Directed construction method of deglycosylated alfalfa saponins and activity evaluation and application thereof

Abstract

The application discloses the technical field of plant extract and animal nutrition, and discloses a method for directional construction of deglycosylated alfalfa saponin, and activity evaluation and application of the deglycosylated alfalfa saponin. The method uses alfalfa as raw material, adopts ultrasonic-assisted extraction combined with response surface method optimization process, and obtains alfalfa saponin (AS) through macroporous adsorption resin purification, and then uses beta-glucosidase from aspergillus niger to directionally hydrolyze the C-3 and C-28 sugar groups of AS, so as to obtain deglycosylated alfalfa saponin. The application also establishes a multi-dimensional activity evaluation system covering in-vitro chemical evaluation, cell model evaluation, mouse in-vivo evaluation and rumen fermentation evaluation. The results show that the antioxidant and antibacterial activities of the deglycosylated alfalfa saponin are significantly enhanced compared with AS, the deglycosylated alfalfa saponin can improve lipid metabolism disorder of high-fat diet mice, inhibit rumen methane production, and protect rumen epithelial cells from oxidative stress and inflammation damage, and the application provides a basis for research and development of natural functional feed additives.

Description

Technical Field

[0001] This invention belongs to the field of plant extracts and animal nutrition technology, specifically involving the targeted deglycosylation modification of alfalfa saponins using β-glucosidase to enhance their bioactivity, and the uses of the resulting products. Background Technology

[0002] In existing technologies, alfalfa (Medicago sativa L.) is a perennial herbaceous plant belonging to the legume family. It possesses wide ecological adaptability and excellent nutritional value, prefers warm and semi-arid climates, and has a long history of cultivation. Currently, alfalfa is widely cultivated in North America, Europe, and Asia, occupying an important position in global livestock farming and providing high-quality feed resources for cattle, sheep, pigs, and poultry. Furthermore, alfalfa has an excellent nitrogen-fixing capacity, making it an important green manure crop for soil improvement, playing a positive role in improving soil structure and reducing the use of chemical fertilizers.

[0003] Alfalfa's secondary metabolites have attracted widespread attention due to their rich bioactivity. Related studies show that alfalfa contains bioactive components such as flavonoids, saponins, alkaloids, coumarins, leaf proteins, and polysaccharides, as well as photosynthetic pigments such as chlorophyll, xanthophyll, carotene, and carotenoids. These secondary metabolites participate in the regulation of plant growth and development and defense mechanisms, while also exhibiting antioxidant, anti-inflammatory, antibacterial, and antitumor bioactivities, making them valuable for applications in pharmaceuticals, health products, and cosmetics research and development.

[0004] Alfalfa saponins (AS), as one of the important active components of alfalfa, possess significant pharmacological activities such as antitumor, anti-inflammatory, immunomodulatory, and antibacterial effects. However, the maintenance mechanism of AS's bioactivity and dose-response relationship in production applications remain unclear. Furthermore, abnormal increases in saponin content during silage processing can lead to rumen bloat in ruminants, the molecular regulatory mechanism of which is not yet understood. Currently, there are still many gaps in research on the molecular mechanisms of AS, including the elucidation of its physicochemical properties, the changes in activity and structural stability during silage, strategies for improving bioavailability, and the regulatory mechanisms on ruminant metabolism—key scientific issues that urgently require systematic investigation.

[0005] With the rising incidence of metabolic diseases, the regulatory role of plant-derived saponins in lipid metabolism has received increasing attention. Multiple studies have shown that diosgenin inhibits SREBP-1c expression by activating the AMPK signaling pathway, while downregulating ACC and upregulating CPT-1 expression to promote fatty acid oxidation; bupleuronide D inhibits SREBP-1c-mediated fatty acid and cholesterol synthesis by activating PPAR-α and upregulating INSIG1 / 2 expression; and gymnema sylvestre saponins reduce lipid accumulation by inhibiting LXR expression. These studies provide important references for the application of saponins in metabolic regulation.

[0006] However, natural saponins generally suffer from low bioavailability, primarily because the presence of glycosylated structures in the saponin molecule limits their transmembrane permeability and target binding efficiency. Deglycosylation modification is considered an effective structural optimization strategy, as removing sugar chains can significantly improve the lipid solubility and bioactivity of saponins. However, current methods for the targeted deglycosylation of alfalfa saponins are still imperfect, and the systematic evaluation of the bioactivity of deglycosylated products and their application potential in animal nutrition require further in-depth research.

[0007] Therefore, developing an efficient method for the targeted construction of alfalfa saponins through deglycosylation and systematically evaluating their bioactivity and application value is of great theoretical significance and practical guiding value for promoting the optimization of efficient utilization processes of alfalfa saponins and the construction of a precise nutritional regulation system for ruminants. Summary of the Invention

[0008] This invention aims to provide a method for the directional construction of deglycosylated alfalfa saponins and their activity evaluation and application, in order to solve the technical problems of low bioavailability, insufficient extraction rate, single activity evaluation system, and unclear metabolic regulation mechanism in the rumen of ruminants caused by the glycosylation structure of existing alfalfa saponins.

[0009] Therefore, the objective of this invention is achieved as follows: a method for the directional construction of deglycosylated alfalfa saponins, comprising the following steps:

[0010] (1) Extraction and purification: Using alfalfa as raw material, the extraction process was optimized by ultrasonic-assisted extraction combined with response surface methodology. The extraction solvent was ethanol-water solution to obtain crude alfalfa saponin extract, which was then purified by macroporous adsorption resin to obtain alfalfa saponin AS with a purity ≥95%.

[0011] (2) Directed deglycosylation: AS was dissolved in sodium acetate buffer, and β-glucosidase from Aspergillus niger was added. The reaction was carried out at 35-37℃ with shaking for 12-24h to enzymatically hydrolyze the C-3 and C-28 glycosyl groups of AS.

[0012] (3) Post-treatment: The reaction solution was inactivated by a 100°C water bath, cooled in an ice bath, centrifuged to obtain the supernatant, purified by a 300Da molecular weight cutoff dialysis bag and freeze-dried to obtain deglycosylated alfalfa saponin DAS.

[0013] A further improvement of the present invention is that the process parameters optimized by the response surface methodology in step (1) are: a solvent-to-raw material mass ratio of (10-14):1, an extraction time of 2.7-2.9 h, an extraction temperature of 70-80 °C, and an ethanol volume fraction of 60%-80%. The preferred solvent-to-raw material mass ratio is 13:1, the extraction time is 2.8 h, and the extraction temperature is 72.8 °C.

[0014] A further improvement of the present invention is that the macroporous adsorption resin in step (1) is DM-130 type, the elution conditions are 50% ethanol solution, flow rate 2.0 BV / h, column temperature 60℃; the mass ratio of β-glucosidase to AS in step (2) is (10-12):1, and the pH of the reaction system is 5.0.

[0015] A further improvement of the present invention is that, before extraction and purification, a silage pretreatment step is performed: alfalfa is cut into sections and wilted to a moisture content of 65%, a fermentation agent is added, and the mixture is sealed and stored at 25°C for 60 days to obtain silage alfalfa. Then, the extraction and purification in step (1) are performed, and the resulting deglycosylated product is silage-derived deglycosylated saponins SPDS or SSDS.

[0016] This invention also provides a method for evaluating the activity of deglycosylated alfalfa saponins, wherein the deglycosylated alfalfa saponins (DAS or SPDS) prepared by the above method are subjected to two or more of the following activity evaluations:

[0017] Antioxidant activity evaluation: The IC50 of the samples in the concentration range of 0-200 μg / mL was determined using the DPPH free radical scavenging method and the FRAP ferric reduction method. 50 Value and Fe 2+ equivalent;

[0018] Antibacterial activity evaluation: The micro-broth dilution method was used, with Escherichia coli (E. coli) as the indicator bacterium, and the inoculum size was 10. 5 -10 6 CFU / mL, determine the minimum inhibitory concentration (MIC);

[0019] Cell safety evaluation: BEAS-2B cells or goat rumen epithelial cells were cultured at 1×10⁻⁶ using the CCK-8 assay. 5 After seeding at a density of 10 cells / mL, add 5-240 μg / mL of DAS or SPDS and culture for 6 h, then measure cell viability.

[0020] Evaluation of metabolic regulation in vivo: A high-fat diet-induced HFD C57BL / 6 mouse model was constructed. DAS was administered by gavage at a dose of 300 mg / kg / d for 30 consecutive days. Serum total cholesterol (TC), triglycerides (TG), fasting blood glucose, and the activities of antioxidant enzymes SOD, CAT, and GSH-PX were measured. The relative expression levels of LXR, NPC1L1, TNF-α, IL-6, and IL-10 genes in liver tissue were detected by RT-qPCR.

[0021] Evaluation of rumen epithelial protection: An inflammation model was established by stimulating goat rumen epithelial cells with 1 μg / mL lipopolysaccharide (LPS). The cells were then co-cultured with 60 μg / mL SPDS for 6 h. The mRNA expression levels of TLR4, MYD88, NF-κB, IRF3, MMP-9, IL-1β, TNF-α, and IL-10 were detected by RT-qPCR. The intracellular activities of SOD, CAT, GSH-PX, and MDA content were also measured.

[0022] Evaluation of methane emission reduction by rumen in vitro fermentation: In a goat rumen in vitro fermentation system, 3.0 mg of DAS or SPDS was added, and after anaerobic culture at 39 °C for 24 h, methane production and volatile fatty acid composition were determined by gas chromatography, and rumen microbial community structure was analyzed by 16S rRNA high-throughput sequencing.

[0023] The deglycosylated alfalfa saponins obtained by this invention can be used in the preparation of products that improve lipid metabolism disorders in animals. Specifically, they can be used in the preparation of functional foods or feed additives to reduce serum TC and TG levels induced by a high-fat diet and to upregulate the expression of hepatic cholesterol absorption-related genes LXR and NPC1L1.

[0024] The deglycosylated alfalfa saponins prepared by this invention can be used in the preparation of feed additives that protect the rumen epithelial barrier of ruminants and inhibit rumen methane production.

[0025] The deglycosylated alfalfa saponins prepared by this invention can also be used in the preparation of anti-inflammatory and antioxidant agents. These agents maintain cellular redox homeostasis by downregulating the expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β and upregulating the expression of anti-inflammatory factor IL-10.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. Significantly improves the extraction rate of alfalfa saponins. This invention uses ultrasonic-assisted extraction technology combined with response surface methodology to optimize process parameters, achieving an extraction rate of 1.5% for alfalfa saponins, which is significantly higher than that of traditional solvent extraction methods. It also shortens extraction time and reduces solvent consumption, which is beneficial for industrial production.

[0028] 2. Targeted deglycosylation significantly enhances the bioactivity of saponins. This invention utilizes β-glucosidase derived from Aspergillus niger to specifically hydrolyze the β-glycosidic bonds of alfalfa saponins, targeting the removal of glycosyl groups at C-3 and C-28 to obtain deglycosylated alfalfa saponins (DAS). Compared with unmodified alfalfa saponins, DAS exhibits significantly enhanced free radical scavenging and iron ion reducing abilities (P < 0.05), and a 5.2-fold increase in the inhibition rate against Escherichia coli, confirming that glycosylation inhibits saponin activity and that deglycosylation is an effective strategy for enhancing saponin bioactivity.

[0029] 3. A comprehensive, multi-dimensional, cross-model systematic activity evaluation system has been established. This invention constructs a full-chain activity evaluation system, encompassing in vitro chemical evaluation (DPPH free radical scavenging, FRAP iron ion reduction), cell model evaluation (BEAS-2B cell safety, anti-inflammatory and antioxidant effects in goat rumen epithelial cells), in vivo evaluation in small animals (metabolic regulation in high-fat diet C57BL / 6 mice), and rumen fermentation evaluation (methane emission reduction, microbial community analysis). This system can comprehensively and systematically reflect the structure-activity relationship and application potential of deglycosylated alfalfa saponins.

[0030] 4. Deglycosylated alfalfa saponins (DAS) exhibit excellent metabolic regulation. In a high-fat diet-induced C57BL / 6 mouse model, DAS significantly reduced serum total cholesterol (TC) and triglyceride (TG) levels, improved fasting blood glucose, upregulated the expression of anti-inflammatory factor IL-10, and downregulated the expression of pro-inflammatory factors TNF-α and IL-6 (P < 0.05). Simultaneously, it regulated the expression of cholesterol absorption-related genes LXR and NPC1L1, effectively alleviating lipid metabolism abnormalities and chronic inflammatory responses. No reproductive or organ toxicity was observed, demonstrating good safety.

[0031] 5. The synergistic effect of silage and enzymatic hydrolysis further enhances saponin activity. This invention systematically evaluated the saponin activity of 21 different fall-dormant alfalfa varieties after silage, and found that the antioxidant activity of saponins from high fall-dormant varieties (FD9-FD10) after silage was significantly better than that of low fall-dormant varieties (FD4). The synergistic treatment of silage and deglycosylation further enhanced saponin activity, providing a new direction for feed processing and the development of saponin functions.

[0032] 6. Deglycosylated saponins possess dual functions of reducing rumen methane emissions and protecting rumen epithelium. In a goat rumen liquid in vitro fermentation model, the addition of 3.0 mg of deglycosylated saponins significantly inhibited the abundance of methanogenic bacteria, promoted the conversion of hydrogen to propionic acid, and reduced methane production by 30.15%. Simultaneously, in a lipopolysaccharide-induced goat rumen epithelial cell inflammation model, SPDS effectively alleviated oxidative stress and inflammatory damage by inhibiting the activation of the TLR4-MYD88 / NF-κB and TRIF-IRF3 signaling pathways, downregulating the expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β, upregulating the expression of the anti-inflammatory factor IL-10, enhancing the activity of antioxidant enzymes (SOD, CAT, GSH-PX), reducing the level of lipid peroxide MDA, and inhibiting MMP-9 expression, providing a new solution for the health management of ruminants.

[0033] In summary, this invention significantly enhances the bioactivity of alfalfa saponins through deglycosylation-directed construction technology, establishes a comprehensive activity evaluation system, and reveals its multiple application values ​​in metabolic regulation, rumen methane reduction, and rumen epithelial protection, providing a scientific basis and technical support for the research and development of natural functional feed additives. Attached Figure Description

[0034] Figure 1 Phase diagram of bioavailability of alfalfa saponins (AS) under optimal extraction conditions.

[0035] Figure 2 This is a diagram showing the product analysis of alfalfa saponins (AS) after hydrolysis by β-glucosidase.

[0036] Figure 3 A comparison chart showing the final purity of saponins from 21 alfalfa varieties after silage.

[0037] Figure 4 A comparative graph showing the antioxidant activity of saponins (AS) before and after silage in 21 alfalfa varieties. In the graph, (A) shows the half-maximal scavenging concentration (IC50) of DPPH (2,2-diphenyl-1-picrylhydrazine) free radicals. 50 (B) Ferric ion reducing power (FRAP).

[0038] Figure 5 A comparative graph showing the antioxidant activities of enzymatically hydrolyzed saponins from fresh Pegasus (FPDS), fresh Salsa (FSDS), silage Pegasus (SPDS), and silage Salsa (SSDS). Among them, (A) shows the half-maximal concentration (IC50) of 2,2-diphenyl-1-picrylhydrazine (DPPH) free radicals. 50 (B) Ferric reducing power (FRAP).

[0039] Figure 6A comparative graph showing the effects of alfalfa saponins (AS) and deglycosylated saponins (DAS) on serum total cholesterol (TC) and triglycerides (TG) in C57BL / 6 mice.

[0040] Figure 7 A comparative graph showing the effects of alfalfa saponins (AS) and deglycosylated saponins (DAS) on serum prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) in C57BL / 6 mice.

[0041] Figure 8 Bar chart showing the effect of enzymatic saponins (SPDS) from silage pegasis on the relative activity of goat rumen epithelial cells.

[0042] Figure 9 A comparative diagram showing the effects of enzymatic hydrolysed saponins (SPDS) from silage pegasis on the antioxidant properties of goat rumen epithelial cells stimulated by lipopolysaccharide (LPS).

[0043] Figure 10 Effects of enzymatic saponins (SPDS) from silage on the relative mRNA expression levels of inflammation-related genes in goat rumen epithelial cells stimulated by lipopolysaccharide (LPS): (A) NF-κB and IL-6; (B) IL-8 and TNF-α; (C) IL-1β; (D) IL-10; (E) TOLLIP; (FF) TLR4; (G) MYD88; (H) IRF3; (I) MMP-9. Detailed Implementation

[0044] Example 1

[0045] The targeted construction and bioactivity verification of deglycosylated alfalfa saponins were carried out according to the following methods:

[0046] Ultrasonic-assisted extraction technology was employed, with the solvent-to-raw material mass ratio (10-14):1, extraction time (2.7-2.9 h), and extraction temperature (70-80℃) as key variables. A predictive model was established using response surface methodology to optimize the process parameters. The optimal solvent-to-raw material mass ratio of 13:1, extraction time of 2.8 h, and extraction temperature of 72.8℃ significantly improved the extraction rate of Aspergillus oryzae (AS) by 1.5%. Furthermore, the bioavailability of AS was quantitatively assessed using an in vitro simulated digestion model (gastrointestinal stage), providing a scientific basis for its practical application.

[0047] The crude extract was purified using DM-130 macroporous adsorption resin, and the atherosclerotic (AS) component was separated by methanol-water gradient elution (35%-100% methanol). The AS component was identified using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-QTOF / MS). The deglycosylated product DAS was obtained by β-glucosidase hydrolysis, and UPLC-QTOF / MS was used to verify glycosidic bond cleavage and molecular structural changes. A multi-dimensional activity evaluation system was employed, including the determination of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical scavenging capacity, and reducing power analysis, to quantify the difference in antioxidant activity between AS and DAS. The antibacterial activity against *Escherichia coli* (E. coli) was determined by microdilution. Finally, the effects of AS and DAS on the inhibition rate of human lung epithelial cells (BEAS-2B) were evaluated. Through the above structural characterization and functional evaluation, the mechanism by which deglycosylation modification affects the bioactivity of AS was elucidated, and its structure-activity relationship was preliminarily revealed.

[0048] 1. Experimental Methods

[0049] 1-1 In vitro bioavailability of alfalfa saponins

[0050] An in vitro simulated digestion model was established to determine the bioavailability of asthenospermia (AS). Gastric digestion stage: 100 mg AS and 10 mg lecithin were mixed in 14 mL of gastric digestive fluid (containing 6 mM CaCl2, 150 mM NaCl, pH 2.5), and premixed at 37°C with constant shaking (50 r / min) for 10 min. Then, 3 mL of an enzyme mixture containing 170 mg gastric lipase and 15 mg pepsin was added, and the reaction was maintained under the same conditions for 50 min. The pH was monitored every 10 min, and finally adjusted to pH 4.5 with 0.1 M NaHCO3 solution. Intestinal digestion stage: Pre-cooled bile salt secretion fluid (containing 0.1 g lecithin, 0.25 g bile salts, 0.5 mL 325 mM CaCl2, 1.5 mL 3.25 M NaCl, and 10 mL pH 7.5 Tris-maleate buffer) was homogenized on ice for 10 min and then added to the digestion system. The mixture was shaken at 37°C and 200 r / min for 1 min. Then, freshly prepared trypsin extract (0.5 g trypsin dissolved in 3 mL Tris-maleate buffer) was added, and the supernatant was collected by centrifugation. The pH was adjusted to 7.0, and the reaction was continued at 37℃ and 200 r / min for 60 min with shaking. An equal volume of buffer was used as a blank control. Sample processing and detection: The digest was centrifuged at 37℃ and 2688 r / min for 40 min to separate the aqueous phase (micelle phase) and the precipitate phase. The absorbance of the digest (total saponin content) and the aqueous phase sample at 520 nm before centrifugation was measured (using Tris-maleate buffer as a reference). Bioaccessibility was calculated using the following formula:

[0051] Bioavailability (%) = (Saponin content in aqueous phase / Total saponins in digestion medium) × 100%

[0052] In the formula, the content of saponins in the aqueous phase needs to be corrected for blank. The entire experiment was conducted at a constant temperature of 37±0.5℃, and the pH of the system was monitored in real time using a pH meter.

[0053] Purification of 1-2 alfalfa saponins

[0054] Adsorption and desorption purification of saponins (AS) was performed using DM-130 macroporous resin. After activation with 95% ethanol for 24 h, the resin was washed with deionized water until no alcohol odor remained, and then wet-packed (8.0 × 1.0 cm, column volume 6.3 mL). Loading conditions were: AS solution concentration 10 mg / mL (pH 5.0), constant temperature 25±1℃, loading flow rate 2.0 BV / h (12.6 mL / h), and adsorption was considered saturated when the saponin concentration in the effluent reached the initial 10%. After washing with 3 BV of ultrapure water (2.0 BV / h) to remove impurities, 3 BV of preheated 50% ethanol solution (60±1℃) was eluted at the same flow rate, maintaining the column temperature at 60℃ throughout. After elution, the resin was regenerated sequentially with 2 BV of 95% ethanol and deionized water, and finally stored in 20% ethanol (4℃).

[0055] 1-3 Biotransformation and Identification of Alfalfa Saponins

[0056] 1-3.1 β-Glucosidase hydrolyzes alfalfa saponins

[0057] DAS was prepared using a β-glucosidase-catalyzed method. 10 mg of β-glucosidase was dissolved in 0.1 mL of 25 mM NaAc buffer (pH 5.0, sterilized using a 0.22 µm filter) and stored on ice for later use. 10.00 mg of AS powder was weighed and dissolved in 10 mL of NaAc buffer, followed by vortexing (3 min) and sonication-assisted dissolution (40 kHz, 5 min) to prepare a 1.0 mg / mL AS solution. 90 μL of the AS solution was mixed with 10 μL of the enzyme solution in a 1.5 mL centrifuge tube and reacted at 37 ± 0.5 °C and 120 r / min for 24 h. After the reaction was terminated, the enzyme was inactivated in a 100 °C water bath for 10 min, immediately cooled on ice, and centrifuged at 12000 r / min for 5 min to collect the supernatant. The supernatant was filtered through a 0.22 µm organic filter membrane and purified for 48 h using a 300 Da molecular weight cutoff dialysis bag (with ultrapure water replaced every 6 h). The resulting DAS powder was lyophilized, weighed (±0.01 mg), and stored according to the AS storage conditions (shelf life ≤ 6 months).

[0058] 1-3.2 Determination of alfalfa saponins and deglycosylated alfalfa saponin components

[0059] Samples were analyzed using UPLC-QTOF / MS. Samples were diluted 4-fold with ultrapure water containing 2 ng / μL digoxin (internal standard), centrifuged at 19300 r / min for 15 min at 4℃, and the supernatant was injected. Chromatographic conditions: Waters BEH C18 column (50±0.5℃), mobile phases A (0.1% formic acid aqueous solution) and B (0.1% formic acid acetonitrile), flow rate 0.3 mL / min, gradient elution program (0–1 min 25% B, 1–24 min 25%–55% B, 24–26 min 55%–100% B, 26–30 min 25% B equilibration). Mass spectrometry parameters: nano-current electrospray ionization negative ion mode, capillary voltage 3.1 kV, ion source temperature 140℃, desolvation gas (N2) flow rate 700 L / h, temperature 350℃, scan range m / z 150-2000, data-dependent acquisition mode (trigger threshold 1000 counts, collision energy 5-40 eV), real-time mass correction (reference ion m / z 554.2615). Data were acquired using MassLynx 4.2 and peak extraction and normalization were performed using Progenesis QI 3.0.

[0060] Quantification was performed using a Dionex Ultim KB te 3000RS UPLC system (equipped with a 4℃ autosampler) coupled with a Bruker Impact II QTOF mass spectrometer in electrospray ionization (ESI) mode. Chromatographic conditions: Acquity UPLC HSS T3 column (40℃), mobile phases A (5 mM ammonium formate aqueous solution) and B (acetonitrile), flow rate 0.4 mL / min. Mass spectrometry parameters: capillary voltage 3 kV, drying gas (N2) flow rate 6 L / min, nebulizer gas pressure 0.7 bar, drying temperature 200℃, scan range m / z 150–2000, collision energy 20–120 eV (dynamically switchable), Targeted MS / MS acquisition mode (isolation window m / z 2). Pre-analysis calibration was performed using 5 mM sodium formate-50% isopropanol solution (20 μL) (mass error ≤ 1 ppm). Data were processed using Bruker DataAnalysis 5.0 and quantified using the external standard method (standard curve 0.1–100 μg / mL, R² ≥ 0.999). The characteristic mass-to-charge ratio of AS was matched with the Bruker Sumner Metabose plant library version 1.0.

[0061] The monosaccharide components (xylose, glucuronic acid GlcA, glucose Glc, rhamnose Rha, and galactose Gal) in the enzymatic hydrolysis products were quantified using solid-phase extraction-liquid chromatography-mass spectrometry. Samples were purified using a Waters Oasis HLB column (60 mg / 3 mL), activated sequentially with methanol and ultrapure water, and then filtered through a 0.22 μm filter. Impurities were washed with 5% methanol / water, and the target analytes were eluted with 85% methanol / water. The solution was concentrated to 200 μL under nitrogen blowing and 10 μL of ¹³C-labeled monosaccharide internal standard (1 μg / mL) was added. Chromatographic conditions: Waters XBridge Amide column (35℃), mobile phase A (10 mM ammonium formate aqueous solution, pH 4.5) and B (acetonitrile), flow rate 0.3 mL / min, gradient elution program (0-1 min 85% B, 1-10 min 85%-70% B, 10-15 min 70%-50% B, 16-20 min 85% B equilibration). Mass spectrometry was performed in ESI± mode with the following parameters: capillary voltage ±3.5 / 2.8 kV, nebulizer gas pressure 1.5 bar, dry gas (N2) flow rate 8 L / min, temperature 220℃, and scan range m / z 100-500. Selective ion monitoring was performed using characteristic ions of the target monosaccharide ([MH]⁻: Xyl 149.0456, GlcA 193.0350, Glc 179.0561, Rha 163.0612, Gal 179.0561). Quantification was performed using ¹³C isotope internal standards.

[0062] 1-4. Results

[0063] 1-4.1 Bioavailability of alfalfa saponins

[0064] The effect of ultrasound-assisted extraction on the bioavailability of alfalfa saponins was evaluated using an in vitro digestion model. Figure 1 The results showed that ultrasonic treatment significantly improved the release kinetics of alfalfa saponins. With the extension of ultrasonic time, the cumulative release curve of alfalfa saponins conformed to the first-order kinetic model (R² = 0.987). The release process can be divided into a rapid phase (0-100 min, rate constant k1 = 0.015 / min) and a slow phase (100-300 min, rate constant k2 = 0.003 / min), ultimately improving the bioavailability to 18.23%.

[0065] 1-4.2 Identification of Alfalfa Saponins and Deglycosylated Alfalfa Saponins

[0066] Mass spectrometry analysis results show that ( Figure 2The purified AS had a purity of 96.23%. Comparison of the monosaccharide composition of AS and DAS revealed that the xylose content detected in DAS was consistent with the total xylose groups contained in the three saponins MAGA (3-GlcA-28-AraRhaXyl medicagenic acid), MAG1 (3-Glc-28-AraRhaXyl medicagenic acid), and ZAGA (3-GlcA-28-AraRhaXyl zanhic acid) in AS. The glucuronic acid content was comparable to the total glucuronic acid groups contained in MAGA and ZAGA in AS, while the glucose content matched the glucose group content of MAG1 in AS.

[0067] 1-5. Summary

[0068] 1-5.1 The hot ethanol ultrasound-assisted extraction method was used, and the optimal solvent-to-raw material mass ratio of 13:1, extraction time of 2.8 h, and temperature of 72.8℃ were further screened using response surface methodology, which significantly improved the AS extraction rate (1.5%). Ultrasound enhances AS dissolution by disrupting plant cell walls, which is key to improving its bioavailability.

[0069] 1-5.2 β-glucosidase from Aspergillus niger specifically hydrolyzes β-glycosidic bonds in DAS to achieve deglycosylation, which reduces the polarity of DAS molecules and promotes transmembrane permeation.

[0070] 1-5.3 DAS showed enhanced free radical scavenging ability compared to AS, and a 5.2-fold increase in the antibacterial rate against E. coli. Low concentrations activated the antioxidant defense of BEAS-2B cells, while high concentrations induced oxidative stress, exhibiting a dose-dependent bidirectional regulation.

[0071] Example 2

[0072] Effects of silage and enzymatic hydrolysis on the activity and structure of alfalfa saponins Microbial fermentation-driven biotransformation during silage processing can significantly alter saponin composition, for example, more than doubling the total saponin content. This biotransformation process involves the activation of complex enzyme systems and the establishment of microbial-plant interaction networks, mainly including:

[0073] 1) The anaerobic environment of silage promotes the proliferation of specific lactic acid bacteria, which secrete extracellular enzymes to catalyze the modification of saponin structure;

[0074] 2) Plant cell wall degradation releases bound saponins, improving their bioavailability;

[0075] 3) Microbial metabolites interact with saponins to form novel active complexes.

[0076] However, the coupling mechanism between microbial community dynamics and saponin conversion efficiency during the silage stage is still lacking systematic analysis. In particular, the synergistic effect mechanism between silage pretreatment and subsequent enzymatic hydrolysis is still unclear, which restricts the efficient utilization of alfalfa's active ingredients.

[0077] This embodiment investigates the effect of the synergistic effect of silage and enzymatic hydrolysis on the activity and structure of alfalfa saponins through the following experiments.

[0078] 2-1 Experimental Materials

[0079] Varieties include: Fado (dormancy level 4), from France; Instinct (dormancy level 4), from France; Mezzo (dormancy level 4), from France; Salsa (dormancy level 4), from France; Sibemol (dormancy level 4), from France; Vision (dormancy level 4), from France; Paola (dormancy level 5), from Italy; MS-5S43 (dormancy level 5), from the USA; Power 5020 (dormancy level 5), from Canada; Sandili (dormancy level 5), from the USA; Sidili (dormancy level 5), from the USA; Villiger (dormancy level 5), from Canada; Aurora (dormancy level 6), from Australia; Level 6 (dormancy level 6), from the USA; Blue-moon (dormancy level 7), from Italy; Claudia (dormancy level 7), from Italy; and Gannon 5. Varieties No. 5, hibernation level 7, domestically bred; Sardi 7II, hibernation level 7, from Australia; Eureka+, hibernation level 8, from Australia; Pegassis, hibernation level 9, from Australia; Sardi 10, hibernation level 10, from Australia. Hibernation level classification: 4 = semi-dormant; 5-7 = moderately dormant; 8-9 = dormant; 10 = non-dormant.

[0080] The tested alfalfa materials were collected in a standard experimental field on April 28, 2023 (early flowering stage), with a uniform stubble length of 2-3 cm at harvest. The study included 21 alfalfa varieties from Dannon Seed Group (Denmark), Bailv International Grassland Co., Ltd., Pioneer Seeds Inc. (USA), and Gansu Agricultural University. Their fall dormancy (FD) grades ranged from FD4 to FD10 (Table 1), including low-to-medium dormancy types (FD4-FD6) and medium-to-high dormancy types (FD7-FD10). The FD grading standard was based on alfalfa autumn growth rate and low-temperature adaptation characteristics; a higher FD value indicated stronger autumn growth activity but relatively lower cold resistance. Some fresh samples were freeze-dried and pulverized, and crude AS extract was obtained by ultrasonic extraction using an ethanol-water system. Fresh AS powder was then prepared by separation, purification, and freeze-drying (methods as described in 1-3.1 of Example 1). The remaining fresh samples were cut into 1-2 cm pieces and allowed to wilt for 6 hours (moisture content reduced to 65%). Then, the mixture was sprayed evenly and mixed with 5 mg of starter culture per kg of raw material. The mixture was then packed into polyethylene silage tanks at a compacted density of 700 kg / m³ and stored in a sealed container at 25°C for 60 days. After ensiling, the fermentation quality was assessed by measuring key parameters such as pH, lactic acid / acetic acid ratio, and ammonia nitrogen content. Samples meeting the standards were dried and pulverized using the same method (1-3.1) of Example 1 to prepare silage AS powder.

[0081] The corresponding desaccharified AS powder is as follows:

[0082] FPDS (Fresh Pegasis Deglycosylated Saponin): It is derived from fresh (unensiled) alfalfa variety Pegasis (dormant grade FD9), which is extracted and purified to obtain AS, and then subjected to targeted deglycosylation.

[0083] FSDS (Fresh Salsa Deglycosylated Saponin): It is derived from fresh (unensiled) alfalfa variety Salsa (dormant grade FD4), which is extracted and purified to obtain AS, and then subjected to targeted deglycosylation.

[0084] SPDS (Silage Pegasis Deglycosylated Saponin): It is derived from the alfalfa variety Pegasis that has undergone silage pretreatment (cut into sections and wilted to a moisture content of 65%, then sealed and stored at 25°C for 60 days), and is obtained by extracting AS and then enzymatically hydrolyzing to remove the glycosylation.

[0085] SSDS (Silage Salsa Deglycosylated Saponin): It is derived from the Salsa alfalfa variety that has undergone silage pretreatment. The aspartate sugar (AS) is extracted and then enzymatically hydrolyzed to remove the glycosylation.

[0086] The following are some of the saponins used in the comparative experiment:

[0087] FPS (Fresh Pegasis saponin): Original alfalfa saponin (AS) obtained by extraction and purification from fresh (unencumbered) alfalfa variety Pegasis (autumnal FD9).

[0088] FSS (Fresh Salsa saponin): Original alfalfa saponin (AS) obtained by extraction and purification from fresh (unencumbered) alfalfa variety Salsa (dormant grade FD4).

[0089] SPS (Silage Pegasis saponin): The original alfalfa saponin (AS) is obtained by extraction and purification from the alfalfa variety Pegasis, which has undergone silage pretreatment (cut into sections and wilted to a moisture content of 65%, then sealed and stored at 25°C for 60 days).

[0090] SSS (Silage Salsa saponin): Derived from the Salsa alfalfa variety after silage pretreatment, and obtained through extraction and purification of raw alfalfa saponin (AS).

[0091] 2-2 Silage Quality Assessment

[0092] Microbial analysis was performed on silage samples within 24 hours of opening. Specific microbial populations were quantitatively detected using selective culture media: lactic acid bacteria were counted on MRS agar (anaerobic incubation at 37°C for 48-72 h); yeast and mold were counted on Bengal red agar (aerobic incubation at 28°C for 48-72 h); and E. coli was detected on purple bile salt agar (aerobic incubation at 37°C for 18-24 h). The specific procedure was as follows: homogenized samples were serially diluted 10-fold (10... -1 Up to 10 -3 (Gradual dilutions) 100 μL of bacterial suspension at each dilution was evenly spread onto the corresponding culture medium surface, incubated upside down, and identified and counted based on typical colony morphology. Lactic acid bacteria formed milky white, round colonies; yeast formed milky white, smooth colonies; molds showed fluffy or pigmented colonies; and E. coli formed red colonies with a characteristic bile salt precipitation ring. The effective counting range was limited to 30-300 CFU / plate, and the final result was expressed as 1g CFU / g fresh weight. Strict aseptic operation procedures were followed throughout the process. Aerobic stability was assessed by monitoring the temperature change of silage samples exposed to a 25°C environment, with the time required for the sample temperature to rise 2°C above the ambient temperature as the evaluation criterion.

[0093] 2-3 Preparation and Activity Determination of Alfalfa Saponins Before and After Silage

[0094] The separation, purification, and purity of saponins were performed as described above, as were the determinations of DPPH free radical scavenging ability. The ferric reducing antioxidant power (FRAP) determination procedure was as follows: 0.10 mL of sample was mixed with 3 mL of freshly prepared FRAP working solution (containing 0.25 M sodium acetate buffer pH 3.6, 10 mM 2,4,6-tripyridyltriazine, and 20 mM FeCl3·6H2O, volume ratio 10:1:1), reacted at 37℃ for 30 min, and the absorbance was measured at 593 nm. Quantification was performed using the FeSO4·7H2O standard curve. Based on the DPPH free radical scavenging ability and FRAP determination results of 21 alfalfa varieties after silage, two varieties with the highest and two with the lowest antioxidant activity were selected. Their antioxidant activity and silage characteristic parameters were analyzed to elucidate the correlation between antioxidant activity and variety characteristics.

[0095] 2-4 Experimental Results

[0096] The number of lactic acid bacteria decreased with increasing FD (Table 1). The number of yeasts in Pegasis was higher than in Salsa (P < 0.05), while there was no significant difference in yeast count among the other varieties. Pegasis and Sardi 10 had high mold counts (P < 0.05), while Fado, Instinct, Mezzo, Salsa, Sibemol, and Vision had lower mold counts (P < 0.05). Furthermore, the pH after ensiling was below 4.72 in all these varieties, and the lactic acid content was also higher (Vision was 58.97 g / kg DM). E. coli was not detected in any treatment (< 0.1 lg CFU / g). Aerobic stability ranged from 149 to 158 h, with no significant differences among treatments.

[0097] Table 1: Microbial counts and aerobic stability results of 21 alfalfa varieties after silage

[0098]

[0099] The final purity of AS extracted from silage alfalfa ranged from 95.08% to 97.57%. Figure 3 Among the cultivars, Paola (FD5) and Sidili (FD5) had the highest purity, at 97.52% and 97.57%, respectively. The antioxidant activity of all AS varieties increased after ensiling. The DPPH free radical scavenging capacity showed significant differences among the varieties, with Salsa (FD4) having the highest IC50. 50 The value was highest (110.61 μg / mL), while Pegassis (FD9) was the lowest (76.68 μg / mL). IC50 50The value decreased with increasing dormancy level (FD4 to FD9), but FD10 (Sardi10) rebounded (83.07 μg / mL). Figure 4 A). Pegasis (FD9) exhibits the strongest iron-reducing ability (0.35 μM Fe). 2+ Salsa (FD4) had the lowest FRAP value (0.22 μM Fe / g). 2+ / g)(P < 0.05), the FRAP values ​​of the FD5-FD8 groups (Paola to Villiger) were concentrated in the range of 0.23-0.26 μM Fe. 2+ / g ( Figure 4 B). In summary, Pegasis (FD9) also possesses the lowest DPPHIC 50 The value (76.68 μg / mL) and the highest FRAP value (0.35 μM Fe) were compared. 2+ / g), while Salsa (FD4) showed the highest IC. 50 The value (110.61 μg / mL) and the lowest FRAP (ferric reducing power) value (0.22 μM Fe) were observed. 2+ / g). Based on the differences in dormancy range (FD4-FD9) and antioxidant properties, Salsa (FD4) and Pegassis (FD9) were selected as characteristic varieties.

[0100] The xylose content in FPDS (fresh Pegasus deglycosylated saponins) is equivalent to the total xylose groups of MAGA, MAG1, ZATA, and ZAGA in FPS (fresh Pegasus saponins); the glucuronic acid content in FPDS is equivalent to the total glucuronic acid groups of MAGA and ZAGA in FPS; the glucose content in FPDS is equivalent to the glucose group content of MAG1 in FPS; and the rhamnose and galactose content in FPDS is equivalent to the rhamnose and galactose group content of SS in FPS. Figure 5 A). The xylose content in FSDS is equivalent to the total xylose groups of MAGA, MAG1, and ZATA in FSS; the glucuronic acid content in FSDS is equivalent to the glucuronic acid group content of MAGA in FSS; the glucose content in FSDS (fresh Salsa deglycosylated saponins) is equivalent to the glucose group content of MAG1 in FSS (fresh Salsa saponins); the rhamnose and galactose content in FSDS is equivalent to the rhamnose and galactose group content of SS (3-GlcAGalRha soyasapogenol B) in FSS. Figure 5B). The xylose content in SPDS (deglycosylated saponins of silage Pegasis) is equivalent to the total xylose groups of MAGA, ZATA, and ZAGA in SPS (pegasis saponins of silage); the glucuronic acid content in SPDS is equivalent to the total glucuronic acid groups of MAGA and ZAGA in SPS; the rhamnose and galactose content in SPDS is equivalent to the rhamnose and galactose content in SS in SPS. The xylose content in SSDS (deglycosylated saponins of silage Salsa) is equivalent to the total xylose groups of MAGA, ZATA, and ZAGA in SSS (salsa saponins of silage); the glucuronic acid content in SSDS is equivalent to the total glucuronic acid groups of MAGA and ZAGA in SSS; the rhamnose and galactose content in SSDS is equivalent to the rhamnose and galactose content in SS in SSS.

[0101] Summary of 2-5

[0102] 2-5.1 The pH value of all alfalfa varieties after silage was between 4.63 and 4.76. Butyric acid was not detected and lactic acid accounted for a high proportion, indicating that homolactic fermentation was dominant and the sealing process effectively inhibited the activity of harmful microorganisms such as Clostridium.

[0103] 2-5.2 As FD increases, the contents of CP, EE, and WSC decrease significantly, while the proportions of ADF and NDF increase. FD9 (Pegasis) and FD10 (Sardi 10) have low nutritional quality due to their high maturity.

[0104] 2-5.3 The antioxidant activity of saponins in high-FD varieties (FD9-FD10) after silage was significantly better than that in low-FD varieties. Among them, FD9 (Pegasis) had the highest DPPH free radical scavenging rate and FRAP, while FD4 (Salsa) had the lowest antioxidant activity.

[0105] 2-5.4 Based on antioxidant activity, eight saponin components (FPS, FSS, SPS, SSS, FPDS, SPDS, FSDS, SSDS) were selected for subsequent rumen in vitro fermentation experiments to verify their bioactivity and functional differences.

[0106] Example 3

[0107] Regulatory Effects of Saponins and Deglycosylated Saponins on Metabolic Health in High-Fat Mice

[0108] Recent studies have shown that saponins have potential therapeutic value in improving HFD-induced metabolic syndrome (including obesity, insulin resistance, and non-alcoholic fatty liver disease). Research indicates that chronic inflammation induced by HFD can promote insulin resistance by interfering with lipid / energy metabolism pathways; this mechanism has been confirmed as a core pathogenic factor of metabolic disorders. However, the clinical application of traditional saponin derivatives (AS) is limited due to their spermicidal effects. Therefore, the development of novel saponin derivatives is of great significance. This study used a C57BL / 6 male mouse model susceptible to dietary obesity to systematically evaluate the effects of AS and DAS on improving metabolic health in HFD mice. By monitoring body condition parameters, serum antioxidant indices, and the expression levels of metabolism-related genes, the biochemical characteristics and therapeutic potential of AS and DAS were comprehensively analyzed.

[0109] 3-1 In vivo mouse test

[0110] All animal experiments in this study were reviewed and approved by the Animal Experiment Ethics Committee of Yangzhou University (Approval No.: SYXK[Su]2012-0029). Forty 5-week-old specific pathogen-free (SPF) grade C57BL / 6 male mice (initial weight 15.0±0.5 g, purchased from the Experimental Animal Center of Yangzhou University) were used and housed in individually ventilated cages. Environmental parameters in the animal room were strictly controlled at a temperature of 23±1℃, relative humidity of 50±5%, and a 12-hour light / dark cycle. All mice underwent a 7-day acclimatization period. Sterile corncob bedding was used and changed daily during the rearing period, and the experimental animals had free access to food and water. Mice were randomly divided into 5 groups (n = 8) using a completely randomized design: (1) Control group: fed standard diet (10% fat) and administered 300 mg / kg distilled water by gavage from 8:00 to 9:00 daily; (2) High-fat diet model group (HFD): fed high-fat diet (60% fat) and administered 300 mg / kg distilled water by gavage; (3) Positive drug control group (ZY): high-fat diet + 300 mg / kg Zhenyuan capsules (dose referenced from the clinical equivalent dose of Zhang et al.); (4) AS intervention group: high-fat diet + 300 mg / kg AS; (5) DAS intervention group: high-fat diet + 300 mg / kg DAS. Preliminary results showed that no significant toxic side effects occurred during the 14-day acute toxicity observation period at a dose of 300 mg / kg. After conversion by body surface area conversion factor (0.0813), it was equivalent to the recommended human dose of 24.39 mg / kg / day. The formal trial period was 30 days, during which the activity status was monitored.

[0111] 3-2 Body Condition Indicators

[0112] One week before the end of the experiment, the remaining feed weight was accurately weighed daily at 8:00 AM to calculate ADFI (g / d), and fasting body weight was measured and ADG (g / d) was calculated at a fixed time each week (9:00-10:00 AM). Mice were immediately euthanized by cervical dislocation after orbital blood collection, and body length (straight-line distance from nose tip to anus, accuracy 0.01 mm) was measured and final body weight recorded. Subsequently, the heart, kidneys, liver, and intestinal tissue (jejunum to colon) were rapidly separated. After carefully removing blood vessels and connective tissue, the tissues were rinsed three times repeatedly with pre-cooled PBS buffer (0.1 M, pH 7.4, 4℃), blotted dry with filter paper, and weighed. One cm segments of intestinal tissue were harvested from the middle of the jejunum and the terminal colon, longitudinally dissected, and the intestinal contents were scraped out. 1 mL of sterile PBS buffer containing 0.05% sodium thioglycolate was added, and the mixture was vortexed for 1 min, centrifuged at 3000 r / min for 10 min at 4℃, and the supernatant was used for pH determination. Simultaneously, the seminal vesicle tissue was completely dissected and placed in a pre-warmed semen washing solution (114 mM NaCl, 3.16 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 5 mM glucose, 0.1 mM sodium pyruvate, 10 mM lactic acid, 20 mM Hepes acid) at 37°C. Sperm were released by puncture with a sterile needle and incubated at 37°C for 15 min to activate sperm motility. 10 μL of the suspension was then added to a hemocytometer, and five fields of view were randomly selected under a microscope to count the total number of sperm. Live sperm (unstained) and dead sperm (red-stained) were distinguished by eosin-γ staining, and the sperm viability was finally calculated.

[0113] 3-3 Blood Indicators

[0114] Mice were fasted for 24 hours, and fasting blood glucose levels were measured in the tail vein using a glucometer in triplicate. Whole blood samples were then collected via the orbital venous plexus sampling method using pre-cooled (4°C) heparin sodium anticoagulant tubes. Collected blood samples were immediately placed on ice and pretreated within 30 minutes. Whole blood samples were then allowed to coagulate at 25°C for 30 minutes, followed by centrifugation at 1000 rpm for 15 minutes to separate serum. Serum was aliquoted into 1.5 mL low-adsorption EP tubes and stored at -80°C until analysis.

[0115] Serum oxidative stress indicators, including SOD activity, MDA content, CAT activity, and TAOC, were measured strictly according to the respective kit instructions. All testing procedures were conducted in the dark, and absorbance values ​​were automatically read using an ELISA reader, ensuring a correlation coefficient (R²) ≥ 0.995 for the standard curve. Lipid metabolism indicators TC and TG were measured according to the kit instructions. Each ELISA plate included low, medium, and high concentrations of quality control samples to ensure an intra-batch coefficient of variation of less than 5%. Coagulation function tests were performed using a fully automated coagulation analyzer to measure prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT). All reagents and samples were preheated to 37°C before testing.

[0116] 3-4 Experimental Results

[0117] 3-4.1 Serum total cholesterol and triglycerides

[0118] like Figure 6 As shown, HFD (high-fat diet) increased serum TC (total cholesterol) and TG (triglycerides) levels in mice (P < 0.05). The TC level in the HFD group was higher than that in the control group (10.23 mM). After gavage administration of ZY, AS, and DAS, TC decreased to 5.74, 7.39, and 6.28 mM, respectively, but remained higher than the control group (P < 0.05). The TC in the DAS group was only 9.43% higher than that in the ZY group, while the TC in the AS group decreased by 27.70% compared to the HFD group, but remained higher than the DAS group (P < 0.05). The TG level in the HFD group increased by 0.58 mM compared to the control group (P < 0.05). The TG levels in the ZY, AS, and DAS groups decreased to 2.40, 2.61, and 2.51 mM, respectively, but remained higher than the control group. The TG levels in the AS and DAS groups were not significantly different from those in the ZY (Zhenyuan capsules) group, while the TG level in the AS group was 11.84% lower than that in the HFD group.

[0119] 3-4.2 Serum coagulation function

[0120] The coagulation function test results showed ( Figure 7HFD significantly shortened the three coagulation parameters PT, APTT, and TT (P < 0.05). The PT (prothrombin time) in the HFD group was shortened to 7.69 s, which was 12.5% ​​lower than that in the control group (8.79 s) (P < 0.05). There was no significant difference between the DAS group (9.40 s) and the AS group (9.71 s), but both were higher than the HFD group and lower than the ZY group (11.69 s) (P < 0.05). The APTT in the HFD group (16.47 s) was shorter than that in the control group (21.74 s) (P < 0.05). There was no significant difference between the DAS group (24.87 s) and the AS group (25.31 s), but both were longer than the HFD group and shorter than the ZY group (29.24 s) (P < 0.05). The time to clotting (TT) in the HFD group (19.58 s) was shorter than that in the control group (24.78 s) (P < 0.05). The TT in the DAS group (32.46 s) was not significantly different from that in the AS group (34.92 s), but was longer than that in the HFD group and shorter than that in the ZY group (42.26 s) (P < 0.05). The anticoagulant effects of the DAS and AS groups were similar, but both were weaker than those in the ZY group (P < 0.05).

[0121] Summary of 3-5

[0122] 3-5.1 HFD-induced C57BL / 6 male mice successfully mimicked the core phenotypes of metabolic syndrome, including obesity, abnormal blood glucose, dyslipidemia, and oxidative stress, providing a reliable model for mechanistic studies.

[0123] 3-5.2 Multidimensional biological activities and mechanisms of DAS: Significantly reduces serum TC, TG and fasting blood glucose levels; regulates lipid metabolism genes (LXR, NPC1L1) and intestinal absorption genes (GLP-2); enhances SOD, CAT activity and TAOC, reduces MDA levels and reverses oxidative damage; downregulates pro-inflammatory factors such as NF-κB p50, TNF-α, and IL-6, and upregulates anti-inflammatory factors such as IL-4 and IL-10, while simultaneously improving cardiac / renal indices and colonic absorption function; and reduces visceral fat accumulation by suppressing appetite (reducing ADFI and ADG) to achieve weight loss.

[0124] 3-5.3 DAS did not affect sperm survival rate at equivalent doses compared to AS, and had no significant organ toxicity. Its bioavailability was improved by deglycosylation, and its safety profile showed greater potential for clinical application.

[0125] Example 4

[0126] Application of deglycosylated saponins in the preparation of products that reduce oxidative stress and inflammation in goat rumen epithelial cells

[0127] Rumen epithelial cells play a crucial physiological role in the active transport of nutrients, short-chain fatty acid metabolism, and maintaining rumen barrier function. When ruminants are fed a high-concentrate diet for an extended period, the rapid degradation of easily fermentable carbohydrates in the rumen leads to excessive accumulation of volatile fatty acids (VFAs), which in turn induces rumen acidosis. This not only disrupts the rumen microbiota balance but also significantly increases the concentration of luteinizing saline (LPS) in the rumen fluid. As a major component of the cell walls of Gram-negative bacteria, LPS can directly damage the integrity of the rumen epithelial barrier and induce local inflammatory responses by promoting the overexpression of pro-inflammatory cytokines such as IL-1β and TNF-α. Therefore, developing effective intervention strategies to alleviate rumen epithelial cell inflammation is of significant practical importance for ensuring the health and production performance of ruminants.

[0128] In recent years, plant secondary metabolites have become an important research subject in animal nutrition due to their natural origin and multiple bioactivities. Among them, anaerobic saponins (AS) have been clearly demonstrated to have multiple physiological functions, including anti-inflammatory, antioxidant, and immunomodulatory effects. However, the glycosylation structure of AS may limit its bioavailability, while deglycosylation modification to remove sugar chains can significantly improve its lipid solubility and cell membrane permeability, thereby enhancing its bioactivity. Existing studies have shown that saponins, such as diosgenin, can alleviate doxorubicin-induced hepatocyte damage in mice by regulating the Sirt1 / FOXO1 / NF-κB signaling pathway, providing an important reference for the study of the mechanism of action of AS. Although previous in vitro experiments have preliminarily confirmed that DAS has the potential to reduce methane emissions, its regulatory mechanism on the inflammatory response of rumen epithelial cells in ruminants, especially its protective effect under LPS stress and its molecular basis, still need to be further elucidated.

[0129] 4-1 Materials and Methods

[0130] 4-1.1 Main Reagents and Instruments

[0131] Main reagents:

[0132] DMEM / F12 culture medium was purchased from Shanghai Huiying Biotechnology Co., Ltd.

[0133] DMSO cell culture medium was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0134] The GSH-PX activity assay kit (BC1195) and the total RNA extraction kit for cultured cells (R1251) were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0135] Trizol (15596-026) was purchased from Invitrogen Life Sciences, Inc., USA.

[0136] The HiScript III cDNA Synthesis Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0137] 4-1.2 Test Materials

[0138] The immortalized goat rumen epithelial cells were derived from Xuhuai goats and provided by Yimo Biotechnology Co., Ltd.

[0139] 4-1.3 Cell viability

[0140] This experiment was conducted in December 2024, and cell viability was determined using the CCK-8 assay. Goat rumen epithelial cells were cultured at 1.00 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 10 cells / mL in 96-well plates and cultured at 37°C in a 5% CO2 incubator until adherence. After discarding the original culture medium, cells were washed with PBS, and then DMEM / F12 medium containing different concentrations of SPDS (5, 15, 30, 60, 120, 240 μg / mL) was added. SPDS was dissolved in DMSO, with the final DMSO concentration controlled at 1‰ for each group, and six replicates were performed per group. After 6 h of culture, 10 μL of CCK-8 solution was added to each well for an additional 3 h of incubation. Finally, the absorbance at 450 nm was measured using a microplate reader, and the optimal concentration of SPDS was determined by calculating cell viability.

[0141] 4-1.4 Antioxidant Indicators

[0142] Goat rumen epithelial cells were used at a concentration of 5.00 × 10⁻⁶ 5 Cells were seeded at a density of [number] cells / mL in six-well plates. After cell adhesion, the culture medium was discarded and the cells were washed with PBS. Cells were then divided into the following groups: control group (DMEM / F12 basal medium), LPS group (basal medium + 1 μg / mL LPS), SPDS group (basal medium + 60 μg / mL SPDS), and LPS+SPDS group (basal medium + 1 μg / mL LPS + 60 μg / mL SPDS). Cells were cultured at 37°C and 5% CO2 for 6 h, and then collected. GSH-PX, SOD, CAT activities, MDA content, and TAOC were measured using a kit.

[0143] 4-1.5 gene expression levels

[0144] After culturing, discard the culture medium, add 1 mL of Trizol to each well, mix well, and let stand for 5-10 min. Extract total RNA from cells using a kit, determine the concentration and purity, and then reverse transcribe to synthesize cDNA. Store the samples at -20℃ for later use. Primer sources (Table 2).

[0145] Table 2 qPCR primer sequences

[0146]

[0147] Note: F, forward primer; R, reverse primer.

[0148] 4-2 Results and Analysis

[0149] 4-2.1 Effects of deglycosylated saponins on rumen epithelial cell activity

[0150] The effect of SPDS on the viability of goat rumen epithelial cells was systematically evaluated using a concentration gradient assay. Figure 8 The results showed that SPDS improved cell viability within the range of 5-240 μg / mL (P < 0.05), and cell viability initially increased and then decreased with increasing SPDS concentration. The 60 μg / mL SPDS group showed the highest cell viability, significantly better than other concentration groups.

[0151] 4-2.2 Effects of deglycosylated saponins on the oxidative properties of rumen epithelial cells under lipopolysaccharide stimulation

[0152] Compared with the control group and the LPS (lipopolysaccharide) group, the GSH-PX activity was increased in the SPDS group and the LPS+SPDS group (P < 0.05). Figure 9 A. The SPDS group had the highest SOD activity (P < 0.05), while the SOD activity in the LPS+SPDS group was not different from that in the control group. Figure 9 B). The CAT activity in both the SPDS group and the LPS+SPDS group was higher than that in the control group and the LPS group (P < 0.05), and the CAT activity in the LPS group was 28.6% lower than that in the control group. Figure 9 C. The LPS group had the highest MDA content, while the SPDS group had the lowest (P < 0.05). The MDA content in the LPS+SPDS group was 38.2% lower than that in the LPS group (P < 0.05). SPDS treatment significantly increased TAOC activity (P < 0.05), while there was no significant difference between the LPS group and the control group. Figure 9 D).

[0153] 4-2.3 Effects of deglycosylated saponins on gene expression levels in rumen epithelial cells stimulated by lipopolysaccharide

[0154] LPS activated the NF-κB signaling pathway, inducing high expression of pro-inflammatory cytokines IL-6, IL-8, TNF-α, and IL-1β through their downstream targets, while inhibiting the anti-inflammatory cytokine IL-10 (P < 0.05). Figure 10LPS+SPDS intervention reversed the above effects (P < 0.05), inhibiting NF-κB activity to near control levels, reducing pro-inflammatory cytokine expression by 50%-80%, and inducing IL-10 expression to its highest level (P < 0.05). The LPS group upregulated TLR4 and its adaptor protein MYD88 expression and activated the non-canonical pathway molecule IRF3 (P < 0.05), while the SPDS group inhibited the TLR4-MYD88 / IRF3 axis, directly downregulating TLR4 expression and blocking MYD88 phosphorylation and IRF3 nuclear translocation (P < 0.05). LPS significantly reduced the expression of TOLLIP, a negative regulator of TLR4, while SPDS did not restore it to normal levels. LPS induced an increase in MMP-9, while LPS+SPDS reduced its level by 50% (P < 0.05), an effect consistent with the trend of NF-κB activity inhibition. Most indicators in the SPDS group were not significantly different from those in the control group, but the SPDS group could specifically enhance the inhibitory effect of TLR4 under basal conditions.

[0155] 4-3. Summary

[0156] SPDS exhibits a concentration-dependent effect on goat rumen epithelial cells; a dose of 60 μg / mL significantly promotes cell proliferation and enhances cell viability, indicating its potential to promote cell repair.

[0157] In the LPS-induced oxidative stress model, SPDS restored redox homeostasis by enhancing the activities of GSH-PX, SOD, and CAT, reducing the level of lipid peroxidation product MDA.

[0158] SPDS blocks the activation of the TLR4-MYD88-NF-κB and TRIF-IRF3 signaling axes; downregulates pro-inflammatory factors such as IL-1β and TNF-α, upregulates the anti-inflammatory factor IL-10; inhibits MMP-9 expression, and reduces inflammatory tissue damage.

[0159] In summary, as a class of secondary metabolites with both biological activity and metabolic regulatory potential, the application of plant-derived active ingredients (AS) is limited by problems such as low extraction rate, inhibition of activity by glycosylation structure, insufficient bioavailability, and unclear rumen functional mechanisms in ruminants. This study systematically analyzed the structure-activity relationship of AS through ultrasound-assisted extraction optimization (response surface methodology), β-glucosidase-mediated targeted deglycosylation, and cross-species model (high-fat mouse-goat rumen fluid extracorporeal activity evaluation). It also explored its application potential in metabolic health regulation, rumen methane emission reduction, and anti-inflammatory and antioxidant effects, providing theoretical and technical support for the precise development of plant-derived active ingredients and the green transformation of animal husbandry.

[0160] The following conclusions can be drawn from the above Examples 1-4:

[0161] 1. Targeted construction of AS

[0162] The ultrasound-assisted thermal ethanol extraction process was optimized using response surface methodology, increasing the extraction rate of saponins (AS) to 1.5%. An in vitro simulated digestion model confirmed its bioavailability at 18.23%. β-glucosidase from *Aspergillus niger* was used to directionally remove the C-3 and C-28 glycosyl groups from AS. The deglycosylated structure was verified using liquid chromatography-mass spectrometry (LC-MS), elucidating the inhibitory effect of glycan modification on saponin activity. The deglycosylated product (DAS) exhibited significantly enhanced antioxidant and antibacterial activity (*E. coli*), and at a low concentration (62.5 μg / mL), it promoted cell proliferation by activating SOD enzyme activity in human lung epithelial cells, confirming a negative correlation between its biological activity and glycosylation modification.

[0163] 2. Regulation of metabolic syndrome in hyperlipidemic mice by AS and DAS

[0164] The intervention effects of atherosclerosis (AS) and diathermy-associated salivary glands (DAS) were systematically evaluated using a HFD-induced metabolic disorder model in C57BL / 6 mice. The results showed that DAS significantly reduced mouse body weight gain and serum TC and TG levels by inhibiting key lipid synthesis genes and regulating intestinal absorption genes. The study confirmed that DAS has no reproductive toxicity and elucidated its ability to reshape immune homeostasis by inhibiting NF-κB pathway activation and upregulating the expression of the anti-inflammatory factor IL-10.

[0165] 3. Effects of silage on AS activity

[0166] A systematic evaluation of the antioxidant activity (AS) of 21 alfalfa varieties from FD4 to FD10 after silage was conducted, revealing that the antioxidant activity of AS from FD9 to FD10 varieties was significantly superior to that of FD4. Lactic acid-dominated homofermentation remodels saponin components through microbial metabolism. Pegasis (FD9) is a superior germplasm resource for highly active silage AS, and its antioxidant activity can be further enhanced by treatment with β-glucosidase.

[0167] 4. DAS mechanism for methane emission reduction in rumen extra-fluid fermentation

[0168] A study on in vitro rumen fermentation in goats showed that the addition of 3.0 mg FPS and SPDS significantly inhibited the abundance of methanogens and promoted the conversion of hydrogen to propionic acid, resulting in a 30.15% reduction in methane production. FPS enhanced metabolic capacity by upregulating the bisphenol degradation pathway, while SPDS simultaneously regulated the olfactory transduction pathway and the prolactin signaling pathway, inhibiting ovarian steroid production and improving MCP synthesis and DM digestibility. This demonstrates that SPDS possesses the dual functional characteristics of methane emission reduction and nutrient metabolism enhancement.

[0169] 5. Intervention of saponin derivatives on rumen epithelial cell inflammation

[0170] SPDS effectively alleviates LPS-induced oxidative stress and inflammatory damage in rumen epithelial cells, significantly increases the activity of antioxidant enzymes (GSH-PX, SOD, CAT), reduces lipid peroxidation product MDA, inhibits TLR4-MYD88 / TRIF signaling axis activation, downregulates pro-inflammatory factors TNF-α and IL-1β, upregulates anti-inflammatory factor IL-10, and reduces MMP-9 expression. This provides a basis for developing functional feed additives that target and regulate rumen health.

[0171] The deglycosylated alfalfa saponins obtained by this invention can be used in the preparation of products that improve lipid metabolism disorders in animals. Specifically, they can be used in the preparation of functional foods or feed additives to reduce serum TC and TG levels induced by a high-fat diet and to upregulate the expression of hepatic cholesterol absorption-related genes LXR and NPC1L1.

[0172] The deglycosylated alfalfa saponins prepared by this invention can be used in the preparation of feed additives that protect the rumen epithelial barrier of ruminants and inhibit rumen methane production.

[0173] The deglycosylated alfalfa saponins prepared by this invention can also be used in the preparation of anti-inflammatory and antioxidant agents. These agents maintain cellular redox homeostasis by downregulating the expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β and upregulating the expression of anti-inflammatory factor IL-10.

[0174] The abbreviations used in this article are shown in Table 3 below:

[0175]

[0176] Continued from Table 3-1

[0177]

[0178] Continued from Table 3-2

[0179]

[0180] This invention is not limited to the above embodiments. Based on the technical solutions disclosed in this invention, those skilled in the art can make some substitutions and modifications to some of the technical features without creative effort, and all such substitutions and modifications are within the protection scope of this invention.

Claims

1. A method for the directional construction of deglycosylated alfalfa saponins, characterized in that, Includes the following steps: (1) Extraction and purification: Using alfalfa as raw material, the extraction process was optimized by ultrasonic-assisted extraction combined with response surface methodology. The extraction solvent was ethanol-water solution to obtain crude alfalfa saponin extract, which was then purified by macroporous adsorption resin to obtain alfalfa saponin AS with a purity ≥95%. (2) Directed deglycosylation: Dissolve AS in sodium acetate buffer, add β-glucosidase from Aspergillus niger, and shake the reaction at 35-37℃ for 12-24h to enzymatically hydrolyze the C-3 and C-28 glycosyl groups of AS. (3) Post-treatment: The reaction solution was inactivated by a 100°C water bath, cooled in an ice bath, and centrifuged to obtain the supernatant. It was purified by a 300Da molecular weight cutoff dialysis bag and freeze-dried to obtain deglycosylated alfalfa saponin DAS.

2. The method for the directional construction of deglycosylated alfalfa saponins according to claim 1, characterized in that, The process parameters optimized by response surface methodology in step (1) are: solvent to raw material mass ratio of (10-14):1, extraction time of 2.7-2.9h, extraction temperature of 70-80℃, and ethanol volume fraction of 60%-80%.

3. The method for the directional construction of deglycosylated alfalfa saponins according to claim 1, characterized in that, The macroporous adsorption resin mentioned in step (1) is DM-130 type, and the elution conditions are 50% ethanol solution, flow rate 2.0 BV / h, column temperature 60℃; the mass ratio of β-glucosidase to AS mentioned in step (2) is (10-12):1, and the pH of the reaction system is 5.

0.

4. The method for the directional construction of deglycosylated alfalfa saponins according to claim 1, characterized in that, Before extraction and purification, a silage pretreatment step is performed: alfalfa is cut into sections and wilted until the moisture content is 65%. After adding a fermentation agent, it is sealed and stored at 25°C for 60 days to obtain silage alfalfa. Then, the extraction and purification in step (1) are performed. The deglycosylated products obtained are silage-derived deglycosylated saponins SPDS or SSDS. The raw material is taken from the alfalfa variety Pegasis and is deglycosylated saponin SPDS. The raw material is taken from the alfalfa variety Salsa and is deglycosylated saponin SSDS.

5. A method for evaluating the activity of deglycosylated alfalfa saponins, characterized in that, The deglycosylated alfalfa saponins (DAS or SPDS) prepared by any of the methods described in claims 1-4 were subjected to two or more of the following activity evaluations: Antioxidant activity evaluation: The IC50 of the samples in the concentration range of 0-200 μg / mL was determined using the DPPH free radical scavenging method and the FRAP ferric reduction method. 50 Value and Fe 2+ equivalent; Antibacterial activity evaluation: The micro-broth dilution method was used, with Escherichia coli as the indicator bacterium and an inoculum size of 10. 5 -10 6 CFU / mL, determine the minimum inhibitory concentration (MIC); Cell safety evaluation: BEAS-2B cells or goat rumen epithelial cells were cultured at 1×10⁻⁶ using the CCK-8 assay. 5 After seeding at a density of 10 cells / mL, add 5-240 μg / mL of DAS or SPDS and culture for 6 h, then measure cell viability. Evaluation of metabolic regulation in vivo: A high-fat diet-induced HFD C57BL / 6 mouse model was constructed. DAS was administered by gavage at a dose of 300 mg / kg / d for 30 consecutive days. Serum total cholesterol (TC), triglycerides (TG), fasting blood glucose, and the activities of antioxidant enzymes SOD, CAT, and GSH-PX were measured. The relative expression levels of LXR, NPC1L1, TNF-α, IL-6, and IL-10 genes in liver tissue were detected by RT-qPCR. Evaluation of rumen epithelial protection: An inflammation model was established by stimulating goat rumen epithelial cells with 1 μg / mL lipopolysaccharide (LPS). The cells were then co-cultured with 60 μg / mL SPDS for 6 h. The mRNA expression levels of TLR4, MYD88, NF-κB, IRF3, MMP-9, IL-1β, TNF-α, and IL-10 were detected by RT-qPCR. The intracellular activities of SOD, CAT, GSH-PX, and MDA content were also measured. Evaluation of methane emission reduction by rumen in vitro fermentation: In a goat rumen in vitro fermentation system, 3.0 mg of DAS or SPDS was added, and after anaerobic culture at 39 °C for 24 h, methane production and volatile fatty acid composition were determined by gas chromatography, and rumen microbial community structure was analyzed by 16S rRNA high-throughput sequencing.

6. The application of deglycosylated alfalfa saponins prepared by any one of claims 1-4, characterized in that, Application in the preparation of products that improve lipid metabolism disorders in animals.

7. The application of deglycosylated alfalfa saponins according to claim 6, characterized in that, Application in the preparation of functional foods or feed additives for reducing serum TC and TG levels induced by a high-fat diet and upregulating the expression of liver cholesterol absorption-related genes LXR and NPC1L1.

8. The application of deglycosylated alfalfa saponins prepared by any one of claims 1-4, characterized in that, Application in the preparation of feed additives that protect the rumen epithelial barrier of ruminants and inhibit rumen methane production.

9. The application of deglycosylated alfalfa saponins prepared by any one of claims 1-4, characterized in that, In the preparation of anti-inflammatory and antioxidant agents, the agents maintain cellular redox homeostasis by downregulating the expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β and upregulating the expression of anti-inflammatory factor IL-10.

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