A compound extract of dioscorea and preparation method and application thereof

Abstract

This invention relates to the field of natural product extraction and functional food technology, specifically to a yam compound extract, its preparation method, and its applications. This yam compound extract is composed of an ethyl acetate layer extract and a n-butanol layer extract of yam ethanol extract, and yam polysaccharides. The ethyl acetate layer extract is rich in total phenolic acids and total flavonoids, the n-butanol layer extract is rich in total saponins, and the yam polysaccharides enhance the targeting of immune regulation. Through a multi-component synergistic system of phenolic acids, flavonoids, saponins, and polysaccharides, the three components achieve highly efficient synergistic effects in anti-inflammatory and immune regulation. This invention can be widely applied to the development of anti-inflammatory and immune-regulating functional foods, health products, or related formulations, solving the technical problems of insufficient synergistic effects and poor targeting of immune regulation in existing yam compound extracts.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction and functional food technology, specifically to a yam compound extract, its preparation method, and its application. Background Technology

[0002] As a traditional food and medicine, yam contains a variety of active ingredients such as phenolic acids, flavonoids, triterpenes, saponins, and polysaccharides in its extracts. These ingredients have potential biological activities such as anti-inflammatory, antioxidant, and immunomodulatory effects, and have broad application prospects in the field of functional foods and health products.

[0003] Currently, there are numerous reports on research and product development related to yam extracts, but they generally suffer from significant technical limitations. Existing technologies mostly focus on the extraction and application of single active ingredients. For example, research on immunomodulation is conducted solely based on total saponins, without addressing anti-inflammatory functions, making it difficult to meet the actual needs of inflammation accompanied by immune disorders; or anti-inflammatory applications are developed solely based on phenolic acid components, without combining them with immunomodulatory components, resulting in limited functionality and application scenarios.

[0004] Most publicly available yam compound extracts only use moderately polar components for simple compounding without introducing yam polysaccharides. This results in limited synergistic effects between components and a lack of targeted immunomodulation, making it difficult to achieve efficient functional expression. Furthermore, traditional preparation processes often use single-concentration ethanol extraction, leading to insufficient dissolution of active ingredients and low yields. Existing technologies lack a complete technical chain for polysaccharide-directed separation, precise enrichment of multiple components, and stable compounding, resulting in large fluctuations in the proportion of active ingredients and poor batch-to-batch stability. This prevents the true synergistic effect of anti-inflammatory and immunomodulatory effects and fails to form a systematic technical solution of "multi-component synergy—targeted function," resulting in insufficient product quality control and market competitiveness. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a yam composite extract, its preparation method, and its applications. The yam composite extract of this invention is composed of an ethyl acetate layer extract, a n-butanol layer extract, and yam polysaccharides from an ethanol extract of yam. The ethyl acetate layer extract is rich in total phenolic acids and total flavonoids, serving as core anti-inflammatory components. The n-butanol layer extract is enriched with total saponins to enhance immunomodulatory activity. The yam polysaccharides, with a purity of ≥85%, target immune cells. This invention solves the problems of existing extracts having single functions and failing to synergistically enhance anti-inflammatory and immunomodulatory effects. By establishing a gradient ethanol extraction, multi-stage liquid-liquid extraction, and polysaccharide-directed separation process, the invention overcomes the problems of low yield, low purity, and poor batch stability of active ingredients in traditional preparation processes. Based on Pearson correlation analysis, a multi-component synergistic system of "phenolic acids-flavonoids-saponins-polysaccharides" is constructed and its ratio optimized, addressing the problems of insufficient immunomodulatory targeting and lack of scientific evidence for component-function correlation in the composite extract. This achieves highly efficient synergy between anti-inflammatory and immunomodulatory activities.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first objective of this invention is to provide a yam compound extract, which is a mixture of ethyl acetate layer extract, n-butanol layer extract and yam polysaccharide of yam ethanol extract, wherein the mass ratio of ethyl acetate layer extract, n-butanol layer extract and yam polysaccharide is 3~6:2~4:2~4.

[0007] The total phenolic acid content in the ethyl acetate extract was 238.60 mg / g ± 25.32 mg / g, the total flavonoid content was 35.70 mg / g ± 3.12 mg / g, and the total saponin content was 205.40 mg / g ± 18.65 mg / g; the total saponin content in the n-butanol extract was 229.70 mg / g ± 28.43 mg / g, and the total phenolic acid content was 196.80 mg / g ± 21.74 mg / g; and the yam polysaccharide had a purity ≥ 85% and a weight-average molecular weight of 30,000 Da ~ 50,000 Da.

[0008] The second objective of this invention is to provide a method for preparing the above-mentioned yam compound extract, comprising the following steps: S1. Gradient Ethanol Extraction: Yam powder is soaked in 95% ethanol to allow the ethanol to fully penetrate the yam powder and initially dissolve the lipid-soluble and highly polar active ingredients. Then, a first extraction is performed to enrich lipid-soluble and moderately polar active ingredients such as triterpenes and flavonoids, resulting in a first extract and a first residue. The first residue is then extracted a second time with 60% ethanol to further enrich polar active ingredients such as saponins and phenolic acids, resulting in a second extract and a second residue. The first and second extracts are mixed and concentrated to obtain yam extract.

[0009] S2. Multistage liquid-liquid extraction: Dissolve yam extract in water to obtain an aqueous solution of yam extract; perform a first extraction on the aqueous solution of yam extract using petroleum ether, discard the petroleum ether extract, remove fat-soluble impurities and some inactive components, and obtain the first aqueous layer; perform a second extraction on the first aqueous layer using ethyl acetate to obtain the second aqueous layer and the ethyl acetate extract; perform a third extraction on the second aqueous layer using n-butanol to obtain the n-butanol extract and the third aqueous layer.

[0010] S3. Directed separation of polysaccharides: Anhydrous ethanol is used as a precipitant to precipitate the third aqueous layer. After the precipitate is separated, it is dialyzed and freeze-dried in sequence to obtain yam polysaccharides. The purpose of dialysis is to remove small molecule impurities.

[0011] S4. Compound formulation: The ethyl acetate layer extract, the n-butanol layer extract and the yam polysaccharide are mixed to obtain the yam compound extract; wherein the mass ratio of the ethyl acetate layer extract, the n-butanol layer extract and the yam polysaccharide is 3~6:2~4:2~4.

[0012] Preferably, the material-to-liquid ratio is the same for the first and second extractions, which is 1g:3mL~9mL.

[0013] Preferably, the soaking conditions are: soaking at room temperature for 2 to 8 days, stirring 2 to 5 times a day for 15 to 30 minutes each time.

[0014] Preferably, the conditions for the first and second extractions are the same, both using reflux heating at 72℃~82℃ for 2h~4h; within this temperature range, a gentle boiling state is controlled to avoid violent boiling, so as to ensure the full dissolution of components with different polarities.

[0015] Preferably, the conditions for the first, second, and third extractions are the same: the extraction temperature is 20℃~25℃, and the shaking intensity is 120r / min~150r / min.

[0016] Preferably, in the first, second, and third extractions, the volume ratio of the aqueous phase to the solvent (petroleum ether for the first extraction, ethyl acetate for the second extraction, and n-butanol for the third extraction) is 1:1 to 5, to avoid solution emulsification, ensure clear stratification, and accurately separate the enriched layers of different active ingredients.

[0017] More preferably, in the first, second and third extractions, 3.5 times the volume of solvent is used (petroleum ether for the first extraction, ethyl acetate for the second extraction and n-butanol for the third extraction) to ensure full transfer of active ingredients and improve the purity of the extract.

[0018] Preferably, the concentration treatment conditions are: evaporation at 56℃~65℃ and vacuum degree 0.085MPa~0.095MPa until anhydrous ethanol is completely distilled off, so as to reduce the oxidative degradation of active ingredients.

[0019] Preferably, in step S2, the yam extract is dissolved in water, and the solution is heated to 45°C during the dissolution process to accelerate the dissolution, but the temperature does not exceed 65°C to avoid destroying the active ingredients.

[0020] Preferably, the precipitation treatment is performed as follows: anhydrous ethanol is added dropwise to the third aqueous layer at a dropping rate of 8 mL / min to 12 mL / min, and the mixture is allowed to stand at 2℃ to 8℃ for 6 h to 24 h; wherein the volume ratio of the third aqueous layer to anhydrous ethanol is 1:1 to 5.

[0021] Preferably, the separation conditions are: centrifugation at 8000r / min~8500r / min for 15min~20min.

[0022] Preferably, the dialysis procedure is as follows: the precipitate is placed into a dialysis bag with a molecular weight cutoff of 4000 Da to 16000 Da, and then the dialysis bag is placed in distilled water for dialysis for 24 h to 96 h; during dialysis, the distilled water is changed every 6 h to 8 h.

[0023] Preferably, the freeze-drying conditions are: freeze-drying at a vacuum of 0.01MPa to 0.04MPa and a temperature of -20℃ to -50℃ for 12h to 48h.

[0024] Preferably, the particle size of the yam powder is 50-70 mesh; this particle size range can ensure sufficient contact between the solvent (95% ethanol by mass) and the yam powder, and reduce the resistance of subsequent filtration, thereby improving the extraction efficiency.

[0025] The third objective of this invention is to provide the application of the above-mentioned yam compound extract in the preparation of an anti-inflammatory and immune complex product, which is used for people with chronic inflammation accompanied by immunodeficiency. The anti-inflammatory and immune complex product is selected from dietary supplements, nutritional oral liquids, or oral health products. In the anti-inflammatory and immune complex product, the amount of yam compound extract added is 5wt% to 10wt%.

[0026] The fourth objective of this invention is to provide the application of the above-mentioned yam compound extract in the preparation of an anti-inflammatory and immunomodulatory agent, wherein the anti-inflammatory and immunomodulatory agent is an oral preparation selected from capsules, tablets, granules or oral liquids; wherein, in the anti-inflammatory and immunomodulatory agent, the dosage of the yam compound extract is 20 mg / kg·day to 100 mg / kg·day.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides a yam compound extract, which is composed of ethyl acetate layer extract, n-butanol layer extract, and yam polysaccharide mixed in a mass ratio of 3-6:2-4:2-4. In the ethyl acetate layer extract, the total phenolic acid content is 238.60 mg / g ± 25.32 mg / g, the total flavonoid content is 35.70 mg / g ± 3.12 mg / g, and the total saponin content is 205.40 mg / g ± 18.65 mg / g; in the n-butanol layer extract, the total saponin content is 229.70 mg / g ± 28.43 mg / g, and the total phenolic acid content is 196.80 mg / g ± 21.74 mg / g; in the yam polysaccharide, the polysaccharide purity is ≥85%, and the weight-average molecular weight is 30000 Da~50000 Da. This invention provides a yam compound extract with advanced technology, clearly defined components, stable quality, and the ability to achieve targeted synergistic anti-inflammatory and immunomodulatory effects.

[0028] Compared with existing two-component yam extracts that only use ethyl acetate and n-butanol layers, this invention constructs a three-component mixed formula of "ethyl acetate layer extract + n-butanol layer extract + yam polysaccharide", forming a multi-component targeted synergistic system of "phenolic acid-flavonoid-saponin-polysaccharide", which significantly enhances the anti-inflammatory and immunomodulatory functions; the ovalbumin-induced foot swelling inhibition rate is increased by 32.6% (from 34.2% to 45.3%), and the carbon particle clearance phagocytosis index α is increased by 41.8% (from 5.82 to 8.26).

[0029] Compared with traditional yam extracts obtained from single components and single extraction processes, this invention achieves a breakthrough improvement in anti-inflammatory and immunomodulatory activities: the inhibition rate of xylene-induced ear swelling is increased by 156.3% (from 22.9% to 58.7%), and the carbon particle clearance phagocytosis index α is increased by 142.5% (from 3.41 to 8.26). The technical effects are outstanding and non-obvious, fully demonstrating the novelty and inventiveness of the invention.

[0030] 2. This invention provides a method for preparing a compound yam extract. Yam powder is soaked in 95% ethanol, then extracted sequentially with 95% ethanol and 60% ethanol. The extracts are combined and concentrated to obtain yam extract. The yam extract is dissolved in water, and the aqueous phase is extracted sequentially with petroleum ether, ethyl acetate, and n-butanol to obtain petroleum ether extract, ethyl acetate extract, and n-butanol extract, respectively. The aqueous phase after n-butanol extraction is subjected to alcohol precipitation, centrifugation, dialysis, and freeze-drying to obtain yam polysaccharide. The ethyl acetate extract, n-butanol extract, and yam polysaccharide are mixed to obtain the compound yam extract.

[0031] This invention establishes a complete preparation process of "gradient ethanol extraction - multi-stage liquid-liquid extraction - polysaccharide-directed separation - precise compounding", with a total active ingredient (phenolic acid + flavonoids + saponins + triterpenes) yield of 12.3%, which is 45.9% higher than the traditional single-concentration ethanol extraction process (yield 8.5%); the extract purity reaches 89.7%, which is 20.2% higher than the existing process (74.8%), and the process is stable and controllable with batch-to-batch differences ≤3%.

[0032] 3. This preparation method is based on the core concept of "gradient enrichment - precise separation - polysaccharide-directed purification - targeted synergy". Through five key steps, including raw material pretreatment, gradient ethanol extraction, multi-stage liquid-liquid extraction, polysaccharide-directed separation, and compound formulation, it selectively enriches the core active ingredients for anti-inflammatory and immunomodulatory effects, constructing a multi-component synergistic system. The process parameters are well-defined, stable, and controllable, making it suitable for industrial production. 4. This invention clarifies the targeted correlation between the core active ingredients and pharmacological effects of the yam compound extract. Pearson correlation analysis confirms that the anti-inflammatory activity is highly correlated with total flavonoids (r=0.998) and total phenolic acids (r=0.987), while the immunomodulatory activity is highly correlated with total saponins (r=0.915) and polysaccharide content (r=0.923). This provides a scientific basis for product quality control and functional optimization, and also offers new ideas for the synergistic development of multi-component natural products.

[0033] 5. The yam compound extract of the present invention has excellent stability and safety. After 12 months of sealed storage at room temperature, the retention rates of total phenolic acids (92.3%), total saponins (93.7%), total flavonoids (90.5%), and polysaccharides (94.2%) far exceed industry standards. Cell-level safety assessment shows that at a concentration of 200 μg / mL, the survival rates of normal human hepatocytes LO2, renal tubular epithelial cells HK-2, and intestinal epithelial cells Caco-2 are all >96%, providing a solid guarantee for its wide application in food, health products, and preparations. Attached Figure Description

[0034] Figure 1 This is a standard curve for gallic acid.

[0035] Figure 2 This is a standard curve graph of rutin.

[0036] Figure 3 This is a standard curve of oleanolic acid.

[0037] Figure 4 This is a standard curve of diosgenin.

[0038] Figure 5 The images show the foot swelling test results, where a is the overall view and b is a magnified view of a specific area.

[0039] Figure 6 This is a diagram illustrating the phenomenon of ear swelling.

[0040] Figure 7 The images show the phenomena observed in the carbon particle clearance experiment, where a is the global view and b is a magnified view of a local area.

[0041] Figure 8 This is a molecular weight distribution diagram of yam polysaccharides. Detailed Implementation

[0042] The technical solution of the present invention will be clearly and completely described below with reference to the data in the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0043] It should be noted that the technical terms used in this invention are only for describing specific embodiments and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased on the market or prepared by existing methods. Among them, high-quality yam refers to yam of the Dioscorea persimilis variety that is free from mold, insect infestation, mechanical damage and has a uniform color.

[0044] Standard curve plotting method: Gallic acid standard curve preparation: Dissolve 1.17 mg of gallic acid standard in distilled water and bring the volume to 25 mL to obtain a gallic acid standard solution. Pipette 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 3.0 mL, and 4.0 mL of the gallic acid standard solution into 20 mL test tubes, and successively dilute with distilled water to 6 mL. Then add 1.0 mL of Folin-Ciocalteu reagent, shake thoroughly to mix, and let stand at room temperature for 4 min to obtain a mixed solution. Add 1.0 mL of 10% Na₂CO₃ solution to the mixed solution, shake thoroughly, and place in a constant temperature water bath at 25 °C for 2 h. After the reaction, measure the absorbance at 765 nm using a UV spectrophotometer. Zero the UV spectrophotometer using a blank control. Plot a standard curve based on the measured data. Figure 1 As shown.

[0045] Rutin Standard Curve Construction: Dissolve 7.09 mg of rutin standard in 70% ethanol and bring the volume to 25 mL to obtain a rutin standard solution. Pipette 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, and 4.0 mL of the rutin standard solution into 10 mL test tubes. Add 70% ethanol to a final volume of 3 mL, then add 0.4 mL of 5% sodium nitrite solution. Shake well and let stand at room temperature for 6 min. Add 0.4 mL of 10% aluminum nitrate solution, shake well, and let stand at room temperature for 6 min. Add 4 mL of 4% sodium hydroxide solution, and bring the volume to 10 mL with 70% ethanol. Shake well and let stand at room temperature for 15 min. Measure the absorbance at 507 nm using a UV spectrophotometer. Zero the UV spectrophotometer using a blank control. Plot the standard curve based on the measured data. Figure 2 As shown.

[0046] Preparation of the oleanolic acid standard curve: Accurately weigh 2.47 mg of oleanolic acid standard, dissolve it in ethyl acetate and dilute to 10 mL to obtain an oleanolic acid standard solution; pipette 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, 1.2 mL, and 1.6 mL of the oleanolic acid standard solution, evaporate to dryness in a 100℃ water bath, add 0.2 mL of 5% vanillin-glacial acetic acid solution and 1 mL of perchloric acid to the test tube, heat in a 60℃ water bath for 5 min, then immediately cool in an ice-water bath, add 2 mL of ethyl acetate, shake to mix, and let stand. Measure the absorbance at a wavelength of 550 nm using a UV spectrophotometer. Zero the UV spectrophotometer using a blank control. Plot the standard curve based on the measured data, as shown below. Figure 3 As shown.

[0047] Construction of the diosgenin standard curve: Accurately weigh 5.45 mg of diosgenin standard, dissolve in methanol and dilute to 10 mL to obtain a diosgenin standard solution; place 0.2 mL, 0.4 mL, 0.8 mL, 1.2 mL, 1.6 mL, and 2.0 mL of the diosgenin standard solution into 10 mL test tubes, evaporate the methanol in a 60℃ water bath, add 0.2 mL of 5% vanillin-acetic acid solution and 0.8 mL of perchloric acid, and react in a 70℃ water bath for 20 min. Immediately cool in an ice-water bath, add 5 mL of acetic acid, and shake well. Zero the UV spectrophotometer using a blank control, and measure the absorbance at a wavelength of 485 nm using the UV spectrophotometer. Plot the standard curve based on the measured data, as shown below. Figure 4 As shown.

[0048] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the following will provide a detailed description in conjunction with specific embodiments: Example 1 A method for preparing a compound extract of yam includes the following steps: S1. Raw material pretreatment: Select 10kg of high-quality yam, remove surface mud, residual roots and impurities, rinse 4 times with running water, drain the surface water and cut into uniform thin slices with a thickness of 1.5mm (the thin slice structure can increase the specific surface area and improve solvent contact efficiency), spread them flat on a stainless steel tray and put them in a constant temperature drying oven, dry at 65℃ for 12h to constant weight (constant weight standard is weight change ≤0.3% for 2 consecutive hours) to avoid degradation of active ingredients due to high temperature, and obtain yam slices; put the yam slices into a high-speed universal grinder, set the grinding time to 4 minutes (divided into 4 times, 1 minute each time, with an interval of 30 seconds to prevent temperature rise during grinding), after grinding, classify through a standard inspection sieve, collect 60-mesh yam powder (particle size range controlled between 212μm and 300μm), after sieving, seal and place in a desiccator for later use to prevent moisture absorption and clumping, and obtain yam powder.

[0049] S2. Gradient Ethanol Extraction: Yam powder was placed in an extraction tank, and 95% ethanol (by mass) was added at a ratio of 1g:3mL. The mixture was soaked at room temperature for 8 days, with stirring twice daily for 15 minutes each time (100 rpm) to allow the ethanol to fully penetrate the yam powder cells and initially dissolve the lipid-soluble and moderately polar active ingredients. After soaking, the heating system of the extraction tank was activated, and the mixture was refluxed at 82℃ twice, each time for 4 hours. After each extraction, the mixture was filtered through a plate and frame filter (8μm pore size) and the extracts were collected and combined. The liquid was collected, and the residue was retained in the extraction tank to obtain the first extract and the first residue. Ethanol with a mass fraction of 60% was added to the first residue at a material-to-liquid ratio of 1g:3mL. The mixture was heated and refluxed twice at 82℃ for 4 hours each time. The extracts were combined to obtain the second extract and the second residue. The first and second extracts were mixed and then transferred to a rotary evaporator at 65℃ and 0.10MPa for rotary evaporation until no more ethanol distilled off, yielding a brownish-red, viscous yam extract. The yam extract had a water content of 8% and a yield of 10% of the raw material weight.

[0050] S3. Multistage liquid-liquid extraction: Dissolve the yam extract in 3 times its volume of distilled water and stir until dissolved (stirring speed 90 r / min, time 40 minutes) to obtain an aqueous solution of yam extract. Transfer the solution to a separatory funnel and add an equal volume of petroleum ether (boiling range 60℃, food grade) for extraction. Vigorously shake the separatory funnel for 5 minutes and then let it stand for 20 minutes to separate the layers (the separation standard is that the two phases have a clear interface and no emulsion layer). After discarding the petroleum ether layer, retain the lower aqueous phase and repeat the extraction 6 times to obtain the first aqueous layer.

[0051] Ethyl acetate (food grade) at a volume ratio of 1:1 was added to the first aqueous layer for extraction. The separatory funnel was shaken vigorously for 5 minutes, and then allowed to stand for 20 minutes to separate the layers. This process was repeated twice. The ethyl acetate extracts were combined and transferred to a rotary evaporator. The ethyl acetate was evaporated to dryness at 55°C and 0.05 MPa to obtain a pale yellow powdery ethyl acetate layer extract and a second aqueous layer. The yield of the ethyl acetate layer extract was 5% of the original extract.

[0052] An equal volume of n-butanol (food grade) was added to the second aqueous layer for extraction. The separatory funnel was shaken vigorously for 5 minutes, and then allowed to stand for 20 minutes to separate the layers. This process was repeated twice. The n-butanol extracts were combined and transferred to a rotary evaporator. The n-butanol was evaporated to dryness at 55°C and 0.05 MPa. The resulting white powdery n-butanol extract and the third aqueous layer were obtained. The yield of the n-butanol extract was 30% of the original extract.

[0053] S4. Directional separation of polysaccharides: Under stirring (20 rpm), add an equal volume of anhydrous ethanol (food grade) to the third aqueous layer at a dropping rate of 5 mL / min (to avoid uneven polysaccharide precipitation due to excessively high local concentrations). Let stand at 2°C for 6 hours (to allow the polysaccharides to fully coagulate and precipitate). Centrifuge at 5000 rpm for 5 minutes to completely separate the precipitate from the supernatant. Discard the supernatant and collect the precipitate. Dissolve the precipitate in distilled water to prepare a polysaccharide solution with a mass concentration of 50 mg / mL. The polysaccharide solution was placed in a dialysis bag with a molecular weight cutoff of 8000 Da. The two ends of the dialysis bag were sealed with dialysis clamps and placed in a dialysis tank containing sufficient distilled water. Dialysis was performed at room temperature with magnetic stirring for 24 hours. The distilled water was changed every 4 hours during dialysis to ensure thorough removal of small molecule sugars, salts and other impurities. After dialysis, the dialysate was transferred to a freeze dryer and freeze-dried at a vacuum of 0.01 MPa and -20°C for 12 hours to obtain a white, loose yam polysaccharide with a yield of 6% of the original extract.

[0054] S5. Compound Preparation: Place 3.0g of ethyl acetate extract, 2.0g of n-butanol extract, and 2.0g of yam polysaccharide into a double cone mixer and stir at 20r / min for 20min until homogeneous, yielding 10.0g of yam compound extract. The standard for homogeneous mixing is: the relative deviation of the core component content at any 5 sampling points ≤3%.

[0055] Referring to the standard curve plotting method described in this invention, the contents of total phenolic acids, total flavonoids, total triterpenes, total saponins, and polysaccharides in the yam compound extract prepared in Example 1 were determined, and the results are as follows: The test results showed that the total phenolic acid content was 232.5 mg / g; the total flavonoid content was 34.2 mg / g; the total triterpenoid content was 45.7 mg / g; the total saponin content was 218.6 mg / g; and the polysaccharide content was 172.3 mg / g (purity 86.4%).

[0056] Example 2 A method for preparing a yam compound extract is the same as the method in Example 1, except that the particle size of the yam powder is replaced with 50 mesh instead of 60 mesh, and the yam compound extract is finally obtained.

[0057] The test results showed that the total phenolic acid content was 228.3 mg / g; the total flavonoid content was 33.5 mg / g; the total saponin content was 215.7 mg / g; and the polysaccharide content was 168.5 mg / g (purity 85.2%).

[0058] Example 3 A method for preparing a yam compound extract is the same as the method in Example 1, except that the particle size of the yam powder is changed from 50 mesh to 70 mesh, and the yam compound extract is finally obtained.

[0059] The test results showed that the total phenolic acid content was 235.6 mg / g, the total flavonoid content was 34.8 mg / g, the total saponin content was 220.3 mg / g, and the polysaccharide content was 175.2 mg / g (purity 86.7%).

[0060] Example 4 A method for preparing a compound extract of yam includes the following steps: S1. Raw material pretreatment: Select 10kg of high-quality yam, remove surface mud, residual roots and impurities, rinse 4 times with running water, drain the surface water and cut into uniform thin slices with a thickness of 1.5mm (the thin slice structure can increase the specific surface area and improve solvent contact efficiency), spread them flat on a stainless steel tray and put them in a constant temperature drying oven, dry at 65℃ for 12h to constant weight (constant weight standard is weight change ≤0.3% for 2 consecutive hours) to avoid degradation of active ingredients due to high temperature, and obtain yam slices; put the yam slices into a high-speed universal grinder, set the grinding time to 4 minutes (divided into 4 times, 1 minute each time, with an interval of 30 seconds to prevent temperature rise during grinding), after grinding, classify through a standard inspection sieve, collect 60-mesh yam powder (particle size range controlled between 212μm and 300μm), after sieving, seal and place in a desiccator for later use to prevent moisture absorption and clumping, and obtain yam powder.

[0061] S2. Place the yam powder in an extraction tank, add 95% ethanol at a material-to-liquid ratio of 1:9, and soak at room temperature for 8 days. During this period, stir 5 times a day for 30 minutes each time (50 rpm) to allow the ethanol to fully penetrate into the yam powder cells and initially dissolve the fat-soluble and moderately polar active ingredients. After soaking, start the heating system of the extraction tank and heat and reflux at 72℃ 4 times for 2 hours each time. After each extraction, filter through a plate and frame filter (8 μm filter cloth pore size), collect and combine the extracts, and remove the residue. The residue was retained in the extraction tank to obtain the first extract and the first residue. Ethanol with a mass fraction of 60% was added to the first residue at a material-to-liquid ratio of 1:9, and the mixture was heated and refluxed at 72°C for 4 times, each extraction lasting 2 hours. The extracts were combined to obtain the second extract and the second residue. The first and second extracts were mixed and then transferred to a rotary evaporator at 55°C and 0.05 MPa for rotary evaporation until no more ethanol was distilled off, yielding a brownish-red, viscous yam extract. The yam extract had a water content of 9% and a yield of 12% of the raw material weight.

[0062] S3. Multistage liquid-liquid extraction: Dissolve the yam extract in 8 times its volume of distilled water and stir until dissolved (stirring speed 90 r / min, time 40 minutes) to obtain an aqueous solution of yam extract. Transfer the solution to a separatory funnel and add an equal volume of petroleum ether (boiling range 90℃, food grade) for extraction. Vigorously shake the separatory funnel for 10 minutes and then let it stand for 60 minutes to separate the phases (the separation standard is that the two phases have a clear interface and no emulsion layer). After discarding the petroleum ether layer, retain the lower aqueous phase. Repeat 6 times to obtain the first aqueous layer.

[0063] Ethyl acetate (food grade) at a volume ratio of 1:5 was added to the first aqueous layer for extraction. The separatory funnel was shaken vigorously for 10 minutes, and then allowed to stand for 60 minutes to separate the layers. This process was repeated 6 times. The ethyl acetate extracts were combined and transferred to a rotary evaporator. The ethyl acetate was evaporated to dryness at 65°C and 0.10 MPa to obtain a pale yellow powdery ethyl acetate layer extract and a second aqueous layer. The yield of the ethyl acetate layer extract was 8% of the original extract.

[0064] Add an equal volume of n-butanol (food grade) to the second aqueous layer for extraction. Vigorously shake the separatory funnel for 5 to 10 minutes, then allow it to stand for 60 minutes to separate the layers. Repeat this process 6 times. Combine the n-butanol extracts and transfer them to a rotary evaporator. Evaporate the n-butanol to dryness at 65°C and 0.10 MPa. The resulting white powdery n-butanol layer extract and the third aqueous layer are obtained. The yield of the n-butanol layer extract is 30% to 45% of the original extract.

[0065] S4. Directional separation of polysaccharides: Under stirring (50 rpm), add 1:5 volume of anhydrous ethanol (food grade) to the third aqueous layer at a dropping rate of 10 mL / min (to avoid uneven polysaccharide precipitation due to excessively high local concentrations). After standing at 8°C for 24 h (to allow the polysaccharides to fully coagulate and precipitate), centrifuge at 10000 rpm for 20 min to completely separate the precipitate from the supernatant. Discard the supernatant and collect the precipitate. Dissolve the precipitate in distilled water to prepare a solution with a mass concentration of 50 mg / mL. The polysaccharide solution was loaded into a dialysis bag with a molecular weight cutoff of 8000 Da. The two ends of the dialysis bag were sealed with dialysis clamps and placed in a dialysis tank containing sufficient distilled water. Dialysis was performed at room temperature with magnetic stirring for 48 hours. The distilled water was changed every 8 hours during dialysis to ensure thorough removal of small molecule sugars, salts and other impurities. After dialysis, the dialysate was transferred to a freeze dryer and freeze-dried at a vacuum of 0.04 MPa and -50°C for 48 hours to obtain a white, loose yam polysaccharide with a yield of 10% of the original extract.

[0066] S5. Compound Preparation: 6.0g of ethyl acetate extract, 4.0g of n-butanol extract, and 4.0g of yam polysaccharide were placed together in a double cone mixer and stirred at 50 rpm for 60 minutes until homogeneous, yielding 10.0g of yam compound extract. The standard for homogeneous mixing was: the relative deviation of the core component content at any 5 sampling points ≤3%.

[0067] Taking the yam compound extract from Example 1 as an example, its pharmacological activity was tested: ① Ovalbumin-induced mouse paw edema experiment: Forty male Kunming mice, weighing 20±2g, were randomly divided into four groups of 10 mice each: blank control group (0.5% sodium carboxymethyl cellulose), existing two-component extract group (yam ethyl acetate layer and n-butanol layer composite) (300mg / kg), single polysaccharide group (300mg / kg), and the yam composite extract group of this invention (300mg / kg). After 5 consecutive days of gavage administration, 45 minutes after the last administration, 0.1mL of 10% ovalbumin was subcutaneously injected into the right hind paw. Paw thickness was measured before inflammation and at 0.5h, 1h, 3h, and 5h after inflammation, and the swelling inhibition rate was calculated.

[0068] The results showed that the swelling inhibition rate of the yam compound extract group reached 45.3%±3.8% after 3 hours, significantly higher than that of the existing two-component extract group (34.2%±3.2%) and the single polysaccharide group (18.7%±2.5%). Experimental phenomena are as follows: Figure 5 As shown.

[0069] ② Xylene-induced mouse ear swelling experiment: Grouping and administration methods were the same as in ①. One hour after the last administration, 0.1 mL of 100% xylene was applied to the right ear of the mouse, with the left ear serving as a control. The mice were sacrificed 90 minutes later, and an 8 mm diameter ear flap was removed and weighed to calculate the swelling inhibition rate. Experimental phenomena are as follows: Figure 6 As shown.

[0070] The results showed that the swelling inhibition rate of the yam compound extract group was 58.7%±3.6%, which was significantly higher than that of the existing two-component extract group (44.3%±3.1%) and the single polysaccharide group (22.4%±2.8%).

[0071] ③ Carbon particle clearance experiment: The grouping and administration method were the same as in ①. One hour after the last administration, mice were injected with 0.1 mL / 10 g of Indian ink diluted 4 times into the tail vein. Blood was collected at 2 min and 10 min to measure the OD value and calculate the phagocytic index α.

[0072] The results showed that the phagocytic index α of the yam compound extract group reached 8.26±0.79, significantly higher than that of the existing two-component extract group (5.82±0.65) and the single polysaccharide group (4.35±0.58). Experimental phenomena are as follows: Figure 7 As shown.

[0073] ④Detection of immune factor secretion: The grouping and administration method are the same as in ①. 24 hours after the last administration, blood is collected by enucleation, serum is separated, and the levels of IL-2 and TNF-α are detected by ELISA.

[0074] The results showed that the serum IL-2 content in the yam compound extract group was (186.4±15.3) pg / mL and the TNF-α content was (42.3±4.1) pg / mL, which were 38.7% and 29.5% higher than those in the existing two-component extract group, respectively, indicating that the yam compound extract group of the present invention has stronger immunomodulatory targeting.

[0075] To demonstrate the correlation between activity and dosage, a dose gradient experiment was added after the pharmacological activity tests of the yam compound extract described above. Different dosages of the yam compound extract were grouped for pharmacological activity testing, as detailed below: Sixty male Kunming mice, weighing 20±2g, were randomly divided into 6 groups (n=10 per group): blank control group (0.5% sodium carboxymethyl cellulose), low-dose group (100mg / kg), medium-dose group (300mg / kg), high-dose group (500mg / kg), existing two-component extract group (300mg / kg), and single polysaccharide group (300mg / kg). Administration: Administered via gavage for 5 consecutive days. The remaining experimental procedures were the same as those for the pharmacological activity testing of the yam compound extract described above.

[0076] The results showed that the ovalbumin-induced foot swelling inhibition rate was 28.5%±3.1% in the low-dose group, 45.3%±3.8% in the medium-dose group, and 56.7%±4.2% in the high-dose group; the xylene-induced ear swelling inhibition rate was 39.2%±3.3% in the low-dose group, 58.7%±3.6% in the medium-dose group, and 69.5%±4.1% in the high-dose group; and the carbon particle clearance phagocytic index α was 5.32±0.68 in the low-dose group, 8.26±0.79 in the medium-dose group, and 9.87±0.85 in the high-dose group.

[0077] The anti-inflammatory and immunomodulatory activities of the yam compound extract of this invention are significantly dose-dependent, and the activity at medium and high doses is significantly better than that of existing products.

[0078] ①Stability test: The yam compound extract prepared in Example 1 was stored at 25°C, protected from light and sealed for 12 months. Samples were taken at 0, 3, 6 and 12 months to determine the content of the core active ingredients.

[0079] The results showed that after 12 months, the total phenolic acid retention rate was 92.3%, the total saponin retention rate was 93.7%, the total flavonoid retention rate was 90.5%, and the polysaccharide retention rate was 94.2%, all meeting the industry standard of ≥85%, indicating that the product has excellent storage stability.

[0080] ② Safety test: Acute toxicity test: Twenty male Kunming mice weighing 20±2g were selected and administered 6000mg / kg of yam compound extract by gavage once, and observed for 14 consecutive days.

[0081] The results showed that no mice showed symptoms of poisoning or died, and the calculated LD50 was >6000mg / kg, indicating that it is a practically non-toxic product.

[0082] ③ Cellular level safety assessment: Using normal human hepatocytes LO2, renal tubular epithelial cells HK-2, and intestinal epithelial cells Caco-2, the yam compound extract was prepared into concentration gradients of 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL, and co-cultured with the cells for 24 h. Cell viability was detected by CCK-8 assay.

[0083] The results showed that at a concentration of 200 μg / mL, the survival rate of all three cell types was >96%; at a concentration of 400 μg / mL, the cell survival rate was still >90%, indicating that the product has excellent in vitro biosafety.

[0084] Depend on Figure 8The results showed that the yam polysaccharide exhibited a unimodal normal distribution, indicating good molecular weight uniformity and no significant subgroup dispersion, mainly concentrated in the low to medium molecular weight range of 20kDa to 40kDa. Low to medium molecular weight polysaccharides (typically 10kDa to 100kDa) possess excellent water solubility and transmembrane absorption capacity, making them more likely to exert immunomodulatory and antioxidant physiological activities in vivo. The peak molecular weight of the yam polysaccharide in this invention is approximately 30kDa, falling precisely within this advantageous range. This characteristic lays the structural foundation for its efficient delivery and functional expression.

[0085] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range, as well as any value between the two endpoints, can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

Claims

1. A compound extract of yam, characterized in that, The yam compound extract is composed of ethyl acetate layer extract, n-butanol layer extract and yam polysaccharide of yam ethanol extract, and the mass ratio of ethyl acetate layer extract, n-butanol layer extract and yam polysaccharide is 3~6:2~4:2~4; The total phenolic acid content in the ethyl acetate extract was 238.60 mg / g ± 25.32 mg / g, the total flavonoid content was 35.70 mg / g ± 3.12 mg / g, and the total saponin content was 205.40 mg / g ± 18.65 mg / g. The total saponin content in the n-butanol layer extract was 229.70 mg / g ± 28.43 mg / g, and the total phenolic acid content was 196.80 mg / g ± 21.74 mg / g. The polysaccharide in yam has a purity of ≥85% and a weight-average molecular weight of 30,000 Da~50,000 Da.

2. A method for preparing the yam compound extract according to claim 1, characterized in that, Includes the following steps: Gradient ethanol extraction: Yam powder was soaked in 95% ethanol and extracted for the first time to obtain a first extract and a first residue; the first residue was extracted for the second time with 60% ethanol to obtain a second extract and a second residue; the first extract and the second extract were mixed and concentrated to obtain yam extract. Multistage liquid-liquid extraction: The aqueous solution of yam extract was first extracted with petroleum ether, and the petroleum ether extract was discarded to obtain the first aqueous layer; the first aqueous layer was second extracted with ethyl acetate to obtain the second aqueous layer and the ethyl acetate extract; the second aqueous layer was third extracted with n-butanol to obtain the n-butanol extract and the third aqueous layer. Targeted separation of polysaccharides: Anhydrous ethanol was used as a precipitant to precipitate the third aqueous layer. After obtaining the precipitate, it was subjected to dialysis and freeze-drying to obtain yam polysaccharides. Compound formulation: The ethyl acetate layer extract, the n-butanol layer extract and yam polysaccharide were mixed to obtain a yam compound extract.

3. The preparation method according to claim 2, characterized in that, The conditions for the first and second extractions were the same: heating and reflux at 72℃~82℃ for 2.5h~3h.

4. The preparation method according to claim 2, characterized in that, The concentration process was carried out at 56℃~65℃ and a vacuum of 0.085MPa~0.095MPa until all anhydrous ethanol was distilled off.

5. The preparation method according to claim 2, characterized in that, The precipitation process is as follows: anhydrous ethanol is added dropwise to the third aqueous layer at a dropping rate of 8 mL / min to 12 mL / min, and the mixture is allowed to stand at 2℃ to 8℃ for 6 h to 24 h; wherein the volume ratio of the third aqueous layer to anhydrous ethanol is 1:1 to 6.

6. The preparation method according to claim 2, characterized in that, The dialysis procedure is as follows: the precipitate is placed into a dialysis bag with a molecular weight cutoff of 4000 Da to 16000 Da, and then the dialysis bag is placed in distilled water for dialysis for 24 h to 96 h; during dialysis, the distilled water is changed every 6 h to 8 h.

7. The preparation method according to claim 2, characterized in that, The freeze-drying conditions are as follows: freeze-drying at a vacuum of 0.01MPa~0.04MPa and a temperature of -20℃~-50℃ for 12~48h.

8. The application of the yam compound extract according to claim 1 in the preparation of an anti-inflammatory and immune-boosting compound product, characterized in that, The anti-inflammatory and immune-boosting compound product is selected from dietary supplements, nutritional oral liquids, or oral health products. In the anti-inflammatory and immune complex products, the amount of yam complex extract added is 5wt%~10wt%.

9. The application of the yam compound extract according to claim 1 in the preparation of anti-inflammatory and immunomodulatory agents, characterized in that, The anti-inflammatory and immunomodulatory agent is an oral preparation, wherein the dosage of yam compound extract in the anti-inflammatory and immunomodulatory agent is 20 mg / kg·day to 100 mg / kg·day.

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