Method for in-situ preparation of iron oxide nanoparticles and composite material thereof through mediation of radix paeoniae rubra extract and application of iron oxide nanoparticles and composite material

Abstract

The invention discloses a method for mediating in-situ preparation of iron oxide nanoparticles and a composite material thereof by using a red paeony root extract, which is characterized in that the red paeony root extract as a traditional Chinese medicinal material is used as a reducing agent and a stabilizing agent, and mixed-dimensional attapulgite clay with one-dimensional rod-like and two-dimensional sheet-like morphology is used as a carrier; the iron oxide nanoparticles with small in-situ green synthesis size, obvious synergistic hemostatic effect and excellent biocompatibility and the composite material thereof effectively solve the practical application problems of relatively large particle size, relatively low activity, tedious preparation process, poor biocompatibility and the like of a composite material prepared by a non-green in-situ method. Natural traditional Chinese medicine extracts and clay minerals are selected as main raw materials, and the iron oxide nanoparticles and the composite material thereof prepared through a green, clean and simple method have good application prospects in the aspects of hemostasis and wound healing.

Description

Technical Field

[0001] The invention relates to the field of green in-situ synthesis of nanomaterials mediated by Chinese herbal medicine extracts, and in particular to a method and application of preparing iron oxide nanoparticles and composite materials thereof by using red peony root extracts. Background Art

[0002] Iron is an essential catalytic and structural element for proteins, enzymes and transcription factors in the body, and can inhibit or increase their expression levels and activities through conformational changes. About 72% of iron is present in hemoglobin, which is the main component of red blood cells in the blood. Studies have shown that iron can lead to reactive thrombocytosis and promote thrombosis (Arterioscl ThromVas, 2019, 39 (1): 37-47). In the late stage of hemostasis, iron oxide nanoparticles are physically coupled to thrombin, which not only retains the coagulation activity of thrombin, but also further enhances the coagulation activity of thrombin by inhibiting antithrombin III (Biomaterials, 31 (2010) 741-747). Iron is a cofactor for the hydroxylation of hypoxia-inducible factor (HIF-1α). Due to the chelation of iron, HIF-1α expression is upregulated, which increases the expression of growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and stromal cell-derived factor-1 (SDF-1), thereby stimulating angiogenesis and accelerating wound healing (Molecules, 2024, 29 (9), 2050; J. Res. Med. Sci. 2014, 19 (2), 164−174.). Therefore, studying the effect of iron on the body's life activities has become a research hotspot.

[0003] Among the numerous hemostatic and wound healing materials, mineral medicinal materials, as an important component of traditional Chinese medicine, have been used for thousands of years from ancient times to the present. Ochre, an oxide mineral corundum family hematite, mainly contains α-Fe2O3, which has the effects of calming the liver and suppressing yang, calming diarrhea, cooling blood and stopping bleeding, clearing away heat and detoxifying, and enhancing body immunity. In view of this, various composite hemostatic materials with Fe2O3 as the active ingredient have been developed. The methods for synthesizing iron oxide nanoparticles include coprecipitation, microemulsion, sol-gel, solvothermal, thermal decomposition and chemical methods to synthesize iron oxide nanoparticles (Biocatalysis and Agricultural Biotechnology, 2019, 20, 101251). These processes have problems such as the use of toxic chemical reagents and uneconomical reactions. In order to overcome the shortcomings of the above traditional synthesis methods, green in situ synthesis mediated by plant extracts has attracted much attention due to its low cost, environmental friendliness and rapidity.

[0004] Traditional Chinese medicines that are both medicinal and edible have a wide range of sources and low toxicity, and have unique advantages and great potential in hemostasis. The active substances contained in them, such as polyphenols, polysaccharides, saponins, flavonoids, and coenzymes, not only have the effects of promoting wound hemostasis, anti-inflammatory, antibacterial and healing, but also have the functions of reducing, encapsulating and stabilizing nanoparticles. Although there have been reports on the green synthesis of iron oxide nanoparticles using extracts such as camellia sinensis, aloe vera, cycas repens, arisaema root and nepeta tenuifolia fruit (Ahmmad et al., 2013, Mukherjee et al., 2016, Khalil et al., 2017, Narayanan and Sung, 2016, Bishnoi et al., 2018), they are rarely used in wound healing materials. Importantly, single iron oxide nanoparticles are easy to agglomerate and have poor stability, resulting in reduced biological activity.

[0005] Loaded nano-iron oxide composites have become a research trend because they can obtain uniformly dispersed and stable iron oxide nanoparticles. Attapulgite is abundant in nature and is a 2:1 chain-layered hydrated magnesium-aluminum clay mineral with a high specific surface area, unique one-dimensional rod crystal morphology (diameter is about 20-70 nm, length is about 0.5-5 μm), zeolite-like pore structure (size is 0.37 nm × 0.64 nm) and rich surface active groups. It is one of the ideal raw materials for constructing functional composite materials. Mixed-dimensional attapulgite clay is deposited in inland saltwater lake phases and is mainly composed of one-dimensional attapulgite, two-dimensional illite, chlorite, kaolinite and illite-montmorillonite mixed-layer minerals. It is not only a potential excellent hemostatic agent, but also can improve the size and stability of nanoparticles and effectively enhance the biological activity of nanoparticles. In addition, the nano-iron oxide and its composite materials prepared by using mixed-dimensional attapulgite clay as a carrier and plant extracts as a reducing agent, encapsulating agent and stabilizer have the advantages of no pollution, simple operation and low cost.

[0006] Red peony root is a famous wild authentic Chinese medicinal material with the effects of clearing heat and cooling blood, dispersing blood stasis and relieving pain, and has a long history of application. As early as in the "Shennong Bencao Jing", it was recorded that "peony root has a bitter and flat taste. It can treat abdominal pain caused by evil qi, remove blood stasis, break up hard masses, cold and heat hernia, and relieve pain". It mainly contains active ingredients such as paeoniflorin, paeoniflorin lactone glycosides, catechins, ellagic acid and gallic acid, and has the effects of reduction, stabilization, antibacterial and antioxidant (CN202010539277.7, China Food Additives, 2024, 35). Therefore, the use of mixed-dimensional attapulgite clay with authentic Chinese medicinal material red peony root extract and excellent carrier properties and hemostatic properties is expected to prepare iron oxide nanoparticles with higher activity and utilization rate and its composite hemostatic material, and there are no related research reports on this technology, and there is no precedent for its application. Summary of the invention

[0007] The present invention aims to provide a method and application of in situ preparation of iron oxide nanoparticles and composite materials thereof mediated by red peony root extract, wherein the active ingredients in red peony root are used as reducing agents and stabilizers, and iron oxide nanoparticles and mixed-dimensional attapulgite clay-loaded nano-iron oxide composite materials are synthesized in situ by a one-step method, the preparation process is simple and green, and the nanoparticles have good hemostatic properties and biocompatibility.

[0008] 1. Preparation and structure of iron oxide nanoparticles and their composite materials 1. Preparation of iron oxide nanoparticles The method for preparing iron oxide nanoparticles in situ by using red peony root extract comprises the following steps: (1) Soaking red peony root tablets in water, extracting by ultrasonic wave, filtering, and obtaining a red peony root extract solution; the solid-liquid ratio of the red peony root tablets to water is 1:5 to 1:30; the soaking time is 12 to 36 hours; and the ultrasonic extraction time is 0.5 to 2 hours.

[0009] (2) dissolving an iron salt in deionized water under stirring to obtain an iron salt solution; the iron salt is at least one of ferrous sulfate heptahydrate, ferric chloride hexahydrate, and ferric acetate tetrahydrate.

[0010] (3) Adding the red peony root extract solution to the iron salt solution, stirring to obtain a mixed solution, reacting at room temperature to 90°C for 15 to 120 min, centrifuging, washing, and drying to obtain iron oxide nanoparticles. The concentration of the iron salt in the mixed solution is 0.001 to 0.2 mol / L; the volume ratio of the red peony root extract solution to the iron salt solution is 1:1 to 1:5; and drying is performed at 30 to 60°C under vacuum for 10 to 36 h.

[0011] 2. Preparation of iron oxide nanoparticle composites (1) Soaking red peony root tablets in water, extracting by ultrasonic wave, filtering, and obtaining a red peony root extract solution; the solid-liquid ratio of the red peony root tablets to water is 1:5 to 1:30; the soaking time is 12 to 36 hours; and the ultrasonic extraction time is 0.5 to 2 hours.

[0012] (2) Dispersing mixed-dimensional attapulgite clay in deionized water under stirring, adding iron salt to obtain a mixed solution of attapulgite iron salt; the iron salt is at least one of ferrous sulfate heptahydrate, ferric chloride hexahydrate, and ferric acetate tetrahydrate; the mass ratio of the mixed-dimensional attapulgite clay to the iron salt is 1:0.5 to 1:3. The mixed-dimensional attapulgite clay is composed of one-dimensional rod-shaped attapulgite and two-dimensional flaky illite, chlorite, kaolinite, and illite-montmorillonite mixed layer minerals.

[0013] (3) Adding the red peony root extract solution to the attapulgite iron salt mixture, stirring to obtain a mixed solution, reacting at room temperature to 90°C for 15 to 120 min, centrifuging, washing, and drying to obtain an iron oxide nanoparticle / mixed-dimensional attapulgite clay composite material. The concentration of the iron salt in the mixed solution is 0.001 to 0.2 mol / L; the volume ratio of the red peony root extract solution to the attapulgite iron salt mixture is 1:1 to 1:5; and the drying is vacuum drying at 30 to 60°C for 10 to 36 h.

[0014] The red peony root extract used in the present invention contains polyphenol active ingredients such as paeoniflorin, catechins, ellagic acid and gallic acid, and the unreacted active substances help accelerate wound hemostasis. The one-dimensional nanorod crystals and two-dimensional flake morphology in the mixed-dimensional attapulgite clay are used as carriers, and the prepared iron oxide nanoparticles are more uniform and smaller in size, while enhancing the performance of the composite material.

[0015] Figure 1 The XRD diagram of the iron oxide nanoparticles and the composite material thereof prepared by the present invention. Since the synthesized iron oxide nanoparticles are amorphous and small in size, the plant extracts that do not fully participate in the reaction will be wrapped outside the nanoparticles, and XRD fails to detect its characteristic diffraction peaks. In addition to the characteristic diffraction peaks of mixed-dimensional attapulgite clay, the iron oxide nanoparticle composite material shows characteristic diffraction peaks of iron oxide when compared with the standard card, indicating the successful synthesis of the iron oxide nanoparticles.

[0016] Figure 2 The ultraviolet absorption spectrum of the iron oxide nanoparticles synthesized by the present invention and the composite material thereof. Compared with the ultraviolet absorption peaks of pure red peony root extract at 218 nm and 273 nm, the ultraviolet absorption peak of the iron oxide nanoparticles basically retains the absorption peak of the pure extract, but shifts to 220 nm and 285 nm. The disappearance of the absorption peak of polyphenols in the composite material further illustrates that the mixed-dimensional attapulgite clay as a carrier effectively improves the synthesis of iron oxide nanoparticles.

[0017] Figure 3 The TEM image of the synthesized iron oxide nanoparticles and the composite material thereof of the present invention is shown. The prepared iron oxide nanoparticles are spherical and exist in the form of aggregates. However, the introduction of mixed-dimensional attapulgite clay with one-dimensional rod crystals and two-dimensional flaky morphology effectively improves the dispersibility and stability of the iron oxide nanoparticles.

[0018] 2. Properties of Iron Oxide Nanoparticles and Their Composite Materials The hemostatic performance of the iron oxide nanoparticles and the composite materials thereof prepared by the present invention is mainly evaluated by the relative hemoglobin concentration. The relative hemoglobin concentration test method is as follows: according to the ratio of 1:10 (mg / μL) between the sample and the sodium citrate anticoagulated whole blood, the coagulation is started after vortexing evenly, and ultrapure water is added at different time points to terminate the coagulation reaction. The uncoagulated red blood cells absorb water and burst to release hemoglobin, and the absorbance of the supernatant is measured at 540 nm. The lower the absorbance value, the higher the coagulation efficiency of whole blood. The hemostatic effects of 1-5 in Examples are shown in Table 1. Compared with the group without material, the iron oxide nanoparticles and the composite materials prepared by the present invention have a stronger hemostatic effect.

[0019] The biocompatibility of the iron oxide nanoparticles and the composite materials prepared by the present invention is mainly evaluated by cytotoxicity and hemolysis rate. The cytotoxicity test method is as follows: mouse embryonic fibroblasts (NIH-3T3) (density 4×10 4 After the cells were inoculated and attached to the wall, they were cultured with culture medium containing fetal bovine serum, double antibody and material suspension. After culturing in a 37°C 5% CO2 incubator for 24 h, CCK-8 reagent was added and the absorbance value was measured at 405 nm. The control group containing only culture medium was used as the control group, and the relative cell survival rate was calculated according to the following formula: Relative cell viability (%) = absorbance value 样品 / Absorbance value 对照 ×100% The hemolysis rate test method is as follows: Sodium citrate anticoagulated whole blood is centrifuged at low speed to obtain red blood cells, and then diluted with phosphate buffered solution (PBS) to a 5% (v / v) red blood cell solution. Add 780 μL PBS and 200 μL 5% (v / v) red blood cell solution to a test tube containing 20 μL sample suspension. After incubation at 37°C for 1 h, centrifuge, take the supernatant and measure the absorbance at 540 nm to calculate the hemolysis rate. Among them, 0.1% Triton X-100 (+5% red blood cells) and PBS (+5% red blood cells) were used as positive and negative control groups, respectively.

[0020] Hemolysis rate (%) = (absorbance value of material group - absorbance value of negative control) × 100% / (absorbance value of positive control - absorbance value of negative control) Figure 4 and Figure 5 The cytotoxicity and hemolysis rate results of the iron oxide nanoparticles and the composite materials prepared by the present invention are respectively: the cell viability of the iron oxide nanoparticles and the composite materials thereof are both greater than 80%, and the hemolysis rate is both less than 5%, indicating that the materials obtained by the present invention have excellent biocompatibility.

[0021] Therefore, the iron oxide nanoparticles and the composite material thereof prepared by the present invention can be used as a simple and convenient first aid trauma hemostatic material or wound healing material.

[0022] In summary, the present invention uses the extract of Chinese medicinal material red peony root as a reducing agent and stabilizer, and uses mixed-dimensional attapulgite clay with one-dimensional rod-shaped and two-dimensional flaky morphology as a carrier to in-situ green synthesize iron oxide nanoparticles and composite materials with small size, obvious synergistic hemostatic effect and excellent biocompatibility, which effectively solves the practical application problems of non-green in-situ preparation of composite materials, such as large particle size, low activity, cumbersome preparation process and poor biocompatibility. The present invention selects natural Chinese medicine extracts and clay minerals as the main raw materials, and the iron oxide nanoparticles and composite materials prepared by green, clean and simple methods have good application prospects in the field of wound healing.

[0023] Compared with the prior art, the advantages of the present invention are: (1) The medicinal plant resources and mineral resources used in the present invention are easily available and non-toxic and harmless.

[0024] (2) The hemostatic performance of the composite material prepared by using mixed-dimensional attapulgite clay in the present invention is better than that of the materials prepared by single attapulgite, montmorillonite, kaolinite, halloysite, sepiolite, zeolite and the commercially available product Yunnan Baiyao.

[0025] (3) The present invention uses mixed-dimensional attapulgite clay with hemostatic function and carrier properties, and the active ingredients such as paeoniflorin, catechins, ellagic acid and gallic acid in the red peony root extract and the in-situ synthesized iron oxide nanoparticles synergistically significantly enhance the hemostatic performance and biocompatibility of the composite material. The preparation process of the material obtained by the present invention is simple, clean and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The XRD diagram of the iron oxide nanoparticles and the composite material thereof prepared by the present invention; Figure 2 The ultraviolet absorption spectrum of the iron oxide nanoparticles and the composite material thereof prepared by the present invention; Figure 3 TEM images of the iron oxide nanoparticles and the composite materials thereof prepared by the present invention (ac: iron oxide nanoparticles; d: mixed-dimensional attapulgite clay; ef: composite materials); Figure 4 The cytotoxicity diagram of the iron oxide nanoparticles and the composite materials prepared by the present invention; Figure 5 This is a graph showing the hemolysis rate of the iron oxide nanoparticles and their composite materials prepared in the present invention. DETAILED DESCRIPTION

[0027] The following is a specific example to further illustrate the method of the present invention for in-situ preparation of iron oxide nanoparticles and composite materials thereof using red peony root extract.

[0028] Example 1 1) 15 g of red peony root tablets were added into 250 mL of deionized water, soaked for 20 h, and ultrasonically extracted for 1.5 h before filtering to obtain red peony root extract solution; 2) 2.70 g of ferric chloride hexahydrate was dissolved in 70 ml of deionized water under stirring to obtain ferric chloride hexahydrate solution; 3) 30 mL of red peony root extract solution was added into the ferric chloride hexahydrate solution and stirred to obtain a mixed solution, wherein the concentration of ferric chloride hexahydrate in the mixed solution was 0.1 mol / L, the mixed solution was reacted at room temperature for 40 min, centrifuged, washed, and vacuum dried at 45°C for 24 h to obtain iron oxide nanoparticles.

[0029] Example 2 1) 10 g of red peony root tablets were added into 250 mL of deionized water, soaked for 30 h, and ultrasonically extracted for 2.0 h, then filtered to obtain a red peony root extract solution; 2) 1.23 g of ferric acetate tetrahydrate was dissolved in 60 ml of deionized water under stirring to obtain a ferric acetate tetrahydrate solution; 3) 40 mL of red peony root extract solution was added into the ferric acetate tetrahydrate solution, stirred to obtain a mixed solution, the concentration of ferric acetate tetrahydrate in the mixed solution was 0.050 mol / L, the mixed solution was reacted at room temperature for 30 min, centrifuged, washed, and vacuum dried at 50°C for 20 h to obtain iron oxide nanoparticles.

[0030] Example 3 1) 20 g of red peony root tablets were added into 250 mL of deionized water, soaked for 18 h, and ultrasonically extracted for 1.0 h before filtering to obtain red peony root extract solution; 2) 0.139 g of ferrous sulfate was dissolved in 55 ml of deionized water under stirring to obtain ferrous sulfate solution; 3) 45 mL of red peony root extract was added into the ferrous sulfate solution and stirred to obtain a mixed solution, in which the concentration of ferrous sulfate in the mixed solution was 0.005 mol / L. The mixed solution was reacted at 60 °C for 60 min, centrifuged, washed, and vacuum dried at 55 °C for 16 h to obtain iron oxide nanoparticles.

[0031] Example 4 1) 20 g of red peony root tablets were added into 250 mL of deionized water and soaked for 15 h. After ultrasonic extraction for 0.5 h, the red peony root extract solution was obtained by filtration. 2) 0.5 g of mixed-dimensional attapulgite clay was dispersed into 70 ml of deionized water under stirring. After ultrasonic treatment, 0.675 g of ferric chloride hexahydrate was added to obtain a attapulgite iron salt mixture. 3) 30 mL of red peony root extract was added to the attapulgite iron salt mixture so that the concentration of ferric chloride hexahydrate was 0.025 mol / L. Then, the mixture was reacted at 60 °C for 30 min, centrifuged, washed, and vacuum dried at 45 °C for 22 h to obtain an iron oxide nanoparticle / mixed-dimensional attapulgite clay composite material.

[0032] Example 5 1) 25 g of red peony root tablets were added into 250 mL of deionized water, soaked for 18 h, and ultrasonically extracted for 1.0 h before filtering to obtain a red peony root extract solution; 2) 0.5 g of mixed-dimensional attapulgite clay was dispersed into 55 ml of deionized water under stirring, and 0.278 g of ferrous sulfate was added after ultrasonic treatment to obtain a attapulgite iron salt mixture; 3) 45 mL of red peony root extract solution was added to the attapulgite iron salt mixture to make the ferrous sulfate concentration of 0.010 mol / L, then reacted at 60 ℃ for 60 min, centrifuged, washed, and vacuum dried at 660 ℃ for 18 h to obtain an iron oxide nanoparticle / mixed-dimensional attapulgite clay composite material.

Claims

1. A method for in situ preparation of iron oxide nanoparticles mediated by red peony root extract, comprising the following steps: (1) Soaking red peony root tablets in water, extracting by ultrasonic, and filtering to obtain a red peony root extract solution; (2) dissolving an iron salt in deionized water under stirring to obtain an iron salt solution; (3) Add the red peony root extract solution to the iron salt solution, stir to obtain a mixed solution, react at room temperature to 90°C for 15 to 120 min, centrifuge, wash, and dry to obtain iron oxide nanoparticles.

2. The method for in-situ preparation of iron oxide nanoparticles mediated by Chinese herbal medicine extracts as claimed in claim 1, characterized in that: In step (1), the solid-liquid ratio of the red peony root tablets to water is 1:5 to 1:30; the soaking time is 12 to 36 hours; and the ultrasonic extraction time is 0.5 to 2 hours.

3. The method for in-situ preparation of iron oxide nanoparticles mediated by Chinese herbal medicine extracts as claimed in claim 1, characterized in that: In step (2), the iron salt is at least one of ferrous sulfate heptahydrate, ferric chloride hexahydrate, and ferric acetate tetrahydrate.

4. The method for in-situ preparation of iron oxide nanoparticles mediated by Chinese herbal medicine extracts as claimed in claim 1, characterized in that: In step (3), the concentration of the iron salt in the mixed solution is 0.001-0.2 mol / L; the volume ratio of the red peony root extract solution to the iron salt solution is 1:1-1:5; and the drying is performed at 30-60°C under vacuum for 10-36 hours.

5. Use of the iron oxide nanoparticles prepared by the method of claim 1 as a hemostatic material or a wound healing material.

6. A method for in-situ preparation of iron oxide nanoparticle composite materials mediated by red peony root extract, comprising the following steps: (1) Soaking red peony root tablets in water, extracting by ultrasonic, and filtering to obtain a red peony root extract solution; (2) dispersing the mixed-dimensional attapulgite clay in deionized water under stirring, and adding iron salt to obtain an attapulgite iron salt mixed solution; (3) Add the red peony root extract solution to the attapulgite iron salt mixture, stir to obtain a mixed solution, react at room temperature to 90°C for 15 to 120 min, centrifuge, wash, and dry to obtain an iron oxide nanoparticle / mixed-dimensional attapulgite clay composite material.

7. The method for in-situ preparation of iron oxide nanoparticle composite material mediated by Chinese herbal medicine extracts as claimed in claim 6, characterized in that: In step (1), the solid-liquid ratio of the red peony root tablets to water is 1:5 to 1:30; the soaking time is 12 to 36 hours; and the ultrasonic extraction time is 0.5 to 2 hours.

8. The method for in-situ preparation of iron oxide nanoparticle composite material mediated by Chinese herbal medicine extracts as claimed in claim 6, characterized in that: In step (2), the iron salt is at least one of ferrous sulfate heptahydrate, ferric chloride hexahydrate, and ferric acetate tetrahydrate; and the mass ratio of the mixed-dimensional attapulgite clay to the iron salt is 1:0.5 to 1:

3.

9. The method for in-situ preparation of iron oxide nanoparticle composite material mediated by Chinese herbal medicine extracts as claimed in claim 6, characterized in that: In step (3), the concentration of the iron salt in the mixed solution is 0.001-0.2 mol / L; the volume ratio of the red peony root extract solution to the attapulgite iron salt mixed solution is 1:1-1:5; and the drying is performed at 30-60°C in vacuum for 10-36 hours.

10. Use of the iron oxide nanoparticle composite material prepared by the method according to claim 6 as a hemostatic material or wound healing material.

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