spectrometry and nuclear magnetic resonance (NMR) data of isoquercitrin confirmed the molecular structure of this compound. Based on the NMR and MS analysis data, combined with literature data, the compound was finally confirmed as isoquercitrin. The HPLC chromatogram further validated this conclusion. Figure 9 ).

[0090] Example 5: Effects of SSEE-30%D and isoquercitrin on body weight in DIO mice

[0091] (1) Animal model establishment and grouping:

[0092] SPF-grade male C57BL / 6 mice, 6-8 weeks old, were randomly divided into two groups after one week of acclimatization: a normal diet group (NFD group, n=10) fed a standard maintenance diet, and a high-fat diet group fed a high-fat diet (fat energy ratio 60%), for 12 consecutive weeks. A diet-induced obesity (DIO) model was considered successfully established when the body weight of mice in the high-fat diet group exceeded the average body weight of mice in the NFD group by 20%.

[0093] The successfully modeled DIO mice were randomly divided into 6 groups of 10 mice each:

[0094] Model group (HFD group): administered an equal volume of physiological saline by gavage;

[0095] Positive control group (Orlistat group): Orlistat was administered by gavage at a dose of 15.6 mg / kg;

[0096] SSEE-30%D low-dose group (SSL group): SSEE-30%D was administered by gavage at a dose of 50 mg / kg;

[0097] SSEE-30%D high-dose group (SSH group): SSEE-30%D was administered by gavage at a dose of 200 mg / kg;

[0098] Isoquercitrin low-dose group (IQL group): isoquercitrin was administered by gavage at a dose of 25 mg / kg;

[0099] High-dose isoquercitrin group (IQH group): isoquercitrin was administered by gavage at a dose of 100 mg / kg.

[0100] An NFD group (n=10) was set up, and the same volume of physiological saline was administered by gavage as a normal control.

[0101] Mice in each group were administered the drug once daily by gavage for 8 consecutive weeks. During the administration period, mice had free access to food and water, and their body weight, food intake, and water intake were recorded weekly.

[0102] (2) Experimental results:

[0103] like Figure 10 and Figure 11 As shown, the body weight of mice in the HFD group showed a continuous increasing trend, while after drug intervention, the weight gain trend of mice in each treatment group slowed down significantly. After 8 weeks of treatment, compared with the HFD group, the body weight of each treatment group was significantly reduced (P<0.001). The specific reductions in body weight are as follows: the Orlistat group mice reduced their body weight by an average of 13.47g, while the SSL, SSH, IQL, and IQH groups reduced their body weight by 9.69g, 7.96g, 7.37g, and 7.30g, respectively. Figure 12 ).

[0104] Example 6: Effects of SSEE-30%D and isoquercitrin on glucose metabolism and insulin sensitivity in DIO mice

[0105] (1) Fasting blood glucose (FBG) measurement:

[0106] Eight weeks after administration, mice in each group were fasted for 12 hours, blood was collected from the tail tip, and fasting blood glucose (FBG) levels were measured using a blood glucose meter.

[0107] The results are as follows Figure 13 c and Figure 14As shown in Figure c, compared with the NFD group, the FBG level in the HFD group mice was significantly increased (P<0.001), indicating that a long-term high-fat diet led to significant glucose metabolism disorders in the DIO mice. After 8 weeks of treatment, the FBG levels in all treatment groups were significantly reduced. Among them, the orlistat group and the IQ group showed particularly outstanding hypoglycemic effects (P<0.001).

[0108] (2) Oral glucose tolerance test (OGTT):

[0109] Eight weeks after administration, mice in each group were fasted for 12 hours and then administered glucose (2 g / kg) by gavage. Blood glucose levels were measured by collecting blood samples from the tail tip at 0, 15, 30, 60, 90, and 120 minutes, and the area under the blood glucose-time curve (AUC) was calculated.

[0110] The results are as follows Figure 13 a and Figure 14 As shown in figure a, mice in the HFD group exhibited impaired glucose tolerance, with a significantly increased area under the glucose curve (AUC). All treatment groups effectively improved glucose tolerance, with a significant decrease in AUC (P<0.01).

[0111] (3) Insulin Tolerance Test (ITT):

[0112] Eight weeks after administration, mice in each group were fasted for 4 hours and injected intraperitoneally with insulin (0.75 U / kg). Blood glucose levels were measured by collecting blood from the tail tip at 0, 15, 30, 60, 90, and 120 minutes, and the area under the blood glucose-time curve (AUC) was calculated.

[0113] The results are as follows Figure 13 b and Figure 14 As shown in b, the insulin tolerance of mice in the HFD group was significantly reduced, and blood glucose remained high. The decrease in blood glucose was more pronounced in each treatment group, and the AUC was significantly reduced (P<0.001), indicating that the treatment groups enhanced insulin sensitivity.

[0114] (4) Fasting insulin (FINS) and HOMA-IR and HOMA-IS measurements:

[0115] Eight weeks after drug administration, mice in each group were fasted for 12 hours, blood was collected from the orbital sinus, serum was separated, and fasting insulin (FINS) levels were detected using an ELISA kit. The insulin resistance index (HOMA-IR) was calculated as FBG (mmol / L) × FINS (mIU / L) / 22.5, and the insulin sensitivity index (HOMA-IS) was calculated as 1 / (FBG × FINS).

[0116] The results are as follows Figure 13 df and Figure 14As shown in the data, the FINS level in the HFD group mice was significantly increased (P<0.01), the HOMA-IR index was increased (P<0.001), and the HOMA-IS index was decreased (P<0.01), indicating a significant insulin resistance state. After treatment, all treatment groups showed varying degrees of improvement in FINS and HOMA-IR; some treatment groups also showed an increase in HOMA-IS. Orlistat and SSEE-30%D effectively reduced FINS levels (P<0.01), bringing them close to normal levels, and decreased the HOMA-IR index, thus improving insulin sensitivity.

[0117] The above results indicate that SSEE-30%D and isoquercitrin can effectively improve HFD-induced glucose metabolism disorders and insulin resistance in mice.

[0118] Example 7: Effects of SSEE-30%D and isoquercitrin on lipid metabolism in DIO mice

[0119] Eight weeks after administration, mice in each group were fasted for 12 hours, blood was collected from the orbital rim, serum was separated, and the levels of triglycerides (TG), total cholesterol (TC), non-esterified fatty acids (NEFA), glycerol (GL), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in the serum were detected using a fully automated biochemical analyzer or corresponding reagent kits.

[0120] The results are as follows Figure 15 and Figure 16 As shown, compared with the NFD group, the serum levels of TG, TC, NEFA, GL, and LDL-C in the HFD group mice were significantly increased (P<0.01), indicating that the HFD group mice had obvious lipid metabolism disorders. After treatment, the levels of the above indicators decreased, with the orlistat group and the high-dose IQ group showing the best lipid-lowering effects.

[0121] The results showed that SSEE-30%D and isoquercitrin had a significant effect on improving lipid metabolism disorders.

[0122] Example 8: Effects of SSEE-30%D and isoquercitrin on inflammatory response in DIO mice

[0123] Eight weeks after administration, blood was collected from the orbital sinus of mice in each group, serum was separated, and the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-4 (IL-4), monocyte chemoattractant protein-1 (MCP-1), leptin (LEP), and adiponectin (ADPN) in the serum were detected using an ELISA kit.

[0124] The results are as follows Figure 17 and Figure 18As shown, DIO mice exhibited a sharp increase in serum levels of TNF-α, IL-1β, MCP-1, and LEP (P<0.01), and a decrease in IL-4 and ADPN levels (P<0.05), demonstrating typical characteristics of chronic low-grade inflammation. After drug treatment, TNF-α, IL-1β, MCP-1, and LEP levels significantly decreased, while ADPN secretion significantly increased. High-dose IQ showed the most significant anti-inflammatory effect (P<0.001). All drug treatment groups showed some improvement in IL-4 secretion, but there was no significant difference (P<0.001). Figure 17 c and Figure 18 c).

[0125] The above results indicate that SSEE-30%D and isoquercitrin exert their anti-inflammatory effects by inhibiting the secretion of pro-inflammatory factors and upregulating the levels of anti-inflammatory factors.

[0126] Example 9: Effects of SSEE-30%D and isoquercitrin on liver damage and mitochondrial respiratory chain in DIO mice

[0127] (1) Observation of liver appearance and histopathology:

[0128] Eight weeks after drug administration, mice were sacrificed, and liver tissue was collected for observation of liver appearance and H&E staining. Results are as follows: Figure 19 fg and Figure 20 As shown in fg, the livers of mice in the HFD group showed significant leukopenia, indicating substantial lipid deposition; H&E staining revealed significant fatty vacuolation and microvesicular fatty degeneration in hepatocytes. After intervention with SSEE-30%D and isoquercitrin, the liver appearance returned to normal, vacuolation was significantly alleviated, and the liver tissue became closer to normal morphology.

[0129] (2) Assay of mitochondrial respiratory chain complex enzyme activity:

[0130] Liver tissues were collected from mice in each group, mitochondria were extracted, and the enzyme activities of mitochondrial respiratory chain complex I (NADH-CoQ reductase, CI), complex II (succinate-CoQ reductase, CII), complex III (CoQ-cytochrome C reductase, CIII), complex IV (cytochrome C oxidase, COX), and complex V (ATP synthase) were detected using the corresponding kits.

[0131] The results are as follows Figure 19 ae and Figure 20 As shown in ae:

[0132] Complex I: Compared with the NFD group, CI activity was significantly reduced in the HFD group; intervention with SSEE-30%D and isoquercitrin upregulated CI enzyme activity and promoted ATP production.

[0133] Complex II: SSEE-30%D and isoquercitrin can increase CII activity and optimize electron transfer efficiency.

[0134] Complex III: SSEE-30%D and isoquercitrin can upregulate CIII activity and promote electron transport and proton pumping.

[0135] Complex IV: SSEE-30%D and isoquercitrin can enhance COX activity and promote ATP production.

[0136] Complex V: SSEE-30%D and isoquercitrin can significantly enhance the activity of complex V and improve ATP synthesis efficiency.

[0137] The above results suggest that SSEE-30%D and isoquercitrin may alleviate lipid deposition-induced liver damage by enhancing the activity of hepatic mitochondrial respiratory chain enzymes and improving mitochondrial dysfunction.

[0138] Example 10: Effects of SSEE-30%D and isoquercitrin on adipose tissue morphology in DIO mice

[0139] (1) Measurement of adipose tissue weight:

[0140] Eight weeks after administration, the mice were sacrificed, and the epididymal fat (eWAT), inguinal fat (iWAT), and perirenal white adipose tissue (pWAT) were separated, weighed, and the weight of each adipose tissue was calculated.

[0141] The results are as follows Figure 21 As shown in bc, compared with the NFD group, the eWAT, iWAT, and pWAT weights of mice in the HFD group were significantly increased (P<0.001). After intervention with SSEE-30%D and isoquercitrin, the weight of each adipose tissue decreased, with the high-dose SSEE-30%D showing a particularly significant weight loss effect (P<0.01).

[0142] (2) H&E staining of adipose tissue:

[0143] eWAT, iWAT and brown adipose tissue (BAT) of mice in each group were collected and H&E stained to observe changes in adipocyte morphology.

[0144] The results are as follows Figure 21 As shown in the figure, the WAT cells in the NFD group mice were neatly arranged, with uniform cell morphology and size and tight connections, mainly in a round shape; the WAT cells in the HFD group showed obvious hypertrophy, severe vacuolation, and deformed cell morphology. After drug treatment, the adipocytes became smaller, their number increased, and their normal morphology was restored. In addition, SSEE-30%D and isoquercitrin also improved the morphology of BAT tissue, increased the proportion of multilocular small lipid droplets in the cells, and reduced unilocular large lipid droplet-like cells.

[0145] The above results indicate that SSEE-30%D and isoquercitrin exert their weight-loss effects by improving the morphology of WAT and BAT and reducing WAT weight.

[0146] Example 11: Effects of SSEE-30%D and isoquercitrin on organ indices in DIO mice

[0147] Eight weeks after administration, the mice were sacrificed, and the heart, liver, spleen, lungs, kidneys, and pancreas were separated, weighed, and the organ index (organ weight / body weight × 100%) was calculated.

[0148] The results are as follows Figure 22 and Figure 23 As shown, compared with the NFD group, the liver index (P<0.001) and kidney index (P<0.01) of mice in the HFD group were significantly reduced, indicating that HFD induced liver and kidney dysfunction. After drug intervention, the liver and kidney indices of mice in all groups recovered, with high-dose SSEE-30%D, high-dose IQ, and orlistat showing particularly significant improvement. In addition, SSEE-30%D can improve heart failure and metabolic disorders; high-dose SSEE-30%D can also significantly improve the pancreatic index (P<0.01), suggesting its potential role in alleviating insulin resistance and related pancreatic damage.

[0149] Example 12: The ameliorative effect of SSEE-30%D and isoquercitrin on liver and kidney damage in DIO mice

[0150] Eight weeks after administration, blood was collected from the orbital sinuses of mice in each group, serum was separated, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) in the serum were detected using a fully automated biochemical analyzer.

[0151] The results are as follows Figure 24 As shown, compared with the NFD group, the serum levels of ALT, AST, BUN, and Cr in the HFD group mice were significantly increased, indicating that HFD induced liver and kidney damage. After treatment with SSEE-30%D and isoquercitrin, the levels of each indicator decreased (P<0.05), indicating that both had an ameliorative effect on HFD-induced liver and kidney damage.

[0152] Example 13: Effects of SSEE-30%D and isoquercitrin on the expression of mitochondrial autophagy-related proteins in DIO mice

[0153] Eight weeks after administration, mice were sacrificed, BAT tissue was collected, total protein was extracted, and the protein expression levels of peroxisome proliferator-activated receptor-α (PPAR-α), PPAR-γ, PPAR-δ, uncoupling protein-1 (UCP-1), Beclin-1, Bcl-2, Bax, and p62 were detected by Western blot.

[0154] (1) Effects of SSEE-30%D on mitophagy-related proteins:

[0155] The results are as follows Figure 25 As shown, compared with the NFD group, the expression levels of PPAR-α, PPAR-γ, and PPAR-δ proteins were decreased in the HFD group. After treatment with high-dose SSEE-30%D, the expression levels of the above PPARs proteins were significantly upregulated (P<0.05). Furthermore, high-dose SSEE-30%D also significantly upregulated the protein expression levels of UCP-1, LC3-II / LC3-I, Beclin-1, and Bax (P<0.05), while downregulating the protein expression levels of Bcl-2 and p62 (P<0.01).

[0156] (2) Effects of isoquercitrin on mitochondrial autophagy-related proteins:

[0157] The results are as follows Figure 26 As shown, isoquercitrin can also significantly upregulate the protein expression levels of PPAR-α, PPAR-γ and PPAR-δ (P<0.05), significantly upregulate the expression levels of autophagy-related proteins UCP-1, Beclin-1 and Bax (P<0.01), and downregulate the expression levels of Bcl-2 and p62 (P<0.01).

[0158] The above results indicate that SSEE-30%D and isoquercitrin can exert anti-obesity effects by upregulating the expression of PPAR family proteins, activating mitophagy, and promoting thermogenesis in brown adipose tissue.

[0159] Example 14: Pharmaceutical formulation containing SSEE-30%D

[0160] Tablet preparation:

[0161] Take 100g of SSEE-30%D prepared in Example 1, mix it evenly with 200g of microcrystalline cellulose, 100g of lactose and 30g of crospovidone, prepare a soft mass with an appropriate amount of 85% ethanol as a wetting agent, granulate it through a 20-mesh sieve, dry it at 60℃, granulate it, add 5g of magnesium stearate, mix it evenly, compress it into tablets, and you will get SSEE-30%D tablets, each tablet containing 50mg of SSEE-30%D.

[0162] Preparation of capsules:

[0163] Take 100g of SSEE-30%D prepared in Example 1, mix it evenly with 150g of starch and 100g of microcrystalline cellulose, pass it through an 80-mesh sieve, and fill it into hard capsule shells to obtain SSEE-30%D capsules, each containing 50mg of SSEE-30%D.

[0164] It should be understood that numerous specific implementation decisions can be made during the development of any practical implementation, such as in any engineering or design project. Such development efforts may be complex and time-consuming, but for those skilled in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.

[0165] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An active component of Suaeda salsa, characterized in that, The active component is the 30% ethanol elution fraction (SSEE-30%D) obtained by macroporous adsorption resin column chromatography of Suaeda salsa extract.

2. A method for preparing isoquercitrin, using the active component of *Suaeda salsa* as described in claim 1, characterized in that: The preparation steps include the following; S1. First silica gel column chromatography: SSEE-30%D was loaded onto a silica gel column and eluted with a gradient of dichloromethane-methanol mixed solvent. Each fraction was detected by thin-layer chromatography (TLC). Fractions with consistent spot characteristics were combined to obtain component II. S2. Second silica gel column chromatography: Component II was loaded onto a silica gel column and eluted with a gradient of dichloromethane-methanol mixed solvent. The fraction that showed a single spot on TLC and a single peak on HPLC was collected and dried to obtain isoquercitrin monomer.

3. The method according to claim 2, characterized in that: In S1, the volume ratios of dichloromethane to methanol in the gradient elution are 20:1, 15:1, 10:1, 5:1, 3:1, 1:1, and 0:1, respectively. Component II is obtained by combining fractions with similar target spots when using a dichloromethane to methanol eluent in a 20:1 volume ratio for TLC detection, resulting in an Rf value of 0.15-0.

25.

4. The method according to claim 2, characterized in that: In step S2, the volume ratios of dichloromethane and methanol in the gradient elution were 12:1, 10:1, and 0:1, respectively; the resulting isoquercitrin monomer has the molecular formula C2. 21 H 20 O 12 Its molecular weight is 464.

38.

5. The application of the active component of Suaeda salsa according to claim 1 in the preparation of anti-obesity drugs or health products.

6. The application of the active component of Suaeda salsa according to claim 1 in the preparation of drugs or health products for regulating glucose and lipid metabolism disorders.

7. The use of the active ingredient of Suaeda salsa according to claim 1 in the preparation of drugs or health products for improving insulin resistance.

8. The use of the active component of Suaeda salsa according to claim 1 in the preparation of drugs or health products for improving non-alcoholic fatty liver disease.

9. The application of isoquercitrin in the preparation of drugs or health products for anti-obesity, regulation of glucose and lipid metabolism disorders, improvement of insulin resistance, and improvement of non-alcoholic fatty liver disease, characterized in that, The isoquercitrin is prepared by the method described in any one of claims 2-4.

10. A pharmaceutical composition for the prevention and treatment of obesity and disorders of glucose and lipid metabolism, characterized in that, It contains an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is SSEE-30%D and isoquercitrin.

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