Curcumin nano vesicles and application thereof

Abstract

The application relates to a curcuma nano vesicle and application thereof, and a preparation method of the curcuma nano vesicle. The preparation method comprises the following steps: obtaining curcuma supernatant by differential centrifugation of curcuma original slurry at 4 DEG C; reacting the curcuma supernatant with polyethylene glycol overnight; precipitating aggregates by low-speed centrifugation; resuspending and dispersing the precipitated aggregates by using a phosphate buffer, and then dialyzing; mixing with a high-speed centrifugation precipitate resuspension liquid; extracting an organic phase solution from the mixed solution by using an extraction liquid; rotary evaporating the organic phase solution; and finally hydrating the evaporated film to obtain the curcuma nano vesicle. The nano vesicle prepared by the method has good stability, high in-vivo bioavailability, and has an obesity treatment effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a turmeric nanovesicle and its application. Background Technology

[0002] In recent years, the global prevalence of obesity has been increasing at an alarming rate. The problem of overweight and obesity among Chinese residents is becoming increasingly prominent, and has now become a medical and social issue in my country. Traditional chemically synthesized drugs suffer from unstable quality and side effects such as appetite suppression, seriously endangering human health. Therefore, there is an urgent need to develop new products for treating obesity.

[0003] Turmeric is a traditional Chinese medicine that is also used as food. The active ingredient in turmeric, curcuminoids, can reduce insulin resistance and thus aid in weight loss, while avoiding the adverse reactions associated with traditional chemically synthesized weight-loss drugs. However, its poor solubility, instability, low absorption rate, and rapid metabolism hinder its clinical application. Summary of the Invention

[0004] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, one object of this invention is to provide a method for preparing turmeric nanovesicles. The nanovesicles prepared by this method have good stability, high bioavailability in vivo, and exhibit therapeutic effects for obesity.

[0005] Therefore, in one aspect of the present invention, a method for preparing turmeric nanovesicles is proposed, which includes the following steps:

[0006] Step 1: Wash the turmeric, add phosphate buffer solution, juice it, filter it, and obtain turmeric tissue pulp;

[0007] Step 2: Centrifuge the turmeric tissue slurry at 4°C at a differential speed to obtain the turmeric supernatant, and use phosphate buffer to collect the precipitate from the last centrifugation to obtain the precipitate resuspension.

[0008] Step 3: Add polyethylene glycol to the turmeric supernatant, stir well, and incubate overnight at 4°C;

[0009] Step 4: Centrifuge the solution after overnight incubation in Step 3 at low speed, resuspend and disperse the precipitate with phosphate buffer to obtain the initial extract of turmeric extracellular vesicles, and dialyze overnight at 4°C.

[0010] Step 5: Mix the precipitate resuspension from Step 2 with the initial extract of turmeric extracellular vesicles, add the extraction solution for extraction, and collect the organic phase;

[0011] Step 6: Distill the organic phase under reduced pressure to form a thin film, add phosphate buffer to hydrate the film, and obtain turmeric nanovesicles.

[0012] According to an embodiment of the present invention, a method for preparing turmeric nanovesicles is provided. The method collects the precipitate during centrifugation, thereby increasing the concentration of turmeric compounds. At the same time, extraction and rotary evaporation are used to effectively remove impurities such as proteins and water-soluble impurities from the turmeric nanovesicles.

[0013] Compared to traditional nanocarrier-encapsulated curcumin drugs, these curcumin nanovesicles have lower toxicity, excellent biocompatibility, and naturally carry the active molecules from the source plant, which can effectively alleviate obesity in mice.

[0014] In addition, the method for preparing turmeric nanovesicles according to the above embodiments of the present invention may also have the following additional technical features:

[0015] Optionally, in step 2, the differential centrifugation is performed at 4°C, 400g, for 15 min; at 4°C, 1000g, for 15 min; at 4°C, 3000g, for 30 min; at 4°C, 3000g, for 30 min; and at 4°C, 10000g, for 1 h.

[0016] Optionally, in step 3, the concentration of polyethylene glycol is 10% to 15%.

[0017] Optionally, in step 4, the low-speed centrifugation is performed at 4°C, 8000g, for 30 minutes.

[0018] Optionally, in step 5, the precipitate resuspension from step 2 and the initial extract of turmeric extracellular vesicles are mixed and vortexed for 2 minutes at a ratio of mixture:methanol:chloroform = 1:2:1. Then, an equal volume of chloroform is added for extraction, and the organic phase is collected.

[0019] Optionally, in step 6, the organic phase is distilled under reduced pressure at 37°C for 1–2 hours to form a thin film.

[0020] Optionally, the phosphate buffer solution has a concentration of 0.01 M and a pH of 7.4.

[0021] In another aspect of the invention, a turmeric nanovesicle is also provided, which is prepared by the above-described method.

[0022] According to embodiments of the present invention, the drug-loaded extracellular vesicles employ the above-described active drug loading method, which, compared to the passive drug loading method, can increase the drug loading rate of doxorubicin from less than 10% to nearly 60%, significantly improving the problem of low drug loading rate of extracellular vesicles.

[0023] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0024] Figure 1This is a flowchart illustrating the preparation process of turmeric nanovesicles according to an embodiment of the present invention;

[0025] Figure 2 Electron micrographs of turmeric nanovesicles and a control sample according to an embodiment of the present invention;

[0026] Figure 3 The purity of turmeric nanovesicles according to embodiments of the present invention;

[0027] Figure 4 The loading amount of turmeric nanovesicles according to an embodiment of the present invention;

[0028] Figure 5 The above is a high-performance liquid chromatogram of turmeric nanovesicles according to an embodiment of the present invention;

[0029] Figure 6 For in vivo fluorescence imaging in mice according to embodiments of the present invention;

[0030] Figure 7 A staining image of turmeric nanovesicles according to an embodiment of the present invention;

[0031] Figure 8 The change in body weight of mice according to an embodiment of the present invention;

[0032] Figure 9 The change in body fat percentage in mice according to an embodiment of the present invention. Detailed Implementation

[0033] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0034] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0035] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0036] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0037] Example 1

[0038] Preparation of turmeric nanovesicles:

[0039] Step 1: Take an appropriate amount of fresh local turmeric, wash it, add 50% phosphate buffer (0.01M, pH=7.4), place it in a blender and stir into a paste, filter out the plant residue with gauze to obtain turmeric tissue pulp.

[0040] Step 2: Centrifuge the turmeric tissue slurry obtained in Step 1 at different speeds (4℃, 400g, 15min; 4℃, 1000g, 15min; 4℃, 3000g, 30min; 4℃, 3000g, 30min; 4℃, 10000g, 1h) to obtain the turmeric supernatant. Collect the precipitate from the last centrifugation using phosphate buffer.

[0041] Step 3: Add 15% PEG8000 to the turmeric supernatant obtained in Step 2, stir well, and incubate overnight at 4°C.

[0042] Step 4: Centrifuge the solution obtained in Step 3 at low speed (4℃, 8000g, 30min), resuspend and disperse the precipitate with phosphate buffer to obtain the initial extract of turmeric extracellular vesicles, and dialyze overnight at 4℃.

[0043] Step 5: Mix the precipitate resuspension obtained in Step 2 and the initial extract of turmeric extracellular vesicles obtained in Step 4, and vortex mix at a ratio of 1:2:1 (mixture:methanol:chloroform) for 2 min. Then add an equal volume of chloroform for extraction, and wash with phosphate buffer and DDH2O to obtain the organic phase.

[0044] Step 6: The organic phase solution obtained in Step 5 is distilled under reduced pressure at 37°C for 1 hour to form a thin film. The film is then hydrated with phosphate buffer to obtain turmeric nanovesicles. The extraction process is as follows: Figure 1 As shown.

[0045] Comparison product:

[0046] The turmeric was pretreated using the steps described above. The supernatant of the turmeric sample obtained by differential centrifugation in step 2 was centrifuged at 100,000g at 4°C for 70 minutes. The precipitate was resuspended and centrifuged at 10,000g at 4°C for 10 minutes. The supernatant was then collected to obtain the ultracentrifuged turmeric extracellular vesicle control sample.

[0047] Example 2

[0048] 1. The morphology of the turmeric nanovesicles from Example 1 and the turmeric extracellular vesicle control sample was examined by transmission electron microscopy. 10 μL of sample was dropped onto a copper grid and incubated at 37°C. Excess solution was absorbed with filter paper, and then 10 μL of sample was dropped onto the copper grid.

[0049] After staining with 1% phosphotungstic acid for 1-2 minutes, absorb the excess phosphotungstic acid with filter paper and observe its morphology and structure using an HT-7800 transmission electron microscope.

[0050] The results are as follows Figure 2 As shown ( Figure 2 The left side shows turmeric nanovesicles, and the right side shows turmeric extracellular vesicle control samples. The turmeric nanovesicles prepared in Example 1 have a typical teacup-shaped exosome structure.

[0051] 2. The purity and curcumin loading of the turmeric nanovesicles and the turmeric extracellular vesicle control sample from Example 1 were determined by nanoflow cytometry (nFCM) and BCA method. Nanoflow cytometry (nFCM): The Flow Nanoanalyzer was preheated. After the indicator light illuminated, the laser was turned on. High-pressure rinsing and air bubble removal were then performed. The instrument's optical path was adjusted using fluorescent silicon spheres. The sample was diluted to approximately 10... 8 After determining the particle / mL concentration, samples were loaded for detection. BCA: First, solution A and solution B from the BCA protein quantification kit were mixed at a ratio of 50:1 to obtain the BCA reaction working solution. Then, different concentrations of bovine serum albumin (BSA) standard solutions were prepared, ranging from 2 mg / mL to 0.05 mg / mL. 50 μL of the BSA standard solution and 200 μL of the test sample were added to ice-cold acetone, and the mixture was incubated at 20°C for 30 min. After incubation, the mixture was centrifuged using a high-speed refrigerated centrifuge. After the acetone evaporated in the test tube, 50 μL of DDH2O was added to the precipitate. 100 μL of the BCA reaction working solution was added to each BSA standard and test sample, and the mixture was incubated at 65°C for 20 min. The absorbance at 562 nm was then measured using a microplate reader. A standard curve was plotted between BSA concentration and absorbance, and the sample concentration was calculated.

[0052] The results are as follows Figure 3 and 4 As shown, the purity and loading of curcumin-like compounds of the turmeric nanovesicles prepared in Example 1 were both higher than those of the ultracentrifuged turmeric extracellular vesicle control sample.

[0053] 3. The turmeric nanovesicles from Example 1 and the turmeric extracellular vesicle control sample were analyzed for the composition of curcuminoids by high performance liquid chromatography (HPLC). An Agilent SB-C18 column was used, with acetonitrile / 0.25% glacial acetic acid aqueous solution as the mobile phase, a detection wavelength of 420 nm, a column temperature of 30 °C, and a flow rate of 1 mL / min. Curcuminoids were used as standards, and the injection volume of each sample was 10 μL.

[0054] The results are as follows Figure 5 As shown, the turmeric nanovesicles prepared in Example 1 contain three types of curcumin compounds: demethoxycurcumin, demethoxycurcumin, and curcumin.

[0055] Example 3

[0056] In vitro and in vivo uptake capacity study of turmeric nanovesicles:

[0057] 1. The turmeric nanovesicles prepared in Example 1 were administered to mice via gavage and subcutaneous injection, respectively. In vivo fluorescence imaging was used to monitor the retention of the turmeric nanovesicles in the animals. Preparation of DiR-labeled turmeric nanovesicles: A final concentration of 50 μM of the membrane fluorescent dye DiR was added to the turmeric nanovesicles, and the mixture was co-incubated at 37°C for 1 h. In vivo imaging: C57BL / 6 mice were administered the drug at 50 mg / kg via gavage and subcutaneous injection, respectively. Mice were anesthetized at 1, 3, 6, 9, 12, and 24 h for in vivo imaging. After the detection, the mice were dissected, and the fluorescence imaging results of each organ were observed.

[0058] The results are as follows Figure 6 As shown, both administration methods allow the drug to enter the animal's body. In the gavage group, the drug is mainly concentrated in the intestine, while in the subcutaneous injection group, the drug is mainly concentrated in the fat.

[0059] 2. Curcuma nanovesicles were stained with the membrane fluorescent dye DiD. The stained nanovesicles were then added to 3T3-L1 cells and stained with Hoechst and Mitotracker Green dyes, followed by confocal microscopy imaging. The nanovesicles were incubated with DiD at 37°C for 1 hour for membrane fluorescence staining. Dialysis was then performed using dialysis bags with a molecular weight cutoff of 8-12 kDa in PBS buffer at 4°C to remove the free fluorescent dye; the medium was changed three times during this process. 2 × 10⁶ cells were seeded in a dedicated confocal culture dish. 53T3-L1 mouse preadipocytes were cultured overnight in a constant temperature incubator. After cell attachment, DiD was diluted 1000 times to stain the cells. After culturing for 24 hours, Hoechst 33342 and 20 nM Mitotracker Green were added to a final concentration of 5 μg / mL. The cells were co-incubated at 37°C for 30 min and then washed. Cell uptake was observed using a Leica SP-8 confocal microscope.

[0060] The results are as follows Figure 7 As shown, turmeric nanovesicles can be specifically taken up by 3T3-L1 cells.

[0061] 3. The turmeric nanovesicles prepared in the examples were administered to obese mice via gavage and subcutaneous injection, and their weight was recorded. After treatment, the changes in body fat in the mice were detected using MRI. Healthy male C57BL / 6 mice, weighing 20-25g, were randomly divided into two groups: a group fed a diet containing 45% kcal fat (n=9, denoted as the HFD group) and a group fed a standard diet (n=3, denoted as the SD group). Mice were weighed weekly, and weight changes were observed and compared after eight weeks. A significant difference in weight gain between the two groups was considered a successful establishment of the obese mouse model. After successful model establishment, the HFD group mice were randomly divided into three groups: a gavage group, a subcutaneous injection group, and an obese model control group. The SD group continued to be fed a standard diet freely, while the HFD group continued to be fed a diet containing 45% kcal fat freely. The single dose for the treatment groups was 50 mg / kg, administered every two days for 30 days. Mice were weighed every two days during the treatment period. After treatment, mice were subjected to MRI to measure their total body fat and muscle mass. The weight loss of the mice was analyzed by the percentage of fat and muscle mass.

[0062] Weight changes as follows Figure 8 As shown, the obese mice treated with the treatment experienced a significant decrease in body weight; their body fat percentage was as follows: Figure 9 As shown, the ratio of lean to fat tissue in obese mice decreased significantly after treatment.

[0063] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0064] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing turmeric nanovesicles, characterized in that, Includes the following steps: Step 1: Wash the turmeric, add 0.01M phosphate buffer solution with pH 7.4, juice it, filter it, and obtain turmeric tissue pulp. Step 2: Centrifuge the turmeric tissue concentrate at 4°C using differential speed to obtain the turmeric supernatant. Collect the precipitate from the last centrifugation using phosphate buffer to obtain the precipitate resuspension. The differential centrifugation times were: 4°C, 400 g, 15 min; 4°C, 1000 g, 15 min; 4°C, 3000 g, 30 min; 4°C, 3000 g, 30 min; 4°C, 10000 g, 1 h. Step 3: Add 10%~15% polyethylene glycol to the turmeric supernatant, stir well, and incubate overnight at 4°C; Step 4: Centrifuge the solution after overnight incubation in Step 3 at low speed, resuspend and disperse the precipitate with phosphate buffer to obtain the initial extract of turmeric extracellular vesicles, and dialyze overnight at 4°C; centrifuge at 4°C, 8000 g, for 30 min. Step 5: Mix the precipitate resuspension from Step 2 with the initial extract of turmeric extracellular vesicles, and vortex mix at a ratio of mixture:methanol:chloroform = 1:2:1 for 2 min. Then add an equal volume of chloroform for extraction and collect the organic phase. Step 6: Distill the organic phase under reduced pressure at 37°C for 1-2 h to form a thin film, then add phosphate buffer to hydrate the film to obtain turmeric nanovesicles.

2. A turmeric nanovesicle, characterized in that, It is prepared by the preparation method described in claim 1.

3. The application of turmeric nanovesicles as described in claim 2 in the preparation of drugs for weight loss, lowering blood lipids, and anti-inflammation.

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