Litsea lanceolata oil self-nanoemulsion for enhancing anti-inflammatory activity and bioavailability as well as preparation method and application of litsea lanceolata oil self-nanoemulsion

Abstract

The invention relates to the technical field of fructus cinnamoii oil, in particular to a fructus cinnamoii oil self-nanoemulsion for enhancing anti-inflammatory activity and bioavailability as well as a preparation method and application of the fructus cinnamoii oil self-nanoemulsion. The design of the invention is based on the concepts of simplifying the prescription, improving the drug loading capacity and combining medicines with auxiliary medicines, and the self-nanoemulsion drug release system is prepared by taking the fructus cinnamoii oil as the oil phase of the self-nanoemulsion, the castor oil polyoxyethylene ether 40 as the emulsifier and the 1, 2-propylene glycol as the co-emulsifier, so that the oral bioavailability of the fructus cinnamoii oil is enhanced, and the bioavailability of the fructus cinnamoii oil is improved. And a theoretical basis is provided for the application of the fructus cinnamoii oil in the aspects of functional foods, health-care products, medicines, animal feeds and the like. The obtained fructus cinnamoii oil self-nanoemulsion is an O / W type nanoemulsion, the encapsulation efficiency is 93.08%-97.31%, and the encapsulation efficiency is high; good centrifugal stability, dilution stability, temperature stability and long-term stability are realized. Compared with a fructus cinnamoii oil suspension, the fructus cinnamoii oil self-nanoemulsion has the advantages that the bioavailability is higher, and the anti-inflammatory effect is more remarkable.

Description

Technical Field

[0001] The present invention relates to the technical field of Litsea lancifolia oil, and specifically to a Litsea lancifolia oil self-nanoemulsion for enhancing anti-inflammatory activity and bioavailability, its preparation method and application. Background Art

[0002] Litsea lancifolia oil is a volatile oil obtained by steam distillation of the dried fruits of the Lauraceae plant Litsea migao (Camphora migao (H.W.Li) Y.Yang, Bing Liu & ZhiYang), which is included in the Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province (2019 Edition). It has a wide foundation in folk medicine and a solid clinical application foundation. However, there are many problems such as low preparation process level, poor drug stability, and inability to store for a long time, which will increase the clinical medication risk when used. At the same time, the low solubility, strong volatility, and low bioavailability of Litsea lancifolia oil limit its further clinical promotion and development and utilization.

[0003] Patent CN1973858B discloses a preparation method of a Litsea lancifolia oil emulsion. In this preparation, the Litsea lancifolia volatile oil and a lipophilic emulsifier are dissolved in vegetable oil as the oil phase; a hydrophilic emulsifier and / or an isotonic agent are dissolved in water as the water phase; then the oil phase and the water phase are stirred and mixed evenly in a water bath at 20°C to 80°C with a stirrer, and then fully emulsified with an emulsification device to form an emulsion. The emulsion prepared by this method dissolves Litsea lancifolia oil in vegetable oil as the oil phase, increasing the use of excipients and reducing the drug loading. In addition, due to the introduction of equipment such as a high-pressure homogenizer in the preparation process, the production investment cost is relatively high.

[0004] Self-nanoemulsion is a system composed of an oil phase, an emulsifier, and a co-emulsifier, which has a uniform and clear appearance, and is both kinetically and thermodynamically stable. It can spontaneously form an emulsion only with gentle stirring, simplifying the production process. Self-emulsifying nanoemulsion can obtain smaller particle sizes, significantly improving drug solubility and bioavailability; the surfactant dosage in self-emulsifying nanoemulsion is usually lower than that in microemulsion, reducing the risk of cytotoxicity. Summary of the Invention

[0005] In order to solve the above technical problems existing in the prior art, the present invention provides a Litsea lancifolia oil self-nanoemulsion for enhancing anti-inflammatory activity and bioavailability, its preparation method and application. Specifically, it is achieved through the following technical solutions:

[0006] A Litsea lancifolia oil self-nanoemulsion for enhancing anti-inflammatory activity and bioavailability uses Litsea lancifolia oil as the oil phase, castor oil polyoxyethylene ether 40 as the emulsifier, and 1,2-propanediol as the co-emulsifier.

[0007] Furthermore, the mass ratio of the emulsifier to the co-emulsifier is 2.

[0008] Furthermore, the specific proportions of each component are as follows:

[0009] Litsea lancifolia oil 6.67％ Castor oil polyoxyethylene ether 40:1,2 - propanediol (mass ratio 2:1) 13.33％ Distilled water 80.00％

[0010] The preparation method of the Litsea lancifolia oil self-nanoemulsion for enhancing anti-inflammatory activity and bioavailability includes the following steps: According to the proportion, dissolve the emulsifier in the co-emulsifier, slowly add Litsea lancifolia oil, stir until fully mixed, and add distilled water while stirring to obtain a homogeneous, stable, good-fluidity Litsea lancifolia oil self-nanoemulsion with a light blue opalescence.

[0011] The application of the Litsea lancifolia oil self-nanoemulsion for enhancing anti-inflammatory activity and bioavailability in the preparation of functional foods, health products, drugs, and animal feeds.

[0012] Furthermore, the drug is a drug that inhibits the secretion of pro-inflammatory factors.

[0013] Compared with the prior art, the technical effects of the present invention are reflected in:

[0014] (1) Based on the concept of streamlining the prescription, increasing the drug loading, and "drug-excipient integration", the present invention uses Litsea lancifolia oil as the oil phase of the self-nanoemulsion, castor oil polyoxyethylene ether 40 as the emulsifier, and 1,2-propanediol as the co-emulsifier to prepare a self-nanoemulsion drug delivery system to enhance the oral bioavailability of Litsea lancifolia oil and provide a theoretical basis for the application of Litsea lancifolia oil in functional foods, health products, drugs, animal feeds, etc.

[0015] (2) The Litsea lancifolia oil self-nanoemulsion obtained in the present invention is an O / W type nanoemulsion with an encapsulation efficiency of 93.08 - 97.31%, and the encapsulation efficiency is relatively high.

[0016] (3) The Litsea lancifolia oil self-nanoemulsion obtained in the present invention has good centrifugal stability, dilution stability, temperature stability, and long-term stability.

[0017] (4) The Litsea lancifolia oil self-nanoemulsion obtained in the present invention has relatively high drug stability compared with Litsea lancifolia oil.

[0018] (5) Compared with the Litsea lancifolia oil suspension, the nanoemulsion can significantly improve the oral absorption of Litsea lancifolia oil in rats, thereby increasing its oral bioavailability.

[0019] (6) Compared with the Litsea lancifolia oil suspension, the nanoemulsion has a more significant anti-inflammatory effect. Description of the Drawings

[0020] Figure 1It is the methanol chromatogram.

[0021] Figure 2 It is the chromatogram of the mixed reference substances. Note: 3: Cineole; 4: p-Cymene; IS: Cyclohexanone; 5: 4-Terpineol; 7: 4-Isopropylcyclohex-2-en-1-one; 8: α-Terpineol; 11: 2-(4-Methylphenyl)propan-2-ol.

[0022] Figure 3 It is the chromatogram of Litsea lancifolia oil. Note: 3: Cineole; 4: p-Cymene; IS: Cyclohexanone; 5: 4-Terpineol; 7: 4-Isopropylcyclohex-2-en-1-one; 8: α-Terpineol; 11: 2-(4-Methylphenyl)propan-2-ol.

[0023] Figure 4 It is the chromatogram of blank excipients.

[0024] Figure 5 It is the chromatogram of Litsea lancifolia oil nanoemulsion. Note: 3: Cineole; 4: p-Cymene; IS: Cyclohexanone; 5: 4-Terpineol; 7: 4-Isopropylcyclohex-2-en-1-one; 8: α-Terpineol; 11: 2-(4-Methylphenyl)propan-2-ol.

[0025] Figure 6 It is the characteristic peaks of Litsea lancifolia oil nanoemulsion. Note: 3: Cineole; 4: p-Cymene; 5: 4-Terpineol; 7: Isopropylcyclohexenone; 8: α-Terpineol; 11: 2-(4-Methylphenyl)propan-2-ol; A: Blank excipients.

[0026] Figure 7 It is the pseudo-ternary phase diagrams with different Km values.

[0027] Figure 8 It is the appearance quality (A) and particle size distribution diagram (B) of Litsea lancifolia oil nanoemulsion.

[0028] Figure 9 It is the microscopic morphology of Litsea lancifolia oil nanoemulsion.

[0029] Figure 10 It is the results under the microscope. Note: A: Methylene blue; B: Sudan III.

[0030] Figure 11 It is the results of differential scanning calorimetry.

[0031] Figure 12 It is the results of infrared spectroscopy analysis.

[0032] Figure 13 It is the results of fingerprint spectrum for evaluating the encapsulation efficiency. Note: A Excipient peak.

[0033] Figure 14It is the appearance quality of the long-term stability study. Note: A: 0 months; B: 6 months.

[0034] Figure 15 It is the remaining content of 6 components ( n = 3). Note: Compared with 0 months, ***P < 0.001.

[0035] Figure 16 It is the C-t pharmacokinetic curve of 6 components in the nanoemulsion of Litsea lancifolia oil ( n = 6). Note: Low, medium, and high doses of nanoemulsion: CMONEL, CMONEM, CMONEH; high dose of oil: CMOH.

[0036] Figure 17 It is the comparison of the C-t pharmacokinetic curves between Litsea lancifolia oil and nanoemulsion ( n = 6). Note: High dose of nanoemulsion of Litsea lancifolia oil: CMONEH; high dose of Litsea lancifolia oil: CMOH.

[0037] Figure 18 It is the modeling and dosing scheme of the RAW264.7 cell inflammation model

[0038] Figure 19 It is the effect on the secretion of NO, TNF-α, IL-1β, and IL-6 by LPS-induced RAW264.7 cells ( n = 6). Note: Compared with the CON group, ### P < 0.001; compared with the MOD group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared CMONE with the same dose of CMO, a,b,c P < 0.05, aa ,bb ,cc P < 0.01, aaa,bbb ,ccc P < 0.001. Specific embodiments

[0039] The technical solution of the present invention will be further limited in combination with specific embodiments below, but the scope of protection is not limited to the description made.

[0040] 1 Experimental materials

[0041] 1.1 Main drugs and reagents

[0042]

[0043] 1.2 Main instruments

[0044]

[0045] 2 Methods and Results

[0046] 2.1 Determination of the Contents of Multiple Index Components and Fingerprint Study of Litsea lancifolia Oil and Its Nanoemulsion

[0047] 2.1.1 GC Chromatographic Conditions

[0048] Chromatographic column: InterCap WAX capillary column (30 m × 0.32 mm, 0.5 μm); carrier gas: nitrogen; inlet temperature: 230 °C; flame ionization detector temperature: 250 °C; flow rate: 3 mL / min; injection volume: 1 μL; split ratio: 10:1; temperature programming is shown in Table 1.

[0049] Table 1 Temperature Programming

[0050]

[0051] 2.1.2 Preparation of Reference Substance Solutions

[0052] Weigh accurately appropriate amounts of 6 reference substances, i.e., eucalyptol, p-cymene, 4-terpineol, 4-isopropylcyclohex-2-en-1-one, α-terpineol, and 2-(4-methylphenyl)propan-2-ol, place them in a volumetric flask, dissolve with methanol and dilute to the mark, and store at -20 °C for standby use.

[0053] Table 2 Concentrations of Reference Substances

[0054]

[0055] 2.1.3 Preparation of Cyclohexanone Internal Standard Solution

[0056] Weigh accurately an appropriate amount of cyclohexanone, place it in a volumetric flask, dissolve with methanol and dilute to the mark to prepare an internal standard stock solution of cyclohexanone (1.580 mg / mL), and store at -20 °C for standby use.

[0057] 2.1.4 Preparation of Test Solution

[0058] Weigh accurately 100 mg of Litsea lancifolia oil (CMO), place it in a 10 mL volumetric flask, add 1 mL of internal standard solution, dilute to the mark with methanol, and mix well to obtain the test solution of Litsea lancifolia oil.

[0059] Weigh accurately 1.5 g of Litsea lancifolia oil nanoemulsion (CMONE), place it in a 10 mL volumetric flask, add 1 mL of internal standard solution, add an appropriate amount of methanol, ultrasonicate at 4 °C for 10 min, dilute to the mark with methanol, and mix well to obtain the test solution of Litsea lancifolia oil nanoemulsion.

[0060] 2.1.5 Methodology Investigation of the Determination of Multiple Index Contents

[0061] Inject the sample according to the chromatographic conditions in item "2.1.1" and record the chromatogram. The peak shape of the reference substance is good; the nanoemulsion of Litsea lancifolia oil has no interference at each component. Figures 1 - 5 . The stability test shows that the Litsea lancifolia oil solution is stable within 12 h; the results of the repeatability experiment show that the RSDs of the contents of 6 components in the nanoemulsion of Litsea lancifolia oil are all less than 3%, indicating that this method has good repeatability. The results of the recovery experiment show that the average recoveries of each component are 96.44% - 102.6%, and the RSDs are all less than 3%, indicating that this method has good accuracy.

[0062] 2.1.6 Establishment of the fingerprint of the nanoemulsion of Litsea lancifolia oil

[0063] Refer to the chromatographic method in the first section "2.1.1", and use the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" to calculate the similarity; select 11 characteristic peaks, and identify 6 of them ( Figure 6 ), taking the 4th peak (p-cymene) as the reference peak, calculate the relative retention time and relative peak area of the other 10 characteristic peaks. The results of the stability and repeatability tests show that the similarities of each chromatogram exceed 0.998, the RSDs of the relative retention times of each characteristic peak are all lower than 3%, and the RSDs of the relative peak areas are all lower than 3%, meeting the technical requirements of traditional Chinese medicine fingerprints.

[0064] 2.2 Screening of the prescription of the nanoemulsion of Litsea lancifolia oil

[0065] 2.2.1 Screening of the oil phase

[0066] Based on the concept of streamlining the prescription, improving the drug loading capacity and "integration of drug and excipient", the target drug Litsea lancifolia oil is used as the oil phase of the nanoemulsion drug delivery system.

[0067] 2.2.2 Screening of the co-surfactant

[0068] Take 5 mL of Litsea lancifolia oil, and add 2 mL of polyethylene glycol 400, 1,2-propanediol, and glycerol respectively. Measure the dissolution amounts of 6 components in the co-surfactants. The results show that the solubility of 6 components in 1,2-propanediol is the largest, which are 97.66 ± 2.81, 12.96 ± 0.2713, 5.832 ± 0.0492, 5.262 ± 0.1698, 18.03 ± 1.923, 9.647 ± 0.6118 mg / mL respectively. Considering from the perspective of improving the drug loading capacity, 1,2-propanediol is selected as the co-surfactant of the nanoemulsion.

[0069] 2.2.3 Screening of the surfactant

[0070] Literature reports that the hydrophile-lipophile balance (HLB) value of the most suitable surfactant for O / W nanoemulsion is 8-18, so Tween 20, Tween 60, Tween 80, polyethylene glycol-15-hydroxystearate (HS15), castor oil polyoxyethylene ether 20 (EL20), and castor oil polyoxyethylene ether 40 (EL40) were initially selected as surfactants for investigation. Litsea cubeba oil and 1,2-propylene glycol were mixed in a mass ratio of 1:2, vortexed and shaken to mix, and then each surfactant was added dropwise to the mixed system of Litsea cubeba oil and 1,2-propylene glycol, mixed, and 0.1 mL was taken to dilute 100 times, and the turbidity or clarity of the oil / water mixed system was observed, and the particle size and Zeta potential were measured. The minimum amount of surfactant required for each surfactant to form a nanoemulsion was recorded, and the mass fraction of each surfactant in the mixed system was calculated. The results showed that EL40 had the smallest mass (21.30%) and the smallest particle size (29.03±0.62nm) in the system.

[0071] 2.2.4 Determination of Km value

[0072] First, Smix (surfactant and surfactant mixture) with different Km values ​​(mass ratio of surfactant to co-surfactant) was prepared, and the Km values ​​were 0.5, 1, 2, and 3. Then, Litsea cubeba oil and Smix were mixed at mass ratios of 0.5:9.5, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, respectively. After mixing evenly, water was added dropwise to the mixed phase, vortex mixed, and the conductivity-water content curve was drawn to calculate the area under the nanoemulsion region and the total area of ​​the pseudo-ternary phase diagram.

[0073] Results Figure 7 It can be seen that the Km value increases from 0.5 to 2 microemulsion region ( Figure 7 The black area) gradually increases, while the Km value increases from 2 to 3 in the microemulsion area and decreases instead, so the Km value of the prescription is determined to be 2.

[0074] 2.2.5 Optimizing the formulation of nanoemulsions

[0075] It is generally believed that the points near the center of the nanoemulsion region in the pseudo-ternary phase diagram are relatively stable. Therefore, 5 nanoemulsion formulations were initially selected near the center of the nanoemulsion region in the pseudo-ternary phase diagram with a Km value of 2. Three portions of nanoemulsion were prepared in parallel for each formulation. They were placed at 40 °C for 1 week, and the conductivity, particle size, and stability of the initially selected 5 formulations were comprehensively investigated and compared. From the results in Table 3, it can be seen that the conductivities of the nanoemulsions of the initially screened formulations are similar; the results of the stability investigation show that after the nanoemulsions of each formulation were placed at 40 °C for 1 week, formulations 1, 4, and 5 were stratified, indicating poor stability of the formulations; after formulation 3 was placed for 1 week, the particle size increased and the nanoemulsion transformed into a normal emulsion. The optimized formulation was determined to be formulation 2: Litsea lancifolia oil as the oil phase, polyoxyethylene castor oil 40 as the emulsifier, 1,2-propanediol as the co-emulsifier, and the Km value was 2. The specific proportions are as follows:

[0076] Litsea lancifolia oil 6.67%

[0077] Polyoxyethylene castor oil 40:1,2-propanediol (mass ratio 2:1) 13.33%

[0078] Distilled water 80.00%

[0079] Preparation method: According to the formulation proportions, dissolve polyoxyethylene castor oil 40 in the co-emulsifier 1,2-propanediol, slowly add Litsea lancifolia oil, stir until fully mixed, and add distilled water while stirring to obtain a homogeneous, stable, good-flowing Litsea lancifolia oil nanoemulsion with a light blue opalescence.

[0080] Table 3 Investigation results of the conductivity, particle size, and stability of the formulations (x±s, n = 3)

[0081]

[0082] 2.3 Verification of the optimized formulation

[0083] Weigh each auxiliary material according to the optimized formulation proportions, and prepare three batches of samples in parallel by the self-emulsification method. The prepared Litsea lancifolia oil nanoemulsion has a uniform, transparent, and clear appearance, with a light blue opalescence, and the Tyndall phenomenon can be observed ( Figure 8 A). Its pH value is 6.19, the conductivity is 174.33 μS / cm, the Zeta potential is -13.840 mV, and the average particle size is 23.99 nm ( Figure 8 B), and the polydispersity index is 0.139.

[0084] 2.4 Characterization of Litsea lancifolia oil nanoemulsion

[0085] 2.4.1 Transmission electron microscopy observation of the microscopic morphology of Litsea lancifolia oil nanoemulsion

[0086] The nanoemulsion of Litsea lancifolia oil was diluted 20 times with distilled water, negatively stained with 1% phosphotungstic acid, and its microscopic morphology was observed by transmission electron microscopy. The results are as Figure 9 shown. The droplets of the nanoemulsion of Litsea lancifolia oil are spherical, with a round appearance and relatively uniform size.

[0087] 2.4.2 Judgment of the type of nanoemulsion

[0088] The nanoemulsion of Litsea lancifolia oil was dropped on a glass slide and stained once with the prepared water-soluble dye methylene blue and the oil-soluble dye Sudan III respectively, and the dispersion of the dyes was observed under a microscope. The results are as Figure 10 shown. The water-soluble dye methylene blue is uniformly dispersed in the nanoemulsion, while the fat-soluble dye Sudan III is distributed in clusters. It can be seen from this that the nanoemulsion of Litsea lancifolia oil is an O / W (oil-in-water) type nanoemulsion. An appropriate amount of nanoemulsion was taken and a certain amount of water was added to it. The results showed that the nanoemulsion could be diluted by water without changing the appearance and properties. In summary, the nanoemulsion of Litsea lancifolia oil is an O / W type nanoemulsion.

[0089] 2.4.3 Differential scanning calorimetry

[0090] An appropriate amount of Litsea lancifolia oil, its nanoemulsion, and blank excipients (BE, mixed according to the prescription ratio) were taken for differential scanning calorimetry. The test conditions were: the temperature range was 20-250 °C, the heating rate was 10 °C / min, and the nitrogen atmosphere was 20 mL / min. The results are as Figure 11 shown. Litsea lancifolia oil has two obvious endothermic peaks at 135.3 °C and 178.0 °C, and the blank excipients have an obvious endothermic peak at 97.2 °C. In the nanoemulsion of Litsea lancifolia oil, there is only one obvious endothermic peak at 73.5 °C, which indicates that the prepared nanoemulsion is not a simple physical mixture of several substances. It is preliminarily speculated that Litsea lancifolia oil is encapsulated by the material.

[0091] 2.4.4 Infrared spectroscopy analysis

[0092] Litsea lancifolia oil, nanoemulsion, and blank excipients were taken for infrared spectroscopy analysis. The test resolution was 4 cm -1 , the number of scans was 16 times, the test range was 400-4000 cm -1 , and the test temperature range was 18-35 °C and the humidity was less than 50%. After consulting the literature, the compounds contained in Litsea lancifolia oil include characteristic peaks such as benzene rings, hydroxyl groups, and ester groups. In the infrared spectrum of Litsea lancifolia oil, there are mainly C-H stretching vibration peaks on the benzene ring (ν Φ-H , δ = 2964 cm -1 , δ = 2926 cm -1 ), stretching vibration peaks of ester groups (ν C＝O , δ = 1736 cm -1 , δ = 1712 cm-1 ), the in-plane stretching vibration peak of C-H on the benzene ring (β Φ-H , δ = 1362 cm -1 ), the stretching vibration peak of the carbon-oxygen single bond (ν C-O-C , δ = 1017 cm -1 , ν Ar-O-C , δ = 1215 cm -1 ). After embedding, the characteristic peaks of the above functional groups basically disappeared in the infrared spectrum, indicating that the compound was successfully embedded.

[0093] 2.4.5 Investigation of the encapsulation efficiency of Litsea lancifolia oil nanoemulsion

[0094] The ultrafiltration method combined with fingerprint and multi-index content determination was used to evaluate the encapsulation efficiency. The specific operation was as follows: an appropriate amount of Litsea lancifolia oil nanoemulsion was precisely pipetted and placed in a 30 kDa ultrafiltration tube, and centrifuged at 3000 rpm for 10 min. The nanoemulsion retained in the centrifuge tube and the original Litsea lancifolia oil solution were treated in the same way, and injected for analysis under the chromatographic conditions described in "2.1.1". The encapsulation situation was investigated by the similarity of the characteristic peaks of the nanoemulsion and Litsea lancifolia oil retained in the 3 batches of ultrafiltration tubes; the encapsulation efficiency (EE) and drug loading (LC) of the nanoemulsion were calculated by the content of 6 components, and the calculation formulas were as follows:

[0095] EE = Encapsulated Litsea lancifolia oil / Input Litsea lancifolia oil × 100%

[0096] LC = Encapsulated Litsea lancifolia oil / Total mass of nanoemulsion × 100%

[0097] It can be seen from the chromatograms of the nanoemulsion retained in the centrifuge tube and the original Litsea lancifolia oil solution ( Figure 13 ), the number and proportion of the chromatographic peaks of the two were basically the same. Further calculating the similarity, after deducting the solvent peak and excipient peak, the similarity between the nanoemulsion in the centrifuge tube and the original Litsea lancifolia oil solution was greater than 0.997, indicating that most of the components in Litsea lancifolia oil were encapsulated. The results of calculating the encapsulation efficiency and drug loading according to 6 components are shown in Table 4. The encapsulation rate was 93.08 - 97.31%, with a relatively high encapsulation efficiency; the drug loading was 62.05 - 64.88 mg / g. According to the current "Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province" (2019 edition), the oral dosage of Litsea lancifolia oil is 0.05 - 0.45 g / d, and the daily dosage of the nanoemulsion was calculated to be 0.8 - 7 g.

[0098] Table 4 Results of encapsulation efficiency and drug loading ( n = 3)

[0099]

[0100]

[0101] 2.4.6 Investigation on the Stability of Litsea lancifolia Oil Nanoemulsion

[0102] The stability of Litsea lancifolia oil nanoemulsion was investigated under different conditions. For the centrifugal stability, the nanoemulsion was centrifuged at 8000 rpm for 20 min. The results showed that the nanoemulsion remained clear and transparent after centrifugation, without stratification or demulsification. For the dilution stability, the nanoemulsion was diluted with distilled water at ratios of 50, 100, and 200 times, and no stratification was observed after dilution. For the long-term stability, the nanoemulsion was sealed in sample bottles and stored at 4 °C, room temperature, and 30 °C for 6 months. The results showed that the appearance and particle size of the nanoemulsion remained good at 4 °C, room temperature, and 30 °C after 0, 3, and 6 months, without stratification or demulsification, as shown in Table 5 and Figure 14 ; The results of content determination also showed no obvious changes, as shown in Table 6. It is indicated that the nanoemulsion has good centrifugal stability, dilution stability, temperature stability, and long-term stability.

[0103] Table 5 Appearance and Particle Size of Long-Term Stability Investigation( n = 3)

[0104]

[0105] Table 6 Results of Content Determination for Long-Term Stability Investigation of Litsea lancifolia Oil Nanoemulsion (mg / g, n = 3)

[0106]

[0107]

[0108] 2.6 Comparison of the Stability between Litsea lancifolia Oil and Nanoemulsion

[0109] Litsea lancifolia oil and nanoemulsion were placed in sample bottles respectively, and samples were taken after storage at room temperature without sealing for 1 month, then injected for determination, and the remaining amounts of 6 components were calculated to compare the stability between Litsea lancifolia oil and nanoemulsion.

[0110] The results of the remaining amounts of 6 components in Litsea lancifolia oil are shown in Figure 15 . After Litsea lancifolia oil was placed for 1 month, cineole and p-cymene completely volatilized; the contents of 4-terpineol, 4-isopropylcyclohex-2-en-1-one, α-terpineol, and 2-(4-methylphenyl)propan-2-ol decreased significantly (P < 0.001). After the nanoemulsion was placed for 1 month, there was no significant difference in each component. It is indicated that the nanoemulsion has higher drug stability compared with Litsea lancifolia oil.

[0111] 2.7 Bioavailability of Litsea lancifolia Oil Nanoemulsion

[0112] Normal SD rats were intragastrically administered with high (2400 mg / kg), medium (1200 mg / kg), and low doses (600 mg / kg) of Litsea lancifolia oil nanoemulsion and high dose of Litsea lancifolia oil (160 mg / kg, equivalent to the high dose of Litsea lancifolia oil nanoemulsion). Blood was collected from the orbital cavity at different time points. Subsequently, 100 μL of rat plasma was taken and placed in a 1.5 mL centrifuge tube. 100 μL of cyclohexanone solution was added successively, vortexed for 2 min to mix thoroughly, then 200 μL of ethyl acetate solution was added, and vortexed for 2 min to mix thoroughly. Centrifugation was carried out at 4 °C for 10 min (12000 rmp), and the supernatant was taken for standby. Gas chromatography-mass spectrometry (GC-MS) was used to determine the drug concentrations of eucalyptol, p-cymene, 4-terpineol, 4-isopropylcyclohex-2-en-1-one, α-terpineol, and 2-(4-methylphenyl)propan-2-ol in rat plasma. Using the Litsea lancifolia oil suspension as a reference preparation, the pharmacokinetics of Litsea lancifolia oil nanoemulsion in rats after oral administration was studied, and the pharmacokinetic parameters and relative bioavailability were calculated to provide reference and basis for further application research of nanoemulsion.

[0113] 2.7.1 GC-MS Conditions

[0114] Chromatographic conditions: SH-PolarWax capillary column (30 m × 0.32 mm × 0.25 μm); inlet temperature: 220 °C; carrier gas: helium; column flow rate: 2 mL / min; injection volume: 1 μL; splitless injection; The temperature programming is shown in Table 7.

[0115] Table 7 Column Oven Temperature Program

[0116]

[0117]

[0118] Mass spectrometry conditions: ion source temperature 200 °C; interface temperature 230 °C; detection voltage: 0.25 kV; solvent delay 3 min; SIM mode; The detection ion information of 6 components and the internal standard is shown in Table 8.

[0119] Table 8 Mass Spectrometry Conditions of 6 Index Components and the Internal Standard

[0120]

[0121] 2.7.2 Comparison of Pharmacokinetic Parameters of Litsea lancifolia Oil and Nanoemulsion

[0122] The plasma concentration-time curves of normal rats after administration of different doses of Litsea lancifolia oil nanoemulsion are as Figure 16 shown. After being processed by WinNonLin 8.2 software, the relevant pharmacokinetic parameters are shown in Tables 9-11. The results show that the T of each component maxwas 0.33 - 0.68 h, C max was 73.22 - 5474.99 ng / mL, t 1 / 2 was 0.57 - 1.37 h, AUC 0-t was 76.94 - 12609.35 h·ng / mL, CL Z / F was 1750.93 - 32782.30 mL / h / kg. The C max and AUC 0-t of the 6 components in the nanoemulsion of Litsea lancifolia oil both increased with the increase of the oral dose, indicating that the blood drug concentration was dose-dependent.

[0123] The blood drug concentration-time curve of normal rats after administration of Litsea lancifolia oil was as Figure 17 . After being processed by WinNonLin 8.2 software, the relevant pharmacokinetic parameters are shown in Tables 9 - 11. The results showed that compared with the pharmacokinetic parameters of the nanoemulsion and Litsea lancifolia oil at the same dose in rats, the C max , AUC 0-t , V Z / F , CL Z / F and other pharmacokinetic parameters of the 6 components had obvious differences. The C max of the 6 components in the nanoemulsion was 0.78 - 3.83 times that of Litsea lancifolia oil. Except for 4-isopropylcyclohex-2-en-1-one, the C max of other components increased significantly; the AUC 0-t of the 6 components in the nanoemulsion was 1.43 - 5.73 times that of Litsea lancifolia oil, suggesting that the nanoemulsion drug delivery system significantly increased the exposure of the target components in Litsea lancifolia oil; V Z / F and CL Z / F were 0.17 - 0.79 and 0.19 - 0.71 times that of Litsea lancifolia oil respectively, indicating that the nanoemulsion drug delivery system significantly reduced the apparent distribution volume and elimination rate of the target components in vivo. The relative bioavailability of 6 components such as eucalyptol was calculated as follows: eucalyptol 303.95%, p-cymene 532.00%, 4-terpineol 193.31%, isopropylcyclohexene ketone 140.30%, α-terpineol 191.51%, 2-(4-methylphenyl)propan-2-ol 274.70%. In summary, compared with the Litsea lancifolia oil suspension, the nanoemulsion can significantly improve the oral absorption of Litsea lancifolia oil in rats, thereby increasing its oral bioavailability.

[0124] Table 9 Main pharmacokinetic parameters of eucalyptol and p-cymene in rats( n = 6)

[0125]

[0126] Note: Low, medium, and high doses of nanoemulsion: CMONEL, CMONEM, CMONEH; high dose of oil: CMOH; Comparison of CMONE with CMO at the same dose, * P < 0.05; ** P < 0.01, *** P < 0.001

[0127] Table 10 Main pharmacokinetic parameters of 4 - terpineol and 4 - isopropylcyclohex - 2 - en - 1 - one in rats ( n = 6)

[0128]

[0129]

[0130] Note: Low, medium, and high doses of nanoemulsion: CMONEL, CMONEM, CMONEH; high dose of oil: CMOH; Comparison of CMONE with CMO at the same dose, * P < 0.05; ** P < 0.01, *** P < 0.001

[0131] Table 11 Main pharmacokinetic parameters of α - terpineol and 2 - (4 - methylphenyl) propan - 2 - ol in rats ( n = 6)

[0132]

[0133]

[0134] Note: Low, medium, and high doses of nanoemulsion: CMONEL, CMONEM, CMONEH; high dose of oil: CMOH; Comparison of CMONE with CMO at the same dose, * P < 0.05; ** P < 0.01, *** P < 0.001

[0135] 2.8 Comparison of anti - inflammatory activities of Litsea cubeba oil and nanoemulsion

[0136] An in vitro inflammatory model of RAW264.7 cells stimulated by LPS was established to investigate whether the anti - inflammatory activity of Litsea cubeba oil was enhanced after being formulated into nanoemulsion. The experiment was divided into normal group (CON), model group (MOD), positive drug group (dexamethasone, Dex), blank excipient group (BE), high, medium, and low dose groups of Litsea cubeba oil (CMOL, CMOM, CMOH), high, medium, and low dose groups of Litsea cubeba oil nanoemulsion (CMONEL, CMONEM, CMONEH). The administration doses were as Figure 18After 24 h of LPS and drug intervention, the cell supernatant was collected and the expression of NO, TNF-α, IL-6 and IL-1β was determined using Griess and ELISA kits.

[0137] The results are as follows Figure 19 As shown in the results, after LPS stimulation, RAW264.7 cells significantly increased the production of NO, TNF-α, IL-1β, and IL-6 (P<0.001), proving that the cell inflammation model was successfully established; compared with the model group, there was no significant difference in the secretion of inflammatory factors in the blank excipient group, indicating that the blank excipient had no anti-inflammatory effect. Compared with the model group, after intervention with dexamethasone, Litsea cubeba oil, and Litsea cubeba oil nanoemulsion, the expression levels of NO, TNF-α, IL-6, and IL-1β were significantly reduced (P<0.05), and in a dose-dependent manner, indicating that Litsea cubeba oil and Litsea cubeba oil nanoemulsion had good anti-inflammatory effects. Compared with Litsea cubeba oil, the secretion of pro-inflammatory factors was significantly reduced with the same dose of Litsea cubeba oil nanoemulsion (P<0.05). The results showed that Litsea cubeba oil and Litsea cubeba oil nanoemulsion could effectively inhibit the secretion of pro-inflammatory factors, and the anti-inflammatory effect was more significant after being made into nanoemulsion.

[0138] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A Litsea lancifolia oil self-nanoemulsion for enhancing anti-inflammatory activity and bioavailability, characterized in that, Using Litsea lancifolia oil as the oil phase, castor oil polyoxyethylene ether 40 as the emulsifier, and 1,2-propanediol as the co-emulsifier.

2. The Litsea lancifolia oil self-nanoemulsion for enhancing anti-inflammatory activity and bioavailability according to claim 1, wherein The mass ratio of the emulsifier to the co-emulsifier is 2.

3. The Litsea lancifolia oil self-nanoemulsion for enhancing anti-inflammatory activity and bioavailability according to claim 3, characterized in that The specific proportions of each component are as follows:

4. The preparation method of the Litsea lancifolia oil self-nanoemulsion for enhancing anti-inflammatory activity and bioavailability according to claim 1, characterized in that, It includes the following steps: According to the proportion, dissolve the emulsifier in the co-emulsifier, slowly add Litsea lancifolia oil, stir until fully mixed, and add distilled water while stirring to obtain a homogeneous, stable, good-fluidity Litsea lancifolia oil nanoemulsion with a light blue opalescence.

5. Application of the Litsea lancifolia oil self-nanoemulsion with enhanced anti-inflammatory activity and bioavailability according to claim 1 in the preparation of functional foods, health products, drugs, and animal feeds.

6. The application according to claim 5, characterized in that, The drug is a drug that inhibits the secretion of pro-inflammatory factors.

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