Cnidium monnieri extract-mediated synthesis of nanosilver material and preparation method and application thereof

Abstract

The application discloses a Cnidii Fructus extract-mediated synthesized nano-silver material and a preparation method and application thereof. The nano-silver material is in a spherical core-shell structure, the inside of which is aggregated silver ions, and the outside of which is covered with Cnidii Fructus extract. The nano-silver material is synthesized by Cnidii Fructus extract and silver nitrate, and the average particle size is 44.6 nm. The nano-silver material has good antifungal activity on Trichophyton rubrum, Trichophyton mentagrophytes and Candida albicans, and the minimum inhibitory concentrations (MIC 90 ) are 3.125 μg / mL, 3.125 μg / mL and 0.78125 μg / mL, respectively. The Cnidii Fructus extract, which is commonly used in the treatment of tinea, is used to synthesize nano-silver, the synthesis method is simple, the particle size is small, the stability is good, and the nano-silver is highly dispersed. The preparation method is green and environment-friendly, and can be popularized on a large scale as the preparation of high-efficiency antifungal materials.

Description

Technical Field

[0001] This invention relates to the field of nanosilver materials technology, and more specifically, to a method for synthesizing nanosilver materials mediated by Cnidium monnieri extract, and its preparation and application. Background Technology

[0002] Superficial fungal infections, including skin and mucosal skin infections, are a prevalent public health problem caused by dermatophytes, Candida, and Malassezia. Superficial fungal infections typically cause chronic, non-inflammatory lesions, requiring long treatment courses, prone to recurrence, and causing pain and aesthetic issues that impact patients' quality of life. Underlying comorbidities such as diabetes, cancer, immunodeficiency, or peripheral artery disease can increase susceptibility to superficial fungal infections. Trichophyton rubrum, Trichophyton mentagrophytes, and Candida albicans are among the most important pathogens causing most dermatophyte infections. Drug resistance in dermatophytes can lead to treatment failure and persistent infections. To mitigate and prevent the development of drug resistance, researchers are investigating alternative treatments for superficial infections.

[0003] In recent years, nanoparticles have been widely used in various industries. Due to the need to reduce or eliminate the use or generation of toxic and harmful substances in compounds, reduce environmental pollution, and develop more sustainable methods, the biosynthesis of nanoparticles using natural product extracts has been proposed as a harmless, rapid, and efficient alternative synthetic route. Among these, the biosynthesis of silver nanoparticles uses a safe and non-toxic method, utilizing bioactive molecules with reducing, capping, and stabilizing functions to produce biocompatible nanoparticles suitable for numerous medical applications. Using plant extracts to biosynthesize silver nanoparticles allows the active ingredients from the plants to coat the nanoparticles, stabilizing them and enhancing their antifungal activity.

[0004] Nano-silver was synthesized from dried, mature fruit of Cnidium monnieri using a simple biosynthetic method with its extract. The synthesized nano-silver exhibits antifungal activity. The green synthesis of nano-silver using Cnidium monnieri extract is simple, does not use toxic reagents, and is relatively safe and environmentally friendly. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing antifungal properties of nano-silver particle antifungal materials, which need to be further improved, and to provide a method for synthesizing nano-silver materials mediated by Cnidium monnieri extract, as well as its preparation method and antifungal application.

[0006] The first aspect of this invention provides a method for preparing nano-silver materials mediated by Cnidium monnieri extract, the method being as follows:

[0007] Weigh out the Cnidium monnieri herb, soak it in 75% ethanol, heat and reflux to extract, combine the extracts and concentrate under reduced pressure until there is no alcohol odor, and freeze dry to obtain Cnidium monnieri extract; at the same time, dilute the Cnidium monnieri extract with pure water and filter to obtain Cnidium monnieri extract solution.

[0008] The pH of the Cnidium monnieri extract was adjusted using an alkaline solution. The pH-adjusted Cnidium monnieri extract was then slowly added dropwise to a silver nitrate solution with continuous stirring. After the reaction was complete, the extract was centrifuged to precipitate, washed three times, and the precipitate was collected to obtain the nano-silver material mediated by the Cnidium monnieri extract.

[0009] The specific method is as follows:

[0010] The main active components of Cnidium monnieri extract are coumarins, flavonoids, and phenolic acids, which are extracted by the following method: 50g of Cnidium monnieri is weighed and soaked in 500mL of 75% ethanol for 0.5h. The mixture is then heated under reflux and extracted twice, 2h each time. The extracts are combined and concentrated under reduced pressure until no alcohol odor remains. The extract is then freeze-dried to obtain the Cnidium monnieri extract. Simultaneously, the Cnidium monnieri extract is diluted with pure water to 250mL and filtered through a 0.22μm microporous membrane to obtain the Cnidium monnieri extract solution.

[0011] The pH of the above Cnidium monnieri extract was adjusted to 10-12 using NaOH solution (0.1M). The pH-adjusted Cnidium monnieri extract was slowly added dropwise to silver nitrate solution. The mixture was stirred continuously at 700 rpm for 30 min at room temperature using a magnetic stirrer. After the reaction was completed, the mixture was centrifuged to precipitate the precipitate. The precipitate was collected and freeze-dried to obtain nano-silver synthesized mediated by Cnidium monnieri extract.

[0012] Furthermore, in the biosynthesis process of the nano-silver material of the present invention, the mass ratio of Cnidium monnieri extract to silver nitrate is 260:1200 to 13.6:68. A mass ratio exceeding this range will reduce the yield of the nanomaterial and affect the uniformity of its size, thereby reducing its antifungal properties.

[0013] Furthermore, the mass ratio of the Cnidium monnieri extract to silver nitrate is 1200:62.492.

[0014] Silver ions (Ag) + After being reduced by chemical components in plants, it forms silver atoms (Ag). 0 Silver atoms then slowly aggregate into small silver nanoparticles. During this process, phytochemicals restrict AgNPs, preventing the silver from getting close to each other and continuing to grow at the nanoscale, thus forming small-diameter silver nanoparticles.

[0015] A second aspect of the present invention provides a method for synthesizing nano-silver materials mediated by Cnidium monnieri extract prepared by the above method.

[0016] Furthermore, the silver nanoparticles synthesized through the Cnidium monnieri extract have a spherical core-shell structure, with silver nanoparticles inside and Cnidium monnieri extract molecules covering the outside. They are synthesized from Cnidium monnieri extract and silver nitrate.

[0017] Furthermore, the average particle size of the silver nanoparticles synthesized through the Cnidium monnieri extract is 44.6 nm.

[0018] The third aspect of this invention provides the application of the above-mentioned Cnidium monnieri extract-mediated synthesis of nano-silver in the preparation of antifungal products.

[0019] Furthermore, the fungal species used in the antibacterial application of the present invention are one or more of Trichophyton rubrum, Trichophyton mentagrophytes, or Candida albicans.

[0020] The *Trichophyton rubrum* is *Trichophyton rubrum* BNCC340195; the *Trichophyton mentagrophytes* is *Trichophyton mentagrophytes* BNCC340405; and the *Candida albicans* is *Candida albicans* BNCC263676.

[0021] The antifungal concentration of the nanosilver material synthesized by the Cnidium monnieri extract ranges from 0.78125 to 3.125 μg / mL.

[0022] The antifungal effect of nanosilver is achieved through two main mechanisms: firstly, by influencing fungal morphology, causing membrane permeability, and generating reactive oxygen species to disrupt osmotic balance, thereby destabilizing cells and inhibiting fungal growth; and secondly, by releasing silver ions (Ag) with high affinity. + This process inactivates the sulfhydryl groups in the fungal cell wall, forming insoluble compounds, which then destroy membrane-bound enzymes and lipids, ultimately leading to fungal cell lysis. At the same time, the active molecules derived from Cnidium monnieri encapsulated on the periphery of the nano-silver material also have an inhibitory effect on fungi.

[0023] The beneficial effects of this invention are as follows:

[0024] (1) The method for preparing nano-silver mediated by Cnidium monnieri extract provided by the present invention directly uses the active molecules in Cnidium monnieri extract as reducing agent and stabilizer to prepare nano-silver mediated by Cnidium monnieri extract through a simple biosynthesis method. The operation is simple, uses plant extract, is green and environmentally friendly, and will not cause pollution to the environment. In addition, the raw material cost and operating cost of this method are low, the product purity is high, and it can be prepared in batches, which has good application prospects.

[0025] (2) The average particle size of the silver nanoparticles synthesized by the method provided in this invention through the Cnidium monnieri extract is 44.6 nm, with regular morphology and uniform size. The synthesized silver nanoparticles have an inner layer of silver nanoparticles and an outer layer of active ingredients from the Cnidium monnieri extract. Due to the phytochemicals limiting the silver nanoparticles, they cannot approach each other and continue to grow at the nanoscale, thus forming small-sized silver nanoparticles. The silver nanoparticles synthesized by the common tinea treatment herb Cnidium monnieri are used to treat fungal skin diseases.

[0026] (3) The method provided in this invention produces Cnidium monnieri extract mediated by the synthesis of nano-silver with good antifungal effects. It exhibits excellent antifungal activity against Trichophyton rubrum, Trichophyton mentagrophytes, and Candida albicans, with a minimum inhibitory concentration (MIC) of [missing value]. 90 The concentrations were 3.125 μg / mL, 3.125 μg / mL, and 0.78125 μg / mL, respectively. Attached Figure Description

[0027] Figure 1 UV-Vis absorption spectrum of nano-silver synthesized from Cnidium monnieri extract.

[0028] Figure 2 FTIR analysis of silver nanoparticles synthesized by Cnidium monnieri-mediated biosynthesis.

[0029] Figure 3 XRD analysis of silver nanoparticles synthesized by Cnidium monnieri-mediated biosynthesis.

[0030] Figure 4 Transmission electron microscope image of nanosilver synthesized from Cnidium monnieri extract.

[0031] Figure 5 A 96-well plate image showing the antifungal properties of nanosilver synthesized from Cnidium monnieri extract via bio-mediated synthesis. Detailed Implementation

[0032] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto. Example 1

[0033] A nano-silver material synthesized via Cnidium monnieri extract, wherein the nano-silver material is synthesized from Cnidium monnieri extract and silver nitrate, wherein the mass ratio of Cnidium monnieri extract to silver nitrate is 1200:62.492.

[0034] The specific synthesis method is as follows:

[0035] The main active components of Cnidium monnieri extract are coumarins, flavonoids, and phenolic acids, which are extracted by the following method: 50g of Cnidium monnieri is weighed and soaked in 500mL of 75% ethanol for 0.5h. The mixture is then heated under reflux and extracted twice, 2h each time. The extracts are combined and concentrated under reduced pressure until no alcohol odor is detected. The extract is then freeze-dried to obtain Cnidium monnieri extract. Meanwhile, the Cnidium monnieri extract is diluted with pure water to 250mL and filtered through a 0.22μm microporous membrane to obtain Cnidium monnieri extract solution.

[0036] The pH of the above Cnidium monnieri extract was adjusted to 11.75 using NaOH solution (0.1M). 6 mL of the pH-adjusted Cnidium monnieri extract was slowly added dropwise to 20 mL of 18.38 mM silver nitrate solution. The mixture was stirred continuously at 700 rpm for 30 min at room temperature using a magnetic stirrer. After the reaction was completed, the mixture was centrifuged to precipitate the precipitate. The precipitate was then freeze-dried to obtain the nano-silver material mediated by Cnidium monnieri extract.

[0037] Characterization of nano-silver:

[0038] (1) Ultraviolet-Vis (UV-Vis) spectrum

[0039] Plant extracts are used as reducing agents to remove silver ions (Ag) + ) transformed into nano silver (Ag) 0 This yielded nano-silver synthesized via Cnidium monnieri extract. UV-Vis analysis confirmed the synthesis of the nano-silver. Figure 1 The silver nanoparticles synthesized via Cnidium monnieri extract exhibited a surface plasmon resonance (SPR) peak at 420 nm. Since the typical SPR characteristic range for silver nanoparticles is 350–450 nm, this confirms the successful synthesis of silver nanoparticles in this experiment. The synthesis of AgNPs in this experiment utilized pH adjustment, which accelerated the synthesis and reduced the particle size. pH has a certain influence on the synthesis of AgNPs; under alkaline conditions, the hydroxyl groups in the plant extract more readily lose H+. + This results in the entire molecule carrying a negative charge, making the phytochemical more readily react with Ag. + It has a function, and it is more likely to lose electrons and undergo reduction reactions.

[0040] (2) Fourier transform infrared (FT-IR) spectrum

[0041] FTIR analysis was performed to identify the major phytochemicals involved in the biosynthesis and capping of silver nanoparticles. IR spectral peaks confirmed the binding of silver ions to Cnidium monnieri extract. The FTIR spectra of AgNPs biosynthesized by Cnidium monnieri extract were compared with those of Cnidium monnieri extract. Figure 2Biosynthesized AgNPs showed corresponding FTIR signals, OH (3408 cm⁻¹). −1 ), CH (2924cm) −1 ), benzene ring skeleton (1609cm) −1 ), -CH3 (1384cm) −1 ) and -CO (1054cm −1 These signals match the corresponding peaks in the FTIR spectrum of Cnidium monnieri extract. This indicates that many organic functional groups from Cnidium monnieri extract actually remain on the surface of the silver nanoparticles synthesized through Cnidium monnieri extract mediated by the extract.

[0042] (3) X-ray diffraction (XRD) analysis

[0043] The structure of nano-silver synthesized mediated by Cnidium monnieri extract was analyzed by XRD measurement. Figure 3 It can be seen that the main diffraction peaks are located at 38.08°, 44.24°, 64.46° and 77.46°, pointing to the (111), (200), (220) and (311) diffraction planes, respectively. The nanosilver synthesized by Cnidium monnieri extract shows the diffraction peak characteristics of a metal face-centered cubic structure (JCPDS file No. 4–0783), indicating that the nanosilver particles formed in this synthesis are essentially crystalline. From the peak intensity ratio of (111) with other diffraction peaks, it can be concluded that the (111) plane is the main orientation in the silver crystal structure of Cm-AgNPs.

[0044] (4) Transmission electron microscopy (TEM) analysis

[0045] The shape and size of silver nanoparticles synthesized through Cnidium monnieri extract can be directly observed using TEM. Figure 4 As can be seen, the synthesized AgNPs have a diameter of approximately 44.6 nm, are uniformly distributed, and the active ingredients in the extract adhere to the surface of the AgNPs, preventing particle aggregation. Furthermore, the synthesized AgNPs are spherical. Example 2

[0046] A nano-silver material synthesized via Cnidium monnieri extract, wherein the nano-silver material is synthesized from Cnidium monnieri extract and silver nitrate, wherein the mass ratio of Cnidium monnieri extract to silver nitrate is 400:42.5.

[0047] The specific synthesis method is as follows:

[0048] The preparation method of Cnidium monnieri extract is the same as in Example 1.

[0049] The pH of the above Cnidium monnieri extract was adjusted to 12 using NaOH solution (0.1M). 2 mL of the pH-adjusted Cnidium monnieri extract was slowly added dropwise to 20 mL of 12.5 mM silver nitrate solution. The mixture was stirred continuously at 700 rpm for 30 min at room temperature using a magnetic stirrer. After the reaction was completed, the mixture was centrifuged to precipitate the precipitate. The precipitate was then freeze-dried to obtain the nano-silver synthesized mediated by Cnidium monnieri extract.

[0050] Dynamic light scattering revealed that the average particle size of the silver nanomaterial was 75.84 nm and the PDI was 0.3640. UV-Vis analysis showed that the synthesized silver nanomaterial exhibited a surface plasmon resonance peak at 420 nm, confirming the successful synthesis of the silver nanomaterial. Example 3

[0051] An experiment on the inhibitory effect of nano-silver synthesized from Cnidium monnieri extract on dermatophyte pathogens.

[0052] Experimental materials:

[0053] The standard strains of *Trichophyton rubrum* (BNCC340195), *Trichophyton mentagrophytes* (BNCC340405), and *Candida albicans* (BNCC263676) were all purchased from Beijing Beina Chuanglian Biotechnology Research Institute. RPMI-1640 liquid culture medium was purchased from Wuhan Boster Biological Engineering Co., Ltd. Potato dextrose agar plates (PDA) were purchased from Wenzhou Kangtai Biotechnology Co., Ltd. Sabouraud agar plates (SDA) were purchased from Zhengzhou Antu Biotechnology Co., Ltd. Fluconazole and terbinafine were both purchased from the China National Institutes for Food and Drug Control.

[0054] Instruments and equipment:

[0055] Infinite E Plex microplate reader (Tecan, Austria)

[0056] BSC-1360ⅡA2 Biosafety Cabinet (Beijing Donglian Haer Instrument Manufacturing Co., Ltd.)

[0057] BC-J160 Cell Culture Incubator (Shanghai Boxun Medical Bio-Instrument Co., Ltd.)

[0058] Experimental methods:

[0059] This experiment used the Broth Microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) CLSI-M27 and CLSI-M38 documents to prepare a bio-mediated synthesis of nano-silver from Cnidium monnieri extract (25 μg / mL - 0.098 μg / mL).

[0060] According to CLSI-M38, *Trichophyton rubrum* and *Trichophyton mentagrophytes* strains were inoculated onto PDA medium and cultured at 28°C for 7–14 days prior to the experiment. To prepare the bacterial suspension: spore counts were determined using a cell counting chamber, and the suspension was diluted with RPMI-1640 medium to adjust the concentration to approximately 7.1 × 10⁻⁶. 4 –1.1×10 5 CFU / mL. Incubated in a constant temperature incubator at 28℃ for 7 days.

[0061] According to CLSI-M27, *Candida albicans* was inoculated onto SDA medium and incubated at 35°C for 24 h before the experiment. To prepare the bacterial suspension: spore counts were determined using a cell counting chamber, and the suspension was diluted with RPMI-1640 medium to adjust the bacterial concentration to approximately 5.9 × 10⁻⁶. 4 CFU / mL. RPMI-1640 medium was added to 96-well plates, and the samples were diluted to different concentrations using the broth microdilution method, and incubated at 35°C for 24 h.

[0062] Experimental results:

[0063] The results are shown in Table 1. The minimum concentration well with an inhibition rate greater than 90% calculated using the OD value scanned at 630 nm using a microplate reader is denoted as MIC. 90 The results showed that the minimum inhibitory concentrations (MICs) for Trichophyton rubrum, Trichophyton mentagrophytes, and Candida albicans were determined. 90 The concentrations were 3.125 μg / mL, 3.125 μg / mL, and 0.78125 μg / mL, respectively.

[0064] Table 1: MICs of various drugs against Trichophyton rubrum (7 days), Trichophyton mentagrophytes (7 days), and Candida albicans (24 hours) 90 result

[0065]

[0066] Note: Cm-AgNPs are nano-silver synthesized through bio-mediated synthesis from Cnidium monnieri extract.

[0067] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. The application of a nano-silver material synthesized through Cnidium monnieri extract in the preparation of antifungal products, characterized in that, The fungus is one or more of Trichophyton rubrum, Trichophyton mentagrophytes, or Candida albicans. The nano-silver material synthesized through Cnidium monnieri extract has a spherical core-shell structure, with nano-silver inside and covered by Cnidium monnieri extract molecules on the outside. It is synthesized from Cnidium monnieri extract and silver nitrate. The method for preparing nano-silver materials mediated by Cnidium monnieri extract includes the following steps: S1, Preparation of Cnidium monnieri extract Weigh out the Cnidium monnieri herb, soak it in 75% ethanol, heat and reflux to extract, combine the extracts and concentrate under reduced pressure until there is no alcohol odor, and freeze dry to obtain Cnidium monnieri extract; at the same time, dilute the Cnidium monnieri extract with pure water and filter to obtain Cnidium monnieri extract solution. S2, Preparation of silver nanomaterials mediated by Cnidium monnieri extract: The pH of the Cnidium monnieri extract was adjusted using an alkaline solution. The pH-adjusted Cnidium monnieri extract was then slowly added dropwise to a silver nitrate solution with continuous stirring. After the reaction was complete, the extract was centrifuged to precipitate, washed three times, and the precipitate was collected to obtain the nano-silver material mediated by the Cnidium monnieri extract.

2. The application as described in claim 1, characterized in that, The mass ratio of the Cnidium monnieri extract to silver nitrate is (260~1200):(13.6~68).

3. The application as described in claim 2, characterized in that, The mass ratio of the Cnidium monnieri extract to silver nitrate is 1200:62.

492.

4. The application as described in claim 1, characterized in that, The *Trichophyton rubrum* is *Trichophyton rubrum* BNCC340195; the *Trichophyton mentagrophytes* is *Trichophyton mentagrophytes* BNCC340405; and the *Candida albicans* is *Candida albicans* BNCC263676.

5. The application as described in any one of claims 1 to 4, characterized in that, The antifungal concentration of the nanosilver material synthesized by the Cnidium monnieri extract ranges from 0.78125 to 3.125 μg / mL.

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