A method for preparing antibacterial carbon quantum dots based on anthocyanin-rich water extract

Abstract

The present application belongs to the technical field of bacteriostatic materials, and particularly relates to a method for preparing bacteriostatic carbon quantum dots based on water extract rich in anthocyanins. The present application is based on a method for preparing carbon quantum dots from water extract of mulberry or purple sweet potato and determination of bacteriostatic performance of carbon quantum dots under blue light irradiation. The synthesis method is to extract purple red anthocyanins from dried mulberry and prepare carbon quantum dots by a hydrothermal method. The carbon quantum dot aqueous solution is transparent light orange and has weak acidity. The preparation process is green and pollution-free, and the product has high biological safety. Under 405 nm blue light irradiation, the carbon quantum dot solution has a significant inhibitory effect on Escherichia coli, Staphylococcus aureus and Candida albicans, and the bacteriostatic rate reaches 99%. The water extract of purple sweet potato rich in anthocyanins also has a good bacteriostatic effect. By using the by-product (purple sweet potato peel) of deep processing of purple sweet potato to prepare quantum dots, the cost can be greatly reduced.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial nanomaterials technology, specifically relating to a method for preparing fluorescent carbon quantum dots from dried mulberry or purple sweet potato water extract and its application. Background Technology

[0002] Dried mulberries and purple sweet potatoes are rich in anthocyanins, a type of flavonoid compound with multiple phenolic hydroxyl groups, which are natural pigments found in various colored plants. High concentrations of anthocyanins have an inhibitory effect on a variety of harmful bacteria (Staphylococcus aureus, Escherichia coli, Aspergillus niger, Penicillium), making them a natural antibacterial agent.

[0003] In recent years, carbon quantum dots (CQDs) have attracted widespread attention due to their excellent fluorescence properties, extremely small size, and low cytotoxicity. The interaction between CQDs and microorganisms mainly depends on their composition, size, shape, and surface-active groups. Research on the antibacterial applications of CQDs focuses on two main approaches: synergistic use with other materials and surface modification to enhance antibacterial rates. Synergistic use with antibiotics yields the best results, but its widespread application is limited by bacterial resistance. Recent research has focused on developing CQDs for synergistic use with photosensitizers, but organic photosensitizers suffer from water solubility and dispersibility issues, affecting antibacterial efficacy. Existing studies have shown that anthocyanins have a good inhibitory effect on Helicobacter pylori, and the CQD solution is weakly acidic, indicating significant potential for inhibiting the growth of Helicobacter pylori in the stomach.

[0004] Patent CN201710992717.2 discloses a method for synthesizing a carbon quantum dot quaternary ammonium salt cationic surfactant. Using citric acid as the carbon source and a diamino compound with one primary amino group and the other tertiary amino group as the nitrogen source, nitrogen-doped carbon quantum dots are synthesized via a hydrothermal method. These nitrogen-doped carbon quantum dots are then used as raw materials to undergo a quaternization reaction with a normal-formed haloalkane in an ethanol-water solution to obtain the carbon quantum dot cationic surfactant. At a concentration of 500 μg / mL, this carbon quantum dot surfactant exhibits an antibacterial rate of 89.73% against *Escherichia coli*. Patent CN202211421754.5 discloses fluorescent coffee grounds carbon quantum dots, their preparation method, and applications. They exhibit antibacterial activity against *Escherichia coli* and *Staphylococcus aureus*. In summary, while some cationic carbon quantum dots possess strong antibacterial properties and surface activity, their synthesis methods are complex. The application range of carbon quantum dots used in synergy with antibiotics and photosensitizers is limited.

[0005] Patent CN201510929542.1 discloses a method for hydrothermal synthesis of carbon quantum dots based on fruit. The obtained carbon quantum dots have a maximum emission of 425 nm, exhibiting blue light, but the antibacterial properties and photosensitivity of the carbon quantum dots were not investigated. Different raw materials and different hydrothermal reactions affect the particle size and surface groups of carbon quantum dots, thus affecting their properties. The photoluminescence properties of carbon quantum dots are related to both the particle size and surface groups. Hydrothermal time and temperature directly affect the degree of carbonization of carbon quantum dots, thereby affecting the surface groups. This invention studies the photosensitivity and antibacterial properties of carbon quantum dots at different temperatures.

[0006] This invention investigates carbon quantum dots prepared from dried mulberry aqueous extract. The preparation process is simple, and these carbon quantum dots exhibit good biocompatibility and excellent water solubility. Under 405nm blue light irradiation, their antibacterial properties against *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans* are significantly enhanced. Therefore, there is an urgent need to develop carbon quantum dots that possess both antibacterial properties and good water solubility, and whose antibacterial activity can be further improved under photoexcitation. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a method for preparing carbon quantum dots from dried mulberry and purple sweet potato aqueous extracts. By preparing carbon quantum dots from dried mulberry and purple sweet potato aqueous extracts, a material with antibacterial activity, excellent fluorescence properties, and selective antibacterial activity is formed. Furthermore, the prepared carbon quantum dots also exhibit good water solubility and dispersibility, good antibacterial activity under blue light irradiation, low toxicity, and suitability for weakly acidic environments, showing promising application prospects.

[0008] The technical solution adopted by this invention to solve its technical problem is: preparing carbon quantum dots based on dried mulberry and purple sweet potato water extract, wherein the preparation method comprises the following steps:

[0009] (1) Weigh out dried mulberries, add deionized water, and place them in a 50℃ oven for 1 hour, stirring with a glass rod 2-3 times (3-4 minutes each time). After 1 hour, filter to remove the mulberry residue and obtain mulberry anthocyanin aqueous extract (AM);

[0010] (2) After processing fresh purple sweet potatoes with a wall-breaking machine, soak them in a 60% ethanol solution (pH=4.0) for 12 hours, filter, let stand for 24 hours, take the supernatant and evaporate it to remove the filter residue and ethanol to obtain purple sweet potato anthocyanin water extract (AP).

[0011] (3) Place the dried mulberry water extract (AM) or purple sweet potato water extract (AP) in a high-pressure reactor and react at 180°C for 10 hours. After the reaction is completed and cooled to room temperature, filter with a 0.22 μm aqueous filter membrane to obtain a light orange carbon quantum dot solution (AMQDs, APQDs) and store it in a refrigerator for later use.

[0012] Mix the light orange carbon quantum dot solution (AMQDs, APQDs) with PBS buffer and calculate the proportion to prepare a carbon quantum dot antibacterial solution with a concentration ≥500ug / mL.

[0013] Furthermore, the pH of the carbon quantum dot antibacterial solution is preferably 4.

[0014] Furthermore, when using the carbon quantum dot antibacterial solution, at 405nm 20mW / cm 2 Irradiation under blue light promotes the improvement of antibacterial properties.

[0015] The prepared carbon quantum dot (AMQDs, APQDs) antibacterial solutions showed significant inhibitory effects on Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The prepared carbon quantum dots exhibited fluorescence properties with a maximum emission of 545 nm and green light. The prepared carbon quantum dots also possessed photosensitivity and photo-induced antibacterial properties, and their antibacterial performance was enhanced under 405 nm blue-violet light irradiation.

[0016] The beneficial effects of this invention are:

[0017] (1) This invention uses a one-step hydrothermal method to prepare fluorescent carbon quantum dots from dried mulberry and purple sweet potato water extracts. The preparation method is simple, and the materials are widely available. The carbon quantum dots prepared from dried mulberry and purple sweet potato water extracts have antibacterial activity, excellent fluorescence properties, and selective antibacterial activity. In addition to their excellent antibacterial properties against Gram-positive and Gram-negative bacteria, the fluorescent carbon quantum dots also have excellent antibacterial properties against Candida albicans.

[0018] (2) The fluorescent carbon quantum dots prepared by this invention have photosensitivity and can significantly improve their antibacterial properties under 405nm blue light irradiation. Compared with traditional photosensitizers, they have excellent water solubility and dispersibility and can be applied to medical treatment.

[0019] (3) The maximum emission wavelength of the fluorescent carbon quantum dots prepared by this invention changes with the concentration. In practical applications, the fluorescence can be adjusted by changing the concentration as needed. Attached Figure Description

[0020] Figure 1 EDX spectrum of AMQDs;

[0021] Figure 2 Fluorescence emission spectra of AMQDs at different concentrations;

[0022] Figure 3 Transmission electron microscopy image of AMQDs. Detailed Implementation

[0023] Preparation of dried mulberry aqueous extract: Weigh 3.0g of dried mulberries into a beaker, add 30mL of deionized water, and place in a 50℃ oven for 1 hour, stirring 2-3 times (3-4 minutes each time) with a glass rod. After 1 hour, filter to remove the mulberry residue to obtain dried mulberry anthocyanin aqueous extract (AM); process purple sweet potatoes with a high-speed blender, soak in 60% ethanol solution (pH=4.0) for 12 hours, filter, let stand for 24 hours, and take the supernatant for rotary evaporation to remove the residue and ethanol to obtain purple sweet potato anthocyanin aqueous extract (AP).

[0024] Preparation of carbon quantum dots: Mulberry dried aqueous extract was placed in a high-pressure reactor and reacted at 180℃ for 10 h. After the reaction was completed and cooled to room temperature, it was filtered through a 0.22 μm aqueous filter membrane to obtain a light orange carbon quantum dot solution. The mulberry dried aqueous extract carbon quantum dots (AMQDs) were stored in a refrigerator for later use. 1 mL of the carbon quantum dot solution was placed in a beaker and dried in an oven to constant weight, and the concentration was calculated. A series of carbon quantum dot solutions (AMQDs) were prepared at different temperatures and for different times.

[0025] Preparation of carbon quantum dots: The aqueous extract of purple sweet potato was placed in a high-pressure reactor and reacted at 180℃ for 10 h. After the reaction was completed and cooled to room temperature, the solution was filtered through a 0.22 μm aqueous filter membrane to obtain a light orange carbon quantum dot solution. The purple sweet potato aqueous extract carbon quantum dots (APQDs) were stored in a refrigerator for later use. 1 mL of the carbon quantum dot solution was placed in a beaker and dried in an oven until constant weight. The concentration was then calculated.

[0026] Preparation of CQDs: 2g of L-glutamic acid was dissolved in 55mL of deionized water by sonication for 10min. The completely dissolved glutamic acid solution was then placed in a high-pressure reactor and hydrothermally reacted at 230℃ for 12h (or 180℃ for 10h). After the reaction was completed and cooled to room temperature, a pale yellow carbon quantum dot solution was obtained.

[0027] Preparation of carbon quantum dots from fresh mulberry fruit: The operation is the same as above, but the hydrothermal temperature is 180℃ and the hydrothermal time is 10h.

[0028] Preparation of carbon quantum dots for tea infusion: Weigh 20.0g of tea leaves into a beaker, add 30mL of deionized water, and place in a 50℃ oven for 1 hour, stirring 2-3 times (3-4 minutes each time) with a glass rod. After 1 hour, filter to remove the tea leaves, obtaining the tea infusion. Place the tea infusion in a high-pressure reactor and react at 180℃ for 10 hours. After the reaction is complete and cooled to room temperature, filter through a 0.22µm aqueous filter membrane to obtain a light orange carbon quantum dot solution, which is stored in a refrigerator for later use. Take 1mL of the carbon quantum dot solution into a beaker, dry it in an oven to constant weight, and calculate the concentration.

[0029] PBS buffer: Dissolve 2.84g disodium hydrogen phosphate and 1.36g potassium dihydrogen phosphate in 1000mL of water, and adjust the pH with 1% HCl.

[0030] The pH of the mulberry dried aqueous extract (AM) prepared above was 5.6, and the pH of the carbon quantum dot solution (AMQDs) prepared from the mulberry dried aqueous extract was 3.6. The pH of the purple sweet potato aqueous extract (AP) was 5.8; the pH of the carbon quantum dot solution (APQDs) prepared from the purple sweet potato aqueous extract was 3.8; the pH of the antibacterial solution was adjusted to 4.0 using PBS buffer.

[0031] All examples and comparative examples were tested for antibacterial rate according to the quantitative suspension antibacterial test method in WS / T 650-2019 "Evaluation Methods for Antibacterial and Bacteriostatic Effects". (The following examples / comparative examples mainly present the antibacterial data of carbon quantum dots prepared from dried mulberry aqueous extract.)

[0032] Example 1:

[0033] A solution of carbon quantum dots (AMQDs) from dried mulberry water extract and PBS buffer was prepared by mixing and calculating the proportions to obtain a solution of 6 mg / mL with pH = 4.0.

[0034] Example 2:

[0035] A solution of carbon quantum dots (AMQDs) from dried mulberry water extract and PBS buffer was prepared by mixing and calculating the proportions to obtain a solution of 4 mg / mL with pH = 4.0.

[0036] Example 3:

[0037] A solution of carbon quantum dots (AMQDs) from dried mulberry water extract and PBS buffer was prepared by mixing and calculating the proportions to obtain a solution of 2 mg / mL with pH = 4.0.

[0038] Example 4:

[0039] A solution of purple sweet potato aqueous extract carbon quantum dot (APQDs) and PBS buffer was prepared by mixing and calculating the ratio to obtain a solution of 6 mg / mL and pH = 4.0.

[0040] Table 1. Antibacterial rate of carbon quantum dots at different concentrations under no blue light irradiation.

[0041]

[0042] The data in Table 1 show that, under pH 4.0 and without blue light irradiation, when the concentration of these carbon quantum dots is greater than 4 mg / mL, they exhibit an inhibition rate of over 90% against Escherichia coli and Staphylococcus aureus, but show no significant inhibitory effect against Candida albicans. At 2 mg / mL, they show no significant inhibitory effect against any of the three bacteria.

[0043] Example 5:

[0044] Mulberry dried water extract carbon quantum dot (AMQD) solution and PBS buffer were mixed to prepare a solution with a concentration of 6 mg / mL and pH = 4.0. The prepared mixture was then exposed to light for 1 hour. (Light conditions: 405 nm, 20 mW / cm²) 2 (Irradiation under blue light)

[0045] Example 6:

[0046] Mulberry dried water extract carbon quantum dot (AMQD) solution and PBS buffer were mixed to prepare a solution with a concentration of 4 mg / mL and pH = 4.0. The prepared mixture was then exposed to light for 1 hour (light conditions: 405 nm, 20 mW / cm²). 2 (Irradiation under blue light).

[0047] Example 7:

[0048] Mulberry dried water extract carbon quantum dot (AMQD) solution and PBS buffer were mixed to prepare a 500 μg / mL solution with pH = 4.0. The prepared mixture was then exposed to light for 1 hour (light conditions: 405 nm, 20 mW / cm²). 2 (Irradiation under blue light).

[0049] Example 8:

[0050] Mulberry dried water extract carbon quantum dot (AMQD) solution and PBS buffer were mixed to prepare a solution of 400 μg / mL, pH=4.0. The prepared mixture was then exposed to light for 1 hour (light conditions: 405 nm, 20 mW / cm²). 2 (Irradiation under blue light).

[0051] Table 2. Antibacterial rates of carbon quantum dots at different concentrations after 1 hour of blue light irradiation.

[0052]

[0053] Comparing the data in Table 2 with those in Table 1, it can be seen that under pH 4.0 and blue light irradiation, the carbon quantum dots significantly enhanced the inhibition of Candida albicans. At 500 ug / mL, it still exhibited significant antibacterial activity against Escherichia coli and Staphylococcus aureus, but showed no significant antibacterial activity against Candida albicans.

[0054] Comparative Example 1:

[0055] The CQDs solution prepared from the L-glutamic acid was mixed with PBS in the above proportions to prepare a 6 mg / mL pH 4.0 CQDs solution.

[0056] Comparative Example 2:

[0057] The CQDs solution prepared from the L-glutamic acid was mixed with PBS in the above proportions to prepare a 4 mg / mL pH 4.0 CQDs solution.

[0058] Comparative Example 3:

[0059] The CQDs solution prepared from L-glutamic acid was mixed with PBS in a specific ratio to prepare a 6 mg / mL pH 4.0 CQDs solution. The solution was then irradiated for 1 hour (light conditions: 405 nm, 20 mW / cm²). 2 (Irradiation under blue light).

[0060] Comparative Example 4:

[0061] Preparation of CQDs: 2g of L-glutamic acid was dissolved in 55mL of deionized water by sonication for 10min. The completely dissolved glutamic acid solution was then placed in a high-pressure reactor and hydrothermally reacted at 180℃ for 10h. After the reaction was complete and cooled to room temperature, a pale yellow carbon quantum dot solution was obtained. A 500ug / mL CQDs solution with pH=4.0 was prepared by mixing the L-glutamic acid-based CQDs solution and PBS according to the calculated ratio, and then irradiated for 1h (irradiation conditions: 405nm, 20mW / cm²). 2 (Irradiation under blue light).

[0062] Comparative Example 5:

[0063] A solution with pH = 4.0 and 6 mg / mL was prepared by mixing dried mulberry water extract (AM stock solution) and PBS in a specific ratio.

[0064] Comparative Example 6:

[0065] A solution with pH 4.0 of 4 mg / mL was prepared by mixing dried mulberry water extract (AM stock solution) and PBS in a specific ratio.

[0066] Comparative Example 7:

[0067] A solution of 500 μg / mL pH 4.0 was prepared by mixing dried mulberry water extract and PBS according to the calculated ratio, and then irradiated with light for 1 hour (light conditions: 405 nm, 20 mW / cm²). 2 (Irradiation under blue light).

[0068] Comparative Example 8: PBS solution with pH = 4.0.

[0069] Comparative Example 9: PBS solution with pH = 4.0, irradiated for 1 hour, under the following conditions: 405 nm, 20 mW / cm². 2 Irradiation under blue light.

[0070] Comparative Example 10:

[0071] The dried mulberry extract was placed in a high-pressure reactor and reacted at 160°C for 10 hours. After the reaction was completed and cooled to room temperature, it was filtered through a 0.22 μm aqueous filter membrane to obtain a light orange carbon quantum dot solution, namely mulberry dried water extract carbon quantum dots (AMQDs).

[0072] A solution of carbon quantum dots (AMQDs) from dried mulberry aqueous extract and PBS was prepared at a ratio of 500 μg / mL with a pH of 4.0, and then irradiated for 1 hour (light conditions: 405 nm, 20 mW / cm²). 2 (Irradiation under blue light).

[0073] Comparative Example 11:

[0074] Compared with Comparative Example 10, the difference is that carbon quantum dots (AMQDs) of dried mulberry water extract were obtained by reacting at 200℃ for 10h, while other operations were the same.

[0075] Comparative Example 12:

[0076] Compared with Comparative Example 10, the difference is that carbon quantum dots (AMQDs) of dried mulberry water extract were obtained by reacting at 180℃ for 12 hours, while other operations were the same.

[0077] Comparative Example 13:

[0078] Compared with Comparative Example 10, the difference is that carbon quantum dots (AMQDs) of mulberry dried water extract were obtained by reacting at 180℃ for 8 hours, while other operations were the same.

[0079] Comparative Example 14:

[0080] A solution of 500 μg / mL pH 4.0 was prepared by mixing carbon quantum dots obtained from fresh mulberry fruit with PBS according to a calculated ratio. The solution was then irradiated for 1 hour (light conditions: 405 nm, 20 mW / cm²). 2 Its antibacterial properties were tested after irradiation with blue light.

[0081] Comparative Example 15:

[0082] A solution of 500 μg / mL pH 4.0 was prepared by mixing carbon quantum dots obtained from tea leaves with PBS according to a calculated ratio and then irradiated for 1 hour (light conditions: 405 nm, 20 mW / cm²). 2 Its antibacterial properties were tested after irradiation with blue light.

[0083] Comparative Example 16:

[0084] Mulberry dried water extract carbon quantum dot (AMQDs) solution and PBS buffer were mixed to prepare a solution with a concentration of 4 mg / mL and pH = 6.0.

[0085] Table 3 shows the antibacterial rates of a series of CQDs, AM stock solutions, and PBS solutions.

[0086]

[0087] Table 3 shows that CQDs calcined with glutamic acid (230℃ for 12h) showed no antibacterial activity against *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans* at a concentration of 4 mg / mL without blue light irradiation. CQDs calcined with glutamic acid (230℃ for 12h) showed no antibacterial activity against these bacteria at a concentration of 6 mg / mL under blue light irradiation. CQDs calcined with glutamic acid (180℃ for 10h) also showed no antibacterial activity against these bacteria at a concentration of 500 μg / mL under blue light irradiation. Carbon quantum dots calcined from fresh mulberry fruit did not show significant antibacterial activity under blue light irradiation at a concentration of 500 μg / mL. Carbon quantum dots calcined from tea infusion containing anthocyanins showed no antibacterial activity under blue light irradiation at a concentration of 500 μg / mL. The antibacterial activity decreased significantly at pH > 4.

[0088] Data showed that AMQDs prepared at different hydrothermal times and temperatures exhibited lower inhibition rates against Escherichia coli, Staphylococcus aureus, and Candida albicans under 500 μg / ml blue light irradiation compared to AMQDs prepared at 180℃ for 10 h. PBS solution at pH 4.0 showed no inhibitory effect on the three bacteria regardless of blue light irradiation, thus eliminating the influence of pH and blue light irradiation on the growth of these bacteria.

[0089] In summary, AMQDs have abundant, safe, and biocompatible raw materials. They exhibit good antibacterial effects against Escherichia coli and Staphylococcus aureus, with a minimum inhibitory concentration (MIC) of 4 mg / mL. After 1 hour of blue light irradiation, the MIC of AMQDs decreased to 500 μg / mL, and they also showed significant antibacterial activity against Candida albicans at a concentration of 6 mg / mL. Compared to carbon quantum dots used in combination with photosensitizers, AMQDs solve the problems of poor water solubility and dispersibility of photosensitizers.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An application of antibacterial carbon quantum dots prepared from anthocyanin-rich aqueous extract in antibacterial products, characterized by: (1) First, extract anthocyanin aqueous extract from raw materials rich in anthocyanins; the raw materials rich in anthocyanins are dried mulberries or purple sweet potatoes; (2) Place the anthocyanin aqueous extract in a high-pressure reactor and react at 180°C for 10 h. After the reaction is completed, cool to room temperature and filter to obtain a light orange carbon quantum dot solution. (3) Mix the light orange carbon quantum dot solution with PBS buffer to prepare a solution with pH=4.0 to obtain an antibacterial solution; Antibacterial carbon quantum dots, as photosensitizers, exhibit antibacterial effects under 405nm blue light irradiation.

2. The use of the antibacterial carbon quantum dots prepared from the anthocyanin-rich water extract according to claim 1 in antibacterial products, characterized in that: Water extract of anthocyanins from dried mulberries: Weigh dried mulberries into a container, add deionized water, place in an oven and keep warm for a period of time, then filter to remove the mulberry residue to obtain water extract of anthocyanins from dried mulberries. Water extract of anthocyanins from purple sweet potatoes: After processing purple sweet potatoes with a high-speed blender, soak them in 60% ethanol solution at pH=4.0 for 12 hours, filter, let stand, take the supernatant and rotary evaporate to remove the filter residue and ethanol to obtain water extract of anthocyanins from purple sweet potatoes.

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