Preparation method and application of pueraria montana var. lobata carbon dots

Abstract

This invention belongs to the field of carbon nanomaterials technology, specifically disclosing a method for preparing and applying carbon dots from *Pu Di Lan* (a type of medicinal herb) residue. The specific steps are as follows: dried *Pu Di Lan* residue is pulverized using a cell wall breaker to obtain *Pu Di Lan* residue powder; the powder is then reacted using a hydrothermal method; the reaction solution is collected; the reaction solution is centrifuged, and the supernatant is collected; the supernatant is filtered, and the filtrate is collected; the filtrate is dialyzed, and the retaining liquid is collected; the retaining liquid is freeze-dried to obtain the *Pu Di Lan* residue carbon dots. The obtained *Pu Di Lan* residue carbon dots exhibit blue fluorescence under a 365 nm ultraviolet lamp, and their surface is rich in functional groups such as hydroxyl and carboxyl groups, showing good hydrophilicity. The *Pu Di Lan* residue carbon dots promote β-D-glucosidase activity and can be used in food processing; simultaneously, the fluorescence of the *Pu Di Lan* residue carbon dots is highly sensitive to β-D-glucosidase, and β-D-glucosidase activity can be detected by fitting a linear regression equation with a standard solution.

Description

Technical Field

[0001] This invention belongs to the field of carbon nanomaterials technology, and relates to the preparation of carbon dots from traditional Chinese medicine residues, specifically to a method for preparing carbon dots from *Pu Di Lan* (a type of medicinal herb) residues and their application in the detection of β-D-glucosidase activity. Background Technology

[0002] Carbon dots (CDs), as novel nanomaterials, possess excellent water solubility, low toxicity, and biocompatibility, and are widely used in fields such as bioimaging, drug delivery, and electrochemical sensing. The surface states of carbon dots play a crucial role in their photoinduced states; changes in the external environment alter their surface functional groups. Therefore, carbon dots are used as fluorescent probes for the detection of substances such as metal ions, pesticides, and proteins.

[0003] The synthesis methods for carbon dots are mainly divided into two types: bottom-up and top-down. The top-down method involves using physical or chemical methods to cut large carbon precursors into small carbon quantum dots. The bottom-up method uses small molecules as precursors and obtains large carbon quantum dots through a series of chemical reactions. Currently, carbon quantum dots are mainly obtained from small organic molecules, natural products, and carbon-based materials.

[0004] In their article "Research Progress on the Application of Carbon Dots from Traditional Chinese Medicine" (Chinese Modern Traditional and Herbal Drugs, Vol. 25, No. 10, October 2023), Wang Qian et al. disclosed that carbon dots from traditional Chinese medicine are carbon dots made from traditional Chinese medicine as precursors. Their preparation process is similar to that of carbon quantum dots, hence they are also called traditional Chinese medicine-derived carbon quantum dots. Traditional Chinese medicine carbon dots not only have advantages such as wide availability of raw materials, low toxicity, and good biocompatibility, but also possess various biological properties such as hemostasis, anti-inflammatory and analgesic effects, and anti-tumor activity. They can be applied in fields such as bioimaging, biosensing, and the detection of large and small molecules and ions. However, currently, the precursor for the synthesis of CDs from traditional Chinese medicine is traditional Chinese medicine, and there are no reports on whether the residue after extraction still contains some active ingredients, or on the synthesis of CDs from the residue.

[0005] Pudilan is a traditional Chinese medicine compound composed of four herbs: Scutellaria baicalensis, Taraxacum mongolicum, Viola yedoensis, and Isatis indigotica. It possesses the effects of clearing heat and detoxifying, and reducing inflammation and swelling. It is used to treat pharyngitis, boils, lymphadenitis, tonsillitis, and other inflammatory conditions. Most studies on the preparation of carbon dots from herbal sources focus on a single herb; there are virtually no reports of using the residue of a single compound medicine as a material. Pudilan has a large market demand, but the residue of traditional Chinese medicine is mostly treated as organic fertilizer. Preparing Pudilan residue into carbon dots could broaden its applications, give it new commercial value, and significantly promote research on carbon dots from traditional Chinese medicine. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing carbon dots from *Phyllostachys edulis* (a type of medicinal herb residue). This method uses *Phyllostachys edulis* residue as a precursor for CDs synthesis, is simple to operate, and inexpensive. The prepared carbon dots exhibit excellent fluorescence stability and photobleaching resistance, and demonstrate ideal catalytic effects, making them applicable to the detection of biomolecules. The prepared *Phyllostachys edulis* residue carbon dots promote β-D-glucosidase activity and can be used for the detection of β-D-glucosidase in plants.

[0007] This invention is achieved through the following technical solution:

[0008] A method for preparing carbon dots from *Phyllanthus urinaria* residue includes the following steps: pulverizing dried *Phyllanthus urinaria* residue using a cell wall breaker to obtain *Phyllanthus urinaria* residue powder; reacting the powder using a hydrothermal method; collecting the reaction solution; centrifuging the reaction solution and collecting the supernatant; filtering the supernatant and collecting the filtrate; dialyzing the filtrate and collecting the retaining liquid; and freeze-drying the retaining liquid to obtain the carbon dots from the *Phyllanthus urinaria* residue.

[0009] The hydrothermal reaction is carried out at a temperature of 160~220 ℃ for a duration of 4~8 hours.

[0010] A further improvement to the present invention is as follows:

[0011] The ratio of the *Pu Di Lan* herb residue powder, triethylamine, and deionized water is 0.1–1.2 g: 0.1–0.8 mL: 40 mL.

[0012] Furthermore, the reaction solution was centrifuged at a speed of 12000 r / min for 30 min.

[0013] Furthermore, during filtration, a 0.22 μm microporous membrane was used to filter the supernatant.

[0014] Furthermore, when dialyzing the filtrate, a dialysis bag with a molecular weight cutoff of 1000 Da is used, the dialysis time is not less than 12 hours, and the water is changed every 2-4 hours.

[0015] Furthermore, when drying the retained solution, the drying temperature was -70℃ and the drying time was 72 h.

[0016] A further improvement of the present invention is as follows:

[0017] Application of carbon dots from *Pu Di Lan* residue prepared by the above method in the detection of β-D-glucosidase activity.

[0018] Furthermore, the application of carbon dots from *Pyrrosia lingua* residue in the detection of β-D-glucosidase activity includes the following steps: Carbon dots from *Pyrrosia lingua* residue are dissolved in water to prepare a carbon dot stock solution, which is then added to β-D-glucosidase extracted from *Prunus japonica* seeds to detect changes in enzyme activity. Subsequently, the same volume of the carbon dot stock solution is placed in centrifuge tubes, and then β-D-glucosidase standard solutions are added to prepare gradient concentrations of β-D-glucosidase-*Pyrrosia lingua* residue carbon dot mixed solutions. Using 325 nm as the optimal excitation wavelength, the fluorescence intensity emitted in the range of 335–635 nm is measured to obtain a series of fluorescence intensity values ​​for gradient concentrations of β-D-glucosidase-*Pyrrosia lingua* residue carbon dot mixed solutions. A graph is plotted and linearly fitted to obtain a linear regression equation, which is then used to detect the β-D-glucosidase content in plants.

[0019] The beneficial effects of this invention are as follows:

[0020] The carbon dots of the *Pu Di Lan* residue prepared by this invention exhibit blue fluorescence under a 365 nm ultraviolet lamp. The carbon dots are mainly composed of C, O, H, and N elements, and the particle size of the carbon dots is approximately 5–10 nm.

[0021] The method for preparing carbon dots from *Pu Di Lan* medicinal residue provided by this invention is simple, easy to operate, and low in cost.

[0022] The carbon dots of *Pu Di Lan* residue provided by this invention have excellent fluorescence properties and can be used to improve β-D-glucosidase activity, showing good application prospects in the food processing industry.

[0023] The carbon dot fluorescence of *Pu Di Lan* residue provided by this invention has a good response to β-D-glucosidase and has good application in detection. Attached Figure Description

[0024] Figure 1 The ultraviolet absorption spectrum and optimal excitation and emission spectrum of the carbon dots of the *Pu Di Lan* residue prepared in Example 1 of this invention are shown.

[0025] Figure 2 The emission spectra of carbon dots from the *Pu Di Lan* residue prepared in Example 1 of this invention are obtained at different excitation wavelengths.

[0026] Figure 3 The infrared spectrum of carbon dots in the *Pu Di Lan* residue prepared in Example 1 of this invention;

[0027] Figure 4 The image shows the XRD pattern of carbon dots in the residue of *Pu Di Lan* prepared in Example 1 of this invention.

[0028] Figure 5 This is a standard curve of β-D-glucosidase activity versus p-nitrophenol concentration used in Example 1 of this invention;

[0029] Figure 6 This illustrates the effect of carbon dots from different volumes of *Pu Di Lan* residue on the activity of extracted β-D-glucosidase in Example 1 of this invention.

[0030] Figure 7 The fluorescence emission spectra are those of β-D-glucosidase with different activities added in Example 1 of this invention. Detailed Implementation

[0031] The present invention will now be described in detail with reference to specific embodiments.

[0032] Example 1: Preparation of carbon dots from *Pu Di Lan* (a type of medicinal herb) residue

[0033] 1.2 g of *Pu Di Lan* (a type of medicinal herb) residue powder and 0.8 mL of triethylamine were weighed and dissolved in 40 mL of deionized water. After stirring and ultrasonic dissolution, the solution was packaged. The packaged reaction vessel was heated in a 200℃ electric thermostatic drying oven for 8 h and centrifuged at 12000 r / min for 30 min. The supernatant was filtered using a 0.22 μm microporous membrane. The filtrate was dialyzed using a dialysis bag with a molecular weight cutoff of 1000 Da for at least 12 h, with the water changed every 2-4 h. The retained solution was dried at -70℃ for 72 h to obtain carbon quantum dots.

[0034] The results of characterization of the prepared carbon quantum dots are as follows: Figure 1 , 2 As shown in Figures 3 and 4, the prepared carbon quantum dots exhibit good fluorescence properties and are excitation-dependent.

[0035] Example 2: Preparation of carbon dots from *Pu Di Lan* (a type of medicinal herb) residue

[0036] 0.8 g of *Pu Di Lan* (a type of medicinal herb) residue powder and 0.6 mL of triethylamine were weighed and dissolved in 40 mL of deionized water. After stirring and ultrasonic dissolution, the solution was packaged. The packaged reaction vessel was heated in a 180℃ electric thermostatic drying oven for 6 h and centrifuged at 12000 r / min for 30 min. The supernatant was filtered using a 0.22 μm microporous membrane. The filtrate was dialyzed using a dialysis bag with a molecular weight cutoff of 1000 Da for at least 12 h, with the water changed every 2-4 h. The retained solution was dried at -70℃ for 72 h to obtain carbon quantum dots.

[0037] Example 3: Preparation of carbon dots from *Pu Di Lan* (a type of medicinal herb) residue

[0038] 0.4 g of *Pu Di Lan* (a type of medicinal herb) residue powder and 0.4 mL of triethylamine were weighed and dissolved in 40 mL of deionized water. After stirring and ultrasonic dissolution, the solution was packaged. The packaged reaction vessel was heated in a 160℃ electric thermostatic drying oven for 4 h and centrifuged at 12000 r / min for 30 min. The supernatant was filtered using a 0.22 μm microporous membrane. The filtrate was dialyzed using a dialysis bag with a molecular weight cutoff of 1000 Da for at least 12 h, with the water changed every 2-4 h. The retained solution was dried at -70℃ for 72 h to obtain carbon quantum dots.

[0039] Example 4: Preparation of carbon dots from *Pu Di Lan* (a type of medicinal herb) residue

[0040] 0.2 g of *Pu Di Lan* (a type of medicinal herb) residue powder and 0.2 mL of triethylamine were weighed and dissolved in 40 mL of deionized water. After stirring and ultrasonic dissolution, the solution was packaged. The packaged reaction vessel was heated in a 140℃ electric thermostatic drying oven for 8 h and centrifuged at 12000 r / min for 30 min. The supernatant was filtered using a 0.22 μm microporous membrane. The filtrate was dialyzed using a dialysis bag with a molecular weight cutoff of 1000 Da for at least 12 h, with the water changed every 2-4 h. The retained solution was dried at -70℃ for 72 h to obtain carbon quantum dots.

[0041] Example 5: Application of carbon dots from *Pu Di Lan* (a type of medicinal herb) residue in the detection of β-D-glucosidase activity.

[0042] In a 1 mL β-D-glucosidase system, 50 μL of enzyme solution diluted 100-fold and 50 μL of (10 mmol / L) p-NPG were added. The pH 5.5 buffer solution in the system was changed to 500, 400, 300, 200, and 100 μL, and the carbon dot concentration was changed to 100, 200, 300, 400, and 500 μL, respectively. After hydrothermal incubation on a shaker for 30 min, 300 μL of Na₂CO₃ stop solution was added. 100 μL of the solution was pipetted onto a 96-well plate, and the absorbance at 405 nm was measured using a microplate reader. This absorbance value represents the concentration of β-D-glucosidase and substrate at the amount of carbon quantum dots changed. The enzyme activity was calculated by substituting the absorbance value into the standard curve of β-D-glucosidase activity versus p-nitrophenol concentration. Figure 5 Subsequently, its relative enzyme activity was obtained by comparing it with a blank sample, such as... Figure 6 .

[0043] A series of β-Glu standard solutions with activities of (0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0 U / L) were prepared. 40 μL of CDs stock solution, 460 μL of enzyme solutions with different activities, and 1 mL of p-NPG solution (1.0 × 10⁻⁶) were added. -3 Add mol / L of the solution to a 5 mL EP tube, and bring the volume to 3.5 mL with 2 mL of pH 5.5 buffer. Incubate the reaction hydrothermally at 50 °C on a shaker for 30 min. Immediately after the reaction is complete, add 500 μL of Na₂CO₃ solution to terminate the reaction. After cooling to room temperature, measure the fluorescence intensity of the fluorescence emission peak (Em = 400 nm) at an excitation wavelength of 325 nm. The blank solution is I₀, and the test solution is I. Calculate lg(I₀ / I) to obtain the linear relationship between fluorescence intensity and enzyme activity (e.g., ...). Figure 7 The method was applied to the detection of β-D-glucosidase in Prunus japonica seeds, and the results are shown in Table 1.

[0044] Table 1

[0045]

[0046] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent transformations or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. An application of carbon dots from *Pu Di Lan* medicinal residue in the detection of β-D-glucosidase activity, characterized in that... The preparation method of the carbon dots of the *Pu Di Lan* (a type of medicinal herb) residue includes the following steps: The dried *Pu Di Lan* residue is pulverized using a cell wall breaker to obtain *Pu Di Lan* residue powder; the powder is mixed with triethylamine in deionized water, and a hydrothermal reaction is carried out; the reaction solution is collected; the reaction solution is centrifuged, and the supernatant is collected; the supernatant is filtered, and the filtrate is collected; the filtrate is dialyzed, and the retaining liquid is collected; the retaining liquid is freeze-dried to obtain the carbon dots of the *Pu Di Lan* residue. The hydrothermal reaction is carried out at a temperature of 140~220 ℃ for a time of 2~10 h; The ratio of the *Pu Di Lan* herb residue powder, triethylamine, and deionized water is 0.1–1.2 g: 0.1–0.8 mL: 40 mL.

2. The application according to claim 1, characterized in that: The reaction solution was centrifuged at a speed of 12000 r / min for 30 min.

3. The application according to claim 1, characterized in that: During filtration, a microporous membrane with a diameter of 0.22~0.45μm is used to filter the supernatant.

4. The application according to claim 1, characterized in that: When dialyzing the filtrate, a dialysis bag with a molecular weight cutoff of 1000 Da is used, the dialysis time is 8-12 hours, and the water is changed every 2-4 hours.

5. The application according to claim 1, characterized in that: When drying the retention solution, the drying temperature is -50~-80℃ and the time is 36~72 h.

6. The application according to claim 1, characterized in that, The application process includes the following steps: dissolving the carbon dots from *Pyrrosia lingua* residue in water to prepare a carbon dot stock solution, which is then added to β-D-glucosidase extracted from *Prunus japonica* seeds to detect changes in enzyme activity; subsequently, taking the same volume of the carbon dot stock solution and placing it in a centrifuge tube, then adding β-D-glucosidase standard solution to prepare gradient concentrations of β-D-glucosidase-*Pyrrosia lingua* residue carbon dot mixed solutions; using 325 nm as the optimal excitation wavelength, measuring the fluorescence intensity emitted in the range of 335–635 nm to obtain a series of gradient concentrations of β-D-glucosidase-*Pyrrosia lingua* residue carbon dot mixed solutions, plotting and performing linear fitting to obtain a linear regression equation, and detecting β-D-glucosidase activity in plants through the linear regression equation.

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