Nano preparation as well as preparation method and application thereof

Abstract

The invention discloses a nano preparation as well as a preparation method and application thereof. The invention specifically provides a preparation method of a nano preparation and the nano preparation prepared by the method, the preparation method comprises the following steps: step 1, pretreating a pharmaceutical composition, mixing the pharmaceutical composition with a buffer solution, and carrying out ultrasonic centrifugation to obtain a water phase and a lower precipitate; and step 2, preparing the nano preparation from the organic phase and the water phase in the step 1 through a microfluidic method, the flow velocity of the organic phase is 1-5 mL / min, and the flow velocity of the water phase is 6-12 mL / min; the organic phase comprises the lower layer precipitate in the step 1, an alcohol solvent, phospholipid and sterol. The nano preparation disclosed by the invention can be used for treating oral diseases, and can effectively promote the growth of gingival fibroblasts and inhibit the content expression of IL-1alpha and TNF-alpha.

Description

Technical Field

[0001] This invention relates to a nano-formulation, its preparation method, and its application. Background Technology

[0002] Traditional Chinese medicine formulas for nourishing Yin and clearing the lungs suffer from poor water solubility, insufficient stability, and low bioavailability, severely hindering their widespread clinical application. These problems not only challenge the modernization of traditional Chinese medicine but also prompt researchers to explore new solutions. The introduction of nanotechnology offers a new perspective for addressing these issues. Through nano-processing, the solubility, stability, and bioavailability of traditional Chinese medicine can be significantly improved, thereby enhancing its efficacy and reducing side effects. However, despite some progress in the nano-processing of single-herb traditional Chinese medicines, the nano-processing of compound traditional Chinese medicine formulas still faces numerous challenges.

[0003] The complexity of compound traditional Chinese medicines lies in the synergistic effects of their multiple components and targets. This necessitates not only considering the nano-sizing of individual components but also researching how to preserve the synergistic effects of the compound at the nanoscale. Current research largely focuses on the nano-sizing of single herbs or simple extracts, while systematic research and reports on the nano-sizing of compound traditional Chinese medicines are still lacking. Summary of the Invention

[0004] The technical problem this invention aims to solve is to overcome the lack of research on the nano-scale preparation of compound Yin-nourishing and Lung-clearing ointment in the prior art, and to provide a nano-preparation, its preparation method, and its application. This nano-preparation can be used to treat oral diseases, effectively promoting the growth of gingival fibroblasts and inhibiting the expression of IL-1α and TNF-α.

[0005] This invention provides a method for preparing nano-formulations, comprising the following steps:

[0006] Step 1: Pretreatment of the drug composition:

[0007] The drug composition was mixed with a buffer solution and then ultrasonically centrifuged to obtain an aqueous phase and a lower precipitate.

[0008] The pharmaceutical composition comprises the following components in parts by weight: 70-130 parts of Rehmannia glutinosa, 30-90 parts of Ophiopogon japonicus, 50-110 parts of Scrophularia ningpoensis, 10-70 parts of Fritillaria cirrhosa, 10-70 parts of Paeonia lactiflora, 10-70 parts of Paeonia suffruticosa, 15-45 parts of Mentha haplocalyx and 10-30 parts of Glycyrrhiza uralensis.

[0009] Step 2: The organic phase and the aqueous phase from Step 1 are combined using a microfluidic method to prepare the nano-formulation.

[0010] The flow rate of the organic phase is 1-5 mL / min, and the flow rate of the aqueous phase is 6-12 mL / min;

[0011] The organic phase includes the lower precipitate from step 1, alcohol solvents, phospholipids, and sterols.

[0012] In one embodiment, the organic phase is prepared by the following method:

[0013] Step S1: Mix the drug composition with a buffer solution, and centrifuge by sonication to obtain the lower precipitate;

[0014] Step S2: Mix the precipitate with an alcohol solvent, and after ultrasonic centrifugation, obtain the upper layer solution as solution 1;

[0015] Step S3: Mix the solution 1 with the volatile oil to obtain the oil phase;

[0016] Step S4: Mix the oil phase, phospholipids and sterols to obtain the organic phase.

[0017] In one embodiment, the organic phase further includes volatile oils.

[0018] In one embodiment, the nano-formulation comprises the following components in parts by weight: 90-110 parts of Rehmannia glutinosa, 50-70 parts of Ophiopogon japonicus, 70-90 parts of Scrophularia ningpoensis, 30-50 parts of Fritillaria cirrhosa, 30-50 parts of Paeonia lactiflora, 30-50 parts of Paeonia suffruticosa, 15-35 parts of Mentha haplocalyx, and 15-25 parts of Glycyrrhiza uralensis; for example, it may include 100 parts of Rehmannia glutinosa, 60 parts of Ophiopogon japonicus, 80 parts of Scrophularia ningpoensis, 40 parts of Fritillaria cirrhosa, 40 parts of Paeonia lactiflora, 40 parts of Paeonia suffruticosa, 25 parts of Mentha haplocalyx, and 20 parts of Glycyrrhiza uralensis.

[0019] In one embodiment, the pharmaceutical composition in the nano-formulation is prepared by means of the following method: the components are prepared according to the compound preparations and single-herb preparations of the Chinese Pharmacopoeia 2020, Part I.

[0020] In one embodiment, the buffer is a bicarbonate buffer, a HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer, or a PBS buffer (phosphate buffer); for example, a PBS buffer.

[0021] In one embodiment, the alcohol solvent is methanol or ethanol, preferably anhydrous ethanol.

[0022] In one embodiment, the mass-to-volume ratio of the pharmaceutical composition to the buffer solution is (0.5-6) ​​g / L; preferably (1-4) g / L; for example, 2 g / L.

[0023] In one embodiment, the mass-to-volume ratio of the pharmaceutical composition to the volume of the alcohol solvent is (3-8) g / L; preferably (4-7) g / L; for example, 5.7 g / L.

[0024] In one embodiment, the ultrasonic time in the aqueous phase and step S1 is 2-10 min, preferably 5 min.

[0025] In one embodiment, the frequency of the ultrasound in the aqueous phase and step S1 is 35-45 MHz; for example, 40 MHz.

[0026] In step S2, the ultrasound duration is conventional in the art. In one embodiment, the ultrasound duration is until no precipitation occurs.

[0027] In one embodiment, the volume ratio of solution 1 to the volatile oil is (1-6):1; preferably (2-5):1; for example, 3:1.

[0028] In one embodiment, the volatile oil is one or both of peony bark volatile oil and peppermint volatile oil; preferably, the volatile oil is prepared by steam distillation of peony bark and peppermint respectively; more preferably, the mass ratio of peony bark to peppermint is 8:5.

[0029] The phospholipid may be a conventional phospholipid in the art. In one embodiment, the phospholipid is distearylphosphatidylcholine (DSPC), dimyristoyl phosphocholine (DMPC), dioleoyl phosphocholine (DOPC), palmitoyl phosphocholine (DPPC), docosanoyl phosphocholine (DUPC), palmitoyl phosphocholine (POPC), or soy lecithin; for example, soy lecithin.

[0030] The sterol may be a conventional sterol in the art. In one embodiment, the sterol is cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, or α-tocopherol, such as cholesterol.

[0031] In one embodiment, the mass ratio of the phospholipid to the sterol is (0.3-3):1; preferably (0.8-1.5):1; for example, 1:1.

[0032] In one embodiment, the mass-to-volume ratio of the phospholipid to the oil phase is (5-10) g / L; preferably (6-8) g / L; for example, 7.5 g / L.

[0033] In one embodiment, the flow rate of the organic phase is 2-4 mL / min; preferably 3 mL / min.

[0034] In one embodiment, the flow rate of the aqueous phase is 8-10 mL / min; preferably 9 mL / min.

[0035] This invention provides a nano-formulation, which is prepared by the preparation method described above.

[0036] The present invention provides a nano-formulation comprising the following components in parts by weight: 10-30 parts of a pharmaceutical composition, 20-40 parts of phospholipids and 20-40 parts of sterols;

[0037] The pharmaceutical composition comprises the following components in parts by weight: 70-130 parts of Rehmannia glutinosa, 30-90 parts of Ophiopogon japonicus, 50-110 parts of Scrophularia ningpoensis, 10-70 parts of Fritillaria cirrhosa, 10-70 parts of Paeonia lactiflora, 10-70 parts of Paeonia suffruticosa, 15-45 parts of Mentha haplocalyx and 10-30 parts of Glycyrrhiza uralensis.

[0038] In one embodiment, the nanoformulation comprises the following components in parts by weight: 15-25 parts pharmaceutical composition, 25-35 parts phospholipids and 25-35 parts sterols; for example, it comprises the following components in parts by weight: 20 parts pharmaceutical composition, 30 parts phospholipids and 30 parts sterols.

[0039] In one embodiment, the nano-formulation comprises the following components in parts by weight: 90-110 parts of Rehmannia glutinosa, 50-70 parts of Ophiopogon japonicus, 70-90 parts of Scrophularia ningpoensis, 30-50 parts of Fritillaria cirrhosa, 30-50 parts of Paeonia lactiflora, 30-50 parts of Paeonia suffruticosa, 15-35 parts of Mentha haplocalyx, and 15-25 parts of Glycyrrhiza uralensis; or, for example, the following components in parts by weight: 100 parts of Rehmannia glutinosa, 60 parts of Ophiopogon japonicus, 80 parts of Scrophularia ningpoensis, 40 parts of Fritillaria cirrhosa, 40 parts of Paeonia lactiflora, 40 parts of Paeonia suffruticosa, 25 parts of Mentha haplocalyx, and 20 parts of Glycyrrhiza uralensis.

[0040] In one embodiment, the types of phospholipids and sterols are as described above.

[0041] In one embodiment, the average particle size of the nano-formulation is 139-155 nm; for example, 144-147 nm.

[0042] In one embodiment, the PDI (polydispersity index) of the nanoformulation is 0.12 to 0.22, for example, 0.16.

[0043] In one embodiment, the zeta potential of the nanoformulation is -18 to -9 mV, for example -15 to -12 mV.

[0044] This invention provides the application of the nano-formulation described above in the preparation of medicaments for the prevention and / or treatment of oral diseases.

[0045] In one embodiment, the oral disease is a periodontal disease or an oral mucosal disease; the periodontal disease is, for example, gingivitis or periodontitis; the gingivitis and periodontitis are preferably diseases mediated by the inflammatory factors IL-1α and / or TNF-α in gingival fibroblasts.

[0046] The oral mucosal diseases are preferably Sjögren's syndrome, oral lichen planus, or oral ulcers.

[0047] This invention provides the use of a pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of oral diseases;

[0048] The pharmaceutical composition comprises the following components in parts by weight: 70-130 parts of Rehmannia glutinosa, 30-90 parts of Ophiopogon japonicus, 50-110 parts of Scrophularia ningpoensis, 10-70 parts of Fritillaria cirrhosa, 10-70 parts of Paeonia lactiflora, 10-70 parts of Paeonia suffruticosa, 15-45 parts of Mentha haplocalyx and 10-30 parts of Glycyrrhiza uralensis.

[0049] The pharmaceutical composition preferably comprises the following components in parts by weight: 90-110 parts of Rehmannia glutinosa, 50-70 parts of Ophiopogon japonicus, 70-90 parts of Scrophularia ningpoensis, 30-50 parts of Fritillaria cirrhosa, 30-50 parts of Paeonia lactiflora, 30-50 parts of Paeonia suffruticosa, 15-35 parts of Mentha haplocalyx and 15-25 parts of Glycyrrhiza uralensis; for example, it may include 100 parts of Rehmannia glutinosa, 60 parts of Ophiopogon japonicus, 80 parts of Scrophularia ningpoensis, 40 parts of Fritillaria cirrhosa, 40 parts of Paeonia lactiflora, 40 parts of Paeonia suffruticosa, 25 parts of Mentha haplocalyx and 20 parts of Glycyrrhiza uralensis.

[0050] The types of oral diseases are as described above.

[0051] The present invention also provides an inhibitor of the inflammatory factor IL-1α or TNF-α (for use in mammalian organisms or in vitro), comprising the nanoformulations or pharmaceutical compositions as described above.

[0052] The present invention also provides a kit comprising the nanoformulations, pharmaceutical compositions, or inhibitors as described above.

[0053] In one embodiment, the kit provides a rapid detection of the effect of inhibiting inflammatory factors IL-1α or TNF-α.

[0054] In this invention, the nano-formulation is a nanoliposome.

[0055] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0056] The reagents and raw materials used in this invention are all commercially available.

[0057] The positive and progressive effects of this invention are that it has one or two of the following effects:

[0058] (1) The nano-prepared formulation prepared by the present invention has spherical nanoparticles with a particle size of about 145 nm. After being stored at 4℃ and 25℃ for one month, there is no significant difference in particle size and dispersibility of the nano-prepared formulation, which has good stability.

[0059] (2) The nano-formulation of the present invention can significantly inhibit the secretion of inflammatory factors IL-1α and TNF-α by gingival fibroblasts and promote cell migration and healing. Attached Figure Description

[0060] Figure 1 This is the appearance of a nano-sized Yin-nourishing and lung-clearing preparation.

[0061] Figure 2 This is a TEM image of a nano-sized lung-nourishing and yin-clearing preparation.

[0062] Figure 3 SEM image of the Yin-nourishing and Lung-clearing paste.

[0063] Figure 4 This is a SEM image of a nano-sized lung-nourishing and yin-clearing preparation. Detailed Implementation

[0064] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0065] Example 1

[0066] Preparation of Yin-nourishing and Lung-clearing Paste

[0067] Table 1 Prescription

[0068]

[0069] The components in Table 1 were prepared according to the formula and single-herb preparations in the Chinese Pharmacopoeia 2020 edition, Part I.

[0070] Example 2

[0071] Preparation of nano-nourishing Yin and clearing lung preparations

[0072] In a tube, 20 mg of Yangyin Qingfei Ointment was dissolved in 10 mL of PBS and sonicated for 5 min (40 MHz). The sonicated solution was centrifuged, resulting in a light brown aqueous phase on top and a brownish-red precipitate on the bottom. The aqueous phase was transferred to a new tube. 3.5 mL of anhydrous ethanol was added to the brownish-red precipitate in the initial tube, and the mixture was sonicated until no obvious precipitate was observed, resulting in a brownish-red suspension. After centrifugation, the layers separated, with a light yellow organic phase 1 on top and a brownish-red precipitate on the bottom (discarded). Approximately 3 mL of organic phase 1 was transferred to a centrifuge tube, and 1 mL of volatile oil was added to obtain the oil phase.

[0073] The obtained oil phase, 30 mg of soybean lecithin, and 30 mg of cholesterol were mixed to form organic phase 2. This organic phase was then combined with an aqueous phase via microfluidic control to prepare nanoparticles of traditional Chinese medicine. The system parameters for nanoparticle preparation were set as follows: organic phase flow rate 3 mL / min, aqueous phase flow rate 9 mL / min. A clear brown solution was obtained after synthesis, which is the nano-nourishing and lung-clearing preparation.

[0074] The preparation of volatile oils is as follows:

[0075] Distill 40g of peony bark and 25g of peppermint separately using 1000mL of water, collect the distillates, let them stand to separate into layers, and then take the volatile oil components separately and mix them together.

[0076] Example 3

[0077] Morphological observation of nano-nourishing Yin and clearing lung preparation

[0078] (1) The particle size, PDI (polydispersity index), and Zeta potential of the nano-nourishing and lung-clearing preparation were detected by a nanoparticle size and Zeta potential analyzer. After being stored at 4℃ and 25℃ for one month, there was no significant difference in the particle size and dispersibility of the nanoparticles, which showed good stability (Table 2).

[0079] Table 2. Particle size distribution and Zeta potential of nano-sized Yin-nourishing and lung-clearing preparations

[0080]

[0081] (2) The morphology of the nano-nourishing yin and clearing lung particles was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Figure 1 The results showed that the nano-sized Yin-nourishing and lung-clearing particles were spherical, and TEM results showed that ( Figure 2 The particle size is around 145 nm.

[0082] Figure 3 and Figure 4 SEM results showed that the nano-nourishing yin and clearing lung particles were uniformly spherical with good dispersibility. Compared with the particles in the herbal paste, the nano-nourishing yin and clearing lung particles were significantly smaller in size.

[0083] Example 4 Cell viability and migration assay

[0084] To evaluate the growth-promoting effect of the nano-nourishing and lung-clearing preparation on gingival fibroblasts, we used the MTT assay and scratch assay to detect cell viability and cell migration ability, respectively. The experiment was divided into a negative control group, a positive control group (human epidermal growth factor, hEGF), and a sample group, with two concentration gradients of 1.250% and 0.625% set in the sample group.

[0085] (1) Collect cells in the logarithmic growth phase, adjust the cell density, and seed them into 96-well plates, with 100 μL of culture medium per well. After culturing in a CO2 cell incubator for 24 h, aspirate the culture medium and wash the cells twice with PBS buffer. The samples were grouped and administered drugs according to the final concentration (%, V / V) of the prepared solution (10.000, 5.000, 2.500, 1.250, 0.625, 0.313, and 0.156).

[0086] (2) Grouping situation (Table 3)

[0087] Table 3

[0088]

[0089] (3) Drug administration: After drug administration according to the above grouping, the cells were cultured in a CO2 cell incubator for another 24 hours, and then the MTT cell viability assay was performed. The absorbance OD value was read at 490 nm. The cell viability was calculated. Two sample concentrations with cell viability ≥90% were used for subsequent efficacy testing. Only cell culture medium was added to the blank wells.

[0090] (4) MTT cell viability assay: After 24 hours of culture, cells were stained with MTT and their viability was measured. 490 After measuring the absorbance, cell viability was calculated.

[0091] (5) Cell migration detection:

[0092] Adjust the cell density, re-seedle the cells in 96-well plates, and incubate them in a CO2 cell culture incubator for 24 hours before performing the scratching operation. After scratching, gently wash the cells once with PBS, observe and photograph them under an inverted microscope, and take a picture at 0h (initial scratch area).

[0093] After administration of drugs according to grouping in (2), the cells were cultured in a CO2 cell culture incubator for 24 hours. The cells were then gently washed three times with PBS, observed under an inverted microscope, and photographed for 24 hours (scratch area after 24 hours).

[0094] Analysis: Data analysis was performed using Image Pro Plus software.

[0095] (6) Data processing

[0096] Cell viability (%) = OD of sample group 490 / Negative control group OD 490 *100%

[0097] Cell healing capacity (%) = (Initial scratch area - Scratch area after 24 hours) / Initial scratch area * 100%

[0098] Note: Blank holes are deducted from each set of data.

[0099] (7) Results of cell viability test

[0100] Table 4 Results of Cell Viability Assay

[0101]

[0102] The results showed that the cell viability of the sample groups at concentrations of 0.625%, 0.313%, and 0.156% were 101.56%, 109.73%, and 119.46%, respectively, with cell viability ≥90%, meeting the requirements for subsequent efficacy testing (Table 4).

[0103] (8) Results of cell migration experiment

[0104] Table 5. Results of cell migration experiments (concentration gradient of 1.250%)

[0105]

[0106] Table 6. Results of cell migration experiments (concentration gradient of 0.625%)

[0107]

[0108] Scratch assay results showed that the nano-nourishing and lung-clearing preparation significantly promoted the migration and healing of gingival fibroblasts at concentrations of 1.250% and 0.625%. Compared with the negative control group, the cell healing rate treated with the nano-nourishing and lung-clearing preparation was significantly increased (P<0.05) (Tables 5 and 6), and the cell healing rate of the positive control group (hEGF) was also significantly higher than that of the negative control group. This indicates that the sample nano-nourishing and lung-clearing preparation has a growth-promoting effect on gingival fibroblasts (HGF) and has oral cavity repair efficacy.

[0109] Example 5 Anti-inflammatory effect

[0110] To evaluate the anti-inflammatory effect of the nano-nourishing and lung-clearing preparation on gingival fibroblasts, we used a method to detect the levels of inflammatory factors IL-1α and TNF-α induced by bacterial lipopolysaccharide (LPS) in human gingival fibroblasts, and evaluated its efficacy in protecting oral cavity and gums.

[0111] The cells were divided into a negative control group, a positive control group, a model group, and a sample group. The sample group contained three different concentration gradients of 0.625%, 0.313%, and 0.156% for cell viability experiments and cytokine detection.

[0112] (1) Cell viability assay:

[0113] Cells in the logarithmic growth phase were collected and seeded into 96-well plates at adjusted cell density, with 100 μL of culture medium per well. After culturing in a CO2 cell incubator for 24 h, the culture medium was aspirated, and the cells were washed twice with PBS buffer. Sample groups were administered the drug according to the final concentrations (%, V / V) of the prepared drug (20.000, 10.000, 5.000, 2.500, 1.250, 0.625, or 0.313).

[0114] (2) Grouping situation (Table 7)

[0115] Table 7

[0116]

[0117] (3) Drug administration: Discard the culture medium in each well of the plate. Add normal culture medium to the blank wells and negative control wells, and add Dex to the positive control wells. Add the above-mentioned samples containing different concentrations to the sample groups. After drug administration, continue to culture in a CO2 cell incubator for 24 hours, and then perform the MTT cytotoxicity test. Read the absorbance OD value at 490 nm. Calculate the cell viability. Only add culture medium to the blank wells. Take the three sample concentrations with cell viability ≥90% for subsequent efficacy testing.

[0118] (4) Cytokine detection:

[0119] Re-coat the 96-well plates. After 24h±1h, the cells adhered to the plates. Then, administer the drugs according to the grouping in (2) and proceed to the next step of the experiment. Each group had 3 replicates.

[0120] (5) Sample collection: After 24 hours of incubation, collect the supernatant for ELISA detection. Collect 150 μL of supernatant from each well into a 1.5 mL centrifuge tube. Centrifuge at 10000 rpm for 10 min before ELISA detection and collect the supernatant for the experiment.

[0121] (6) ELISA detection: IL-1α and TNF-α cytokines were selected for detection in this experiment.

[0122] (7) Data processing

[0123] Cell viability (%) = OD of sample group 490 / Negative control group OD 490 *100%

[0124] Note: Blank holes are deducted from each set of data.

[0125] Inhibition rate (%) = (1 - sample group / model group) * 100%

[0126] (8) Results of cell viability test

[0127] Table 8

[0128]

[0129] (9) Inhibition rate of inflammatory factors

[0130] Table 9. Inhibition rate of nano-Yin-nourishing and lung-clearing preparations on IL-1α

[0131]

[0132] Table 10 Inhibition rate of nano-Yin-nourishing and lung-clearing preparations against TNF-α

[0133]

[0134] ELISA results showed that the inhibition rates of the nano-nourishing yin and clearing lung preparation against IL-1α at concentrations of 0.625%, 0.313%, and 0.156% were 69.7%±0.4%, 60.4%±0.1%, and 33.5%±0.5%, respectively, which were significantly different from those of the model group (P<0.05) (Table 9), indicating that the nano-nourishing yin and clearing lung preparation of this sample has the effect of inhibiting the expression of IL-1α at this concentration.

[0135] The nano-based lung-nourishing and yin-clearing preparation showed inhibition rates of TNF-α of 82.6%±1.2%, 55.6%±0.39%, and 29.8%±4.8% at concentrations of 0.625%, 0.313%, and 0.156%, respectively, which were significantly different from the model group (P<0.05) (Table 10). This indicates that the nano-based lung-nourishing and yin-clearing preparation has the effect of inhibiting TNF-α expression at these concentrations, suggesting that this sample of the nano-based lung-nourishing and yin-clearing preparation has a gingival protective effect.

Claims

1. A method for preparing a nano-formulation, characterized in that, It includes the following steps: Step 1: Pretreatment of the drug composition: The drug composition was mixed with a buffer solution and then ultrasonically centrifuged to obtain an aqueous phase and a lower precipitate. The pharmaceutical composition comprises the following components in parts by weight: 70-130 parts of Rehmannia glutinosa, 30-90 parts of Ophiopogon japonicus, 50-110 parts of Scrophularia ningpoensis, 10-70 parts of Fritillaria cirrhosa, 10-70 parts of Paeonia lactiflora, 10-70 parts of Paeonia suffruticosa, 15-45 parts of Mentha haplocalyx and 10-30 parts of Glycyrrhiza uralensis. Step 2: The organic phase and the aqueous phase from Step 1 are combined using a microfluidic method to prepare the nano-formulation. The flow rate of the organic phase is 1-5 mL / min, and the flow rate of the aqueous phase is 6-12 mL / min; The organic phase includes the lower precipitate from step 1, alcohol solvents, phospholipids, and sterols.

2. The method for preparing nano-formulations as described in claim 1, characterized in that, It satisfies one or more of the following conditions: (1) The pharmaceutical composition comprises the following components in parts by weight: 90-110 parts of Rehmannia glutinosa, 50-70 parts of Ophiopogon japonicus, 70-90 parts of Scrophularia ningpoensis, 30-50 parts of Fritillaria cirrhosa, 30-50 parts of Paeonia lactiflora, 30-50 parts of Paeonia suffruticosa, 15-35 parts of Mentha haplocalyx and 15-25 parts of Glycyrrhiza uralensis; for example, it comprises 100 parts of Rehmannia glutinosa, 60 parts of Ophiopogon japonicus, 80 parts of Scrophularia ningpoensis, 40 parts of Fritillaria cirrhosa, 40 parts of Paeonia lactiflora, 40 parts of Paeonia suffruticosa, 25 parts of Mentha haplocalyx and 20 parts of Glycyrrhiza uralensis; (2) The flow rate of the organic phase is 2-4 mL / min; preferably 3 mL / min; (3) The flow rate of the aqueous phase is 8-10 mL / min; preferably 9 mL / min; (4) The organic phase also includes volatile oil.

3. The method for preparing nano-formulations as described in claim 1, characterized in that, The organic phase was prepared by the following method: Step S1: Mix the drug composition with a buffer solution, and centrifuge by sonication to obtain the lower precipitate; Step S2: Mix the precipitate with an alcohol solvent, and after ultrasonic centrifugation, obtain the upper layer solution as solution 1; Step S3: Mix the solution 1 with the volatile oil to obtain the oil phase; Step S4: Mix the oil phase, phospholipids and sterols to obtain the organic phase.

4. The method for preparing the nano-formulation as described in claim 3, characterized in that, It satisfies one or more of the following conditions: (1) The buffer solution is a bicarbonate buffer, a 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer, or a phosphate buffer; for example, a phosphate buffer. (2) The mass-to-volume ratio of the pharmaceutical composition to the buffer solution is (0.5-6) ​​g / L; preferably (1-4) g / L; for example, 2 g / L; (3) The alcohol solvent is methanol or ethanol; preferably anhydrous ethanol; (4) The mass-to-volume ratio of the pharmaceutical composition to the volume of the alcohol solvent is (3-8) g / L; preferably (4-7) g / L; for example, 5.7 g / L; (5) The volume ratio of the solution 1 to the volatile oil is (1-6):1; preferably (2-5):1; for example, 3:1; (6) The volatile oil is one or both of peony bark volatile oil and peppermint volatile oil; (7) The phospholipid is distearylphosphatidylcholine, myristoyl phosphocholine, dioleoyl phosphocholine, palmitoyl phosphocholine, docosanoyl phosphocholine, palmitoyl phosphocholine or soybean lecithin; for example, soybean lecithin; (8) The sterol is cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, or α-tocopherol; for example, cholesterol; (9) The mass ratio of the phospholipid to the sterol is (0.3-3):1; preferably (0.8-1.5):1; for example, 1:1; (10) The mass-to-volume ratio of the phospholipid to the volume of the oil phase is (5-10) g / L; preferably (6-8) g / L; for example, 7.5 g / L.

5. A nano-formulation prepared by the method of any one of claims 1 to 4.

6. A nano-formulation, characterized in that, It comprises the following components in parts by weight: 10-30 parts pharmaceutical composition, 20-40 parts phospholipids and 20-40 parts sterols; The pharmaceutical composition comprises the following components in parts by weight: 70-130 parts of Rehmannia glutinosa, 30-90 parts of Ophiopogon japonicus, 50-110 parts of Scrophularia ningpoensis, 10-70 parts of Fritillaria cirrhosa, 10-70 parts of Paeonia lactiflora, 10-70 parts of Paeonia suffruticosa, 15-45 parts of Mentha haplocalyx and 10-30 parts of Glycyrrhiza uralensis. The types of phospholipids and sterols are as described in claim 4.

7. The nanoformulation as described in claim 6, characterized in that, It satisfies one or more of the following conditions: (1) The nano-formulation comprises the following components in parts by weight: 15-25 parts of pharmaceutical composition, 25-35 parts of phospholipids and 25-35 parts of sterols; The pharmaceutical composition preferably comprises the following components in parts by weight: 90-110 parts of Rehmannia glutinosa, 50-70 parts of Ophiopogon japonicus, 70-90 parts of Scrophularia ningpoensis, 30-50 parts of Fritillaria cirrhosa, 30-50 parts of Paeonia lactiflora, 30-50 parts of Paeonia suffruticosa, 15-35 parts of Mentha haplocalyx and 15-25 parts of Glycyrrhiza uralensis. (2) The average particle size of the nano-formulation is 139~155 nm; for example, 144~147 nm; (3) The PDI of the nano-formulation is 0.12~0.22; for example, 0.16; (4) The zeta potential of the nano-formulation is -18 to -9 mV; for example, -15 to -12 mV.

8. The use of a nanoformulation as described in any one of claims 5-7 or a pharmaceutical composition as described in claim 1 or 2 in the preparation of a medicament for the prevention and / or treatment of oral diseases; The oral disease is preferably a periodontal disease or an oral mucosal disease; the periodontal disease is, for example, gingivitis or periodontitis; the gingivitis and periodontitis are preferably diseases mediated by the inflammatory factors IL-1α and / or TNF-α in gingival fibroblasts. The oral mucosal diseases are preferably Sjögren's syndrome, oral lichen planus, or oral ulcers.

9. An inhibitor of the inflammatory cytokine IL-1α or TNF-α, characterized in that, It includes the nanoformulations as described in any one of claims 5-7 or the pharmaceutical compositions as described in claims 1 or 2.

10. A reagent kit, characterized in that, It includes the nanoformulations as described in any one of claims 5-7, the pharmaceutical composition as described in claim 1 or 2, or the inhibitor as described in claim 9.

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