Method for separating and purifying walnut male flower polyphenol based on macroporous adsorption resin and application

Abstract

The present application belongs to the field of food science and technology, and particularly relates to a method for separating and purifying walnut male flower polyphenols based on macroporous adsorption resin and application. Research shows that the X-5 type resin has the best performance in adsorbing and desorbing walnut male flower polyphenols, the adsorption process conforms to the Temkin isothermal model and the pseudo-second-order kinetic model, indicating that it is a spontaneous exothermic physical adsorption process. After process optimization (80% ethanol, 2 BV / h flow rate, 10 BV elution volume), the polyphenol content is significantly improved. The purified product detects 26 kinds of phenolic acid compounds and 22 kinds of flavonoids, the main components are astragalin, isquercitrin, protocatechuic acid, gallic acid and quercetin, accounting for 89.91% of the total phenol content. The antioxidant activity of the purified polyphenol extract is significantly enhanced, and it has obvious bacteriostatic effect on Staphylococcus aureus. The present application provides a theoretical basis for the high-value utilization of walnut male flower resources and its application in the fields of food and health care.

Description

Technical Field

[0001] This invention belongs to the field of food science and technology, and specifically relates to a method for separating and purifying polyphenols from male walnut flowers. Background Technology

[0002] Walnut( Juglans regia Walnuts (L.) are widely cultivated woody plants with a long history of edible and medicinal use. Walnut kernels are renowned for their nutritional value, while walnut male flowers, despite their enormous annual production (reaching billions of kilograms dry weight annually in China alone), are often discarded as agricultural waste, causing not only resource waste but also environmental pressure. Therefore, realizing the high-value utilization of walnut male flowers and exploring their potential nutritional and functional value has become one of the current research hotspots in the field of deep processing of agricultural and forestry products.

[0003] Modern pharmacological studies have shown that walnut male flowers are rich in polyphenols, exhibiting broad application prospects in functional foods, health products, cosmetics, and pharmaceuticals. To fully utilize the bioactivity of plant polyphenols, efficient purification processes are crucial. Patent CN111616362B discloses a method for preparing antioxidants from walnut male flowers, employing a process of high-voltage pulsed electric field cell disruption + ethyl acetate extraction + compound enzymatic hydrolysis to improve the extraction rate of antioxidants. However, it does not explicitly target polyphenols as the core purification objective, nor does it specifically optimize the purity of polyphenols. Patent CN118976056A discloses a method for extracting polyphenols from walnut male flowers. This method obtains a crude polyphenol extract through steps such as soaking in a specific treatment agent, low-temperature freeze-drying, and ultrasonic extraction. However, it lacks separation and purification, resulting in low purity that cannot meet the application requirements of functional products and pharmaceuticals. Among existing purification technologies, macroporous adsorption resins have become one of the mainstream methods for polyphenol enrichment due to their advantages such as good selectivity, large adsorption capacity, mild operating conditions, and ease of scale-up. They have been successfully used for the purification of polyphenols from grape skins, sugarcane skins, boletus, and mung bean skins.

[0004] However, to date, there is a lack of systematic and in-depth research on the adsorption behavior, mechanism of action, and component changes of walnut male flower polyphenols by macroporous adsorption resins. Based on these shortcomings, there is an urgent need to develop a simple, efficient, and low-cost method for the separation and purification of walnut male flower polyphenols, significantly improving their purity and bioactivity, and possessing the potential for large-scale application. This would provide a theoretical basis for the efficient enrichment and resource utilization of walnut male flower polyphenols, and simultaneously provide a material foundation and technical support for the development of functional natural food additives. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method and application for separating and purifying polyphenols from walnut male flowers based on macroporous adsorption resin.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention aims to systematically explore the purification mechanism and efficiency of macroporous adsorption resins for polyphenols from walnut male flowers. The main components include: screening for the optimal resin suitable for purifying polyphenols from walnut male flowers; elucidating the adsorption behavior and mechanism of polyphenols on the resin through adsorption kinetics, isotherms, and thermodynamic analysis; optimizing dynamic desorption conditions to establish an efficient and stable purification process; comprehensively analyzing the dynamic changes in polyphenol composition before and after purification using ultra-high performance liquid chromatography-high resolution mass spectrometry (LC-MS); and systematically evaluating the in vitro antioxidant activity and inhibitory effect on foodborne pathogens of the purified product.

[0008] On one hand, this invention provides a method for separating and purifying polyphenols from walnut male flowers based on macroporous resin, the steps of which are as follows:

[0009] (1) Add ethanol solution to walnut male flowers, mix and extract in an ultrasonic cell disruptor, then vacuum filter, collect the filtrate, and concentrate by rotary evaporation to obtain crude extract of walnut male flowers.

[0010] (2) Mix the crude extract of walnut male flowers with macroporous adsorption resin and stir to adsorb;

[0011] (3) After washing and filtering the saturated macroporous adsorption resin, add ethanol for elution and collect the eluent, which is the purified walnut male flower polyphenol solution.

[0012] Preferably, in step (1) above, the concentration of ethanol is 50-70%, the ratio of walnut male flowers to ethanol solution is 1:20-25 g / mL, and the extraction conditions are extraction at 200-400 W for 60-120 min and rotary evaporation concentration at 40-60℃.

[0013] Preferably, in step (2) above, the ratio of macroporous adsorption resin to crude extract of walnut male flowers is 2:20-25 g / mL, and the stirring conditions are 25-45℃ and 150-200 r / min for 18-30 h.

[0014] Preferably, the macroporous adsorption resin in step (2) above is X-5 macroporous resin or D101 macroporous resin. X-5 resin performs best, with an adsorption rate of 62.91% and a desorption rate of 96.29% for polyphenols from walnut male flowers. X-5 and D101 have similar specific surface areas but different adsorption capacities, indicating that the adsorption performance of the resin is the result of the combined effect of its polarity and physical structure.

[0015] The adsorption process conforms to the Temkin isotherm model and the pseudo-second-order kinetic model. The adsorption process is dominated by physical action, and may be mainly through van der Waals adsorption, indicating that it is a spontaneous exothermic physical adsorption process.

[0016] Preferably, in step (3) above, the volume fraction of ethanol is 70-95% v / v, the elution temperature is 20-30℃, the elution volume is 8-12 BV, and the elution flow rate is 1-4 BV / h.

[0017] Secondly, this invention applies to protect the walnut male flower polyphenol extract prepared using the above-described method.

[0018] Preferably, the above-mentioned walnut male flower polyphenol extract contains astragaloside, isoquercitrin, protocatechuic acid, gallic acid and quercetin, and the total content of the above five components in the extract is ≥80%.

[0019] Thirdly, this invention application protects the use of the above-mentioned walnut male flower polyphenol extract in the preparation of products with antioxidant and antibacterial functions.

[0020] Preferably, the above-mentioned antibacterial activity is against Staphylococcus aureus, and the product can be a functional food, dietary supplement, or natural preservative.

[0021] The present invention has the following beneficial effects:

[0022] This invention provides a method for separating and purifying polyphenols from walnut male flowers based on macroporous resin, and establishes an efficient purification process for walnut male flower polyphenols using macroporous adsorption resin. Four commonly used macroporous adsorption resins were selected to compare their adsorption / desorption performance of polyphenols from walnut male flowers. X-5 resin showed the best performance, with adsorption and desorption rates of 62.91% and 96.29%, respectively, indicating that the resin's adsorption performance is the result of the combined effect of its polar characteristics and physical structure. The adsorption process conformed to the Temkin isotherm model and pseudo-second-order kinetic model, indicating that it is a spontaneous exothermic physical adsorption process. After process optimization (80% ethanol, 2 BV / h flow rate, 10 BV elution volume), the polyphenol content increased from 2.08 mg / mL to 6.23 mg / mL. LC-MS / MS identification of the purified products revealed 26 phenolic acid compounds and 22 flavonoids, with the main components being astragaloside (1853.49 mg / kg), isoquercitrin (1324.90 mg / kg), protocatechuic acid (514.56 mg / kg), and gallic acid (485.10 mg / kg). The purified polyphenol extract contained 102.92 mg / kg of walnut male flowers and quercetin (102.92 mg / kg), accounting for 89.91% of the total phenol content. The DPPH scavenging rate, ABTS scavenging rate, and FRAP iron reducing capacity of the purified polyphenol extract were significantly enhanced, and the antioxidant capacity was significantly improved compared with the crude extract. It also showed a significant antibacterial effect against Staphylococcus aureus. This application provides a theoretical basis for the high-value utilization of walnut male flower resources and their application in the food and health care fields. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The adsorption and desorption amounts of crude extract of walnut male flowers on resin are represented.

[0025] Figure 2 Linear fitting of the adsorption kinetics of walnut male flower polyphenols in X-5 macroporous resin at the same temperature (A), pseudo-first-order model (B), pseudo-second-order model (C), and intraparticle diffusion model (D).

[0026] Figure 3 Linear fitting of the adsorption isotherms of walnut male flower polyphenols on X-5 adsorption resin at different temperatures (A), Langmuir model (B), Freundlich model (C), and Temkin model (D).

[0027] Figure 4 The graphs show the relationship between ln(Qe / Ce) and Qe at different temperatures (A), the relationship between lnK and 1 / T (B), the effect of ethanol concentration on desorption rate (C), and the effect of elution volume and flow rate on elution effect (D).

[0028] Figure 5 The antioxidant activity of X-5 resin before and after purification was analyzed; Figure A shows the DPPH free radical scavenging rate, B shows the ABTS free radical scavenging rate, C shows the FRAP iron reducing capacity, and D shows a schematic diagram of the inhibition zone.

[0029] Figure 6 The values ​​represent the relative content of polyphenols before and after purification.

[0030] Figure 7 Total ion chromatogram of purified polyphenols. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below with reference to 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.

[0032] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0033] 1. The experimental materials and reagents used in this invention are as follows:

[0034] The male flowers of the walnut tree were collected in April 2025 from Lushi County, Henan Province. They were dried, ground, and passed through a 60-mesh sieve. Folin-Ciocalteu reagent, gallic acid standard, anhydrous ethanol, anhydrous sodium carbonate, NaOH, HCl, 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), rutin standard, vitamin C, sodium acetate, glacial acetic acid, FeCl3·6H2O, and FeSO4·7H2O were purchased from Shanghai Maclean Biochemical Technology Co., Ltd. AB-8 macroporous resin, X-5 macroporous resin, D101 macroporous resin, and NKA macroporous resin were all purchased from Hangzhou Dongpeng Chemical Co., Ltd. All chemicals and reagents used were analytical grade.

[0035] 2. Data Statistical Analysis

[0036] Statistical analysis and correlation analysis of the experimental data were performed using Excel 2024, Origin 2024 and SPSS 26.0. All data are expressed as mean (mean) ± standard deviation (SD) of three measurements.

[0037] Example 1: Resin screening for purifying crude extract

[0038] (1) Extraction of polyphenols from walnut flowers

[0039] Extraction was performed at 60% ethanol concentration and a solid-liquid ratio of 1:25 (g / mL) using an ultrasonic cell disruptor at 300 W for 90 min (5 s start-up, 5 s stop-down). The extract was then vacuum filtered through a Buchner funnel to obtain the filtrate. The residue was further extracted, and the filtrates were combined and concentrated by rotary evaporation at 50 °C to obtain the polyphenol extract.

[0040] (2) Determination of total polyphenol content in walnut flowers

[0041] The total polyphenol content (TPC) was determined by colorimetric method. In simple terms, 25 μL of the sample solution was taken and diluted to 2 mL with distilled water. This was then mixed with 0.8 mL of Folin-Ciocalteu solution. After reacting for 8-9 min, 4 mL of 7.5% Na₂CO₃ solution was added to terminate the reaction. The mixture was then incubated in a 50℃ water bath in the dark for 10 min. The absorbance at 765 nm was measured, establishing a standard curve for gallic acid in the range of 0-100 μg / mL (A = 0.3148C + 0.0462, where A and C are absorbance and concentration, respectively). The correlation coefficient (…) R 2 The value is 0.9954.

[0042] (3) Determination of total flavonoid content in male walnut flowers

[0043] Take 25 μL of sample, add 60% ethanol to make up to 2.00 mL, add 0.4 mL of 5% NaNO2 solution, shake well, and let stand at 25℃ for 6 min; add 0.40 mL of 10% AlNO3 solution, shake well, and calculate the total flavonoid content in the sample. A standard curve for rutin in the range of 0-100 μg / mL was established (A=0.0139C-0.0026, where A and C are absorbance and concentration, respectively), and the correlation coefficient ( R 2 The value was 0.9937. The results showed that the phenolic acid content (16.8 mg / g) in the sample was significantly higher than the flavonoid content (5.47 mg / g). Given that phenolic acids are the main component of total polyphenols, phenolic acid content was consistently used as the characterization index for total polyphenol content in subsequent experiments.

[0044] (4) Screening of macroporous adsorption resins

[0045] 2.0 g of activated macroporous adsorption resin was added to 25 mL of diluted crude walnut male flower extract in a 50 mL Erlenmeyer flask. The adsorption capacity and adsorption rate were determined by stirring at 25 °C for 24 h (200 r / min). After the fully adsorbed macroporous adsorption resin was washed and filtered, 40 mL of 95% ethanol was added, and the mixture was stirred at 25 °C for 12 h before calculating the desorption amount. Finally, the macroporous adsorption resin with the highest adsorption rate, adsorption capacity, and desorption rate was selected.

[0046] Q (mg / g) = P (mg / g) = (1)

[0047] C0 and C1 are the concentrations of polyphenol solutions before and after adsorption (mg / mL); C1 is the concentration of polyphenol solution after adsorption (mg / mL); C2 is the concentration of polyphenol solution after desorption (mg / mL); V is the solution volume (mL); W is the resin mass (g); Q and P are the adsorption and desorption capacities of the resin, respectively (mg / g).

[0048] The physicochemical properties of resins, such as polarity, pore size, and specific surface area, are key factors affecting their adsorption and desorption performance. Furthermore, the adsorption behavior of the same resin for extracts from different plant sources varies. This application selected four commonly used macroporous adsorption resins (AB-8 macroporous resin, X-5 macroporous resin, NKA macroporous resin, and D101 macroporous resin) and compared their adsorption / desorption performance of walnut male flower polyphenols. The results are shown in […]. Figure 1 .

[0049] Polyphenolic compounds contain multiple phenolic hydroxyl groups and can bind to resins through mechanisms such as hydrogen bonding, hydrophobic interactions, and π-π conjugation. Therefore, they are generally more easily adsorbed by resins with higher polarity. Figure 1The results showed that X-5 and D101 resins had relatively high adsorption capacities for crude walnut male flower extract, at 4.67 mg / g and 4.07 mg / g, respectively, while AB-8 and NKA had relatively low adsorption capacities, at 3.80 mg / g and 3.86 mg / g, respectively. Considering the polarity parameters of the resins in Table 1 (X-5 and D101 have different polarities, as do AB-8 and NKA), this indicates that adsorption capacity is not entirely determined by resin polarity; other physical properties must also be considered.

[0050] Table 1. Polar parameters of macroporous adsorption resins

[0051]

[0052] Factors such as pore size, particle size, and specific surface area of ​​the resin all affect its adsorption capacity. AB-8 and NKA exhibited low adsorption capacities for polyphenols from walnut male flowers, possibly related to their smaller specific surface areas (Table 1), resulting in fewer available adsorption sites and thus limiting polyphenol adsorption. This result is consistent with research findings on the adsorption of phenolic substances by macroporous resins, namely, resins with larger specific surface areas generally have higher adsorption potential. Notably, X-5 and D 101 have similar specific surface areas but different adsorption capacities, further indicating that the adsorption performance of the resin is the result of the combined effect of its polar characteristics and physical structure.

[0053] In the desorption experiment, X-5 resin exhibited the highest desorption rate, which may be attributed to its large specific surface area and abundant pore structure, providing more active adsorption sites and facilitating the release of phenolic substances during elution. Considering both adsorption and desorption performance, X-5 resin performed best among the four macroporous resins, with adsorption and desorption rates of 62.91% and 96.29% for walnut male flower polyphenols, respectively. Therefore, X-5 was selected for subsequent adsorption kinetic studies.

[0054] Example 2: Adsorption kinetics test

[0055] 2.0 g of activated macroporous adsorption resin was weighed and added to 25 mL of diluted crude extract of walnut male flowers in a 50 mL Erlenmeyer flask. The mixture was stirred at 150 rpm and 25 °C for 12 hours. The total phenol concentration in the supernatant was determined using the Folin-Ciocalteu reagent method at time intervals of 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, and 7 hours. The test results were measured in triplicate.

[0056] The adsorption kinetics of polyphenols on the resin were evaluated using pseudo-first-order model equation (4) and pseudo-second-order model equation (5). To further determine the rate-controlling step, an intraparticle diffusion model equation (6) was also introduced.

[0057] (4)

[0058] (5)

[0059] (6)

[0060] Q t (mg / g) represents the amount of polyphenols adsorbed on the resin at time t minutes; k1 and k2 represent the rate constants of the pseudo-first-order model and the pseudo-second-order model, respectively; k i C represents the interparticle diffusion rate constant; i It is a constant characterizing the thickness of the boundary layer.

[0061] Adsorption kinetics reflects the change in the adsorption rate of adsorbates on adsorbents over time, and is of great significance for process design and scale-up. For example... Figure 2 As shown in Figure A, the adsorption of polyphenols from walnut male flowers by X-5 resin increased rapidly within the first 2 hours, and then leveled off between 2 and 7 hours. The rapid adsorption phase may be related to the abundant effective adsorption sites on the resin surface. Subsequently, due to the gradual saturation of surface adsorption and the increase in mass transfer resistance, the adsorption rate slowed down, eventually reaching adsorption equilibrium.

[0062] To further elucidate the adsorption process and mechanism, this study employed pseudo-first-order, pseudo-second-order, and intraparticle diffusion models to fit the kinetic data. Figure 2 (BD). Pseudo-first-order models are often used to describe the initial stage of the adsorption process, while pseudo-second-order models are more suitable for predicting the behavior of the entire adsorption process. As shown in Table 2, the adsorption capacity (Q) predicted by the pseudo-second-order model... e =4.3498 mg / g) is closer to the experimental value (Q). e =4.673 mg / g), compared to the pseudo-first-order model prediction (Q e =2.86 mg / g) is higher. The R-squared value of the pseudo-second-order model is higher. 2 The value (0.9876) is higher than the R of the pseudo-first-order model. 2 The value (0.9776) indicates that the pseudo-second-order model better describes the adsorption process, suggesting that chemisorption may be the main rate-determining step.

[0063] Table 2. Equations and parameters of adsorption kinetics of walnut polyphenols on X-5 resin

[0064]

[0065] Generally, the adsorption process typically includes membrane diffusion (boundary layer diffusion), intraparticle diffusion, and a final equilibrium stage. Intraparticle diffusion models are often used to identify the rate-limiting step in the adsorption process. Fitting results show that ( Figure 2D), Qt and t¹ / ² show a multilinear relationship, and the fitted line does not pass through the origin, indicating that the adsorption process is simultaneously controlled by intraparticle diffusion and boundary layer diffusion, verifying the existence of multi-step mass transfer resistance in the adsorption process.

[0066] Example 3: Adsorption isotherm test

[0067] The selected macroporous adsorption resin was mixed with 25 mL of polyphenol aqueous solution in an Erlenmeyer flask. At different polyphenol concentrations, the mixture was shaken at 150 rpm for 12 hours at 25, 35, and 45 °C, respectively. The polyphenol content and the equilibrium adsorption capacity of the macroporous adsorption resin were then calculated.

[0068] The equilibrium data were modeled using the Langmuir equation (7), the Freundlich equation (8), and the Temkin equation (9).

[0069] (7)

[0070] (8)

[0071] (9)

[0072] Q m (mg / g) represents the theoretical maximum adsorption capacity; Q e Actual adsorption capacity; K L K T K F represent the equilibrium constants of the Langmuir model, Temkin model, and Freundlich model, respectively; 1 / n indicates that the adsorption strength of the adsorbent is a constant, and B T (J / mol) represents the Temkin constant related to the heat of adsorption of the resin.

[0073] Adsorption isotherm fitting

[0074] Adsorption isotherms describe the relationship between the equilibrium concentration of the adsorbent and the equilibrium adsorption amount at adsorption equilibrium. They help to understand the interaction between the adsorbate and the adsorbent, optimize performance parameters, and save energy. For example... Figure 3 As shown in Figure A, at the same initial concentration, the equilibrium adsorption capacity of X-5 resin for walnut male flower polyphenols gradually decreased with increasing temperature (25℃, 35℃, 45℃), indicating that the adsorption process may be exothermic, and high temperature is unfavorable for adsorption. The amount of walnut male flower polyphenols adsorbed on X-5 resin increased with increasing equilibrium concentration (C... e The data increases with the increase of , reaching a saturation state; the Langmuir, Freundlich, and Temkin models were used to fit the experimental data respectively. Figure 3 BD).

[0075] Table 3. Adsorption isotherm equations and parameters of walnut polyphenols on X-5 resin

[0076]

[0077] The Langmuir model assumes homogeneous adsorption in a monolayer, with equal energy at all sites and no intermolecular interactions between adsorbent molecules. Table 3 shows that the maximum adsorption capacity (Q) in the Langmuir model increases with temperature. m The gradual decrease in K further illustrates that the adsorption of polyphenols from walnut male flowers by X-5 resin is exothermic. Furthermore, K... L The value decreases slightly with increasing temperature, indicating that phenolic substances adhere better to the resin surface, which is consistent with the adsorption results of walnut male flower polyphenols on X-5 resin. In the Freundlich model, the adsorption surface is non-uniform, and each adsorption site has a unique binding energy. When the value of 1 / n is greater than 1, it indicates that the adsorption is multilayer adsorption. As shown in Table 3, the 1 / n value of the Freundlich model at different temperatures is approximately 1.4, indicating that this adsorption is more consistent with the characteristics of multilayer adsorption. The Temkin model assumes that the heat of adsorption decreases linearly with increasing adsorption amount, and its goodness of fit ( R 2 =0.9876) is higher than Langmuir ( R 2 = 0.9550) and Freundlich ( R 2 = 0.9553) model, indicating that the Temkin model is best suited to describe this adsorption system, and its parameter B>0, further confirming that adsorption is an exothermic process.

[0078] Example 4: Adsorption Thermodynamics

[0079] Calculation of adsorption thermodynamic parameters:

[0080] The Gibbs free energy change (ΔG) is calculated using equation (10). Ignoring the influence of reaction temperature on enthalpy and entropy changes, the enthalpy change (ΔH) and entropy change (ΔS) are calculated using equation (11).

[0081] (10)

[0082] (11)

[0083] R (8.314 J / mol K) represents the gas constant, and T (K) and K refer to the absolute temperature and equilibrium constant, respectively.

[0084] Table 4. Thermodynamic parameters of walnut polyphenol adsorption on X-5 resin

[0085]

[0086] Note: T: Temperature, K; ΔG: Gibbs free energy change, kJ / mol; ΔH: Enthalpy change, kJ / mol; ΔS: Entropy change.

[0087] By plotting ln(Q) e / C e) Q e The relationship curve, and Q e Extrapolating to zero, the adsorption equilibrium constant K at different temperatures (25, 35, 45℃) was obtained. c .like Figure 4 As shown in A, K c The value decreases with increasing temperature, indicating that heating is unfavorable for the adsorption of polyphenols from walnut male flowers by X-5 resin. Thermodynamic parameter calculations (Table 4) show that the Gibbs free energy change ΔG is negative in all cases, indicating that adsorption can occur spontaneously. The absolute value of ΔG decreases with increasing temperature, indicating that the degree of spontaneity of adsorption is higher at lower temperatures. It was found that lnK... c It has a good linear relationship with 1 / T (Van 't Hoff equation). R 2 =0.9812), therefore, ΔS and ΔH are determined by calculating the intercept and slope respectively. Figure 4 B). In Table 4, the enthalpy change ΔH is negative (-3.6193 kJ / mol), confirming that adsorption is an exothermic process, consistent with the conclusions of the adsorption isotherm. The entropy change ΔS is positive (11.5033 J / mol K), indicating an increase in the disorder of the solid-liquid interface during adsorption. Furthermore, the absolute value ΔH < 40 kJ / mol suggests that the adsorption process is dominated by physical forces, possibly primarily through van der Waals adsorption.

[0088] Example 5: Optimization of Dynamic Desorption Process

[0089] To investigate the effects of ethanol volume fraction and elution volume on the desorption process, the selected resin was packed into a glass column with a height-to-diameter ratio of 6.5:1 and a packed bed volume (BV) of 30 mL. The saturated macroporous adsorption resin was washed with distilled water to remove surface impurities, and then eluted with ethanol under different conditions.

[0090] (1) Effect of ethanol volume fraction on purification

[0091] The saturated macroporous adsorption resin was desorbed at an adsorption temperature of 25℃, an elution volume of 10 BV, and an elution flow rate of 2 BV / h. Eluent was added at volume fractions of 70% (v / v), 75% (v / v), 80% (v / v), 85% (v / v), and 90% (v / v) ethanol, respectively. The content of polyphenols in walnut male flowers was determined, and the desorption rate was calculated.

[0092] (2) Effect of elution flow rate on purification

[0093] The macroporous adsorption resin was saturated with adsorption at an adsorption temperature of 25℃ and an ethanol concentration of 80%. Eluent was added at elution flow rates of 1 BV / h, 2 BV / h, and 4 BV / h, and the content of walnut male flower polyphenols in the eluent was measured at different time points to investigate the effect of elution flow rate and eluent volume on desorption efficiency.

[0094] Dynamic adsorption was performed on an X-5 resin column to load crude walnut male flower extract to 5 BV (2.0 mg / mL of phenolic compounds). The effects of ethanol concentration (70%-95%) and elution flow rate (1, 2, 4 BV / h) on desorption were investigated, with the final colorless eluent as the endpoint. Figure 4 As shown in Figure C, an 80% ethanol / water solution is most suitable for dissolving phenolic compounds bound to X-5 resin, achieving the highest elution efficiency (79.51%). Both excessively low and high ethanol concentrations lead to a decrease in elution efficiency. The eluent volumes consumed to reach the elution endpoint at different flow rates were 8, 10, and 12 BV, respectively, indicating that ( Figure 4 (D) Although a lower flow rate (1 BV / h) is beneficial for complete desorption, the longer process time is not conducive to industrial applications. At a flow rate of 2 BV / h, using 10 BV of 80% ethanol achieves efficient elution, balancing efficiency and time cost. Through the above process optimization, the content of polyphenols from walnut male flowers increased from the initial 2.08 mg / mL to 6.23 mg / mL, demonstrating a significant purification effect.

[0095] Example 1: Antioxidant Activity Analysis of Walnut Male Flower Polyphenol Extract

[0096] (1) DPPH

[0097] Mix 50 µL of a suitable concentration of sample with 100 µL of DPPH solution in a 96-well microplate, react in the dark for 30 min, and measure the absorbance at 492 nm (An). The reaction system using 70% ethanol solution instead of the sample is the blank group (Ac), and the reaction system using ethanol instead of DPPH is the sample control group (Ab). Calculate the IC50 (IC50) of DPPH free radical scavenging. 50 Value. The formula for calculating the DPPH free radical scavenging rate is:

[0098] Clearance rate = (12)

[0099] A n A represents the light absorbance of the experimental sample. b A represents the absorbance value of the sample control group. c The light absorption value of the blank group

[0100] (2) ABTS

[0101] Take 50 µL of sample of appropriate concentration into a 96-well microplate, add 200 µL of ABTS solution, react at room temperature for 5 min, and measure the absorbance of the solution after reaction at 405 nm. Use ethanol instead of ABTS solution as sample blank and ethanol instead of sample as control. The formula for calculating the ABTS free radical scavenging rate is (13).

[0102] Clearance rate = (13)

[0103] In the formula, A0: absorbance of the blank control group using purified water instead of the test solution; A1: absorbance of the test solution.

[0104] (3) Determination of iron reducing power

[0105] Vitamin C (V C Using ferrous sulfate as a standard and ferrous sulfate as a control (A = 118.56C + 82.092, where A and C are absorbance and concentration, respectively), a standard curve was plotted, and the correlation coefficient was calculated. R 2 The value is 0.9953.

[0106] The antioxidant activity of plant phenolic compounds plays a positive role in regulating and reducing the risk of chronic diseases. This study evaluated the antioxidant activity of polyphenol extracts before and after macroporous resin purification using DPPH radical scavenging, ABTS radical scavenging, and FRAP iron reducing power assays. Figure 5 As shown in AC, within the concentration range of 10-100 μg / mL, the DPPH scavenging rate of the purified polyphenols increased from 2.46% to 42.05%, and the ABTS scavenging rate increased from 16.24% to 56.07%; within the concentration range of 2-20 μg / mL, the FRAP iron reducing capacity increased from 240 μg / mL to 1000 μg / mL. The DPPH and ABTS scavenging rates of the purified samples were also shown. 50 The values ​​were 343.14 and 117.58 μg / mL, respectively, still higher than the Trolox positive control (100.53 μg / mL and 84.65 μg / mL), but its antioxidant capacity was significantly higher than that of the crude extract. This may be related to the increased total polyphenol (TPC) content and the synergistic effect among the components.

[0107] Example 2: Antibacterial activity test of polyphenol extract from walnut male flowers

[0108] Strain activation: Mix 50 µL of Staphylococcus aureus with 4 mL of LB and incubate for 3-4 hours. Measure the absorbance at 570 nm (A = 0.3 ± 0.03). At this point, the bacterial concentration is 10. 7 CFU / mL, proceed to the next step of plating the bacterial strain.

[0109] Spreading and inhibition zone: 50 µL of the activated bacterial solution was spread onto the surface of a solid culture medium, and uniform pores were formed on the medium using a perforation method. 50 µL of purified walnut male flower polyphenol sample solution (50 mg / mL and 100 mg / mL) filtered through a 0.22 µm microporous membrane was added to each pore. Simultaneously, 50 µg / mL kanamycin was set as a positive control, 50% ethanol as a solvent control, and PBS as a control. Figure 5 D). Place all plates in a constant temperature incubator and incubate at 37℃ for 10 h, then observe the results.

[0110] This application uses Staphylococcus aureus as an indicator strain to evaluate the antibacterial activity of polyphenol extract from walnut male flowers. The inhibition zone diameter of 100 mg / mL polyphenol extract was 17.8 mm, close to the 19.2 mm of the positive control group (kanamycin), while no inhibition zone was observed in the negative control group. Walnut male flower polyphenols have a clear antibacterial effect against Staphylococcus aureus.

[0111] Example 3: Identification of polyphenol components after purification of walnut male flower polyphenol extract

[0112] An AB SCIX ultra-high performance liquid chromatography-mass spectrometry (UPLC) system equipped with a Waters ACQUITY UPLCHSS T3 column (100 mm × 2.1 mm, 1.8 μm, Waters, Shimadzu, Japan) and an electrospray ionization source (ESI, SCIEX, Foster City, CA, USA) was used to analyze the types of phenolic compounds in the purified samples. The mobile phase consisted of 0.1% formic acid (v / v) water (A) and acetonitrile (containing 0.1% formic acid, B). The column temperature was 40 °C, the flow rate was 0.4 mL / min, the injection volume was 4 μL, and the linear gradient program was: 0–3.9 min, 95–75% A; 4–4.5 min, 1% A; 4.5–6 min, 95% A. The phenolic compounds in the samples were qualitatively identified using a matching database.

[0113] Phenolic compounds were quantitatively identified using ultra-high performance liquid chromatography (UHPLC) and high-resolution mass spectrometry (HMS). The chromatographic conditions were as follows: column: Waters HSS T3 (50 × 2.1 mm, 1.8 μm); mobile phase: phase A was ultrapure aqueous solution (containing 0.1% formic acid), and phase B was acetonitrile solution (containing 0.1% formic acid); flow rate: 0.3 mL / min; column temperature: 40℃; injection volume: 2 μL; elution gradient: 0 min A / B phase (90:10, v / v); 2 min A / B phase (90:10, v / v); 6 min A / B phase (40:60, v / v); 9 min A / B phase (40:60, v / v); 9.1 min A / B phase (90:10, v / v); 12 min A / B phase (90:10, v / v). Mass spectrometry data were acquired using a Thermo Q Exactive high-resolution mass spectrometry system. Equipped with an electrospray ionization (ESI) source, sheath gas 40 arb, auxiliary gas 10 arb, ion spray voltage -2800 V, temperature 350℃, and ion transmission tube temperature 320℃. The scanning mode was Full scan-ddMS2, and the scanning method was negative ion. The primary mass spectrometry scan range (m / z range) was 100-900. Throughout the analysis, the sample was placed in an autosampler at 4℃.

[0114] Table 5. Phenolic compounds identified in the purified extracts

[0115]

[0116]

[0117] The purified extract was qualitatively and quantitatively analyzed by LC-MS, and a total of 48 phenolic compounds were identified, including 26 phenolic acids and 22 flavonoids (Table 5). The relative contents of phenolic acids are shown in the table below. Figure 6 The external standard method was used to quantitatively analyze 26 phenolic acid compounds (Table 6), and the ion chromatograms are shown in [Table 6]. Figure 7 .Depend on Figure 6As shown in Table 6, compared with the unpurified crude extract, the purified walnut male flower polyphenols showed more significant changes in the content of phenolic acids such as astragaloside, isoquercitrin, protocatechuic acid, gallic acid, and quercetin. The relative content of astragaloside increased from 16.87% to 38.95%, and its content increased from 97.55 mg / kg to 1853.49 mg / kg. The relative content of isoquercitrin increased from 12.06% to 27.84%, and its content increased from 69.73 mg / kg to 1324.90 mg / kg. The relative content of protocatechuic acid increased from 4.68% to 10.81%, and its content increased from 69.7 mg / kg to 1324.90 mg / kg. Although the relative contents of gallic acid and quercetin decreased slightly, from 12.13% to 10.19% and from 5.67% to 2.16%, respectively, quantitative analysis revealed that the content of gallic acid increased from 70.13 mg / kg to 485.10 mg / kg, and that of quercetin increased from 32.79 mg / kg to 102.92 mg / kg. Compared with the unpurified sample, the contents of astragaloside, isoquercitrin, and protocatechuic acid increased by more than 20 times, while the contents of protocatechuic acid, dihydroquercetin, 4-hydroxybenzoic acid, naringenin, and naringin chalcone increased by 2-5 times. These results indicate that the main phenolic compounds in the purified sample are astragaloside (1853.49 mg / kg), isoquercitrin (1324.90 mg / kg), protocatechuic acid (514.56 mg / kg), gallic acid (485.10 mg / kg), and quercetin (102.91 mg / kg), accounting for 89.91% of TPC. This demonstrates that X-5 resin has a good selective purification effect on these phenolic substances. The content of a few components still decreased slightly, such as vanillin from 7.52 mg / kg to 2.04 mg / kg and syringaldehyde from 9.04 mg / kg to 5.83 mg / kg. This may be related to the polymerization of the resin and the chemical properties of the phenolic compounds.

[0118] Table 6. Quantitative comparison of polyphenols in crude extract and purified extract.

[0119]

[0120] This invention uses walnut male flowers as raw material to prepare crude polyphenol extract through ultrasound-assisted extraction. The purification performance of four macroporous adsorption resins was systematically compared, and X-5 type macroporous adsorption resin was selected as the optimal adsorption material. The adsorption process conforms to pseudo-second-order kinetics and the Temkin isotherm model, and thermodynamic analysis indicates that the process is a self-exothermic physical adsorption. After optimization of the dynamic adsorption-desorption process, under conditions of 80% ethanol, a flow rate of 2 BV / h, and an elution volume of 10 BV, the polyphenol content was significantly increased from 2.08 mg / mL to 6.23 mg / mL. Component analysis showed that the main polyphenols were astragaloside, isoquercitrin, protocatechuic acid, gallic acid, and quercetin, accounting for 89.91% of the total phenol content. Activity experiments showed that the antioxidant and antibacterial activities of the purified extract were significantly enhanced. This invention provides more systematic theoretical support for the high-value utilization and functional product development of walnut male flowers.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for separating and purifying polyphenols from walnut male flowers using macroporous resin, characterized in that, The steps are as follows: (1) Add ethanol solution to walnut male flowers, mix and extract in an ultrasonic cell disruptor, then vacuum filter, collect the filtrate, and concentrate by rotary evaporation to obtain crude extract of walnut male flowers. (2) Mix the crude extract of walnut male flowers with macroporous adsorption resin and stir to adsorb; (3) After washing and filtering the saturated macroporous adsorption resin, add ethanol for elution and collect the eluent, which is the purified walnut male flower polyphenol solution.

2. The method according to claim 1, characterized in that: In step (1), the concentration of ethanol is 50-70%, the ratio of walnut male flowers to ethanol solution is 1:20-30 g / mL, and the extraction conditions are: extraction at 200-400 W for 60-120 min, and rotary evaporation concentration at 40-60℃.

3. The method according to claim 2, characterized in that: In step (2), the ratio of macroporous adsorption resin to crude extract of walnut male flowers is 2:20-25 g / mL, and the stirring conditions are 25-45℃ and 150-200 r / min for 18-30h.

4. The method according to claim 3, characterized in that: In step (2), the macroporous adsorption resin is either X-5 macroporous resin or D101 macroporous resin.

5. The method according to claim 4, characterized in that: The adsorption process in step (2) conforms to the Temkin isotherm model and the pseudo-second-order kinetic model, and is a spontaneous exothermic physical adsorption process.

6. The method according to claim 5, characterized in that: In step (3), the volume fraction of ethanol is 70-95% v / v, the elution temperature is 20-30℃, the elution volume is 8-12 BV, and the elution flow rate is 1-4 BV / h.

7. The walnut male flower polyphenol extract prepared by the method according to any one of claims 1-6.

8. The walnut male flower polyphenol extract according to claim 7, characterized in that: The walnut male flower polyphenol extract contains astragaloside, isoquercitrin, protocatechuic acid, gallic acid and quercetin, and the total content of the five components in the extract is ≥80%.

9. The use of the walnut male flower polyphenol extract according to claim 8 in the preparation of products with antioxidant and antibacterial functions.

10. The application according to claim 9, characterized in that: The antibacterial effect refers to the inhibition of Staphylococcus aureus.

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