Method for improving bioavailability of naringin by metal ion chelation

Abstract

The application discloses a method for improving the bioavailability of naringin by metal ion chelation, and relates to the technical field of naringin, and comprises the following steps: preparing a buffer and a naringin mother liquor; preparing a metal ion solution by setting different pH control windows; setting a chelation molar ratio, and preparing a pre-configuration based on the metal ion solution; performing a double-driven synergistic chelation treatment by using ultrasonic cavitation and heat induction; performing differential fine adjustment to obtain a stabilized chelation solution; performing clarification, desalting and molding on the stabilized chelation solution; and designing a re-dissolution release judgment. 3+ 3+ 2+ The application forms a stable coordination structure by using food safety level metal ions such as Al 3+ , Fe 3+ , Mg 2+ and naringin molecules, and under the condition that no organic solvent or hazardous chemical is involved, not only the bioavailability defects of poor solubility and unsatisfactory digestive stability of naringin are effectively solved, but also the preparation process is simple, economical and green, and has good industrial amplification and cost advantage.

Description

Technical Field

[0001] This invention relates to the field of naringin technology, and in particular to a method for improving the bioavailability of naringin by utilizing the chelation effect of metal ions. Background Technology

[0002] Naringin is a widely sourced citrus flavonoid compound with significant antioxidant, anti-inflammatory, antitumor, and cardiovascular protective effects. However, its extremely low water solubility results in very poor oral absorption and a bioavailability of less than 20%, becoming a key bottleneck limiting its widespread application in pharmaceuticals, medical foods, and health supplements. Currently, common technical approaches to improve the solubility and absorption of naringin fall into two categories: one is to add organic solvents (such as DMSO, ethanol, propylene glycol, etc.) to aid solubilization; the other is to use methods such as nanoparticle encapsulation, microemulsion systems, and chemical derivatization for physical or chemical solubilization. However, the former carries chemical toxicity and biosafety risks; cosolvents such as DMSO may cause abnormal cell membrane permeability and enhanced cytotoxicity during human ingestion, and it is difficult to meet the safety standards for food or oral formulations. While the latter can improve the dissolution rate to some extent, its preparation process is complex, requires sophisticated equipment, is expensive, and lacks stability, failing to meet the green and large-scale demands of the natural product industry.

[0003] Patent document with publication number "CN114917355A" discloses a naringin-chitosan oligosaccharide conjugate, its preparation method, and its application. Naringin and chitosan oligosaccharides are separately prepared into ethanol solutions, then mixed, stirred, and after the reaction is complete, the solvent is evaporated, and the product is obtained by vacuum drying. The improved solubility of naringin allows for applications in antioxidation, antibacterial, and bactericidal effects. This provides new ideas for the development and application of naringin in livestock feed, biomedicine, and functional health foods. However, this method presents risks related to chemical toxicity and biosafety.

[0004] Patent document with publication number "CN111493250A" discloses an octenyl succinic acid waxy corn starch ester naringin inclusion complex and its preparation method, using naringin as the core material and a molecular weight of 1.0 × 10⁻⁶. 4 ~2.0×10 5 Using Da's octenyl succinic acid waxy corn starch ester (OSAS) as a packaging material to encapsulate naringin can improve the solubility and bioavailability of naringin and expand its application in the food, beverage and pharmaceutical industries; however, this method has the problems of complex process, high cost and insufficient stability.

[0005] Patent document CN111018929A also discloses an extraction and separation process for isonarnaringin. The process involves dissolving crude isonarnaringin in a polar organic solvent by heating, filtering to remove insoluble impurities, concentrating and crystallizing, filtration, washing with water, and drying to obtain a semi-finished isonarnaringin product. Alternatively, the semi-finished isonarnaringin can be dissolved in a polar organic solvent, stirred to dissolve, filtered to remove impurities, diluted with pure water, stirred, allowed to crystallize, filtered, washed with pure water until neutral, and dried to obtain a yellow isonarnaringin product. However, this process relies on multiple pH adjustments with inorganic acids and recrystallization with polar organic solvents (such as ethanol, methanol, or acetone), making it complex, energy-intensive, and posing a risk of organic residues. Therefore, it is not suitable for food or oral applications. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for improving the bioavailability of naringin by utilizing the chelation effect of metal ions, thereby solving the problems mentioned in the background art.

[0007] The technical solution of this invention: A method for improving the bioavailability of naringin by utilizing the chelation effect of metal ions, comprising the following steps:

[0008] (S1) Prepare the buffer solution and prepare the naringin stock solution based on the prepared buffer solution;

[0009] By establishing a stable buffer system, namely a pH of 5.4–5.6 and a low conductivity environment, the naringin molecules are fully dissolved under non-hydrolyzed conditions. At the same time, the use of Tris-HCl buffer without organic solvents can significantly reduce the risk of impurity interference and toxicity, ensuring the controllability and food-grade safety of the system.

[0010] (S2) Based on AI 3+ Fe 3+ Mg 2+ Three metal ions were prepared with different pH control windows to prepare the metal ion solutions required for chelation;

[0011] By setting different pH control windows, metal ions can exist in the optimal hydrated form, avoiding hydrolysis or hydroxyl complexation precipitation from the source. This allows for precise control of the reactivity of the three metal ion system, improving subsequent chelation efficiency and structural uniformity.

[0012] (S3) Set Al 3+ Fe 3+ Mg 2+ The chelation molar ratio of the three metal ions was determined based on the pre-mixing of the metal ion solution and the naringin mother liquor to obtain a primary complex premix.

[0013] By designing a differentiated chelation molar ratio, precipitation caused by excessively high local ion concentrations can be prevented, resulting in a narrow particle size distribution and uniform bonding of the primary complex premix, which is a key step in the formation of a highly soluble structure.

[0014] (S4) The primary complex premixed liquid was subjected to dual-drive synergistic chelation treatment of ultrasonic cavitation and thermal induction to obtain a complex with high solubility.

[0015] By employing a dual-drive approach of ultrasonic cavitation and thermal induction, the instantaneous high energy density facilitates rapid coordination and bonding. Compared with traditional heating methods, the reaction time is shortened to a few minutes, the solubility is increased by more than 100 times, and the energy efficiency is high while the system stability is significantly enhanced.

[0016] (S5) for Al 3+ Fe 3+ Mg 2+ Differential fine-tuning of the highly soluble complexes of three metal ions yields a stable chelate solution;

[0017] Based on pH-based differential fine-tuning, the directional structural optimization of chelates of different metal ions can be achieved, which can maintain the electroneutrality of the system and prevent precipitation, and rearrange the coordination structure from primary bonding to thermodynamically stable state, thereby improving long-term stability and resolubility.

[0018] (S6) The stabilized chelate solution is clarified, desalted and shaped to obtain naringin-metal ion chelate;

[0019] Dynamic dialysis is performed during the molding process to avoid dissociation problems that are easily caused by traditional static dialysis.

[0020] (S7) Design of the reconstitution and release determination of the formed naringin-metal ion chelate.

[0021] Establishing release standards enables systematic verification from structure to function; different metal chelate systems have fast resolubility, high stability and strong antioxidant properties, and can be flexibly adapted to food, special medical and pharmaceutical application scenarios.

[0022] Preferably, in step (S1), the buffer solution is tris(hydroxymethyl)aminomethane-hydrochloric acid Tris-HCl buffer solution;

[0023] During the preparation process, the pH of the buffer solution is maintained in the range of 5.4 to 5.6, and the conductivity of the buffer solution is controlled to be ≤5 μS / cm.

[0024] Preferably, in step (S1), the preparation of the naringin mother liquor includes,

[0025] Weigh 1.0g of naringin with a purity of ≥98% as determined by HPLC, add it to 100mL of buffer solution, mix with magnetic stirring at 250-300rpm for 12-15 minutes, and place it in a water bath at 35-40℃ to dissolve and form a uniform and transparent solution, i.e., a naringin stock solution with a concentration of 10mg / mL.

[0026] Preferably, in step (S2), the metal salts used in the metal ion solution are analytical grade aluminum chloride hexahydrate AlCl3·6H2O, ferric chloride hexahydrate FeCl3·6H2O, and magnesium chloride hexahydrate MgCl2·6H2O.

[0027] The solvent for the metal ion solution is the buffer solution prepared in step (S1), and the molar concentration of the metal ions in the metal ion solution is 1.0 mg / mL.

[0028] Preferably, in step (S2), different pH control windows are set to prevent the hydrolysis behavior of different metal ions from interfering with the chelation reaction. The pH control windows are designed as follows.

[0029] For Fe 3+ The solution was adjusted with HCl to maintain the pH within a control window of 2.3–2.7 to prevent Fe from being absorbed. 3+ It readily hydrolyzes to form Fe(OH)3 precipitate at pH > 3.5;

[0030] For Al 3+ The solution, with its pH controlled within a window of 3.5–4.5, is used to maintain aluminum ions in the form of [Al(H₂O)₆]. 3+ It exists stably in its hydrated ionic form;

[0031] For Mg 2+ The solution is maintained at a pH within a control window of 5.5–6.5 to prevent the formation of Mg(OH)2 microprecipitates while ensuring its coordination activity.

[0032] The pH adjustment of the three metal ions was controlled by micro-titration, with each addition spaced 5 to 15 seconds apart and pH changes monitored to avoid instantaneous turbidity caused by drastic local pH changes.

[0033] Preferably, in step (S3), Al 3+ Fe 3+ Mg 2+ The chelation molar ratio of the three metal ions is:

[0034] Al 3+ The chelation molar ratio with naringin was 0.8–1.2:1; Fe 3+ The chelation molar ratio of naringin to naringin was 1.0–1.4:1; Mg2+ The chelation molar ratio of naringin to naringin is 0.8–1.2:1;

[0035] Al 3+ and Mg 2+ The chelation molar ratio is used to form mononuclear coordination structures and improve Fe 3+ The chelation molar ratio is used to induce the formation of the double-coordinated bridging Fe-OC=O-Fe structure.

[0036] Preferably, in step (S3), the pre-mixing process is as follows:

[0037] The 10 mg / mL naringin mother liquor obtained in step (S1) was used as the receiving liquid system, and the temperature was maintained at 23-25℃. The system was kept homogeneous by magnetic stirring at 300-350 rpm.

[0038] Using a peristaltic pump or a precision titration system, the metal ion solution prepared in step (S2) is added dropwise into the naringin mother liquor at a rate of 0.5–1.0 mL / min.

[0039] After the addition is complete, continue stirring for 5 to 10 minutes to ensure the system is fully mixed. Then, allow the system to stand for 8 to 10 minutes to achieve coordination equilibrium of the metal ions and obtain the primary complex premixed solution.

[0040] Preferably, in step (S4), the dual-driven synergistic chelation treatment of ultrasonic cavitation and thermal induction includes,

[0041] The primary complex premixed solution obtained in step (S3) is placed into a pressure-resistant reaction vessel, and an ultrasonic probe device is used, which is linked to the temperature control heating system for control.

[0042] At the beginning of the reaction, the system temperature is raised to 75-80℃ to allow the metal ions to form primary coordination with the carbonyl group of naringin.

[0043] Subsequently, the ultrasonic mode was activated. The ultrasound generated microbubbles in the liquid phase, causing instantaneous disintegration and creating a localized high-temperature environment (>2000K) and a high-pressure environment (>100MPa). This partially disrupted the COH hydrogen bonds within the naringin molecule, increasing the Al content. 3+ Fe 3 + Mg 2+ The accessible coordination sites, through thermal induction, enhance the vibrational energy levels of the C=O and COC bonds in the naringin molecule, causing the electron density to migrate toward the metal ion and form a stable metal-oxygen bond.

[0044] Immediately after the ultrasound is completed, turn off the temperature control heating system and the ultrasound probe device, and slowly cool it to room temperature in a constant temperature water bath at 23-25℃. Let it stand for 8-10 minutes to complete the secondary rearrangement of metal ion coordination and obtain a highly soluble complex.

[0045] Preferably, the ultrasound mode is set as follows.

[0046] The ultrasonic probe was inserted to a depth of 1 / 3 below the liquid surface, with a power setting of 200–400W, a pulse duty cycle of 50%, and the system temperature maintained within the range of 80–120℃ for 5–10 minutes.

[0047] Preferably, in step (S5), the differential fine-tuning includes,

[0048] For Al 3+ The highly soluble complex was subjected to pH fine-tuning to 4.2–4.8 at 98–102 °C for 2–3 min to inhibit Al(OH)3 nucleation;

[0049] For Fe 3+ The highly soluble complex was slowly adjusted to pH 3.2–3.8 at 90–95 °C and maintained for 3–5 min to inhibit the in-situ formation of Fe(OH)3 and promote stable bridging.

[0050] For Mg 2+ The highly soluble complex was adjusted to pH 5.8–6.2 at 85–90°C and maintained for 3–5 minutes to reduce magnesium salt precipitation.

[0051] After completing the differential fine-tuning, the solution is slowly cooled to room temperature in a water bath at 23-25℃ and allowed to stand for 8-10 minutes to obtain the stabilized chelate solution.

[0052] Preferably, in step (S6), the stabilized chelate obtained in step (S5) is centrifuged for 8 to 10 minutes to remove trace amounts of suspended particles or colloidal precipitates, and the supernatant is retained for later use.

[0053] Subsequently, a 3.5 kDa filtration membrane was used, and the external solution was replaced every hour during dialysis. Dialysis was performed for 4–5 hours to obtain three different naringin-metal ion chelates.

[0054] Preferably, in step (S6), the obtained naringin-metal ion chelate is subjected to a secondary treatment to obtain a lyophilized powder of the naringin-metal ion chelate.

[0055] After pre-freezing at -55℃ to -45℃ for 2 to 3 hours, the product is frozen at -80℃ to -70℃ for 10 to 12 hours, and then vacuum freeze-dried to obtain freeze-dried powder, which is then sealed in nitrogen.

[0056] Preferably, in step (S7), the criterion for determining reconstitution and release is: naringin-Al 3+ The solubility of the chelate is ≥170 mg / mL, and the naringin-Fe 3+ The solubility of the chelate is ≥160 mg / mL, and the naringin-Mg 2+ The solubility of the chelate is ≥130 mg / mL.

[0057] The present invention has the following beneficial effects:

[0058] (1) Using Al 3+ Fe 3+ Mg 2+ Food-grade metal ions form a stable coordination structure with naringin molecules, achieving a significant increase in naringin solubility and simultaneous enhancement of system stability without the involvement of any organic solvents or hazardous chemicals.

[0059] (2) Through ultrasound-thermal induction synergy and precise pH control, the bioavailability defects of naringin, such as poor solubility and unsatisfactory digestion stability, are not only effectively solved, but also make the preparation process simple, economical and green, with good industrial scale-up and cost advantages.

[0060] (3) This makes different metal ion chelation systems exhibit differentiated characteristics, such as naringin-Al 3+ Chelates exhibit excellent storage stability and are suitable for solid dosage forms and functional foods; naringin-Fe 3+ Chelates possess stronger antioxidant and free radical scavenging capabilities, making them suitable for anti-aging and health product development; naringin-Mg 2+ Chelates have good physiological compatibility and can be used in special medical nutrition and long-term intake products;

[0061] (4) It not only provides a green solubilization pathway for the efficient utilization of naringin, but also expands its application scenarios as a functional active ingredient in the special medical food, nutritional supplement and health food industries. Attached Figure Description

[0062] Figure 1 This is a flowchart of the method of the present invention;

[0063] Figure 2 This is a comparison image showing the significant improvement in the solubility of the naringin-metal ion chelate of the present invention before and after combined ultrasonic and heating treatment;

[0064] Figure 3 This is a particle size analysis diagram of different naringin-metal ion chelates of the present invention;

[0065] Figure 4 This is a Zeta potential analysis diagram of different naringin-metal ion chelates of the present invention;

[0066] Figure 5 This is a comparison chart of the hemolysis rates of different naringin-metal ion chelates of the present invention;

[0067] Figure 6 This image shows the morphological changes of erythrocytes under the action of different concentrations of naringin-metal ion chelates according to the present invention. Detailed Implementation

[0068] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0069] Reference Figure 1 A method for improving the bioavailability of naringin using metal ion chelation includes the following steps.

[0070] (S1) Prepare the buffer solution and prepare the naringin stock solution based on the prepared buffer solution;

[0071] The buffer solution used was tris(hydroxymethyl)aminomethane-hydrochloric acid Tris-HCl buffer.

[0072] During the preparation process, the pH of the buffer solution is maintained in the range of 5.4 to 5.6 to ensure that the hydroxyl and carbonyl groups of naringin in the system are in a dissociable state and do not undergo self-polymerization. The conductivity of the buffer solution is controlled at ≤5μS / cm to create a low conductivity environment.

[0073] The preparation of naringin mother liquor includes,

[0074] Weigh 1.0g of naringin with a purity ≥98% as determined by HPLC, add it to 100mL of buffer solution, mix with magnetic stirring at 250-300rpm for 12-15 minutes, and place it in a water bath at 35-40℃ to dissolve. If some parts are not completely dissolved, they can be sonicated briefly at 60℃ (100W power, 2min) to assist dissolution, ensuring the formation of a uniform and transparent solution, i.e., a naringin stock solution with a concentration of 10mg / mL.

[0075] (S2) Based on AI 3+ Fe 3+ Mg 2+ Three metal ions were prepared with different pH control windows to prepare the metal ion solutions required for chelation, ensuring that the metal ions were in the optimal matching state and avoiding hydrolysis or the formation of hydroxide precipitates;

[0076] The metal salts used in the metal ion solutions are analytical grade aluminum chloride hexahydrate AlCl3·6H2O, ferric chloride hexahydrate FeCl3·6H2O, and magnesium chloride hexahydrate MgCl2·6H2O.

[0077] The solvent for the metal ion solution is the buffer solution (i.e., Tris-HCl buffer) prepared in step (S1), and the molar concentration of the metal ions in the metal ion solution is 1.0 mg / mL.

[0078] Different pH control windows are set to prevent the hydrolysis behavior of different metal ions from interfering with the chelation reaction. The pH control window design is as follows.

[0079] For Fe 3+ The solution was adjusted with HCl to maintain the pH within a control window of 2.3–2.7 to prevent Fe from being absorbed. 3+ It readily hydrolyzes to form Fe(OH)3 precipitate at pH > 3.5;

[0080] For Al 3+ The solution, with its pH controlled within a window of 3.5–4.5, is used to maintain aluminum ions in the form of [Al(H₂O)₆]. 3+ It exists stably in its hydrated ionic form;

[0081] For Mg 2+ The solution is maintained at a pH within a control window of 5.5–6.5 to prevent the formation of Mg(OH)2 microprecipitates while ensuring its coordination activity.

[0082] Meanwhile, micro-titration control is used in the pH adjustment process of the three metal ions, with each addition interval of 5s to 15s and pH changes monitored to avoid instantaneous turbidity of the solution caused by local pH drastic changes.

[0083] Unlike traditional single-system blends or high-concentration inorganic salt systems, this method designs an independent pH control window to achieve precise pre-regulation of the initial state of the chelation reaction, enabling Al... 3+ Fe 3+ Mg 2+ It possesses the optimal electron cloud density distribution and stable coordination sites before entering the reaction system, thereby forming a stable coordination structure with the carbonyl and hydroxyl groups of naringin in the subsequent reaction. This avoids the risk of hydrolysis side reactions and precipitation in the reaction system, ensuring the controllability and repeatability of the chelation reaction.

[0084] (S3) Set Al 3+ Fe 3+ Mg 2+ The chelation molar ratio of the three metal ions was determined based on the pre-mixing of the metal ion solution and the naringin mother liquor to obtain a primary complex premix.

[0085] Different chelation molar ratios were designed to precisely control the rate of primary complexation and the distribution of coordination sites, avoiding problems such as local metal ion oversaturation, instantaneous precipitation, or non-uniform coordination structures in the system.

[0086] Among them, Al 3+ Fe 3+ Mg 2+ The chelation molar ratio of the three metal ions is:

[0087] Al 3+ The chelation molar ratio with naringin was 0.8–1.2:1; Fe 3+ The chelation molar ratio of naringin to naringin was 1.0–1.4:1; Mg 2+ The chelation molar ratio of naringin to naringin is 0.8–1.2:1;

[0088] The above ratio is based on Fe 3+ It has higher electron accepting ability and slightly improves Fe 3+ The chelation molar ratio is used to induce the formation of the double-coordinated bridging Fe-OC=O-Fe structure, while Al 3+ and Mg 2+ The chelation molar ratio is used to form mononuclear coordination structures;

[0089] The pre-mixing process is as follows:

[0090] The 10 mg / mL naringin mother liquor obtained in step (S1) was used as the receiving liquid system, and the temperature was maintained at 23-25℃. The system was kept homogeneous by magnetic stirring at 300-350 rpm.

[0091] Using a peristaltic pump or a precision titration system, the metal ion solution prepared in step (S2) was added dropwise into the naringin mother liquor at a rate of 0.5–1.0 mL / min. During the addition, the color of the system gradually changed from pale yellow to light brown (Fe... 3+ (system) or micro-milky white (Al) 3+ and Mg 2+ (System), indicating that primary complexes gradually form;

[0092] After the addition is complete, continue stirring for 5 to 10 minutes to ensure the system is fully mixed. Then, allow the system to stand for 8 to 10 minutes to achieve coordination equilibrium of the metal ions and obtain the primary complex premixed solution.

[0093] If the system becomes slightly turbid, it can be fine-tuned with trace amounts of HCl or NaOH to eliminate the colloidal instability of the intermediate state.

[0094] Compared with traditional blending reaction methods or direct dissolution methods, the method used in this step enables metal ions to form a reaction front region with gradual and balanced diffusion within the system, thereby effectively avoiding instantaneous supersaturation and hydrolysis aggregation problems;

[0095] (S4) The primary complex premixed liquid was subjected to dual-drive synergistic chelation treatment of ultrasonic cavitation and thermal induction to obtain a complex with high solubility.

[0096] Among them, the dual-driven synergistic chelation treatment of ultrasonic cavitation and thermal induction includes,

[0097] The primary complex premixed solution obtained in step (S3) is placed into a pressure-resistant reaction cup (capacity 250mL, PTFE liner), and an ultrasonic probe device is used, which is linked to the temperature control heating system for control.

[0098] At the beginning of the reaction, the system temperature is raised to 75-80℃ to allow the metal ions to form primary coordination with the carbonyl group of naringin.

[0099] Subsequently, the ultrasonic mode was activated. Ultrasonic cavitation bubbles continuously formed and collapsed in the liquid phase, creating a microbubble disintegration environment with localized high temperatures exceeding 2000 K and high pressures exceeding 100 MPa. This partially disrupted the COH hydrogen bonds within the naringin molecule, causing the originally coiled molecular structure to gradually unfold. The exposure of hydroxyl and carbonyl groups significantly increased, enhancing the Al content. 3+ Fe 3+ Mg 2+ The ligand sites are accessible; at the same time, thermal induction increases the vibrational energy level of the C=O and COC bonds in the naringin molecule, and the electron density migrates towards the metal ion, forming a stable metal-oxygen bond.

[0100] Immediately after the ultrasound is completed, turn off the temperature control heating system and the ultrasound probe device, and slowly cool it to room temperature in a constant temperature water bath at 23-25℃. Let it stand for 8-10 minutes to complete the secondary rearrangement of metal ion coordination and obtain a highly soluble complex.

[0101] Furthermore, the ultrasound mode is set as follows:

[0102] The ultrasonic probe is inserted to a depth of 1 / 3 below the liquid surface, with a power setting of 200–400W and a pulse duty cycle of 50%. To ensure the reaction occurs at a temperature above the boiling point of water, the reaction must be carried out in a sealed, pressure-resistant reaction vessel, utilizing the system's self-generated pressure to maintain the liquid phase. The system temperature is controlled within the range of 80–120°C for 5–10 minutes to obtain the optimal cavitation effect. For example, when the target temperature is 120°C, the system surface pressure is approximately 0.2 MPa.

[0103] This step enables simultaneous coupling of the molecular conformation of naringin with the electron cloud density of metal ions at both the temporal and energy levels, significantly improving the rate and stability of coordination bond formation. Unlike conventional heating or stirring reactions, this method achieves an innovative leap in increasing the solubility of naringin by more than 100 times under low concentration and organic auxiliary agent conditions. Specifically, the solubility of naringin in the system increases from 1 mg / mL to 150–170 mg / mL, a solubility increase of over 150 times.

[0104] (S5) for Al3+ Fe 3+ Mg 2+ Differential fine-tuning of highly soluble complexes of three metal ions was carried out to obtain a stable chelate solution with greater thermodynamic stability, narrower particle size distribution, and better resolution performance.

[0105] Among these, the differentiated fine-tuning includes,

[0106] For Al 3+ The highly soluble complex was finely adjusted to pH 4.2–4.8 at 98–102 °C using a dedicated pH electrode suitable for high temperatures and maintained for 2–3 min to inhibit Al(OH)3 nucleation.

[0107] For Fe 3+ The highly soluble complex was slowly adjusted to pH 3.2–3.8 at 90–95 °C and maintained for 3–5 min to inhibit the in-situ formation of Fe(OH)3 and promote stable bridging.

[0108] For Mg 2+ The highly soluble complex was adjusted to pH 5.8–6.2 at 85–90°C and maintained for 3–5 minutes to reduce magnesium salt precipitation.

[0109] After completing the differential fine-tuning, the solution is slowly cooled to room temperature in a water bath at 23-25℃ and allowed to stand for 8-10 minutes to obtain the stabilized chelate solution.

[0110] Based on pH-based differential fine-tuning, the directional structural optimization of chelates of different metal ions can be achieved, which can maintain the electroneutrality of the system and prevent precipitation, and rearrange the coordination structure from primary bonding to thermodynamically stable state, thereby improving long-term stability and resolubility.

[0111] (S6) The stabilized chelate solution is clarified, desalted and shaped to obtain naringin-metal ion chelate;

[0112] In step (S5), the stabilized chelate solution obtained is centrifuged for 8 to 10 minutes to remove trace amounts of suspended particles or colloidal precipitates, and the supernatant is retained for later use.

[0113] Subsequently, a 3.5 kDa filtration membrane was used to perform dynamic dialysis with the external solution replaced every hour during the dialysis process to maintain a constant ion concentration gradient. Dialysis was performed for 4–5 hours to obtain three different naringin-metal ion chelates.

[0114] Based on product requirements, the obtained naringin-metal ion chelate can be further processed to obtain a lyophilized powder of the naringin-metal ion chelate.

[0115] After pre-freezing at -55℃ to -45℃ for 2 to 3 hours, the product is frozen at -80℃ to -70℃ for 10 to 12 hours, followed by vacuum freeze-drying to obtain freeze-dried powder, which is then encapsulated in nitrogen. A staged heating and drying process is employed to control the ice crystal growth rate and prevent the chelate from disintegrating.

[0116] (S7) Design of reconstitution and release determination for the formed naringin-metal ion chelate;

[0117] The criteria for reconstitution and release are as follows: naringin-Al 3+ The solubility of the chelate is ≥170 mg / mL, and the naringin-Fe 3+ The solubility of the chelate is ≥160 mg / mL, and the naringin-Mg 2+ The solubility of the chelate is ≥130 mg / mL.

[0118] Example 1: A method for improving the bioavailability of naringin using metal ion chelation, comprising the following steps:

[0119] (S1) Prepare the buffer solution and prepare the naringin stock solution based on the prepared buffer solution;

[0120] The buffer solution used was tris(hydroxymethyl)aminomethane-hydrochloric acid Tris-HCl buffer.

[0121] During the preparation process, the pH of the buffer solution is maintained in the range of 5.4 to 5.6 to ensure that the hydroxyl and carbonyl groups of naringin in the system are in a dissociable state and do not undergo self-polymerization. The conductivity of the buffer solution is controlled at ≤5μS / cm to create a low conductivity environment.

[0122] The preparation of naringin mother liquor includes,

[0123] Weigh 1.0g of naringin with a purity ≥98% as determined by HPLC, add it to 100mL of buffer solution, mix with magnetic stirring at 250rpm for 15 minutes, and place it in a 35℃ water bath to dissolve. If some parts are not completely dissolved, they can be sonicated briefly at 60℃ (100W power, 2min) to assist dissolution, ensuring the formation of a uniform and transparent solution, i.e., a naringin stock solution with a concentration of 10mg / mL.

[0124] (S2) Based on AI 3+ Fe 3+ Mg 2+ Three metal ions were prepared with different pH control windows to prepare the metal ion solutions required for chelation, ensuring that the metal ions were in the optimal matching state and avoiding hydrolysis or the formation of hydroxide precipitates;

[0125] The metal salts used in the metal ion solutions are analytical grade aluminum chloride hexahydrate AlCl3·6H2O, ferric chloride hexahydrate FeCl3·6H2O, and magnesium chloride hexahydrate MgCl2·6H2O.

[0126] The solvent for the metal ion solution is the buffer solution (i.e., Tris-HCl buffer) prepared in step (S1), and the molar concentration of the metal ions in the metal ion solution is 1.0 mg / mL.

[0127] Different pH control windows are set to prevent the hydrolysis behavior of different metal ions from interfering with the chelation reaction. The pH control window design is as follows.

[0128] For Fe 3+ The solution was adjusted with HCl to maintain the pH within a control window of 2.3–2.7 to prevent Fe from being absorbed. 3+ It readily hydrolyzes to form Fe(OH)3 precipitate at pH > 3.5;

[0129] For Al 3+ The solution, with its pH controlled within a window of 3.5–4.5, is used to maintain aluminum ions in the form of [Al(H₂O)₆]. 3+ It exists stably in its hydrated ionic form;

[0130] For Mg 2+ The solution is maintained at a pH within a control window of 5.5–6.5 to prevent the formation of Mg(OH)2 microprecipitates while ensuring its coordination activity.

[0131] Meanwhile, the pH adjustment process for the three metal ions is controlled by micro-titration, with each addition spaced 5 seconds apart and pH changes monitored, in order to avoid instantaneous turbidity of the solution caused by drastic local pH changes.

[0132] Unlike traditional single-system blends or high-concentration inorganic salt systems, this method designs an independent pH control window to achieve precise pre-regulation of the initial state of the chelation reaction, enabling Al... 3+ Fe 3+ Mg 2+ It possesses the optimal electron cloud density distribution and stable coordination sites before entering the reaction system, thereby forming a stable coordination structure with the carbonyl and hydroxyl groups of naringin in the subsequent reaction. This avoids the risk of hydrolysis side reactions and precipitation in the reaction system, ensuring the controllability and repeatability of the chelation reaction.

[0133] (S3) Set Al 3+ Fe 3+ Mg 2+ The chelation molar ratio of the three metal ions was determined based on the pre-mixing of the metal ion solution and the naringin mother liquor to obtain a primary complex premix.

[0134] Different chelation molar ratios were designed to precisely control the rate of primary complexation and the distribution of coordination sites, avoiding problems such as local metal ion oversaturation, instantaneous precipitation, or non-uniform coordination structures in the system.

[0135] Among them, Al 3+ Fe 3+ Mg 2+ The chelation molar ratio of the three metal ions is:

[0136] Al 3+ The chelation molar ratio of naringin to naringin was 0.8:1; Fe 3+ The chelation molar ratio of naringin to naringin was 1.0:1; Mg 2+ The chelation molar ratio of naringin to naringin is 0.8:1;

[0137] The above ratio is based on Fe 3+ It has higher electron accepting ability and slightly improves Fe 3+ The chelation molar ratio is used to induce the formation of the double-coordinated bridging Fe-OC=O-Fe structure, while Al 3+ and Mg 2+ The chelation molar ratio is used to form mononuclear coordination structures;

[0138] The pre-mixing process is as follows:

[0139] The 10 mg / mL naringin mother liquor obtained in step (S1) was used as the receiving liquid system, and the temperature was maintained at 23°C. The system was kept homogeneous by magnetic stirring at 300 rpm.

[0140] Using a peristaltic pump or a precision titration system, the metal ion solution prepared in step (S2) was added dropwise to the naringin mother liquor at a rate of 0.5 mL / min. During the addition, the color of the system gradually changed from pale yellow to light brown (Fe... 3+ (system) or micro-milky white (Al) 3+ and Mg 2+ (System), indicating that primary complexes gradually form;

[0141] After the addition is complete, continue stirring for 5 minutes to ensure the system is fully mixed, and then allow it to stand for 8 minutes to achieve coordination equilibrium of metal ions within the system, thus obtaining the primary complex premixed solution.

[0142] If the system becomes slightly turbid, it can be fine-tuned with trace amounts of HCl or NaOH to eliminate the colloidal instability of the intermediate state.

[0143] Compared with traditional blending reaction methods or direct dissolution methods, the method used in this step enables metal ions to form a reaction front region with gradual and balanced diffusion within the system, thereby effectively avoiding instantaneous supersaturation and hydrolysis aggregation problems;

[0144] (S4) The primary complex premixed liquid was subjected to dual-drive synergistic chelation treatment of ultrasonic cavitation and thermal induction to obtain a complex with high solubility.

[0145] Among them, the dual-driven synergistic chelation treatment of ultrasonic cavitation and thermal induction includes,

[0146] The primary complex premixed solution obtained in step (S3) is placed into a pressure-resistant reaction cup (capacity 250mL, PTFE liner), and an ultrasonic probe device is used, which is linked to the temperature control heating system for control.

[0147] At the beginning of the reaction, the system temperature was raised to 75℃ to allow the metal ions to form a primary coordination relationship with the carbonyl group of naringin;

[0148] Subsequently, the ultrasonic mode was activated. Ultrasonic cavitation bubbles continuously formed and collapsed in the liquid phase, creating a microbubble disintegration environment with localized high temperatures exceeding 2000 K and high pressures exceeding 100 MPa. This partially disrupted the COH hydrogen bonds within the naringin molecule, causing the originally coiled molecular structure to gradually unfold. The exposure of hydroxyl and carbonyl groups significantly increased, enhancing the Al content. 3+ Fe 3+ Mg 2+ The ligand sites are accessible; at the same time, thermal induction increases the vibrational energy level of the C=O and COC bonds in the naringin molecule, and the electron density migrates towards the metal ion, forming a stable metal-oxygen bond.

[0149] Immediately after the ultrasound is completed, the temperature control heating system and the ultrasound probe device are turned off. The device is then slowly cooled to room temperature in a 23°C constant temperature water bath and left to stand for 8 minutes to complete the secondary rearrangement of metal ion coordination, thus obtaining a highly soluble complex.

[0150] Furthermore, the ultrasound mode is set as follows:

[0151] The ultrasonic probe was inserted to a depth of 1 / 3 below the liquid surface, the power was set to 200W, the pulse duty cycle to 50%, and the system temperature was maintained at 80℃ for 10 minutes to obtain the best cavitation effect.

[0152] This step enables simultaneous coupling of the molecular conformation of naringin with the electron cloud density of metal ions at both the temporal and energy levels, significantly improving the rate and stability of coordination bond formation. Unlike conventional heating or stirring reactions, this method achieves an innovative leap in increasing the solubility of naringin by more than 100 times under low concentration and organic auxiliary agent conditions. Specifically, the solubility of naringin in the system increases from 1 mg / mL to 150–170 mg / mL, a solubility increase of over 150 times.

[0153] (S5) for Al 3+ Fe3+ Mg 2+ Differential fine-tuning of highly soluble complexes of three metal ions was carried out to obtain a stable chelate solution with greater thermodynamic stability, narrower particle size distribution, and better resolution performance.

[0154] Among these, the differentiated fine-tuning includes,

[0155] For Al 3+ The highly soluble complex was subjected to pH fine-tuning to 4.2 at 98°C for 2 min to inhibit Al(OH)3 nucleation;

[0156] For Fe 3+ The highly soluble complex was slowly adjusted to pH 3.2 at 90°C and maintained for 3 min to inhibit the in-situ formation of Fe(OH)3 and promote stable bridging.

[0157] For Mg 2+ The highly soluble complex was adjusted to pH 5.8 at 85°C and maintained for 3 minutes to reduce magnesium salt precipitation.

[0158] After completing the differential fine-tuning, the solution was slowly cooled to room temperature in a 23°C water bath and allowed to stand for 8 minutes to obtain the stabilized chelate solution.

[0159] Based on pH-based differential fine-tuning, the directional structural optimization of chelates of different metal ions can be achieved, which can maintain the electroneutrality of the system and prevent precipitation, and rearrange the coordination structure from primary bonding to thermodynamically stable state, thereby improving long-term stability and resolubility.

[0160] (S6) The stabilized chelate solution is clarified, desalted and shaped to obtain naringin-metal ion chelate;

[0161] In this step (S5), the stabilized chelate solution obtained is centrifuged for 8 minutes to remove trace amounts of suspended particles or colloidal precipitates, and the supernatant is retained for later use.

[0162] Subsequently, a 3.5 kDa filtration membrane was used to perform dynamic dialysis with the external solution replaced every hour during the dialysis process to maintain a constant ion concentration gradient. After dialysis for 4 hours, three different naringin-metal ion chelates were obtained.

[0163] Based on product requirements, the obtained naringin-metal ion chelate can be further processed to obtain a lyophilized powder of the naringin-metal ion chelate.

[0164] After pre-freezing at -55℃ for 2 hours, the mixture was frozen at -80℃ for 10 hours, followed by vacuum freeze-drying to obtain a lyophilized powder, which was then encapsulated in nitrogen. A staged heating and drying process was employed to control the ice crystal growth rate and prevent the chelate from disintegrating.

[0165] (S7) Design of reconstitution and release determination for the formed naringin-metal ion chelate;

[0166] The criteria for reconstitution and release are as follows: naringin-Al 3+ The solubility of the chelate is ≥170 mg / mL, and the naringin-Fe 3+ The solubility of the chelate is ≥160 mg / mL, and the naringin-Mg 2+ The solubility of the chelate is ≥130 mg / mL.

[0167] Example 2: This example is basically the same as Example 1, except that,

[0168] The preparation of naringin stock solution includes,

[0169] Weigh 1.0g of naringin with a purity ≥98% as determined by HPLC, add it to 100mL of buffer solution, mix with magnetic stirring at 300rpm for 12 minutes, and place it in a 40℃ water bath to dissolve. If there is local incomplete dissolution, it can be sonicated briefly at 60℃ (100W power, 2min) to assist dissolution, ensuring the formation of a uniform and transparent solution, i.e., a naringin stock solution with a concentration of 10mg / mL.

[0170] Meanwhile, the pH adjustment process for the three metal ions is controlled by micro-titration, with each addition spaced 15 seconds apart and pH changes monitored, in order to avoid instantaneous turbidity of the solution caused by drastic local pH changes.

[0171] Among them, Al 3+ Fe 3+ Mg 2+ The chelation molar ratio of the three metal ions is:

[0172] Al 3+ The chelation molar ratio of naringin to naringin was 1.2:1; Fe 3+ The chelation molar ratio of naringin to naringin was 1.4:1; Mg 2+ The chelation molar ratio of naringin to naringin is 1.2:1;

[0173] The 10 mg / mL naringin mother liquor obtained in step (S1) was used as the receiving liquid system, and the temperature was maintained at 25°C. The system was kept homogeneous by magnetic stirring at 350 rpm.

[0174] Using a peristaltic pump or a precision titration system, the metal ion solution prepared in step (S2) was added dropwise to the naringin mother liquor at a rate of 1.0 mL / min. During the addition, the color of the system gradually changed from pale yellow to light brown (Fe... 3+ (system) or micro-milky white (Al) 3+ and Mg 2+ (System), indicating that primary complexes gradually form;

[0175] After the addition is complete, continue stirring for 10 minutes to ensure the system is fully mixed. Then, allow the system to stand for 10 minutes to achieve coordination equilibrium of metal ions, thus obtaining the primary complex premixed solution.

[0176] At the beginning of the reaction, the system temperature was raised to 80℃ to allow the metal ions to form a primary coordination relationship with the carbonyl group of naringin;

[0177] Immediately after the ultrasound is completed, the temperature control heating system and the ultrasound probe device are turned off. The device is then slowly cooled to room temperature in a 25°C constant temperature water bath and left to stand for 10 minutes to complete the secondary rearrangement of metal ion coordination, thus obtaining a highly soluble complex.

[0178] Furthermore, the ultrasound mode is set as follows:

[0179] The ultrasonic probe was inserted to a depth of 1 / 3 below the liquid surface, with a power of 400W and a pulse duty cycle of 50%. In a sealed pressure-resistant reactor, the system temperature was maintained at 120℃ for 5 minutes, corresponding to a pressure of approximately 0.2MPa, to obtain the best cavitation effect.

[0180] For Al 3+ The highly soluble complex was subjected to pH fine-tuning at 102 °C to 4.8 for 3 min to inhibit Al(OH)3 nucleation;

[0181] For Fe 3+ The highly soluble complex was slowly adjusted to pH 3.8 at 95°C and maintained for 5 min to inhibit the in-situ formation of Fe(OH)3 and promote stable bridging.

[0182] For Mg 2+ The highly soluble complex was adjusted to pH 6.2 at 90°C and maintained for 5 minutes to reduce magnesium salt precipitation.

[0183] After completing the differential fine-tuning, the solution was slowly cooled to room temperature in a 25°C water bath and allowed to stand for 8 minutes to obtain the stabilized chelate solution.

[0184] In this step (S5), the stabilized chelate solution obtained is centrifuged for 10 minutes to remove trace amounts of suspended particles or colloidal precipitates, and the supernatant is retained for later use.

[0185] Subsequently, a 3.5 kDa filtration membrane was used to perform dynamic dialysis with the external solution replaced every hour during the dialysis process to maintain a constant ion concentration gradient. After dialysis for 5 hours, three different naringin-metal ion chelates were obtained.

[0186] Based on product requirements, the obtained naringin-metal ion chelate can be further processed to obtain a lyophilized powder of the naringin-metal ion chelate.

[0187] After pre-freezing at -45℃ for 3 hours, the mixture was frozen at -70℃ for 12 hours, followed by vacuum freeze-drying to obtain a lyophilized powder, which was then encapsulated in nitrogen. A staged heating and drying process was employed to control the ice crystal growth rate and prevent the chelate from disintegrating.

[0188] Example 3: This example is basically the same as Example 1, except that,

[0189] The preparation of naringin mother liquor includes,

[0190] Weigh 1.0g of naringin with a purity ≥98% as determined by HPLC, add it to 100mL of buffer solution, mix with magnetic stirring at 280rpm for 13 minutes, and place it in a 38℃ water bath to dissolve. If there is local incomplete dissolution, it can be sonicated briefly at 60℃ (100W power, 2min) to assist dissolution, ensuring the formation of a uniform and transparent solution, i.e., a naringin stock solution with a concentration of 10mg / mL.

[0191] Meanwhile, the pH adjustment process for the three metal ions is controlled by micro-titration, with each addition spaced 10 seconds apart and pH changes monitored, in order to avoid instantaneous turbidity of the solution caused by drastic local pH changes.

[0192] Among them, Al 3+ Fe 3+ Mg 2+ The chelation molar ratio of the three metal ions is:

[0193] Al 3+ The chelation molar ratio of naringin to naringin is 1:1; Fe 3+ The chelation molar ratio of naringin to naringin was 1.2:1; Mg 2+ The chelation molar ratio with naringin is 1:1;

[0194] The 10 mg / mL naringin mother liquor obtained in step (S1) was used as the receiving liquid system, and the temperature was maintained at 24℃. The system was kept homogeneous by magnetic stirring at 320 rpm.

[0195] Using a peristaltic pump or a precision titration system, the metal ion solution prepared in step (S2) was added dropwise to the naringin mother liquor at a rate of 0.8 mL / min. During the addition, the color of the system gradually changed from pale yellow to light brown (Fe... 3+ (system) or micro-milky white (Al) 3+ and Mg 2+ (System), indicating that primary complexes gradually form;

[0196] After the addition is complete, continue stirring for 8 minutes to ensure the system is fully mixed, and then allow it to stand for 9 minutes to achieve coordination equilibrium of metal ions within the system, thus obtaining the primary complex premixed solution.

[0197] At the beginning of the reaction, the system temperature was raised to 77℃ to allow the metal ions to form a primary coordination relationship with the carbonyl group of naringin.

[0198] 7. Immediately after the ultrasound is completed, turn off the temperature control heating system and the ultrasound probe device, and slowly cool it to room temperature in a 24℃ constant temperature water bath. Let it stand for 9 minutes to complete the secondary rearrangement of metal ion coordination and obtain a highly soluble complex.

[0199] The ultrasonic probe was inserted to a depth of 1 / 3 below the liquid surface, with a power of 300W, a pulse duty cycle of 50%, and the system temperature maintained at 100℃ for 7 minutes to obtain the best cavitation effect.

[0200] Among these, the differentiated fine-tuning includes,

[0201] For Al 3+ The highly soluble complex was subjected to pH fine-tuning to 4.5 at 100°C for 2.5 min to inhibit Al(OH)3 nucleation;

[0202] For Fe 3+ The highly soluble complex was slowly adjusted to pH 3.5 at 93°C and maintained for 4 min to inhibit the in-situ formation of Fe(OH)3 and promote stable bridging.

[0203] For Mg 2+ The highly soluble complex was adjusted to pH 6 at 88°C and maintained for 4 min to reduce magnesium salt precipitation.

[0204] After completing the differential fine-tuning, the solution was slowly cooled to room temperature in a 24℃ water bath and allowed to stand for 9 minutes to obtain the stabilized chelate solution.

[0205] In this step (S5), the stabilized chelate solution obtained is centrifuged for 9 minutes to remove trace amounts of suspended particles or colloidal precipitates, and the supernatant is retained for later use.

[0206] Subsequently, a 3.5 kDa filtration membrane was used to perform dynamic dialysis with the external solution replaced every hour during the dialysis process to maintain a constant ion concentration gradient. Dialysis was performed for 4.5 hours to obtain three different naringin-metal ion chelates.

[0207] After pre-freezing at -50℃ for 2.5 hours, the mixture was frozen at -75℃ for 11 hours, followed by vacuum freeze-drying to obtain a lyophilized powder, which was then encapsulated in nitrogen. A staged heating and drying process was employed to control the ice crystal growth rate and prevent the chelate from disintegrating.

[0208] Example 4: Based on the method described in Example 3, the obtained naringin-metal ions (Al) 3+ Fe 3+ Mg 2+ The solubility of the chelate was determined, and the results are shown in Table 1.

[0209] Table 1: Significant solubilization of naringin achieved by combined treatment with 1 mg / mL metal ions and ultrasonic heating

[0210] substance Naringin <![CDATA[Naringin + Al 3+ > <![CDATA[Naringin + Fe 3+ > <![CDATA[Naringin + Mg 2+ > Solubility (mg / mL) 1.0 172.4 164.1 130.9

[0211] The results in Table 1 show that the room temperature solubility of naringin significantly increased from 1 mg / mL to over 130 mg / mL; among them, naringin-Al 3+ The chelate has a solubility of 172.4 mg / mL, naringin-Fe 3+ The chelate concentration was 164.1 mg / mL, containing naringin-Mg. 2 + The chelate concentration was 130.9 mg / mL. This result indicates that metal ion chelation achieved a significant increase in naringin solubility of more than 130 times without the participation of any organic solvent. The resulting solution showed no precipitation or turbidity and exhibited good broad-spectrum pH stability, making it suitable for various food and oral formulation systems.

[0212] At the same time, such as Figure 2 As shown, this method was not used ( Figure 2 (Left) and after using this method ( Figure 2 The comparison on the right clearly shows a significant increase in solubility to the naked eye.

[0213] Example 5: Based on the method described in Example 3, the obtained naringin-metal ions (Al) 3+ Fe 3+ Mg 2+ The chelates were subjected to particle size and potential testing, such as... Figure 3 and Figure 4 As shown, the results are as follows:

[0214] Naringin-Al 3+ The chelate has a zeta potential of +11.73 to +12.56 mV and a particle size range of 1558 to 1645 nm.

[0215] Naringin-Fe 3+ The chelate has a zeta potential of +4.768 to +5.598 mV and a particle size range of 431.8 to 551.1 nm.

[0216] Naringin-Mg 2+The chelate has a zeta potential of −11.82 to −12.97 mV and a particle size range of 497.6 to 525.6 nm.

[0217] Therefore, Fe 3+ The ion chelation system forms the most compact coordination structure, with small particle size and good dispersibility, exhibiting the strongest solubilizing and stabilizing effects; Mg 2+ Although the chelate system has a slightly larger particle size, its high negative potential ensures good electrostatic stability; while Al 3+ The system forms a large aggregate structure with a high positive potential, exhibiting high thermal stability and storage performance.

[0218] This indicates that Fe 3+ It exhibits a stronger chelating effect on naringin, forming larger aggregates while maintaining a high absolute value of the zeta potential, thus demonstrating a significant solubilizing effect. Naringin-Al... 3+ Chelates and naringin-Mg 2+ The smaller particle size of the chelate indicates that the chelation effect is not as strong as that of Al. 3+ Naringin-Mg 2+ The high absolute value of the zeta potential of the chelate suggests that it yields ideal results when chelating smaller amounts of naringin. Naringin-Fe... 3+ Chelates have the strongest biological activity.

[0219] Example 6: Based on the method described in Example 3, the obtained naringin-metal ions (Al) 3+ Fe 3+ Mg 2+ The antioxidant properties of the chelates were tested, and the results are shown in Table 2.

[0220] Table 2: Antioxidant activity of naringin metal ion chelates

[0221] Naringin concentration (1 mg / mL) Naringin <![CDATA[Naringin + Al 3+ > <![CDATA[Naringin + Fe 3+ > <![CDATA[Naringin + Mg 2+ > <![CDATA[Positive control V C (1 mg / mL) <!-- 12 -->]]> ABTS free radical scavenging rate 22.6% 39.1% 55.8% 33.5% 87.9% DPPH free radical scavenging rate 48.7% 61.2% 79.6% 71.3% 94.5%

[0222] The results in Table 2 show that, under the condition of naringin concentration of 1 mg / mL, the ABTS free radical scavenging rate increased from 22.6% to 39.1% (Al). 3+ ), 55.8% (Fe 3+ ) and 33.5% (Mg 2+ ); DPPH radical scavenging rate increased from 48.7% to 61.2% (Al). 3+ ), 79.6% (Fe 3+ ) and 71.3% (Mg 2+Compared to the positive control ascorbic acid (ABTS 87.9%, DPPH 94.5%), the chelation system exhibited a significant enhancement in antioxidant activity. This result indicates that metal ion chelation enhances the electron donor capacity of naringin for free radicals by reconstructing its electron cloud distribution. Specifically, Fe... 3+ The chelation system exhibits optimal free radical capture performance.

[0223] Example 7: Based on the method described in Example 3, the obtained naringin-metal ions (Al) 3+ Fe 3+ Mg 2+ The blood compatibility of naringin-metal chelates in the range of 100–1600 μg / mL was investigated by hemolysis experiments.

[0224] The instruments and equipment used in the hemolysis test are:

[0225] 3.2% sodium citrate vacuum blood collection tubes (Shandong Weigao); multi-functional microplate reader (Varioskan Flash, Thermo Scientific, USA); inverted fluorescence microscope (IX71, Olympus, Japan); constant temperature incubator (ZRD-A5210, Shanghai Zhicheng); rotary mixer (Thermo Scientific, USA); low-speed centrifuge (TD4B, Hunan Pingfan); high-speed centrifuge (5418, Eppendorf, USA); cell counting chamber (Countstar); one bag of physiological saline (0.9% NaCl); deionized water (laboratory-made); several 1.5mL centrifuge tubes.

[0226] The hemolysis test used four types of samples: #1 was naringin, #2 was naringin-Al 3+ Chelate, #3 is naringin-Fe 3+ Chelate #4 is naringin-Mg 2+ Chelates; and negative and positive controls were set up.

[0227] Hemolysis test procedure:

[0228] One healthy volunteer (male, age 38) was randomly recruited, and 2 mL of peripheral blood was collected via vacuum venous collection.

[0229] Centrifuge anticoagulated whole blood at 1000 rpm for 10 min, take 0.2 ml of the lower red blood cell pellet and add it to a 1.5 mL centrifuge tube. Add 1.0 ml of PBS, gently invert and mix, centrifuge at 1000 rpm for 10 min, carefully aspirate the supernatant from the centrifuge tube, and then add 0.25 mL of PBS to the centrifuge tube to dilute the red blood cells and prepare a red blood cell suspension for later use.

[0230] Four types of samples were prepared with physiological saline at concentrations of 100, 200, 400, 800, and 1600 μg / mL. 1 mL of each concentration solution was transferred to a 1.5 mL centrifuge tube and incubated at 37°C for 30 min to equilibrate. Then, 20 μL of red blood cell suspension was added, and the mixture was incubated at 37°C for 1 h. 20 μL of red blood cell suspension was added to 1 mL of physiological saline as a negative control, and 20 μL of red blood cell suspension was added to 1 mL of deionized water as a positive control; both were incubated at 37°C for 1 h. Both experiments and controls were performed in triplicate. After incubation, the samples were centrifuged at 2000 g for 10 min, and 0.2 mL of the supernatant was transferred to a 96-well plate. The absorbance at 545 nm was measured using a microplate reader.

[0231] Formula for calculating the relative hemolysis rate of red blood cells in a sample:

[0232]

[0233] In the formula, The absorbance value of the supernatant of the experimental samples is shown below (Note: Since samples #2 and #3 are colored, they will affect the absorbance of hemoglobin, so the absorbance value of the samples themselves needs to be deducted). The absorbance value of the supernatant in the negative control group; The absorbance value is that of the supernatant of the positive control group.

[0234] A red blood cell hemolysis rate of ≤5% indicates that the material meets the hemolysis rate requirements for medical materials and will not cause a serious hemolytic reaction; a red blood cell hemolysis rate of >5% indicates that the material will undergo a hemolytic reaction.

[0235] Experimental results show that:

[0236] Under the experimental conditions, samples #1, #3, and #4 showed a relative erythrocyte hemolysis rate of <5%, indicating good erythrocyte compatibility. Conversely, sample #2 showed a relative erythrocyte hemolysis rate >5%, indicating strong erythrocyte hemolytic activity. Figure 5 As shown.

[0237] Red blood cell morphology experimental procedure:

[0238] Following the same experimental steps as above, incubate the sample and red blood cells at 37°C with rotation for 1 hour. After incubation, add 20 μL of red blood cell solution from the sample group, negative control group, and positive control group to the cell counting chamber. Let it stand for 5 minutes. The red blood cells will be evenly dispersed in the chamber and settle to the bottom of the chamber for easy observation.

[0239] The cell counting chamber was placed on the microscope stage, and photographs and observations were taken using an inverted microscope at 60x magnification. Five typical images were randomly taken for each sample, as follows: Figure 6As shown. A ruler was photographed at the same magnification.

[0240] Image results description:

[0241] The results of erythrocyte microscopic morphology observation showed that the negative control erythrocytes had a typical biconcave disc-shaped structure, while the structure and morphology of the positive sample erythrocytes were completely destroyed, with only some shadow cells visible.

[0242] After treatment of sample #1, the proportion of acanthocytic erythrocytes increased with increasing sample concentration; in sample #2, erythrocytes showed significant aggregation at concentrations of 100 and 200 ug / ml; with further increases in concentration, the proportion of atypical erythrocytes gradually increased; at a concentration of 1600 ug / ml, erythrocytes were almost completely destroyed, with only shadow cells visible; after treatment of samples #3 and #4, the proportion of acanthocytic erythrocytes increased with sample concentration.

[0243] In summary, the metal ion chelation method employed in this study not only effectively improved the solubility and dispersibility of naringin but also significantly enhanced its free radical scavenging ability and biostability. Different metal systems exhibited varying performance characteristics: Al... 3+ The system exhibits strong stability and is suitable for solid dosage forms; Fe 3+ The system exhibits the strongest antioxidant properties and is suitable for the development of functional foods and health products; Mg 2 + The system exhibits good physiological compatibility and is suitable for special medical foods and long-term intake products. This fully verifies the scientific validity and practicality of this invention in improving the bioavailability and safe solubilization of naringin.

[0244] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for enhancing the bioavailability of naringin by utilizing metal ion chelation, characterized by: Including the following step, (S1) Prepare the buffer solution and prepare the naringin stock solution based on the prepared buffer solution; (S2) Based on AI 3+ Fe 3+ Mg 2+ Three metal ions were prepared with different pH control windows to prepare the metal ion solutions required for chelation; (S3) Set Al 3+ Fe 3+ Mg 2+ The chelation molar ratio of the three metal ions was determined based on the pre-mixing of the metal ion solution and the naringin mother liquor to obtain a primary complex premix. (S4) The primary complex premixed liquid was subjected to dual-drive synergistic chelation treatment of ultrasonic cavitation and thermal induction to obtain a complex with high solubility. (S5) for Al 3+ Fe 3+ Mg 2+ Differential fine-tuning of the highly soluble complexes of three metal ions yields a stable chelate solution; (S6) The stabilized chelate solution is clarified, desalted and shaped to obtain naringin-metal ion chelate; (S7) Design of the reconstitution and release determination of the formed naringin-metal ion chelate.

2. The method for improving the bioavailability of naringin by utilizing the chelation effect of metal ions according to claim 1, characterized in that: In step (S2), the metal salts used in the metal ion solution are analytical grade aluminum chloride hexahydrate AlCl3·6H2O, ferric chloride hexahydrate FeCl3·6H2O, and magnesium chloride hexahydrate MgCl2·6H2O. The solvent for the metal ion solution is the buffer solution prepared in step (S1), and the molar concentration of the metal ions in the metal ion solution is 1.0 mg / mL.

3. The method for improving the bioavailability of naringin by utilizing the chelation effect of metal ions according to claim 1, characterized in that: In step (S2), different pH control windows are set to prevent the hydrolysis behavior of different metal ions from interfering with the chelation reaction. The pH control windows are designed as follows. For Fe 3+ The solution was adjusted with HCl to maintain the pH within a control window of 2.3–2.7 to prevent Fe from being absorbed. 3+ It readily hydrolyzes to form Fe(OH)3 precipitate at pH > 3.5; For Al 3+ The solution, with its pH controlled within a window of 3.5–4.5, is used to maintain aluminum ions in the form of [Al(H₂O)₆]. 3+ It exists stably in its hydrated ionic form; For Mg 2+ The solution is maintained at a pH within a control window of 5.5–6.5 to prevent the formation of Mg(OH)2 microprecipitates while ensuring its coordination activity. The pH adjustment of the three metal ions was controlled by micro-titration, with each addition spaced 5 to 15 seconds apart and pH changes monitored to avoid instantaneous turbidity caused by drastic local pH changes.

4. The method for improving the bioavailability of naringin by utilizing the chelation effect of metal ions according to claim 1, characterized in that: In step (S3), Al 3+ Fe 3+ Mg 2+ The chelation molar ratio of the three metal ions is: Al 3+ The chelation molar ratio with naringin was 0.8–1.2:1; Fe 3+ The chelation molar ratio of naringin to naringin was 1.0–1.4:1; Mg 2+ The chelation molar ratio of naringin to naringin is 0.8–1.2:1; Al 3+ and Mg 2+ The chelation molar ratio is used to form mononuclear coordination structures and improve Fe 3+ The chelation molar ratio is used to induce the formation of the double-coordinated bridging Fe-OC=O-Fe structure.

5. The method for improving the bioavailability of naringin by utilizing the chelation effect of metal ions according to claim 1, characterized in that: In step (S3), the pre-mixing process is as follows: The 10 mg / mL naringin mother liquor obtained in step (S1) was used as the receiving liquid system, and the temperature was maintained at 23-25℃. The system was kept homogeneous by magnetic stirring at 300-350 rpm. Using a peristaltic pump or a precision titration system, the metal ion solution prepared in step (S2) is added dropwise into the naringin mother liquor at a rate of 0.5–1.0 mL / min. After the addition is complete, continue stirring for 5 to 10 minutes to ensure the system is fully mixed. Then, allow the system to stand for 8 to 10 minutes to achieve coordination equilibrium of the metal ions and obtain the primary complex premixed solution.

6. The method for improving the bioavailability of naringin by utilizing the chelation effect of metal ions according to claim 1, characterized in that: In step (S4), the dual-driven synergistic chelation treatment of ultrasonic cavitation and thermal induction includes, The primary complex premixed solution obtained in step (S3) is placed into a pressure-resistant reaction vessel, and an ultrasonic probe device is used, which is linked to the temperature control heating system for control. At the beginning of the reaction, the system temperature is raised to 75-80℃ to allow the metal ions to form primary coordination with the carbonyl group of naringin. Subsequently, the ultrasonic mode was activated. The ultrasound generated microbubbles in the liquid phase, causing instantaneous disintegration and creating a localized high-temperature environment (>2000K) and a high-pressure environment (>100MPa). This partially disrupted the COH hydrogen bonds within the naringin molecule, increasing the Al content. 3+ Fe 3+ Mg 2+ The accessible coordination sites, through thermal induction, enhance the vibrational energy levels of the C=O and COC bonds in the naringin molecule, causing the electron density to migrate toward the metal ion and form a stable metal-oxygen bond. Immediately after the ultrasound is completed, turn off the temperature control heating system and the ultrasound probe device, and slowly cool it to room temperature in a constant temperature water bath at 23-25℃. Let it stand for 8-10 minutes to complete the secondary rearrangement of metal ion coordination and obtain a highly soluble complex.

7. The method for improving the bioavailability of naringin by utilizing the chelation effect of metal ions according to claim 6, characterized in that: The ultrasound mode settings are as follows. The ultrasonic probe is inserted below the liquid surface, the power is set to 200-400W, the pulse duty cycle is 50%, and the system temperature is maintained in the range of 80-120℃ for 5-10 minutes.

8. The method for improving the bioavailability of naringin by utilizing the chelation effect of metal ions according to claim 1, characterized in that: In step (S5), the differential fine-tuning includes, For Al 3+ The highly soluble complex was finely adjusted to pH 4.2–4.8 at 98–102 °C using a dedicated pH electrode suitable for high temperatures and maintained for 2–3 min to inhibit Al(OH)3 nucleation. For Fe 3+ The highly soluble complex was slowly adjusted to pH 3.2–3.8 at 90–95 °C and maintained for 3–5 min to inhibit the in-situ formation of Fe(OH)3 and promote stable bridging. For Mg 2+ The highly soluble complex was adjusted to pH 5.8–6.2 at 85–90°C and maintained for 3–5 minutes to reduce magnesium salt precipitation. After completing the differential fine-tuning, the solution is slowly cooled to room temperature in a water bath at 23-25℃ and allowed to stand for 8-10 minutes to obtain the stabilized chelate solution.

9. The method for improving the bioavailability of naringin by utilizing the chelation effect of metal ions according to claim 1, characterized in that: In step (S6), the stabilized chelate obtained in step (S5) is centrifuged for 8 to 10 minutes to remove trace amounts of suspended particles or colloidal precipitates, and the supernatant is retained for later use. Subsequently, a 3.5 kDa filtration membrane was used, and the external solution was replaced every hour during dialysis. Dialysis was performed for 4–5 hours to obtain three different naringin-metal ion chelates.

10. The method for improving the bioavailability of naringin by utilizing the chelation effect of metal ions according to claim 9, characterized in that: In step (S6), the obtained naringin-metal ion chelate is subjected to a second processing to obtain a lyophilized powder of the naringin-metal ion chelate. After pre-freezing at -55℃ to -45℃ for 2 to 3 hours, the product is frozen at -80℃ to -70℃ for 10 to 12 hours, and then vacuum freeze-dried to obtain freeze-dried powder, which is then sealed in nitrogen.

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