New use of lotus root pdns

Abstract

The application discloses a new use of lotus root PDNs and belongs to the technical field of biological medicines. The preparation method of the lotus root PDNs is as follows: fresh lotus root is squeezed to obtain juice, the first centrifugation is carried out to remove the precipitated plant cells, the filtrate is subjected to the second centrifugation to remove the precipitated dead cells, the filtrate is subjected to the third centrifugation to remove the precipitated cell fragments, and the filtrate is subjected to the fourth centrifugation to obtain the precipitate, which is the lotus root PDNs. The prepared lotus root PDNs can prevent the change of inflammatory factors caused by LPS stimulation, so that the effect of improving intestinal cell inflammation is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to new applications of lotus root PDNs. Background Technology

[0002] Lotus root is the edible rhizome of a plant in the Nelumbo nucifera family (Nelumbo nucifera Gaertn.), widely cultivated as an aquatic economic crop in the middle and lower reaches of the Yangtze River in my country. Lotus root has a unique flavor and texture, and is rich in nutrients such as carbohydrates, protein, vitamins, and minerals. According to traditional Chinese medicine texts, it has effects such as clearing heat and removing blood stasis, promoting body fluid production and relieving nausea, quenching thirst, strengthening the spleen and replenishing qi, and nourishing blood and promoting tissue regeneration. Lotus root combines the characteristics of both vegetables and fruits, integrating nutrition and health benefits, making it a food with medicinal properties. Currently, lotus root in my country is mainly consumed fresh and cooked, with a small amount processed into products such as lotus root powder, lotus root juice, preserved products, and braised products. The added value of these products is not high, failing to fully utilize the unique characteristics and advantages of lotus root resources.

[0003] Plant-derived nanoparticles (PDNs) are spherical nanovesicles with a diameter of 30-150 nm, composed of lipid membranes and secreted by plant cells. PDNs possess various biological activities and have significant potential for treating diseases, as well as for drug delivery. PDNs have direct therapeutic effects on various diseases, including inflammatory diseases, cancer, metabolic disorders, and immune regulation. For example, PDNs extracted from aloe vera peel can reduce the level of the pro-inflammatory cytokine TNF-α, thereby inhibiting the differentiation of myofibroblasts; PDNs extracted from avocado can also inhibit the expression of TNF-α, thereby inhibiting the development of atherosclerosis.

[0004] The gut is not only the primary site for nutrient digestion and absorption, but also the first line of defense against intestinal pathogens and external pathogens. Therefore, gut health is inextricably linked to the overall health of an animal. The intestinal mucosal barrier consists of mechanical, chemical, immune, and microbial barriers. This barrier prevents the invasion of pathogenic microorganisms and endotoxins within the intestinal lumen, and the integrity of its structure and function is crucial for gut health. Gram-negative bacteria (such as desulfovibrio, Escherichia coli, and Streptococcus) and other intestinal pathogens can lead to an increase in LPS (lipopolysaccharide), a component of gut microbiota. LPS can damage the intestinal epithelial cell barrier through a series of inflammatory pathways, thereby hindering intestinal growth and development. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, the first technical problem this invention aims to solve is to provide the application of lotus root PDNs in the preparation of anti-inflammatory drugs, thereby improving intestinal inflammation by reversing changes in inflammatory factors induced by LPS stimulation. The second technical problem this invention aims to solve is to provide a simple and convenient method for preparing lotus root PDNs, and the prepared lotus root PDNs can be used to prepare anti-inflammatory drugs. The third technical problem this invention aims to solve is to provide lotus root PDNs that have excellent therapeutic effects on intestinal inflammation while being non-toxic to intestinal epithelial cells.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] Application of lotus root PDNs in the preparation of anti-inflammatory drugs.

[0008] Furthermore, the concentration of the lotus root PDNs used in anti-inflammatory drugs does not exceed 0.3 mg / mL·Protein.

[0009] Furthermore, the concentration of the lotus root PDNs used in the anti-inflammatory drug is 0.2 mg / mL·Protein.

[0010] Furthermore, the anti-inflammatory drugs prepared from the lotus root PDNs are used to treat intestinal inflammation.

[0011] Furthermore, the preparation method of the lotus root PDNs is as follows: fresh lotus root is juiced, centrifuged for the first time to remove the precipitated plant cells, the filtrate is centrifuged for the second time to remove the precipitated dead cells, the filtrate is centrifuged for the third time to remove the precipitated cell fragments, and the filtrate is centrifuged for the fourth time. The resulting precipitate is the lotus root PDNs.

[0012] The preparation method of lotus root PDNs involves juicing fresh lotus root, centrifuging it once to remove the precipitated plant cells, centrifuging the filtrate a second time to remove the precipitated dead cells, centrifuging the filtrate a third time to remove the precipitated cell fragments, and centrifuging the filtrate a fourth time. The resulting precipitate is the lotus root PDNs.

[0013] Furthermore, the centrifugal forces for the first, second, third, and fourth centrifugations are 300g, 2000g, 10000g, and 100000g, respectively.

[0014] Furthermore, the centrifugation time for the first and second centrifugations was 10 min, and the centrifugation time for the third and fourth centrifugations was 30 min and 70 min, respectively.

[0015] The lotus root PDNs prepared by the method are described.

[0016] The application of lotus root PDNs in the preparation of anti-inflammatory drugs.

[0017] Beneficial effects: Compared with the prior art, the advantages of this invention are:

[0018] (1) The present invention obtains lotus root PDNs by juicing fresh lotus root and centrifuging it four times. The preparation method is simple and can be industrialized on a large scale.

[0019] (2) The lotus root PDNs prepared in this invention can improve intestinal inflammation by preventing changes in inflammatory factors caused by LPS stimulation. It has excellent therapeutic effect on intestinal inflammation and is non-toxic to intestinal epithelial cells. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the process for preparing lotus root PDNs according to the present invention;

[0021] Figure 2 A schematic diagram of NTA detection in lotus root PDNs;

[0022] Figure 3 Figure showing the particle size analysis results of lotus root PDNs;

[0023] Figure 4 Morphological feature image of lotus root PDNs detected by TEM;

[0024] Figure 5 The protein standard curve of lotus root PDNs;

[0025] Figure 6 A graph showing the toxicity analysis of different concentrations of lotus root PDNs on normal human colonic epithelial cells (NCM460).

[0026] Figure 7 A graph showing the uptake analysis of lotus root PDNs by normal human colonic epithelial cells (NCM460);

[0027] Figure 8 Figure showing the results of constructing the LPS cell model;

[0028] Figure 9 Figure showing the effect of lotus root PDNs on cell activity;

[0029] Figure 10 The image shows the anti-inflammatory test results of lotus root PDNs on LPS-stimulated colonic epithelial cells (NCM460).

[0030] Figure 11 A schematic diagram illustrating the uptake of lotus root PDNs by normal mice after oral administration.

[0031] Figure 12Figure showing the test results of lotus root PDNs improving intestinal damage in IBD mice;

[0032] Figure 13 Figure showing the test results of lotus root PDNs improving intestinal inflammation in IBD mice. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments.

[0034] Example 1

[0035] The preparation method of lotus root PDNs is as follows:

[0036] Fresh lotus root juice was extracted and centrifuged for the first time (300g, 10min) to remove precipitated plant cells. The filtrate was then centrifuged a second time (2000g, 10min) to remove precipitated dead cells. The filtrate was then centrifuged a third time (10000g, 30min) to remove precipitated cell debris. Finally, the filtrate was centrifuged a fourth time (100000g, 70min). The resulting precipitate is lotus root PDNs. The preparation process is as follows: Figure 1 As shown.

[0037] Example 2

[0038] NTA method for detection of PDNs and particle size analysis in lotus root

[0039] Nanoparticle tracking analysis (NTA) is used to obtain the particle size distribution of samples in liquid suspensions by utilizing the properties of light scattering and Brownian motion. A laser beam passes through the sample chamber and along the path of the scattered light through the particles in the suspension. The particles are visualized using a long-working-distance magnifying microscope. An NTA schematic diagram is shown below. Figure 2 As shown. Then, software was used to track multiple particles individually, and the hydrodynamic diameter of the particles was calculated using Einstein's equations to further analyze the diameter of the lotus root nanoparticles. (See figure.) Figure 3 As shown.

[0040] Figure 2 The diagram shows the NTA detection of lotus root PDNs. As can be seen from the figure, the present invention obtained nanoscale particulate PDNs.

[0041] Figure 3 The figure shows the particle size analysis results of lotus root PDNs. As can be seen from the figure, the diameter of lotus root PDNs is 82.9±16.8nm, while the nanoparticles secreted by normal plants are 30-150nm.

[0042] Example 3

[0043] morphological feature detection of lotus root PDNs

[0044] The morphological characteristics of lotus root PDNs were observed using transmission electron microscopy (TEM), and the results are as follows: Figure 4 As shown.

[0045] Figure 4 The image shows the morphological characteristics of lotus root PDNs detected by TEM. As can be seen from the image, lotus root PDNs have a phospholipid bilayer membrane structure, proving that the nanoparticles derived from lotus root have been successfully obtained.

[0046] Example 4

[0047] Identification and analysis of contents of lotus root PDNs

[0048] Protein concentration was determined using the BCA method. First, bovine serum albumin (BSA) was serially diluted as a standard. Then, BCA working solution was prepared. Protein standards at various dilutions and the protein sample to be tested were added to microplates or test tubes, and BCA working solution was added and mixed thoroughly. After sealing, the plates were incubated at 37°C for 30–60 min, then cooled to room temperature. Using a blank as a control, the absorbance of the sample was measured near 562 nm. The average absorbance of the blank standard at 562 nm was then subtracted from the absorbance of each standard and the protein sample at 562 nm. The results are as follows: Figure 5 As shown.

[0049] Figure 5 This is a protein standard curve for lotus root PDNs, with the horizontal axis representing protein concentration and the vertical axis representing absorbance. As shown in the graph, by comparing with the standard curve, it was found that 9×10⁻⁶… 9 The protein content in lotus root PDNs with particle number / mL was 1.205922 mg, indicating that lotus root PDNs are rich in protein.

[0050] Example 5

[0051] Toxicity analysis of lotus root PDNs on normal human colonic epithelial cells (NCM460)

[0052] The lotus root PDNs prepared in Example 1 were formulated into solutions with concentrations of 0, 0.025, 0.05, 0.1, 0.2, and 0.3 mg / mL Protein. NCM460 cells were cultured in 96-well plates at a density of 1 × 10⁶ cells per well. 5Cells were cultured in a controlled environment with iron walls, and different concentrations of PDNs were added. After incubation for 24 hours, 10 μL of CCK-8 solution was added to each well, ensuring no air bubbles were generated. The cells were then incubated for another hour, and the absorbance at 450 nm was measured using a microplate reader. Cell viability was assessed using a CCK-8 kit; a cell viability exceeding 90% was considered non-toxic and the optimal concentration. NCM460 cells were cultured in Dulbecco modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS, 10% v / v), streptomycin (100 μg / mL), and penicillin (100 U / mL). Cells were cultured at 37°C under controlled humidity (5% CO2, 95% O2). Results are shown below. Figure 6 As shown.

[0053] Figure 6 The figure shows the toxicity analysis of different concentrations of lotus root PDNs on normal human colonic epithelial cells (NCM460). As can be seen from the figure, when the concentration of lotus root PDNs is higher than 0.2 mg / mL·Protein, the cell viability is <90%, indicating cytotoxicity. Therefore, a concentration of 0.2 mg / mL·Protein for lotus root PDNs was selected as the safe dosage for subsequent cell experiments.

[0054] Example 6

[0055] Analysis of uptake of lotus root PDNs by normal human colonic epithelial cells (NCM460)

[0056] Lotus root PDNs with a concentration of 0.2 mg / mL Protein and 1 mM DIO were incubated at 37°C for 30 min for staining. Residual dye was removed by centrifugation at 100,000 g for 2 h, yielding DIO-labeled PDNs. The labeled PDNs were then resuspended in human normal colonic epithelial cell (NCM460) culture medium and incubated for 0 h, 1.5 h, 3 h, and 6 h, respectively. The uptake of lotus root PDNs by cells was then observed using laser confocal microscopy. Results are as follows: Figure 7 As shown.

[0057] Figure 7 This diagram illustrates the uptake analysis of lotus root PDNs by normal human colonic epithelial cells (NCM460). DIO is a lipophilic fluorescent dye that exhibits very weak fluorescence before entering the cell membrane. Once inside the cell, it binds to the cell membrane and emits a distinct green fluorescence. DAPI, on the other hand, is a nuclear dye and appears blue. The diagram shows that lotus root PDNs can be taken up by NCM460 cells in a time-dependent manner.

[0058] Example 7

[0059] Establishment of LPS-stimulated human normal colonic epithelial cell (NCM460) cell model and determination of drug concentration

[0060] LPS concentrations of 0, 0.5, 1, 10, 20, and 100 μg / mL were prepared, and cells were cultured for 12 h. Cell viability was measured using a CCK8 assay, and 10 μg / mL was ultimately determined to be the optimal concentration for cell modeling. The results are as follows: Figure 8 As shown.

[0061] Lotus root PDNs (0.2 mg / mL Protein) were co-cultured with NCM460 cells for 12 h, followed by culturing for another 12 h with 10 μg / mL LPS. Cell viability was then measured using the CCK8 assay. The results are as follows: Figure 9 As shown.

[0062] Figure 8 The result of constructing the LPS cell model is shown in the figure. Figure 9 The figure shows the effect of lotus root PDNs on cell activity. As can be seen from the figure, the administration of PDNs at a concentration of 10 μg / mL for modeling with LPS and 0.2 mg / mL Protein had no significant effect on cell activity.

[0063] Example 8

[0064] Anti-inflammatory test of lotus root PDNs on LPS-stimulated colonic epithelial cells (NCM460)

[0065] NCM460 cells were stimulated with LPS for 12 h, resulting in a significant increase in the pro-inflammatory factor TNF-α. Pretreatment with different doses of lotus root PDNs (0.1 mg / mL Protein and 0.2 mg / mL Protein) for 12 h yielded the following results: Figure 10 As shown.

[0066] Figure 10 The figure shows the anti-inflammatory test results of lotus root PDNs on LPS-stimulated colonic epithelial cells (NCM460). As can be seen from the figure, lotus root PDNs can prevent changes in inflammatory factors caused by LPS stimulation, and high doses of lotus root PDNs have a significant effect on improving the inflammatory response.

[0067] Example 9

[0068] Oral uptake of lotus root PDNs in normal mice

[0069] Lotus root PDNs were stained with DIO and then administered to normal mice via gavage at a concentration of 0.2 mg / mL Protein before examination. The results are as follows: Figure 11 As shown.

[0070] Figure 11 This diagram illustrates the uptake of lotus root PDNs by normal mice after oral administration. As shown in the diagram, lotus root PDNs are mainly enriched in the gastrointestinal tract.

[0071] Example 10

[0072] Tests on the improvement of intestinal damage in IBD mice by lotus root PDNs

[0073] Three days after normal mice were administered lotus root PDNs at a gavage of 0.2 mg / mL Protein, on the fourth day, the mice were given 0.25% DSS to induce a dysplasia for 7 days. During this period, the mice were continuously treated with lotus root PDNs. On the tenth day, the mice were sacrificed, and intestinal damage was observed. Results are as follows: Figure 12 As shown.

[0074] Figure 12 The figure shows the test results of lotus root PDNs improving intestinal damage in IBD mice. As can be seen from the figure, the lotus root PDNs of the present invention significantly restored colon length and reduced intestinal pathological damage.

[0075] Example 11

[0076] Tests on the improvement of intestinal inflammation in IBD mice by lotus root PDNs

[0077] After normal mice were administered lotus root PDNs at a dose of 0.2 mg / mL Protein by gavage for 3 days, starting on the fourth day, the mice were given 0.25% DSS to induce inflammatory cytokine production for 7 days. During this period, the mice were continuously treated with lotus root PDNs. On the tenth day, blood was collected from the orbital cavity, and the mice were sacrificed. The blood was centrifuged at 3000 rpm, and the supernatant was collected to detect the levels of inflammatory factors in the serum. The results are as follows: Figure 13 As shown.

[0078] Figure 13 The figure shows the test results of lotus root PDNs improving intestinal inflammation in IBD mice. As can be seen from the figure, the lotus root PDNs of the present invention significantly reduced the content of inflammatory factors.

Claims

1. Application of lotus root PDNs in the preparation of anti-inflammatory drugs; wherein the PDNs are plant-secreted nanovesicles.

2. The application according to claim 1, characterized in that, The concentration of lotus root PDNs used in anti-inflammatory drugs does not exceed 0.3 mg / mL·Protein.

3. The application according to claim 2, characterized in that, The concentration of lotus root PDNs used in anti-inflammatory drugs is 0.2 mg / mL·Protein.

4. The application according to claim 1, characterized in that, The anti-inflammatory drug prepared from lotus root PDNs is used to treat intestinal inflammation.

5. The application according to claim 1, characterized in that, The method for preparing lotus root PDNs is as follows: fresh lotus root is juiced, centrifuged for the first time to remove the precipitated plant cells, the filtrate is centrifuged for the second time to remove the precipitated dead cells, the filtrate is centrifuged for the third time to remove the precipitated cell fragments, and the filtrate is centrifuged for the fourth time. The resulting precipitate is lotus root PDNs.

6. The application according to claim 5, characterized in that, The centrifugal forces for the first, second, third, and fourth centrifugations were 300g, 2000g, 10000g, and 100000g, respectively.

7. The application according to claim 5, characterized in that, The centrifugation time for the first and second centrifugations was 10 min, and the centrifugation time for the third and fourth centrifugations was 30 min and 70 min, respectively.

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