A composition of alginate oligosaccharide-nano-selenium-chlorogenic acid, its preparation method and application

Abstract

This invention discloses an alginate oligosaccharide-nanoselenium-chlorogenic acid composition, its preparation method, and its applications, belonging to the fields of functional nanomaterials, biomedicine, and food science. The composition comprises alginate oligosaccharide-modified nanoselenium (AOS-SeNPs) and chlorogenic acid (CGA) in a 1:1 mass ratio. AOS-SeNPs are prepared via an in-situ reduction-modification coupling method and then compounded with CGA in a precise ratio. This composition exhibits a significant synergistic antioxidant effect in ABTS⁺ free radical scavenging and achieves a functional leap from bacteriostasis to bactericidal activity in antibacterial applications, reducing the viable count of Staphylococcus aureus from approximately 10⁵ CFU / mL after treatment with AOS-SeNPs alone to below the detection limit (<10 CFU / mL), overcoming the plateau limitation of single-component bactericidal ability. Furthermore, the composition is structurally stable, has a simple and controllable preparation process, and exhibits good adaptability to hydrogel formulations, making it applicable to antioxidant functional foods, medical dressings, and antibacterial coatings.

Description

Technical Field

[0001] This invention belongs to the fields of functional nanomaterials, biomedicine and food science and technology, specifically relating to a composition based on alginate oligosaccharide-modified nano-selenium and chlorogenic acid, and particularly to a nanocomposite with synergistic antioxidant and antibacterial functional crossover, its preparation method and application. Background Technology

[0002] Selenium nanoparticles (SeNPs) have become a research hotspot in the functional food and biopharmaceutical fields due to their low toxicity, high bioavailability, and excellent antioxidant, antibacterial, and immunomodulatory activities. Compared to inorganic selenium (selenite, selenate) and organic selenium compounds, selenium nanoparticles exhibit lower acute toxicity and higher safety. However, selenium nanoparticle colloidal dispersions are prone to aggregation and precipitation during storage and use, and their long-term stability issues limit their practical application, necessitating the use of stabilizers or dispersants for protection.

[0003] Alginate oligosaccharide (AOS) is a degradation product of the natural marine polysaccharide alginic acid, composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G) linked by 1,4-glycosidic bonds, with a weight-average molecular weight typically below 10,000 Da. Compared to high molecular weight sodium alginate, AOS exhibits superior water solubility and lower solution viscosity, and its densely distributed carboxyl and hydroxyl groups on its molecular chain endow it with excellent coordination ability for metal ions and nanoparticles. Existing studies have reported that AOS can be used as a stabilizer for nano-selenium, but the functions of the constructed AOS-SeNPs system are mainly limited to the activity of a single component, and there are no reports of synergistic effects achieved by combining AOS-modified nano-selenium with chlorogenic acid.

[0004] Chlorogenic acid (CGA), chemically known as 5-O-caffeoylquinic acid, is a phenolic acid compound widely found in natural plants such as Eucommia ulmoides, honeysuckle, and coffee. It possesses various pharmacological activities, including antioxidant, antibacterial, anti-inflammatory, and hepatoprotective effects. However, chlorogenic acid exhibits poor stability in solution and is easily oxidized and degraded by factors such as pH, temperature, and light. Furthermore, its antibacterial activity is primarily bacteriostatic rather than bactericidal, making it difficult to effectively reduce viable bacterial counts below the detection limit when used alone.

[0005] Chinese invention patent CN118557546A (publication date: August 30, 2024) discloses a method for preparing human serum albumin-selenium nanoparticles loaded with chlorogenic acid. The method described in this patent involves the composite of chlorogenic acid, human serum albumin, and selenium nanoparticles, and its process involves animal-derived protein components, but does not involve the synergistic effect of alginate oligosaccharides. Furthermore, Yang et al. (Yang L, Wang N, Zheng G. Enhanced effect of combining chlorogenicacid on selenium nanoparticles in inhibiting amyloid β aggregation and reactive oxygen species formation in vitro. Nanoscale Res Lett, 2018, 13:303.) reported the preparation of chlorogenic acid-modified selenium nanoparticles (CGA@SeNPs). This system uses sodium borohydride to reduce sodium selenite to construct an alkaline synthesis environment, and then adds chlorogenic acid to the reaction system to complete the surface modification of the nanoparticles; this preparation system does not use oligosaccharides such as alginate oligosaccharides as stabilizers. This literature mainly focuses on inhibiting Aβ amyloid protein aggregation and neuroprotection, without addressing the evaluation of antibacterial properties.

[0006] While the preparation of alginate oligosaccharide-stabilized selenium nanoparticles and their single-component activities have been reported in existing technologies, there are no reports of synergistic antioxidant and bactericidal compositions formed by combining alginate oligosaccharide-modified selenium nanoparticles with chlorogenic acid. In particular, there is currently no technological inspiration to combine AOS-SeNPs and CGA in a specific ratio to achieve a significant order-of-magnitude improvement in bactericidal effect. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides an alginate oligosaccharide-nanoselenium-chlorogenic acid composition, its preparation method, and its applications. This composition uses alginate oligosaccharide-modified nanoselenium (AOS-SeNPs) and chlorogenic acid (CGA) as active ingredients. The two active ingredients exhibit structural compatibility and synergistic function, significantly enhancing antioxidant and antibacterial properties. This invention also provides a controllable preparation method for this composition and formulations comprising this composition.

[0008] In this invention, alginate oligosaccharides not only act as colloidal stabilizers and surface modifiers for nano-selenium, but also participate in the efficacy composition as an active component. Specifically, the carboxyl and hydroxyl functional groups distributed on the alginate oligosaccharide molecular chain are bound to the surface of nano-selenium particles through coordination and hydrogen bonding, preventing the nano-selenium particles from agglomerating and forming a uniformly sized and stably dispersed colloidal system. Furthermore, they form functional complementarity with chlorogenic acid at the molecular level, resulting in synergistic effects.

[0009] In a first aspect, the present invention provides an alginate oligosaccharide-nano selenium-chlorogenic acid composition.

[0010] The active components of this composition include alginate oligosaccharide-modified selenium nanoparticles (AOS-SeNPs) and chlorogenic acid (CGA). The mass ratio of AOS-SeNPs to CGA is 1:1; the weight-average molecular weight of the alginate oligosaccharides is 1000–4000 Da. The AOS-SeNPs are amorphous selenium nanoparticles, and their X-ray diffraction patterns show broad, diffuse peaks at 2θ = 22°–24°, without the characteristic diffraction peaks of trigonal selenium.

[0011] Preferably, the AOS-SeNPs have an average hydration kinetic particle size of 90–120 nm, a polydispersity index (PDI) ≤ 0.25, and a zeta potential of -50 mV to -30 mV.

[0012] Secondly, the present invention provides a method for preparing the composition, comprising the following steps:

[0013] (1) Dissolve alginate oligosaccharides with a weight-average molecular weight of 1000-4000 Da in deionized water and stir until completely dissolved;

[0014] (2) Add anhydrous sodium selenite to the solution obtained in step (1) and stir at room temperature until completely dissolved;

[0015] (3) Under light-protected conditions, the reducing agent is dissolved in deionized water and added dropwise to the mixture obtained in step (2). The mixture is stirred and reacted for 3 to 6 hours at room temperature under light-protected conditions to obtain AOS-SeNPs colloidal dispersion.

[0016] (4) The AOS-SeNPs colloidal dispersion obtained in step (3) is purified by dialysis, and the purified AOS-SeNPs colloidal dispersion is collected; freeze-dried to obtain AOS-SeNPs solid powder.

[0017] (5) Add the AOS-SeNPs solid powder obtained in step (4) and chlorogenic acid to deionized water at a mass ratio of 1:1, stir to completely dissolve chlorogenic acid and uniformly disperse AOS-SeNPs, and the composition is obtained.

[0018] Preferably, the concentration of alginate oligosaccharide in deionized water in step (1) is 3-5 mg / mL; the mass ratio of anhydrous sodium selenite to alginate oligosaccharide in step (2) is 0.43:1-0.44:1; and the reducing agent in step (3) is L-ascorbic acid, with a molar ratio of 2:1 to anhydrous sodium selenite.

[0019] Preferably, the dialysis purification in step (4) uses a dialysis bag with a molecular weight cutoff of 1 kDa, and uses deionized water as the external solution for dialysis in the dark for 24 hours, during which the water is changed 3 times.

[0020] Thirdly, the present invention provides a formulation comprising the above-described composition and a pharmaceutically or food-grade acceptable carrier. Preferably, the formulation is one or more of a hydrogel, film, spray, or coating.

[0021] Preferably, the hydrogel is a polysaccharide-based hydrogel. Compared with the prior art, the present invention has the following beneficial effects:

[0022] (a) Significant synergistic antioxidant effect

[0023] This invention combines alginate oligosaccharide-modified selenium nanoparticles (AOS-SeNPs) with chlorogenic acid (CGA) at a mass ratio of 1:1. The two components exhibit a statistically significant synergistic effect in ABTS⁺ free radical scavenging. ABTS⁺ free radical scavenging experiments show that, under the condition that the final concentration of the reaction system is 25 μg / mL, the scavenging rate of AOS-SeNPs alone is 58.6% ± 2.7%, the scavenging rate of CGA alone is 72.8% ± 3.2%, while the scavenging rate of the composition of this invention is as high as 95.2% ± 1.6%. Synergistic evaluation based on the Bliss independent-action model shows a synergistic coefficient K = 1.07, with a 95% confidence interval of (1.04, 1.11), and a lower limit greater than 1, confirming a statistically significant synergistic mechanism between the two components. This synergistic effect is not a simple sum of the activities of each component, but rather stems from the functional complementarity and structural adaptation at the molecular level among the alginate oligosaccharide modification layer, the nano-selenium core, and chlorogenic acid.

[0024] (ii) Achieving a functional leap from antibacterial to bactericidal

[0025] The composition of this invention achieves a breakthrough in antibacterial effects that cannot be achieved by a single component. Plate count results show that the viable count of AOS-SeNPs alone at a final concentration of 25 μg AOS-SeNPs / mL is (1.3±0.2)×10⁻¹⁰. 5 CFU / mL, and even when the concentration was doubled to a final concentration of 50 μg AOS-SeNPs / mL, the viable count was (1.1±0.2)×10⁻⁶. 5 CFU / mL remains at approximately 10. 5At the CFU / mL level, no significant decrease was observed, indicating that its bactericidal ability had reached a plateau within this concentration range. At the same low concentration (AOS-SeNPs 25 μg / mL + CGA 25 μg / mL), the viable count of the composition of this invention decreased to below the detection limit (<10 CFU / mL) in three independent experiments in three separate experiments, achieving a reduction from (1.3±0.2)×10⁻⁶ CFU / mL. 5 A significant reduction of more than four orders of magnitude from CFU / mL to <10 CFU / mL was observed. Transmission electron microscopy further confirmed that the bacteria treated with the composition exhibited irreversible damage, including widespread cell wall rupture, loss of cell membrane integrity, and leakage of cytoplasmic contents, while single-component treatment only caused slight local shrinkage. These results demonstrate that the composition of this invention overcomes the plateau limitation of the bactericidal ability of single AOS-SeNPs, achieving a substantial leap from bacteriostatic to bactericidal effects, an effect that cannot be expected through the conventional superposition of single-component activities. It should be noted that plate count is the gold standard for directly measuring the number of culturable viable bacteria, reflecting the bactericidal endpoint effect; while OD... 600 The method reflects changes in light scattering of the total biomass in the culture system, primarily demonstrating the inhibitory effect on proliferation. The determination of "functional leap" as described in this invention is based on the plate count method, OD... 600 The antibacterial effect reflected by the plate count method and the bactericidal effect reflected by the plate count method belong to different evaluation dimensions, and their conclusions are complementary rather than conflicting.

[0026] (iii) The composition has a well-defined structure and excellent performance.

[0027] This invention utilizes an in-situ reduction-modification coupling method to prepare AOS-SeNPs in the presence of alginate oligosaccharides. The carboxyl and hydroxyl functional groups distributed on the alginate oligosaccharide molecular chain are bound to the surface of selenium nanoparticles through coordination and hydrogen bonding, both spatially preventing the aggregation and growth of the selenium nanoparticles and endowing the particles with excellent surface chemical properties. The prepared AOS-SeNPs have an average hydration kinetic particle size of 107.33±3.75 nm, a polydispersity index (PDI) ≤0.25, and a Zeta potential of -40.82±7.88 mV, exhibiting good monodispersity and electrostatic stability. The AOS-SeNPs of this invention exist in an amorphous state, and their X-ray diffraction pattern shows a broad diffuse peak only at 2θ = 22°~24°. The surface modification of the alginate oligosaccharides effectively inhibits the transformation of selenium nanoparticles to a crystalline state, maintaining the amorphous morphology of the product. It is generally believed that amorphous selenium nanoparticles have higher surface reactivity than crystalline selenium, which provides a favorable structural basis for the antioxidant and antibacterial functions of the composition of this invention. Transmission electron microscopy (TEM) images show that the AOS-SeNPs prepared in this invention have a uniform spherical or near-spherical morphology, uniform particle size, good monodispersity, and no aggregation, further confirming the excellent stability and dispersibility of alginate oligosaccharides.

[0028] (iv) The preparation process is simple and controllable, and is suitable for large-scale production.

[0029] The preparation method provided by this invention employs an in-situ reduction-modification coupling method, using L-ascorbic acid as a reducing agent and alginate oligosaccharide as a stabilizing modifier. AOS-SeNPs can be obtained in a single reaction under light-protected conditions at room temperature. These AOS-SeNPs are then compounded with CGA in a precise ratio to obtain the final composition. The entire process is simple and mild, requiring no complex separation and purification steps. Dialysis purification effectively removes small molecule impurities, ensuring precise control of the active component ratio, making it suitable for industrial-scale production.

[0030] (v) Good dosage form adaptability, facilitating application in multiple scenarios

[0031] The composition of this invention exhibits good loading adaptability in hydrogel carriers. After loading the composition onto a polysaccharide-based hydrogel matrix, the particle size and zeta potential of the released AOS-SeNPs showed no significant difference compared to before loading, indicating that the structural integrity of the AOS-SeNPs was not disrupted during the hydrogel loading process. The loaded composition also maintained synergistic antioxidant activity and bactericidal ability to reduce viable bacterial counts below the detection limit, demonstrating the good dosage form adaptability of the composition. The hydrogel matrix and the alginate oligosaccharides in the composition share structural homology, which is beneficial for the uniform dispersion and stable maintenance of the active components in the matrix, providing a technical basis for the application of the composition in functional foods, health products, pharmaceuticals, and topical formulations. Attached Figure Description

[0032] Figure 1 The particle size distribution diagram is shown for AOS-SeNPs prepared in Example 1 of this invention.

[0033] Figure 2 Transmission electron microscope (TEM) image of AOS-SeNPs prepared for Example 1 of the present invention.

[0034] Figure 3 The X-ray diffraction (XRD) pattern of AOS-SeNPs prepared in Example 1 of this invention.

[0035] Figure 4 The Fourier transform infrared (FTIR) spectrum of AOS-SeNPs prepared in Example 1 of this invention. Detailed Implementation

[0036] The present invention will be further illustrated by specific embodiments below, but the present invention is not limited to these embodiments. All technical solutions implemented based on the above content of the present invention fall within the scope of the present invention.

[0037] All reagents used in the embodiments of this invention are commercially available analytical grade reagents. Chlorogenic acid (CGA, purity ≥98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., sodium selenite (anhydrous) was purchased from Anhui Zesheng Technology Co., Ltd., and L-ascorbic acid was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0038] The preparation method of alginate oligosaccharides (AOS) used in this embodiment of the invention is as follows: Sodium alginate with a weight-average molecular weight of 200-500 kDa was dissolved in deionized water to prepare a 2% (w / v) solution. 30 mM H₂O₂ and 10 mM vitamin C were added, and the solution was degraded at 60°C for 4 hours. After the reaction, the degradation solution was sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 10 kDa, 5 kDa, 3 kDa, and 1 kDa for fractionation. The retentates of the corresponding molecular weight cutoffs were collected and freeze-dried to obtain AOS of different molecular weight ranges. The weight-average molecular weights of the three AOS components obtained were determined by gel permeation chromatography (GPC) to be approximately 1000 Da, 3000 Da, and 4000 Da, respectively. All of the above molecular weight ranges of AOS can be used in the preparation of AOS-SeNPs of this invention. Unless otherwise specified in subsequent embodiments, the weight-average molecular weight of the AOS used is approximately 3000 Da.

[0039] In the following embodiments, unless otherwise stated, the sample concentration refers to the working solution concentration (i.e., the concentration of ABTS). + (Concentration of each component in the reaction system before mixing with equal volumes of working solution or bacterial culture). The actual concentration of each component in the reaction system after mixing is half the concentration of the working solution. All activity data were measured under this reaction system.

[0040] For ease of reference in subsequent embodiments, the ABTS of a single component was measured under the same experimental conditions at different working solution concentrations. + Free radical scavenging rate and growth inhibition rate (OD) 600 The results of the plate viable count (method) and the plate viable count are summarized in Table 1.

[0041] Table 1. Activity baseline data of single components at different working solution concentrations (mean ± SD, n = 3 independent experiments)

[0042] Single CGA 25 12.5 38.5±2.5 45.0±2.0 <![CDATA[(1.1±0.1)×10 6 ]]> Single CGA 50 25 72.8±3.2 91.2±1.9 <![CDATA[(2.6±0.3)×10 3 ]]> Single CGA 100 50 85.0±1.5 96.0±1.0 <![CDATA[(2.8±0.2)×10 2 ]]> Single AOS-SeNPs 25 12.5 32.0±2.0 30.0±2.0 <![CDATA[(1.0±0.1)×10 7 ]]> Single AOS-SeNPs 50 25 58.6±2.7 62.7±2.5 <![CDATA[(1.3±0.2)×10 5 ]]> Single AOS-SeNPs 100 50 61.0±2.0 63.5±2.0 <![CDATA[(1.1±0.2)×10 5 ]]>

[0043] Note: Each sample was first prepared with working solution concentration and an equal volume of ABTS. + When the working solution or bacterial solution is mixed, the final concentration of the reaction system is half the concentration of the working solution. The corresponding final concentration of the reaction system in the table is calculated accordingly.

[0044] The methods for determining the baseline data in Table 1 above are as follows:

[0045] (1) Determination of the baseline value of ABTS⁺ free radical scavenging rate: Referring to the method in Example 3 of this invention, single CGA aqueous solution and single AOS-SeNPs dispersion with different working solution concentrations were prepared respectively, and mixed with an equal volume of ABTS⁺ free radical scavenging rate. + After mixing the working solutions (final concentrations of the reaction system are shown in Table 1), the mixture was reacted at 25°C in the dark for 6 minutes, and the absorbance at 734 nm was measured. The scavenging rate was calculated according to the formula shown in Example 3. Three parallel samples were prepared for each concentration, and the results are expressed as mean ± standard deviation.

[0046] (2) Growth inhibition rate (OD) 600 (Method) Determination of reference value: Referring to the method in Example 4 of this invention, a suspension of Staphylococcus aureus in the logarithmic growth phase was taken and the concentration was adjusted to approximately 1.2 × 10⁻⁶ using MH broth medium. 6 CFU / mL. Equal volumes of single CGA aqueous solution or single AOS-SeNPs dispersion with different working solution concentrations were added to achieve the final concentrations shown in Table 1. A corresponding background control well was included to subtract SeNPs scattering interference. After incubation at 37°C with shaking for 6 hours, OD was measured. 600 The growth inhibition rate was calculated using the formula shown in Example 4. Three parallel samples were prepared for each concentration, and the results are expressed as mean ± standard deviation.

[0047] (3) Determination of plate viable count baseline value: Based on the above OD... 600After the incubation was completed, the bacterial culture of each group was serially diluted appropriately, and 100 μL was spread on LB agar plates and incubated upside down at 37°C for 18 hours. Viable bacteria count (CFU / mL) was performed, and the results are expressed as mean ± standard deviation.

[0048] Table 1 shows that the growth inhibition rate of single AOS-SeNPs at a working solution concentration of 25 μg AOS-SeNPs / mL was 30.0%, and the viable count was (1.0±0.1)×10⁻⁶. 7 CFU / mL; at a working solution concentration of 50 μg AOS-SeNPs / mL, the growth inhibition rate increased to 62.7%, and the viable count decreased to (1.3±0.2)×10⁻⁶. 5 CFU / mL. At a working solution concentration of 100 μg AOS-SeNPs / mL, the growth inhibition rate was 63.5%, and the viable count was (1.1±0.2)×10⁻⁶. 5 The number of viable bacteria at a working solution concentration of 50 μg AOS-SeNPs / mL was (1.3±0.2)×10⁻¹⁰ CFU / mL. 5 There was no statistically significant difference in CFU / mL (P>0.05), indicating a clear plateau, meaning that increasing the concentration of AOS-SeNPs alone to above 50 μg AOS-SeNPs / mL did not further reduce the viable bacterial count. Its ABTS + The free radical scavenging rate at a working solution concentration of 100 μg AOS-SeNPs / mL was essentially the same as that at 50 μg AOS-SeNPs / mL (61.0% vs 58.6%), indicating that the active binding sites on the surface of AOS-SeNPs tend to be saturated within this concentration range. The scavenging rates of specific molecular weight AOS-SeNPs and the corresponding viable cell counts in each example are listed in the tables of each example.

[0049]

Example 1

[0050] 200 mg of alginate oligosaccharide with a weight-average molecular weight of approximately 3000 Da was dissolved in 50 mL of deionized water (4 mg / mL) and stirred until completely dissolved. 86.5 mg of anhydrous sodium selenite (Na₂SeO₃, molecular weight 172.94, representing 43.25% of the alginate oligosaccharide mass) was added to the above solution, and stirring was continued at room temperature for 30 minutes until completely dissolved, forming a solution containing selenite ions. Under light-protected conditions, 176 mg of L-ascorbic acid (molar ratio to anhydrous sodium selenite 2.0:1) was dissolved in 10 mL of deionized water and added dropwise to the above mixture. After the addition was complete, the reaction was continued at 25°C under light-protected conditions with stirring for 4 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag (molecular weight cutoff MWCO 1 kDa), and dialyzed against light for 24 hours using deionized water as the external solution, with the external solution replaced every 8 hours (a total of 3 water changes) to obtain a purified AOS-SeNPs colloidal dispersion. An appropriate amount of the purified colloidal dispersion was filtered through a 0.22 μm microporous membrane, and the filtrate was used for subsequent characterization and analysis. High-performance liquid chromatography (HPLC) confirmed that no L-ascorbic acid residue was present in the purified colloidal dispersion. The purified AOS-SeNPs colloidal dispersion was then freeze-dried to obtain AOS-SeNPs solid powder. The freeze-dried AOS-SeNPs solid powder exhibited good resolubility and could be directly dispersed in deionized water for use.

[0051] The average hydration kinetic particle size (Z-average) was determined using dynamic light scattering (DLS) (see [reference]). Figure 1 The wavelength range (λ) was 107.33 ± 3.75 nm, the polydispersity index (PDI) was 0.18 ± 0.05, and the zeta potential was -40.82 ± 7.88 mV. Transmission electron microscopy (TEM) images (see...) Figure 2 The nano-selenium exhibits a uniform spherical or near-spherical morphology with good monodispersity; X-ray diffraction (XRD) analysis (see...) Figure 3 The results showed that the product exhibited only a broad, diffuse peak at 2θ = 22°–24°, without any characteristic diffraction peaks of crystalline selenium (the characteristic diffraction peaks of trigonal selenium at 2θ = 23.5°, 29.7°, 41.3°, 43.6°, 45.4°, and 51.7° were not observed), confirming that selenium exists as amorphous nanoparticles. Fourier transform infrared spectroscopy (FTIR) (see...) Figure 4 Analysis showed that, compared with free AOS, the νas(COO) of AOS in the complex was higher. - ) by 1613 cm -1 Move to 1600 cm -1 ,νs(COO - ) by 1416 cm -1Moved to 1405 cm -1 Meanwhile, the OH stretching vibration peak decreased from 3420 cm⁻¹. -1 Moved to 3395 cm -1 The aforementioned infrared spectral shifts, combined with the significant negative shift in Zeta potential and particle size stability data, indicate that the carboxyl and hydroxyl groups on the AOS molecular chain are bound to the SeNPs surface through coordination and / or hydrogen bonding.

[0052] To verify the rationality of the preferred weight-average molecular weight range of 1000–4000 Da in this invention, the inventors further prepared SeNPs using AOS with weight-average molecular weights of approximately 1000 Da and 4000 Da under the same conditions. The particle size, polydispersity index, and zeta potential of the obtained AOS-SeNPs were all within the preferred range defined by this invention (Z-average of 109.3 nm and 113.1 nm, PDI of 0.19 and 0.23, and zeta potential of -39.5 mV and -43.5 mV, respectively), which were basically consistent with the performance of the typical samples mentioned above. The inventors further verified that when SeNPs prepared from AOS with three representative molecular weights of 1000 Da, 3000 Da, and 4000 Da, tested within the above-mentioned 1000–4000 Da range, were combined with CGA, the lower limit of the 95% confidence interval of the ABTS⁺ free radical scavenging synergistic coefficient K was greater than 1 (n=3 independent experiments). This indicates that AOS within this molecular weight range can effectively stabilize SeNPs and provide a structural basis for the subsequent synergistic effect with CGA.

[0053] The AOS-SeNPs solid powder obtained in Example 1 was analyzed by ICP-MS, and the total selenium content was determined to be 16.5% ± 1.2% (w / w). Based on this, the AOS-SeNPs concentration in the release solution was calculated using the following formula: AOS-SeNPs concentration (μg / mL) = Selenium element concentration (μg / mL) ÷ (16.5% ± 1.2%).

[0054]

Example 2

[0055] The AOS-SeNPs solid powder obtained in Example 1 and chlorogenic acid (CGA) were added together to deionized water at a mass ratio of 1:1. The mixture was stirred until the chlorogenic acid was completely dissolved and the AOS-SeNPs were uniformly dispersed, preparing a stock solution with a concentration of 200 μg / mL for both AOS-SeNPs and CGA. This solution was then used in ABTS. +When determining free radical scavenging and antibacterial activity, the solution should be diluted with deionized water or appropriate culture medium to the required working solution concentration, according to the specific requirements of each embodiment. For example, in Examples 3 and 4, the stock solution was diluted 4 times to obtain a working solution with both AOS-SeNPs and CGA concentrations of 50 μg / mL, which was then mixed with an equal volume of ABTS. + When the working solution or bacterial culture is mixed, the final concentrations of AOS-SeNPs and CGA in the reaction system are both 25 μg / mL.

[0056]

Example 3

[0057] Refer to ABTS + Standard method for free radical scavenging, preparation of ABTS + The working solution was prepared to achieve an absorbance of 0.70 ± 0.02 at 734 nm. The blank control consisted of an equal volume of deionized water instead of the sample, and was prepared with ABTS. + Working fluid mixing for monitoring ABTS + The absorbance was corrected for spontaneous decay. The working solution was mixed with equal volumes of the following three samples: (a) a single AOS-SeNPs dispersion (working solution concentration 50 μg / mL, final reaction system concentration 25 μg / mL, without CGA); (b) a single CGA aqueous solution (working solution concentration 50 μg / mL, final reaction system concentration 25 μg / mL, without AOS-SeNPs); and (c) the working solution of the composition prepared in Example 2 (working solution containing 50 μg / mL AOS-SeNPs and 50 μg / mL CGA, final reaction system concentration 25 μg / mL AOS-SeNPs + 25 μg / mL CGA). After mixing, the mixture was reacted at 25°C in the dark for 6 minutes, and then the absorbance of each group was measured at 734 nm. To eliminate the influence of sample color or scattering on absorbance, a corresponding background control group (i.e., using an equal volume of deionized water instead of ABTS) was set up for each sample. + (Working solution, all other operations are the same) Sample calibration absorbance A S Correction=A S - Background. The background absorbance of all samples was measured to be less than 0.01. After subtraction, the clearance rate differed from the original value by no more than 0.5 percentage points, and the results and conclusions remained unchanged. The data reported below are clearance rates after background correction. Three parallel samples were set up for each group, and the results are expressed as mean ± standard deviation. Three independent experiments were conducted, each with three technical replicates.

[0058] The formula for calculating the clearance rate (%) is: [(A0 - A S [A0] × 100%, where A0 is the absorbance of the blank control reaction after replacing the sample with deionized water, and AS The absorbance of the sample group was corrected.

[0059] The synergistic effect was evaluated using the Bliss independent-action model combined with the Bootstrap resampling method (n=10000). The synergistic coefficient K was defined as the ratio of the measured clearance rate to the theoretical prediction (Epred) of the Bliss model. K=1 indicates an additive effect, K>1 indicates a synergistic effect, and K<1 indicates an antagonistic effect. If the lower limit of the 95% confidence interval (CI) of K is greater than 1, it is considered a statistically significant synergistic effect. The experimental results are shown in Table 2.

[0060] Table 2 Evaluation of ABTS⁺ free radical scavenging rate and synergistic effect of different samples (mean ± SD, n=3 independent experiments)

[0061] Single CGA 25 μg / mL 72.8±3.2 — Single AOS-SeNPs 25 μg / mL 58.6±2.7 — The composition of the present invention (Example 2) AOS-SeNPs 25 μg / mL + CGA 25 μg / mL 95.2±1.6** 1.07(1.04,1.11)

[0062] Note: **P<0.01, compared with the single CGA group and the single AOS-SeNPs group (one-way ANOVA, Tukey HSD post-hoc test).

[0063] Based on the Bliss model, the theoretical predicted value E pred Calculate using the following formula:

[0064] E pred = 1 - (1 - E A ) × (1 - E B )

[0065] Among them, E A For the clearance rate of a single CGA, E B The clearance rate for a single AOS-SeNP.

[0066] Substitute the corresponding data from Table 2 into the table:

[0067] E pred = 1 - (1-0.728)×(1-0.586) = 1 - 0.272×0.414 = 0.8874 (i.e. 88.74%).

[0068] Measured clearance rate E obs = 0.952 (i.e., 95.2%), synergy coefficient K = E obs / E pred = 95.2% / 88.7% = 1.07 > 1.

[0069] The 95% confidence interval for K, calculated using the Bootstrap method, is (1.04, 1.11), with a lower limit greater than 1, indicating that the synergistic effect is statistically significant.

[0070] [Example 4] Antibacterial activity assay (OD) 600 (Verification using a combination of growth inhibition and plate counting methods)

[0071] Take a suspension of Staphylococcus aureus (S. aureus, ATCC 25923) in the logarithmic growth phase and adjust the concentration to approximately 1.2 × 10⁻⁶ with fresh MH broth. 6 CFU / mL. Equal volumes of the following treatment working solutions were added: (a) single AOS-SeNPs (working solution concentration 50 μg / mL); (b) single CGA (working solution concentration 50 μg / mL); (c) working solution of the combination from Example 2 (working solution containing 50 μg / mL AOS-SeNPs + 50 μg / mL CGA). After mixing each group with an equal volume of bacterial solution, the actual concentration of each component in the reaction system was half the concentration of the working solution, i.e., the final concentration of the single AOS-SeNPs group was 25 μg / mL, the final concentration of the single CGA group was 25 μg / mL, and the final concentration of the combination group was 25 μg / mL AOS-SeNPs + 25 μg / mL CGA. A separate nanoparticle background control well (culture medium containing an equal concentration of SeNPs but no bacteria) was provided to subtract the effect of SeNPs scattered light on OD. 600 To mitigate interference, the single CGA group, which contains no SeNPs, had its OD value unaffected by nanoparticle scattering, and therefore the measured OD value was used directly. For both the single AOS-SeNPs group and the combined group, the absorbance values ​​of the wells containing the same concentration of SeNPs but no bacteria were subtracted (OD sample correction = OD sample – OD background). Positive control wells were also included (using an equal volume of sterile PBS instead of the sample, with the remaining procedures identical). All groups were incubated at 37℃ with shaking for 6 hours. Under these experimental conditions, the viable cell count on the plate after 6 hours of incubation in the positive control group was (1.2 ± 0.3) × 10⁻⁶. 8 The CFU / mL reading indicates that the experimental system is effective. After incubation, the optical density (OD) at 600 nm was measured in each well. 600 The corrected OD values ​​for each well were calculated as follows: For the single CGA group, the measured OD value was used directly (OD sample correction = OD sample); for the single AOS-SeNPs group and the combined group, the formula OD sample correction = OD sample - OD background was used, where OD background is the absorbance value of the well containing the same concentration of SeNPs but no bacteria. The growth inhibition rate (%) was calculated as: [1 - (OD sample correction / OD positive control correction)] × 100%, where OD positive control correction = OD positive control - OD background (the positive control does not contain SeNPs, and OD background is the background value of the culture medium without SeNPs).

[0072] Meanwhile, after incubation, the bacterial suspensions from each group were serially diluted appropriately, and 100 μL was spread onto LB agar plates and incubated upside down at 37°C for 18 hours for viable cell counting (CFU / mL). Under these experimental conditions, the lower limit of detection for plate counting was defined as 10 CFU / mL, which is the original bacterial suspension concentration corresponding to the detection of one colony on a 100 μL plate. If the colony count on the plate was 0, it was reported as below the detection limit (<10 CFU / mL). This statement is not equivalent to absolute sterility, but rather means that no culturable viable bacteria were detected under the current detection conditions. Three parallel samples were set up for each group, and the results are expressed as mean ± standard deviation. Three independent experiments were conducted, each with three technical replicates. The experimental results are shown in Table 3.

[0073] Table 3 Antibacterial activity (OD) of different treatment groups 600 Method and slab counting method, mean ± SD, n=3 independent experiments)

[0074] Single CGA 25 μg / mL 91.2±1.9 — <![CDATA[(2.6±0.3)×10 3 ]]> Single AOS-SeNPs 25 μg / mL 62.7±2.5 — <![CDATA[(1.3±0.2)×10 5 ]]> The composition of the present invention (Example 2) AOS-SeNPs 25 μg / mL + CGA 25 μg / mL 99.6±0.3** 1.03(0.98,1.08) <10 CFU / mL

[0075] Note: **P<0.01, compared with the single CGA group and the single AOS-SeNPs group (one-way ANOVA) and Tukey HSD post-hoc test; no colonies were detected in each technique replicate plate of the three independent experiments (0 CFU / 100 μL plating solution), and the viable count was below the detection limit.

[0076] Transmission electron microscopy morphological observation: To verify whether the damage to bacteria caused by the composition was reversible, after the above incubation, the bacterial solutions of each group were centrifuged at 3000 rpm for 10 minutes, the bacterial pellets were collected, fixed with 2.5% glutaraldehyde, dehydrated by ethanol gradient, embedded in epoxy resin, and ultra-thinly sectioned before observation under a transmission electron microscope. The results showed that the cell walls and cell membranes of the bacteria treated with the single CGA group (final concentration 25 μg / mL) and the single AOS-SeNPs group (final concentration 25 μg / mL) were basically intact, with only slight local shrinkage; while the bacteria in the composition-treated group showed extensive cell wall rupture, loss of cell membrane integrity, and leakage of cytoplasmic contents, indicating that the composition caused irreversible inactivation of bacteria through multiple layers of damage.

[0077] (1) OD 600 Legal Results Analysis

[0078] Based on the Bliss model, the theoretical predicted value E pred Calculate using the following formula:

[0079] E pred =1 - (1 - E A ) × (1 - E B )

[0080] Among them, EA OD for a single CGA 600 Growth inhibition rate, E B OD for a single AOS-SeNPs 600 Growth inhibition rate.

[0081] Substitute the corresponding data from Table 3 into the table:

[0082] E pred = 1 - (1-0.912)×(1-0.627) = 1 - 0.088×0.373 = 0.9672 (i.e. 96.72%).

[0083] Measured inhibition rate E obs = 0.996 (i.e., 99.6%)

[0084] Coordination coefficient K = E obs / E pred = 99.6% / 96.72% = 1.03.

[0085] The 95% confidence interval for K, calculated using the Bootstrap method, is (0.98, 1.08). This interval includes 1, indicating that there is no statistically significant synergistic effect between the two on this detection dimension. According to the Bliss model criteria, an interval containing 1 at the 95% confidence level is considered an additive effect.

[0086] (2) Analysis of plate counting results

[0087] Plate count results showed that the composition of the present invention reduced the number of viable Staphylococcus aureus from (1.2±0.3)×10⁻⁶ in three independent experiments. 8 The CFU / mL concentration was reduced to below the detection limit (<10 CFU / mL). Three technical replicates were set up for each independent experiment, and no culturable colonies were detected in any of the nine plates (0 CFU / 100 μL of plating solution), indicating that the viable count had been reduced below the detection limit. In contrast, CGA alone (final concentration of 25 μg / mL) reduced the viable count to (2.6 ± 0.3) × 10⁻⁶. 3 CFU / mL, with a single AOS-SeNP (final concentration of 25 μg / mL), reduced the viable count to (1.3±0.2)×10⁻⁶. 5 The CFU / mL values ​​were all above the detection limit.

[0088] (3) Discussion of results

[0089] As shown in Table 1, the viable cell counts of single AOS-SeNPs on plates were approximately 10 at both working solution concentrations of 50 μg / mL and 100 μg / mL (corresponding to final concentrations of 25 μg / mL and 50 μg / mL).5 The CFU / mL concentration showed a clear plateau; however, the composition of this invention, under the conditions of a final reaction system concentration of AOS-SeNPs 25 μg / mL + CGA 25 μg / mL, increased the viable bacterial count from (1.3±0.2)×10⁻⁶. 5 The CFU / mL (single AOS-SeNPs plateau value) decreased to <10 CFU / mL, a reduction of more than four orders of magnitude, exceeding the expectation of the simple summation of the single component activities. Transmission electron microscopy morphological observation also confirmed that the composition induced widespread irreversible damage such as cell wall rupture and leakage of cytoplasmic contents, while single component treatment only caused slight local shrinkage, indicating that the composition achieved a functional leap in bactericidal performance.

[0090] It should be noted that OD 600 The evaluation dimensions of the plate counting method and the slab counting method are different: OD 600 The light scattering method reflects the change in total biomass (including viable bacteria, dead bacteria, and cell debris) in the culture system, mainly demonstrating an inhibitory effect on bacterial proliferation. It cannot distinguish between the inhibition and killing of viable bacteria, thus its response to the degree of bactericidal activity is not sensitive enough and tends to be additive. The plate count method, on the other hand, directly measures the number of culturable viable bacteria and is the gold standard for evaluating bactericidal efficacy. The composition of this invention achieves a reduction in viable bacterial count by more than four orders of magnitude in plate counts, breaking through the plateau period of bactericidal ability of single components. Therefore, at the level of inhibition (OD... 600 The additive effect of the method and the breakthrough of the bactericidal level (plate counting method) are not contradictory. They belong to different dimensions of action and together constitute the functional breakthrough of the composition of the present invention.

[0091] It is important to note that the plate count method measures the absolute number of culturable viable bacteria, and the results are expressed as CFU / mL. This method is not suitable for calculating the synergistic coefficient in the Bliss independent-action model (which requires data in percentage form). Therefore, the order-of-magnitude breakthrough in bactericidal effect in this invention is based on a qualitative judgment made from the perspective of effect, based on the reduction of viable bacteria count after composition treatment by several orders of magnitude compared to the plateau level of a single component, and the irreversible damage morphology observed by transmission electron microscopy, rather than a statistical judgment based on the synergistic coefficient K. In summary, OD 600 The antibacterial effect reflected by the plate count method is additive, while the bactericidal effect reflected by the plate count method is a breakthrough across orders of magnitude. The two methods evaluate antibacterial activity from the two levels of antibacterial and bactericidal effects, respectively, and the conclusions are complementary rather than conflicting.

[0092]

Comparative Example 1

[0093] To investigate the effect of the AOS-SeNPs:CGA compound ratio on the synergistic antioxidant and antibacterial effects, the inventors further set up control groups with AOS-SeNPs:CGA mass ratios of 1:2 and 2:1. The working solutions of the composition were prepared according to the same method as in Example 2, and the ABTS⁺ free radical scavenging rate and plate viable count were determined according to the methods in Examples 3 and 4.

[0094] ABTS in different proportions + Free radical scavenging rates are shown in Table 4.

[0095] Table 4 ABTS at different ratios + Free radical scavenging rate

[0096] Single CGA CGA 25 μg / mL 72.8±3.2 — Single AOS-SeNPs AOS-SeNPs 25 μg / mL 58.6±2.7 — AOS-SeNPs:CGA = 1:2 AOS-SeNPs 12.5 + CGA 25 μg / mL 86.3±2.5* 1.06(1.01,1.11) AOS-SeNPs:CGA = 2:1 AOS-SeNPs 25 + CGA 12.5 μg / mL 78.1±2.8* 1.05(0.99,1.10) AOS-SeNPs:CGA = 1:1 AOS-SeNPs 25 + CGA 25 μg / mL 95.2±1.6** 1.07(1.04,1.11)

[0097] Note: *P<0.05, compared with the single component; **P<0.01, compared with the single component and the 1:2 and 2:1 groups (one-way ANOVA, Tukey HSD post-hoc test).

[0098] (2) Table 5 shows the viable cell counts of plates with different proportions.

[0099] Table 5. Viable cell counts on plates with different proportions.

[0100] Positive control — <![CDATA[(1.2 ± 0.3) × 10 8 ]]> Single CGA CGA 25 μg / mL <![CDATA[(2.6 ± 0.3) × 10 3 ]]> Single AOS-SeNPs AOS-SeNPs 25 μg / mL <![CDATA[(1.3 ± 0.2) × 10 5 ]]> AOS-SeNPs:CGA = 1:2 AOS-SeNPs 12.5 μg / mL + CGA 25 μg / mL <![CDATA[(1.2 ± 0.3) × 10 2 ]]> AOS-SeNPs:CGA = 2:1 AOS-SeNPs 25 μg / mL + CGA 12.5 μg / mL <![CDATA[(8.5 ± 0.5) × 10 3 ]]> AOS-SeNPs:CGA = 1:1 AOS-SeNPs 25 μg / mL + CGA 25 μg / mL < 10

[0101] (3) Discussion of results

[0102] The synergistic coefficient was calculated based on the baseline values ​​of the single components at their corresponding final concentrations in Table 1: for the 1:2 group, the final concentrations of AOS-SeNPs were 12.5 μg / mL (scavenging rate 32.0%) and CGA were 25 μg / mL (scavenging rate 72.8%); for the 2:1 group, the final concentrations of AOS-SeNPs were 25 μg / mL (scavenging rate 58.6%) and CGA were 12.5 μg / mL (scavenging rate 38.5%). These results indicate that the AOS-SeNPs:CGA ratio has a significant impact on the synergistic effect. When AOS-SeNPs:CGA=1:1, the ABTS⁺ clearance rate reached 95.2%, significantly higher than that of the 1:2 group (86.3%) and the 2:1 group (78.1%) (P<0.01). Although the 1:2 group also showed statistical synergy in ABTS⁺ clearance (K=1.06, 95% CI lower limit>1), its absolute clearance rate (86.3%) was significantly lower than that of the 1:1 group (95.2%). More importantly, only the 1:1 ratio could reduce the viable count of Staphylococcus aureus to below the detection limit (<10 CFU / mL), while the viable count at the 1:2 ratio was (1.2±0.3)×10⁻⁶. 2CFU / mL, at a 2:1 ratio, is (8.5±0.5)×10 3 The CFU / mL values ​​were all above the detection limit, failing to achieve the breakthrough in bactericidal function described in this invention. These results confirm that AOS-SeNPs:CGA = 1:1 is the key ratio for achieving synergistic antioxidant effects and a breakthrough in bactericidal function; deviations from this ratio will significantly weaken or even eliminate the breakthrough effect of this invention at the bactericidal level.

[0103] [Example 5] Loading and activity verification of the composition in a hydrogel carrier

[0104] To verify the adaptability of the synergistic antioxidant and bactericidal effects of the composition of the present invention in the dosage form carrier, AOS-SeNPs and CGA were loaded into a polysaccharide-based hydrogel at a mass ratio of 1:1, and their activity after release was investigated.

[0105] (1) Preparation of the loaded composition hydrogel

[0106] The AOS-SeNPs solid powder obtained in Example 1 and chlorogenic acid (CGA) were added to deionized water at a mass ratio of 1:1. After stirring and mixing, a working solution of the composition with a concentration of 0.25 mg / mL for both AOS-SeNPs and CGA was obtained. 0.1 g of sodium alginate (SA) and 0.1 g of carboxymethyl chitosan (CMCS) were dissolved in 5 mL of deionized water and stirred at room temperature until completely dissolved. 5.36 mL of the above working solution was added to the SA solution and gently vortexed for 30 seconds. Then, the SA / composition mixture was rapidly mixed with the CMCS solution, and 1 mL of 2% CaCl2 solution was added as a crosslinking agent. After rapid vortexing for about 5 seconds, the mixture was immediately poured into a cylindrical silicone mold and allowed to stand at 37°C for 24 hours until complete crosslinking, thus obtaining a hydrogel loaded with the composition of the present invention. A portion of the hydrogel was taken and 5 mL of 50 mM sodium citrate solution was added. The mixture was shaken at 37°C for 2 hours to completely dissolve the calcium alginate and disrupt its crosslinking. After this gel disruption treatment, the loading rates of AOS-SeNPs and CGA in the hydrogel were determined by HPLC and ICP-MS to be 97.2%±1.5% and 96.5%±2.1%, respectively, which were basically consistent with the theoretical feed amount, indicating that the crosslinking and curing process had a high loading efficiency for the active ingredients.

[0107] Based on the feed ratio, the total mass of AOS-SeNPs and CGA in the stock solution is 5.36 mL × 0.25 mg / mL = 1.34 mg. With the measured total gel mass Mtotal = 13.40 g, the theoretical content of both AOS-SeNPs and CGA in the hydrogel is 1.34 mg, or approximately 100 μg / g (based on a hydrogel density of ≈ 1 g / mL, approximately 100 μg / mL).

[0108] (2) Release of active ingredients and preparation of working solution

[0109] Weigh approximately 1 g of the hydrogel containing the above-mentioned loading composition (denoted as m gel), place it in a centrifuge tube, add 2 mL of phosphate-buffered saline (PBS, pH 7.4), and incubate at 37°C with shaking for 2 hours to release the hydrogel. After release, remove the hydrogel, rinse the surface with a small amount of PBS, combine the rinsing solution and release medium, and transfer to a volumetric flask to a final volume of 2 mL. Shake well and centrifuge at 5000 rpm for 5 minutes, collecting the supernatant.

[0110] In this embodiment, the theoretical total amount of active ingredients contained in 1 g of hydrogel is calculated by mass ratio as: mgel / Mtotal × 1.34 mg. After the above operation completely transfers the active ingredients and brings the volume to 2 mL, the theoretical concentration of the released solution is approximately 50 μg / mL (based on mgel = 1.0 g and Mtotal = 13.40 g, the theoretical release concentration = (1.0 / 13.40 × 1.34 mg) / 2 mL ≈ 0.05 mg / mL = 50 μg / mL).

[0111] To accurately quantify the concentrations, the actual concentrations of the released supernatant were determined: CGA concentration was measured by HPLC, and selenium concentration was measured by ICP-MS. The results showed that the CGA concentration in the released solution was 49.8 μg / mL, and the selenium concentration was equivalent to 50.2 μg / mL for AOS-SeNPs, consistent with the theoretical complete release concentration (50 μg / mL), confirming that the active ingredient was essentially completely released within 2 hours. This released solution was directly used as the working solution for activity testing. Ion chromatography (IC) verification confirmed that no selenite ions (SeO32-) were detected in the released solution. 2- The presence of selenium residue (detection limit 0.1 μg / mL) indicates that the selenium in the released solution exists in the form of nano-selenium, and the method of converting it into AOS-SeNPs concentration is reasonable and reliable.

[0112] Mix the working solution with an equal volume of ABTS + After mixing the working solution or bacterial culture, the final concentrations of AOS-SeNPs and CGA in the reaction system were both 25 μg / mL, which was completely consistent with the test concentrations in Examples 2 and 3, allowing for direct comparison of synergistic effects.

[0113] (3) Preparation of single-component hydrogels and determination of reference values

[0114] To accurately evaluate the synergistic effect, it is necessary to obtain the activity baseline value of a single component under the same release system.

[0115] Single AOS-SeNPs hydrogel: Take 2.68 mL of AOS-SeNPs dispersion (0.50 mg / mL), add 2.68 mL of deionized water, and perform the remaining operations as in step (1). Prepare the release working solution according to the same operation as in step (2) of "removing the gel, rinsing, and adjusting the volume to 2 mL". The concentration of AOS-SeNPs in the release solution was measured and found to be 50.1 μg / mL. Single CGA hydrogel: Take 2.68 mL of CGA aqueous solution (0.50 mg / mL), add 2.68 mL of deionized water, and perform the remaining operations as in step (1). Prepare the release working solution according to the same operation as in step (2). The concentration of CGA in the release solution was measured and found to be 49.5 μg / mL.

[0116] The two release working solutions mentioned above were respectively mixed with an equal volume of ABTS. + The working solution or bacterial culture was mixed to achieve final concentrations of CGA 25 μg / mL and AOS-SeNPs 25 μg / mL, respectively, and their ABTS was determined. + Free radical scavenging rate, OD 600 Growth inhibition rate and plate viable count. The baseline values ​​at this final concentration obtained from three independent experiments are as follows:

[0117] Single CGA release working solution: ABTS + Sweep rate 70.5±2.0%, OD 600 Inhibition rate: 90.5 ± 1.5%; viable cell count on plate: (2.4 ± 0.3) × 10⁻⁶. 3 CFU / mL.

[0118] Single AOS-SeNPs release working solution: ABTS + Sweep rate 55.0±2.2%, OD 600 Inhibition rate: 61.0 ± 2.0%; viable cell count on plate: (1.3 ± 0.2) × 10⁻⁶ 5 CFU / mL.

[0119] The above values ​​will serve as the basis for evaluating the synergistic effect under the release system. The baseline values ​​under the above release system differ slightly from those of the direct solution, which may be due to the slight interference of trace excipients (polysaccharide fragments or cross-linking agents) in the hydrogel release solution on the color development system or bacterial culture environment. However, the difference is small and does not affect the determination of the synergistic effect.

[0120] (4) Elimination of interference from blank hydrogel

[0121] Background subtraction of blank hydrogel: Take 1 g of blank hydrogel without the composition, add 2 mL of PBS, shake to release under the same conditions, and remove the gel in the same manner. Make up to 2 mL, centrifuge, and collect the supernatant. Take this supernatant and an equal volume of ABTS.+ The working solution was mixed, and its absorbance at 734 nm was measured. The scavenging rate was calculated to be 1.1 ± 0.3%, indicating that the hydrogel matrix itself effectively removes ABTS. + The scavenging effect of free radicals is negligible.

[0122] Blank hydrogel antibacterial negative control: The supernatant of the above blank hydrogel was mixed with an equal volume of Staphylococcus aureus suspension, incubated at 37°C for 6 hours, and plate counts were performed. The viable count was (1.1±0.2)×10⁻⁶. 8 CFU / mL, compared with the positive control group (1.2±0.3)×10 8 There was no significant difference in CFU / mL (P>0.05), confirming that the hydrogel matrix component had no antibacterial or bactericidal activity.

[0123] (5) Structural integrity verification

[0124] The release solution of the hydrogel of the loaded composition obtained in step (2) was centrifuged at 5000 rpm for 5 minutes. The supernatant was then rapidly filtered through a 0.45 μm microporous membrane to remove any possible residual trace gel fragments. The particle size and Zeta potential of AOS-SeNPs in the filtrate were determined by DLS. The results showed that the average hydration kinetic particle size of AOS-SeNPs was 108.5 ± 5.0 nm, and the Zeta potential was -39.5 ± 6.0 mV. There was no significant difference compared with that before loading (107.33 ± 3.75 nm, -40.82 ± 7.88 mV) (particle size P = 0.72, potential P = 0.68, paired Student's t-test, n = 3), proving that the preparation, cross-linking and release processes of the hydrogel did not damage the particle size distribution and surface properties of AOS-SeNPs.

[0125] (6) Validation of antioxidant and antibacterial activities

[0126] Take the release working solution of the hydrogel of the loaded composition obtained in step (2) (the concentrations of AOS-SeNPs and CGA were measured to be approximately 50 μg / mL) and perform the determination according to the methods of Example 3 and Example 4.

[0127] ABTS⁺ Free Radical Scavenging Experiment: Release of Working Solution and ABTS +The working solutions were mixed in equal volumes (final reaction concentration: AOS-SeNPs 25 μg / mL + CGA 25 μg / mL), reacted at 25°C in the dark for 6 minutes, and the scavenging rate was measured to be 94.2 ± 1.8% after background subtraction using the same method as in Example 3. Based on the single-component baseline values ​​measured in this release system, the Bliss independent action model predicted the scavenging rate Epred = 1 – (1–0.705)×(1–0.550) = 0.867 (86.7%). The synergistic coefficient K = 94.2% / 86.7% = 1.09, and its 95% confidence interval (1.05, 1.13, calculated using the Bootstrap resampling method, n=10000) has a lower limit greater than 1, indicating that the composition still exhibits a statistically significant synergistic antioxidant effect after loading.

[0128] Antibacterial activity (OD) 600 Method): After the working solution and bacterial culture were mixed in equal volumes, the growth inhibition rate was 98.8±0.5%, and the Bliss model predicted a value of 1 – (1–0.905)×(1–0.610) = 96.3%, K = 1.03, with a 95% confidence interval (0.98, 1.08, Bootstrap method, n=10000), showing an additive effect, consistent with the results of Example 4.

[0129] Antibacterial activity (plate count method): After treatment with the working solution, no culturable colonies were detected on LB agar plates in three independent experiments (100 μL of the incubated bacterial suspension was serially diluted and plated onto LB agar plates; detection limit <10 CFU / mL), meaning the viable count increased from approximately 10⁻⁶ CFU / mL initially. 8 The CFU / mL level was reduced to below the detection limit (<10 CFU / mL), achieving a functional leap in sterilization level, which is completely consistent with the conclusion of Example 4.

[0130] The above results demonstrate that the composition of the present invention, after being loaded onto a polysaccharide-based hydrogel, not only maintains the integrity of the nanostructure of AOS-SeNPs, but also fully preserves its synergistic antioxidant activity and ability to kill Staphylococcus aureus, exhibiting good formulation adaptability.

[0131] [Example 6] Application of the composition in medical dressings and related fields

[0132] The compositions of this invention can be used to prepare antioxidants that scavenge free radicals, bacteriostatic agents or bactericides that inhibit Gram-positive bacteria (especially Staphylococcus aureus), and can also be used as functional food additives or active ingredients in health products. Based on the synergistic antioxidant and bactericidal activity demonstrated in the hydrogel of the loaded composition in Example 5 above, the compositions can be further prepared into medical dressings, wound repair materials or antibacterial coatings for medical devices in the form of hydrogels, films, sprays or coatings, for biomedical applications requiring both antioxidant and antibacterial functions.

[0133] For example, the AOS-SeNPs solid powder obtained in Example 1 and chlorogenic acid (CGA) were added to deionized water at a mass ratio of 1:1. After stirring and mixing, a working solution (5.36 mL) of the composition with a concentration of 0.25 mg / mL for both AOS-SeNPs and CGA was obtained. This working solution was then mixed with 5 mL of sodium alginate solution (containing 0.1 g SA) and 5 mL of carboxymethyl chitosan solution (containing 0.1 g CMCS), and 1 mL of 2% CaCl2 solution was added for crosslinking and curing. The total mass of the gel after crosslinking was approximately 13.40 g, thus obtaining the composite hydrogel dressing loaded with the composition of the present invention. Based on the loading amount and the total mass of the gel, the theoretical concentrations of AOS-SeNPs and CGA in this hydrogel are both approximately 100 μg / mL, consistent with Example 5.

[0134] Similarly, the compositions of the present invention can be mixed with film-forming materials (such as polyvinyl alcohol, chitosan, etc.) to prepare antibacterial films by casting; or dispersed in a solution containing a propellant to prepare a spray; or an antibacterial coating can be constructed on the surface of medical devices by impregnation or spraying. These application methods do not change the synergistic activity of the composition.

[0135] It should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention and are not intended to constitute any limitation. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or legally equivalent substitutions can be made to some technical features; all such modifications or substitutions should not cause the essence of the corresponding technical solutions to deviate from the spirit and scope defined by the technical solutions of the embodiments of the present invention.

Claims

1. A composition of alginate oligosaccharide-nano-selenium-chlorogenic acid, characterized in that, The composition comprises alginate oligosaccharide-modified selenium nanoparticles (AOS-SeNPs) and chlorogenic acid (CGA); the mass ratio of AOS-SeNPs to CGA is 1:1; the weight-average molecular weight of the alginate oligosaccharide is 1000–4000 Da; the AOS-SeNPs are amorphous selenium nanoparticles, and their X-ray diffraction patterns show broad diffuse peaks at 2θ = 22°–24°, and no characteristic diffraction peaks of trigonal selenium are observed.

2. The composition according to claim 1, characterized in that, The AOS-SeNPs have an average hydration kinetic particle size of 90–120 nm, a polydispersity index (PDI) ≤ 0.25, and a zeta potential of -50 mV to -30 mV.

3. A method for preparing the composition according to claim 1, characterized in that, Includes the following steps: (1) Dissolve alginate oligosaccharides with a weight-average molecular weight of 1000-4000 Da in deionized water and stir until completely dissolved; (2) Add anhydrous sodium selenite to the solution obtained in step (1) and stir at room temperature to completely dissolve it to form a solution containing selenite ions; (3) Under light-protected conditions, the reducing agent is dissolved in deionized water and added dropwise to the mixture obtained in step (2). The mixture is stirred and reacted for 3 to 6 hours at room temperature under light-protected conditions to obtain AOS-SeNPs colloidal dispersion. (4) The AOS-SeNPs colloidal dispersion obtained in step (3) was purified by dialysis, and the purified AOS-SeNPs colloidal dispersion was collected; the AOS-SeNPs solid powder was obtained by freeze drying. (5) Add the AOS-SeNPs solid powder obtained in step (4) and chlorogenic acid to deionized water at a mass ratio of 1:1, stir to completely dissolve chlorogenic acid and uniformly disperse AOS-SeNPs, and the composition is obtained.

4. The preparation method according to claim 3, characterized in that, The concentration of alginate oligosaccharide in deionized water in step (1) is 3-5 mg / mL; the mass ratio of anhydrous sodium selenite to alginate oligosaccharide in step (2) is 0.43:1-0.44:1; the reducing agent in step (3) is L-ascorbic acid, and the molar ratio of L-ascorbic acid to anhydrous sodium selenite is 2:

1.

5. The preparation method according to claim 3, characterized in that, The dialysis purification in step (4) uses a dialysis bag with a molecular weight cutoff of 1 kDa, and uses deionized water as the external solution for dialysis in the dark for 24 hours, during which the water is changed 3 times.

6. A formulation comprising the composition of claim 1 and a pharmaceutically or food-grade acceptable carrier.

7. The formulation according to claim 6, characterized in that, The formulation is one or more of hydrogels, films, sprays, or coatings.

8. The formulation according to claim 7, characterized in that, The hydrogel is a polysaccharide-based hydrogel.

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