A modified amber particle, a pickering milk containing oil of aquisgrass and a formulation of aconite beads

Abstract

This invention provides modified amber particles, prepared by adding fine amber powder to molten PEG6000, followed by melting and drying. The weight ratio of PEG6000 to amber is 1-4 parts PEG6000 and 4 parts amber. This invention also provides a Pickering emulsion containing Acorus tatarinowii oil, and a Lingzhu San preparation containing this Pickering emulsion. This invention introduces particle design technology to successfully modify amber particles using PEG6000 as a modifier via a melt method. Using the modified amber particles as a stabilizer, an O / W type Pickering emulsion can be successfully prepared. This improves the stability of the volatile oil of Acorus tatarinowii in the Lingzhu San formula, aligning with the "medicine and excipient integration" philosophy of traditional Chinese medicine, and also provides a new option for improving the stability of volatile oils in other oil-containing solid Chinese medicine preparations.

Description

Technical Field

[0001] This invention relates to modified amber particles, Pickering emulsion containing calamus oil, and Lingzhu powder preparations, belonging to the field of pharmaceutical preparations. Background Technology

[0002] Lingzhu San is a traditional Chinese medicine preparation composed of finely powdered antelope horn, artificial bezoar, cinnabar, pearl powder, amber, and other medicinal materials, as well as volatile oil from Acorus tatarinowii. It is primarily used to treat febrile convulsions in children. The Acorus tatarinowii volatile oil in the formula is applied via spraying, which presents challenges during storage and use due to the easy loss of volatile components, affecting the stability and efficacy of the medicine. Therefore, there is an urgent need to introduce a technology or method to improve the stability of the Acorus tatarinowii volatile oil in Lingzhu San.

[0003] Application No. 202210923905.0, Invention Title: A Pickering Emulsion for Improving the Stability of Acorus tatarinowii Oil. This invention, based on the concept of "drug and excipient integration," develops a Pickering emulsion containing Acorus tatarinowii oil, pearl powder, and water. Experimental results show that this Pickering emulsion can significantly improve the stability of Acorus tatarinowii oil. The pearl powder used in this Pickering emulsion is an original ingredient in the Lingzhu San formula, without introducing any additional excipients. The pearl powder-stabilized Pickering emulsion can improve the dispersion, volatilization, and oxidation of Acorus tatarinowii volatile oil. This patent also discloses that other fine powdered medicinal materials in Lingzhu San are unsuitable Pickering emulsion stabilizers, which affects the promotion and development of Lingzhu San preparations.

[0004] Lingzhu Pulvis contains amber in its raw materials. Amber is a fossil formed from the resin of ancient pine trees (Pinus genus) buried underground for many years. It has the effects of calming the nerves, promoting blood circulation, removing blood stasis, and promoting urination. The inventors have investigated the properties of the powdered medicinal slices in Lingzhu Pulvis. Among them, Pickering emulsion prepared from amber is of the W / O type (Peng, L., et al., Improvement of thermal stability of Acorus tatarinowii volatile oil of pediatric drug Lingzhu Pulvis by Pickering emulsion technology based on concept of “combination of medicine and adjuvant”. Chinese Traditional and Herbal Drugs 2023, 54(02), 544-552.), which cannot solve the problem of the stability of the volatile oil components. Summary of the Invention

[0005] To overcome the above-mentioned technical difficulties, the technical solution of the present invention is to provide a modified amber particle. Another technical solution of the present invention is to provide Pickering emulsion containing Acorus tatarinowii oil and Lingzhu powder preparation.

[0006] This invention provides modified amber particles, which are prepared by adding fine amber powder to molten PEG6000, melting and drying the mixture, wherein the weight ratio of PEG6000 and amber is:

[0007] PEG6000 1-4 parts, amber 4 parts.

[0008] More preferably, the weight ratio of PEG6000 and amber is:

[0009] PEG6000 2 parts, amber 4 parts.

[0010] The modification conditions are as follows: PEG6000 is placed in an evaporating dish, transferred to a heating mantle, the voltage of the heating mantle is set to 100-150 volts, the heating mantle is preheated for 10 minutes, after the PEG6000 is heated and melted, fine amber powder is added, the glass rod is quickly stirred and melted for 5 minutes, the evaporating dish is removed, the dish is dried at room temperature for 24 hours, and then ground to obtain the product.

[0011] The moisture content of the amber powder is ≤5%.

[0012] The modified particles have a contact angle of 72-74°; under scanning electron microscopy (SEM), the surface of the modified amber particles is covered in clusters.

[0013] This invention provides a Pickering emulsion containing Acorus calamus oil, which contains the aforementioned modified amber particles, Acorus calamus oil, and water. The O / W type Pickering emulsion is prepared using a high-speed shearing method. The content of modified amber particles and Acorus calamus oil is as follows:

[0014] The modified amber particles are 2.5-25 μg / mL, and the oil phase volume fraction is 45-85%.

[0015] More preferably, the modified amber particles are 7.5 μg / mL and the oil phase volume fraction is 65%.

[0016] The conditions for the high-speed shearing method are: shearing speed 5000-15000 rpm / min and shearing time 1-4 min.

[0017] The conditions for the high-speed shearing method are: shearing speed 11000 rpm / min and shearing time 2 min.

[0018] This invention provides a Lingzhu powder preparation, wherein the raw materials are in the following weight parts: 100-200 parts antelope horn, 100-200 parts pearl, 250-400 parts bezoar, 250-400 parts silkworm, 100-200 parts cinnabar, 250-350 parts amber, 550-650 parts Arisaema cum Bile, 20-40 parts borneol, and 10-30 parts Acorus tatarinowii oil, wherein the amber and Acorus tatarinowii oil are the Pickering emulsions described in claims 5-8.

[0019] More preferably, the weight parts of the raw materials are:

[0020] 150 parts antelope horn, 150 parts pearl, 300 parts bezoar, 300 parts silkworm, 150 parts cinnabar, 300 parts amber, 600 parts bile arisaema, 30 parts borneol, and 20 parts calamus oil.

[0021] This invention introduces particle design technology to successfully modify amber particles using a melt method with PEG6000 as a modifier. Using the modified amber particles as a stabilizer, O / W type Pickering emulsions can be successfully prepared. This improves the stability of the volatile oil of Acorus tatarinowii in the Lingzhu San formula, aligning with the "medicine and excipient integration" philosophy of traditional Chinese medicine manufacturing. It also provides a new option for improving the stability of volatile oils in other oil-containing solid dosage forms of traditional Chinese medicine. Attached Figure Description

[0022] Figure 1 Comparison of physical mixtures, PEG6000, and modified amber particle stabilized emulsion before and after centrifugation;

[0023] Figure 2 Centrifuged emulsion height of Pickering emulsion stabilized by modified amber particles under different heating mantle voltages;

[0024] Figure 3 Centrifuged emulsion height of Pickering emulsion stabilized by modified amber particles at different modification times;

[0025] Figure 4 Centrifuged emulsion height of Pickering emulsion stabilized by modified amber particles under different modifiers;

[0026] Figure 5 Centrifuged emulsion height of Pickering emulsion stabilized by modified amber particles under different modifier ratios;

[0027] Figure 6 Comparison of Pickering emulsion before and after centrifugation for molten amber, molten PEG6000, and modified amber particles stabilized;

[0028] Figure 7 Visual properties of raw amber powder (A) and modified amber powder particles (B);

[0029] Figure 8 Droplet dilution diagram of modified amber particle-stabilized Pickering emulsion (A: water droplet dilution; B: emulsion droplet dilution; C: calamus oil droplet dilution);

[0030] Figure 9 SEM images of amber (A), PEG6000 (B), physical mixture (C), and modified amber particles (D);

[0031] Figure 10 FT-IR images of amber (a), PEG6000 (b), physical mixture (c), and modified amber particles (d);

[0032] Figure 11 Single-factor investigation of the preparation process of Pickering emulsion (A. Results of single-factor investigation of dosage; B. Results of single-factor investigation of oil phase volume fraction; C. Results of single-factor investigation of shear rate; D. Results of single-factor investigation of shear time);

[0033] Figure 12 Characterization of Pickering milk (A. Methylene blue staining results of Pickering milk; B. Sudan III staining results of Pickering milk; C. Particle size distribution of Pickering milk; D. Near-infrared spectra of Pickering milk (a), suspension of modified amber particles (b), and Acorus calamus oil (c)).

[0034] Figure 13 Retention rates, peroxide content, and malondialdehyde (MDA) content of Acorus calamus oil, physical mixtures, and Pickering emulsions under thermal treatment (A. Retention rates of Acorus calamus oil in the three groups after 1, 3, and 8 hours of heat treatment; B. Peroxide content in the three groups after 1, 3, and 8 hours of heat treatment; C. MDA standard curve; D. Comparison of MDA content in the three groups with the Acorus calamus oil group after 1, 3, and 8 hours of heat treatment: *P<0.05, **P<0.01, ***P<0.001).

[0035] Figure 14 GC-MS total ion chromatogram of Acorus calamus oil;

[0036] Figure 15 Average thermal maps of the relative content of the overall components of Acorus calamus oil, physical mixture, and Pickering emulsion at different time points during heat treatment;

[0037] Figure 16 Volcano plots showing the differences in components between untreated Acorus calamus oil and Acorus calamus oil treated for 1, 3, and 8 hours (A: Volcano plot of differences in components between crude Acorus calamus oil and oil treated for 1 hour; B: Volcano plot of differences in components between crude Acorus calamus oil and oil treated for 3 hours; C: Volcano plot of differences in components between crude Acorus calamus oil and oil treated for 8 hours).

[0038] Figure 17 Differential composition and total PCA plot of Acorus calamus oil, physical mixture, Pickering emulsion under heat treatment (1h, A; 3h, B; 8h, C) (D);

[0039] Figure 18 Line graphs showing the differences in composition among three groups: Acorus calamus oil, physical mixtures, and Pickering emulsion;

[0040] Figure 19 Analysis of the physicochemical properties of the differential components (petal plot of the physicochemical properties of the differential components in the AUP group; petal plot of the physicochemical properties of the differential components in the BDOWN group; radar comparison plot of the physicochemical properties of the differential components in the CUP group and DOWN group; PCA plot of the differential components in the DUP group and DOWN group);

[0041] Figure 20 Screening of the components of Acorus calamus oil under heat treatment (A. Stacked GC-MS chromatograms of Acorus calamus oil, physical mixture, and Pickering emulsion after 8 hours of heat treatment; B. Upset chromatograms of volatile components of Acorus calamus crude oil and Acorus calamus oil after 1, 3, and 8 hours of heat treatment; C. Upset chromatograms of volatile components of Acorus calamus crude oil and Acorus calamus oil, physical mixture, and Pickering emulsion after 8 hours of heat treatment).

[0042] Figure 21 Stacked heatmaps of relative content of qualitative change components (A: Stacked heatmaps of relative content of disappearing components; B: Stacked heatmaps of relative content of newly generated components). Detailed Implementation

[0043] Example 1: Preparation method of modified amber particles

[0044] Weigh 2.00g of PEG6000 and 4.00g of amber. Place the PEG6000 in a 100ml evaporating dish and transfer it to a heating mantle. Set the voltage of the heating mantle to 150V and preheat the heating mantle for 10 minutes. After the PEG6000 melts, add the amber and stir rapidly with a glass rod for 5 minutes to melt it. Remove the evaporating dish and dry it at room temperature for 24 hours. Grind it, pass it through a No. 5 sieve, and store it in a plastic bag for later use.

[0045] Example 2: Preparation of Pickering emulsion containing Acorus calamus oil

[0046] Measure 13 mL of Acorus calamus oil and 7 mL of purified water. Add 7.5 μg / mL of modified amber particles. Prepare a Pickering emulsion using a high-speed shearing machine with a shearing speed of 11000 rpm / min and a shearing time of 2 min.

[0047] Example 3: Preparation of Lingzhu Powder Formulation

[0048] Weigh out 150g of antelope horn, 150g of pearl, 300g of bezoar, 300g of silkworm pupae, 150g of cinnabar, 300g of amber, 600g of Arisaema cum Bile, 30g of borneol, and 20g of Acorus tatarinowii oil.

[0049] In this process, amber and calamus oil were prepared into Pickering emulsions according to the methods of Examples 1 and 2.

[0050] Except for antelope horn powder, pearl powder, silkworm pupae, arisaema cum bile, and calamus oil, cinnabar is water-milled or pulverized into an extremely fine powder. The remaining three ingredients, including bezoar, are ground into fine powder and sieved. Arisaema cum bile and silkworm pupae are decocted twice with water, one hour each time. The decoctions are combined, allowed to stand, and the supernatant is concentrated into a thick paste. This paste is then mixed with antelope horn powder, pearl powder, bezoar, and amber, dried, pulverized into a fine powder, and then ground with borneol and cinnabar. Pickering emulsion is sprayed in, sieved, and mixed well to obtain the final product.

[0051] Example 4: Process Screening Test of Modified Amber Particles

[0052] 1. Instruments and Materials

[0053] 1.1 Instruments

[0054] MH-3000 temperature-controlled electric heating mantle (Beijing Kewei Yongxing Instrument Co., Ltd.); DHG-9140A electric heating forced-air drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.); IKA T18 digital high-speed disperser (Shanghai Tusen Vision Technology Co., Ltd.); TENSOR-27 Fourier transform infrared spectrometer (Bruker GmbH, Germany); K100C-KRUSS fully automatic surface tension and contact angle meter (KRUSS GmbH, Germany); TYM-8L cell-level ultrafine pulverizer (Jinan Tianyu Special Equipment Co., Ltd.); 416 low-speed centrifuge (Gene Co., Ltd.); DF-101S heat-collecting constant-temperature magnetic stirrer (Gongyi Yuhua Instrument Co., Ltd.); 769YP-15A powder tablet press (Tianjin Keqi High-tech Co., Ltd.); Fokas planetary ball mill (Hunan Fokas Experimental Instrument Co., Ltd.).

[0055] 1.2 Materials

[0056] Amber (batch number: 220901-220922) was provided by Lei Yunshang Pharmaceutical Group Co., Ltd.; Acorus tatarinowii oil (batch number: D230310) was purchased from Xi'an Deshengyuan Biotechnology Co., Ltd.; PEG400 (batch number: 20210402), PEG2000 (batch number: 20210306), PEG4000 (batch number: 20220710), and PEG6000 (batch number: 20221121) were all purchased from Tianjin Kemio Chemical Reagent Co., Ltd.; water was Wahaha purified water; all other reagents were of analytical grade.

[0057] 2. Methods and Results

[0058] 2.1 Particle design technology for targeted control of amber powder particle size screening to select suitable O / W type stabilizers

[0059] 2.1.1 Preparation of amber ultrafine powder samples

[0060] Take an appropriate amount of fine amber powder (moisture content ≤5%), place it in an ultra-fine pulverizer and pulverize it (circulating temperature -20℃, medium filling rate 80%, amplitude 5.5mm), take samples intermittently (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 min), place it in a sealed bag and dry it for later use.

[0061] 2.1.2 Examination of Milk Formation Type

[0062] Measure 15 mL each of Acorus tatarinowii volatile oil and purified water into 50 mL centrifuge tubes, add 1.0 g each of amber ultrafine powder at each time point, and use a high-speed disperser to shear at 13000 r / min for 2 min to form an emulsion. The emulsion type was determined by the filter paper method, and the results were all W / O type.

[0063] 2.1.3 Contact Angle Measurement

[0064] Take 0.2g of the above sample particles and compress them into tablets using an infrared tablet press (set the pressure to 6N). Use a K100C-KRUSS fully automatic surface tension and contact angle meter (using water as the test solution) to measure the contact angle of the sample particles. The results are shown in the table below.

[0065] Table 1. Results of contact angle measurement of amber ultrafine powder at different ultrafine grinding time periods (n=3, )

[0066]

[0067] 2.2 Particle design technology for targeted regulation of amber powder wettability and screening of suitable O / W type stabilizers

[0068] 2.2.1 Mechanical grinding and dispersion method

[0069] 2.2.1.1 Investigation of preparation method and milk formation type

[0070] Ethanol (2%), PEG400 (5%), and PEG400 (10%) were used as modifiers and mixed with amber powder in equal volumes. The mixtures were then ground in an ultrafine pulverizer (circulating temperature -20℃, media filling rate 80%, amplitude 5.5mm), with intermittent sampling (0, 5, 10, 20, 30, 40, 50 min). The samples were then dried and stored in sealed bags for later use. Following the section "2.1.2 Emulsion Type Discrimination," the emulsion type of amber particles under different modifiers and modification times was investigated, and all results showed a W / O type.

[0071] 2.2.1.2 Contact Angle Measurement

[0072] Take 0.2g of the above sample and follow the procedure under “2.1.3 Contact Angle Measurement” to measure the contact angle of each modified sample.

[0073] Table 2. Contact angle results of modified amber particles under mechanical grinding and dispersion method (n=3, )

[0074]

[0075] 2.2.2 Magnetic stirring method

[0076] 2.2.2.1 Investigation of preparation method and milk formation type

[0077] 2% PEG400, PEG2000, PEG4000, and PEG6000 were used as modifiers, respectively. Ethanol was added to the final volume, and the mixture was ultrasonically dissolved. 10g of fine amber powder was weighed and placed in a 250mL flat-bottomed flask, and 200mL of 10% anhydrous ethanol solution was added. Two magnetic stirrers were used. The flat-bottomed flask was fixed on a magnetic stirrer, and the temperature was set to 70℃ with a constant stirring speed. 1mL of the modification solution was added every 5 minutes for a total modification time of 30 minutes. After the modification process was completed, the flat-bottomed flask was removed, cooled, filtered, and dried in an oven to obtain the prepared modified amber particles. According to section "2.1.2 Emulsion Type Discrimination," the emulsion type of the amber particles modified with different modifiers was investigated, and all results showed a W / O type.

[0078] 2.2.2.2 Contact Angle Measurement

[0079] Take 0.2g of the above sample and measure the contact angle of each sample according to the procedure under “2.1.3 Contact Angle Measurement”.

[0080] Table 3. Contact angle results of modified amber particles under magnetic stirring method (n=3, )

[0081]

[0082]

[0083] 2.2.3 Wet ball milling modification

[0084] 2.2.3.1 Polyethylene glycol series compounds

[0085] 5% PEG400, PEG2000, PEG4000, and PEG6000 were respectively weighed and ultrasonically dissolved in a certain amount of 10% anhydrous ethanol solution as modifiers. These modifiers were then placed together with amber in a 250mL agate ball mill jar, and 50 grinding balls were added. The milling speed was set to 400 rpm / min for 10 min. After modification, the mixture was dried in an oven. Following the procedure in "2.1.2 Emulsion Type Discrimination," the emulsion type of amber particles modified with different modifiers was investigated, and all results showed a W / O type.

[0086] 2.2.3.2 Polyvinylpyrrolidone (PVP K30)

[0087] 2.5% and 5% PVP K30 were ultrasonically dissolved in a certain amount of purified water as modifiers. These modifiers were then placed together with amber in a 250mL agate ball mill jar, along with 50 grinding balls. The milling speed was set to 400 rpm / min for 60 minutes. After modification, the mixture was dried in an oven. Following the procedure in "2.1.2 Emulsion Type Discrimination," the emulsion type of amber particles modified with different modifiers was investigated, and all results showed a W / O type.

[0088] 2.2.3.3 Contact Angle Measurement

[0089] Take 0.2g of the above sample and measure the contact angle of each sample according to the procedure under “2.1.3 Contact Angle Measurement”.

[0090] Table 4. Results of contact angle determination of amber particles modified with different modifiers under magnetic stirring method.

[0091]

[0092] 2.2.4 Melt Modification

[0093] 2.2.4.1 Preparation of Modified Amber

[0094] Weigh 4.00g each of PEG6000 and fine amber powder. Place the PEG6000 in a 100ml evaporating dish, transfer it to a heating mantle, set the heating mantle voltage to 150V, preheat the heating mantle for 10min, heat and melt the PEG6000, then add the amber, stir rapidly with a glass rod for 5min to melt, remove the evaporating dish, dry at room temperature for 24h, grind, and set aside. Measure 10mL each of Acorus tatarinowii oil and purified water, place them in a 50mL centrifuge tube, add 0.1g of modified amber, and prepare a Pickering emulsion using a high-speed shearing method (13000rpm / min, 2min), successfully preparing an O / W type Pickering emulsion.

[0095] 2.2.4.2 Evaluation of the advantages of modified amber

[0096] However, PEG6000 is a common O / W type co-emulsifier. To avoid the influence of PEG6000 and highlight the importance of modified amber particles, this study compares the differences between PEG6000, physical mixtures of PEG6000 and amber, and modified amber particle stabilizing emulsions to clarify the advantages and importance of modified amber particles.

[0097] Modified amber particles, a physical mixture (PEG6000: amber 4:4, ground for 5 min), and PEG6000 were used as described in section 2.2.4.1, and an emulsion was prepared according to the above method. PEG6000 and modified amber particles successfully prepared an O / W type emulsion. The centrifugal stability of both was then investigated. The centrifugation speed was set at 13000 rpm / min for 15 min. It was observed that the Pickering emulsion prepared from modified amber showed a distinct emulsion layer after centrifugation, with only a small amount of oil separating; while the PEG6000-stable emulsion showed complete oil-water separation after centrifugation. These results demonstrate that using PEG6000 as a modifier and the melt method as the modification method can successfully prepare O / W type modified amber particles with significant advantages (see...). Figure 1 ).

[0098] 2.2.4.3 Single-factor investigation of amber particle modification process

[0099] Based on a heating mantle voltage of 150 volts, a modification time of 5 min, PEG6000 as the modifier, and a PEG6000 to amber ratio of 4:4, single-factor experiments were conducted, using the height of the emulsion layer after centrifugation of the Pickering emulsion stabilized by modified amber particles as the evaluation index. The effects of heating mantle voltage (100, 150 volts), modification time (1, 2, 3, 4, 5, 6 min), modifier (PEG400, PEG2000, PEG4000, PEG6000), and PEG6000 to amber ratio (1:4, 2:4, 3:4, 4:4) on the preparation process of modified amber particles were investigated. All experiments were performed in triplicate.

[0100] (1) Investigation of heating mantle voltage (melting temperature)

[0101] Weigh 4.00 g each of PEG6000 and fine amber powder. Place the PEG6000 in a 100 ml evaporating dish and transfer it to a heating mantle. Set the voltage to 100 V and 150 V respectively. Preheat the heating mantle for 10 min. After the PEG6000 melts, add the fine amber powder and stir rapidly with a glass rod for 5 min to melt. Remove the evaporating dish and dry at room temperature for 24 h. Grind the powder and set aside. Conduct the experiment according to "2.2.4.1 Preparation of Modified Amber". Use the height of the emulsion layer after centrifugation as an indicator to screen the appropriate heating mantle voltage (melting temperature). Repeat the above experiment three times.

[0102] Results: When the heating mantle voltage was 100 volts, only one flocculent emulsion layer remained after centrifugation, with a height of 5.24 ± 0.48 mm; when the heating mantle voltage was 150 volts, the emulsion layer height after centrifugation was 16.57 ± 0.20 mm, and only a small amount of oil precipitated after centrifugation. The difference in emulsion layer height between the two methods was highly significant (P < 0.001). Therefore, the heating mantle voltage was determined to be 150 volts, at which point the melting temperature was approximately 230℃ (see...). Figure 2 ).

[0103] (2) Melting time investigation

[0104] Weigh 4.00 g each of PEG6000 and fine amber powder, in six parallel portions. Place the PEG6000 in a 100 ml evaporating dish, transfer it to a heating mantle, set the voltage to 150 volts, and preheat the mantle for 10 minutes. After the PEG6000 melts, add the amber and stir rapidly with a glass rod for 1, 2, 3, 4, 5, and 6 minutes. Remove the evaporating dish, dry at room temperature for 24 hours, grind, and set aside. Conduct the experiment according to "2.2.4.1 Preparation of Modified Amber," using the height of the emulsion layer after centrifugation as an indicator to screen the melting time. Repeat the above experiment three times.

[0105] Results: When the melting time was 1 or 2 min, the prepared modified amber particles could form an emulsion, but the oil and water completely separated after centrifugation. When the melting time was 3 min or more, the Pickering emulsion prepared from the modified amber particles all had a distinct emulsion layer after centrifugation, and the height of the emulsion layer increased continuously with the extension of the melting time. When the melting time increased to 5 min, the height of the emulsion layer no longer showed a rapid increasing trend. Therefore, the melting time was determined to be 5 min (see...). Figure 3 ).

[0106] (3) Investigation of Modifiers

[0107] Measure 4.00 mL of PEG400, weigh 4.00 g of PEG2000, PEG4000, PEG6000, and four 4.00 g portions of fine amber powder. Place PEG400, PEG2000, PEG4000, and PEG6000 into separate 100 mL evaporating dishes, transfer them to a heating mantle, set the voltage to 150 volts, preheat the mantle for 10 min, and heat the above PEG compounds until melted. Add amber, stir rapidly with a glass rod for 5 min to melt, remove the evaporating dishes, dry at room temperature for 24 h, grind, and set aside. Conduct experiments according to "2.2.4.1 Preparation of Modified Amber," using the height of the emulsion layer after centrifugation as an indicator to screen modifiers. Repeat the above experiments three times.

[0108] Results: The emulsion prepared from PEG400-modified amber particles was unstable, and only a thin emulsion layer remained after centrifugation; the stable Pickering emulsions prepared from PEG2000, PEG4000, and PEG6000-modified amber particles showed a clear emulsion layer after centrifugation, and the emulsion layer height showed an increasing trend. Considering that PEG6000 is the most commonly used compound in the polyethylene glycol series, with low toxicity, stable chemical properties, and compatibility with various drugs, PEG6000 was ultimately selected as the modifier (see...). Figure 4 ).

[0109] (4) Investigation of the proportion of modifier

[0110] Weigh out 1.00, 2.00, 3.00, and 4.00 g of PEG6000 and four portions of 4.00 g amber. Place the PEG6000 in separate 100 ml evaporating dishes, transfer them to a heating mantle, set the voltage to 150 volts, preheat the mantle for 5 minutes, melt the PEG6000, add the amber, and stir rapidly with a glass rod for 5 minutes. Remove the evaporating dishes, dry at room temperature for 24 hours, grind, and set aside. Conduct experiments according to "2.2.4.1 Preparation of Modified Amber," using the height of the emulsion layer after centrifugation as an indicator to screen the carrier ratio.

[0111] Results: When the carrier ratio was 1:4, the emulsion after centrifugation was loose and the emulsion height was significantly lower; when the carrier ratio was 2:4 to 4:4, the emulsion height after centrifugation was more consistent. To reduce the use of excipients, a carrier ratio of 2:4 was determined (see...). Figure 5 ).

[0112] 2.2.4.4 Preparation process and verification of modified amber particles

[0113] Weigh 2.00g of PEG6000 and 4.00g of amber, repeating the process three times. Place the PEG6000 in a 100ml evaporating dish, transfer it to a heating mantle, set the voltage to 150V, preheat the mantle for 10min, and after the PEG6000 melts, add the amber. Stir rapidly with a glass rod for 5min to melt the mixture. Remove the evaporating dish, dry at room temperature for 24h, grind, pass through a No. 5 sieve, and store in a sealed plastic bag for later use. Conduct the experiment according to "2.2.4.1 Preparation of Modified Amber", and measure the emulsion height after centrifugation. The results show that the emulsion height of the three batches of samples is consistent, at 16.25±0.08, proving the reproducibility of the process.

[0114] 2.2.4.5 Verification of the advantages of modified amber

[0115] The effects of the melting method on PEG6000 and amber were investigated to further highlight the advantages of modified amber particles. 2.00g of PEG6000 and 4.00g of fine amber powder were weighed separately and melted according to "2.2.4.4 Preparation Process and Verification of Modified Amber Particles". Melted amber, molten PEG6000, and modified amber were weighed out in proportion, and experiments were conducted according to "2.2.4.1 Preparation of Modified Amber" to clarify the advantages of modified amber particles. Figure 6 It is evident that after melting treatment, amber and PEG6000 completely separated into oil and water layers after centrifugation, with only the Pickering emulsion stabilized by modified amber particles exhibiting a distinct emulsion layer after centrifugation. This indicates that the melting operation has little effect on individual amber samples and PEG6000; only when both amber samples and PEG6000 are present can the melting method successfully prepare modified amber particles, thereby producing O / W type Pickering emulsions.

[0116] 2.3 Characterization of Modified Amber Particles

[0117] 2.3.1 Appearance

[0118] Raw amber powder is yellowish-brown in appearance, while modified amber powder is brownish-brown (see...). Figure 7 ).

[0119] 2.3.2 Investigation of Emulsion Type - Drop Dilution Method

[0120] Take two petri dishes, add one drop each of the modified amber-stabilized Pickering emulsion prepared using the above-determined process, then add 3-4 drops each of purified water and calamus oil to each dish, and observe the dilution. Figure 8 It is evident that when calamus oil was added, the morphology of the modified amber-stabilized Pickering emulsion droplets did not change significantly, indicating that the continuous phase of the emulsion was a non-oil phase. However, with the addition of water droplets, the droplets quickly dispersed, indicating that the continuous phase of the modified amber-stabilized Pickering emulsion was an aqueous phase, i.e., the emulsion type of the modified amber-stabilized Pickering emulsion was O / W (see...). Figure 8 ).

[0121] 2.3.3 Contact Angle Characterization

[0122] Three batches of modified amber sample particles (0.2g each) were taken for process verification and compressed into tablets using a powder tablet press (pressure set at 6N). Water was used as the test solution, and the wettability of each sample particle was determined using KRUSS (optical principle) assay. The determination was repeated three times. The contact angle of the fine amber powder was 87.27±1.33°, while that of the modified amber particles was 72.11±1.68°. A significant difference was observed between the two (P<0.001), indicating that the wettability of the modified amber particles was significantly improved compared to the fine amber powder.

[0123] 2.3.4 Scanning Electron Microscopy

[0124] Small amounts of amber, PEG6000, a physical mixture of the two, and modified amber particles were respectively adhered to conductive adhesive, sputter-coated with gold under vacuum, and then the surface morphology of the samples was observed under a scanning electron microscope. Figure 9 As can be seen, the amber powder is in strip-like form with a relatively smooth surface; PEG6000 is in irregular block form with some fine particles or protrusions on the surface; the physical mixture is a simple mixture of the two; the modified amber particles are clustered on the surface, indicating that the amber and PEG6000 interact in the molten state, which may be the key to the successful preparation of O / W type Pickering emulsion from modified amber.

[0125] 2.3.5 Infrared Spectroscopy (FT-IR)

[0126] Take appropriate amounts of amber, PEG6000, a physical mixture of the two, and modified amber particles, respectively. Grind them finely and thoroughly in an agate mortar with dry potassium bromide at a ratio of 1:100. Compress the mixture into tablets and measure the infrared spectrum of the samples. The scanning range is 4000–400 cm⁻¹. -1 The results are shown below. Figure 10 The IR spectrum of the modified amber particles is quite similar to that of PEG6000, but at 1700 cm⁻¹... -1 Characteristic peaks of amber particles are present on both sides, while PEG6000 does not have these absorption peaks. In the spectrum of the physical mixture, the absorption peaks of amber particles and PEG6000 are clearly visible, indicating a simple superposition of their spectra. FT-IR shows that the surface composition of the modified amber particles is quite similar to that of PEG6000, meaning that PEG6000 successfully modified the surface of amber particles via a melt method.

[0127] Example 5: Screening test of the preparation process of Pickering emulsion containing Acorus calamus oil

[0128] 1. Materials and Methods

[0129] 1.1 Materials and Instruments

[0130] Amber (batch number: 220901-220922) was provided by Lei Yunshang Pharmaceutical Group Co., Ltd.; Acorus tatarinowii oil (batch number: D230310) was purchased from Xi'an Deshengyuan Biotechnology Co., Ltd.; PEG6000 (batch number: 20221121), Sudan III (batch number: 20190710), and disodium EDTA (20211109) were purchased from Tianjin Kemio Chemical Reagent Co., Ltd.; trichloroacetic acid... Purchased from Shanghai Shanpu Chemical Co., Ltd.; Methylene blue (batch number: L11D10H105480), 2-thiobarbituric acid (batch number: J28GS156151, AR, 98.5%), and 1,1,3,3-tetraethoxypropane (batch number: O12GS163530, BR, 95%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; n-Docosane (G171809, 99.6%) was purchased from LGC Labor GmbH; water was purified water, and all other reagents were of analytical grade.

[0131] IKA T18 digital high-speed disperser (Shanghai Tusen Vision Technology Co., Ltd.); DHG-9140A electric heating drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.); Microtrac S35000 laser particle size analyzer (Microtrac, USA); MH-3000 temperature-controlled electric heating mantle (Beijing Kewei Yongxing Instrument Co., Ltd.); N-300M biological microscope (Jinan Taiyi Biotechnology Co., Ltd.); 416 low-speed centrifuge (Gene Co., Ltd.); ANTIRIS II Fourier transform near-infrared spectrometer (Thermo Fisher Scientific, USA); UV-6100 DOUBLE BEAM ultraviolet-visible spectrophotometer (Shanghai Meipuda Instrument Co., Ltd.).

[0132] 1.2 Experimental Methods

[0133] 1.2.1 Preparation process of modified amber particles (prepared according to the method in Example 1)

[0134] 1.2.2 Investigation of the preparation process of Pickering emulsion

[0135] 1.2.2.1 Evaluation Indicators

[0136] (1) Emulsion Stability Index (ESI):

[0137] A 30 μL sample of Pickering emulsion was pipetted into a 10 mL volumetric flask and diluted to the mark with 0.1% SDS solution. The mixture was shaken thoroughly. The flask was zeroed using 0.1% SDS solution as a blank. The absorbance (A0) at 500 nm was measured using a UV spectrophotometer. After standing for 10 min, the absorbance (A0) at 500 nm was measured again. 10ESI is calculated using the following formula: ESI / % = A 10 *100 / A0

[0138] A0 is the initial absorbance of the emulsion; A 10 The absorbance of the emulsion after standing for 10 minutes is shown.

[0139] (2) Centrifuged emulsion height

[0140] The prepared Pickering emulsion was centrifuged at 4000 rpm for 15 min, and the emulsion height was measured using vernier calipers.

[0141] 1.2.2.2 Single-factor experiment

[0142] Based on the modified amber particle addition of 5 μg / mL, an oil phase volume fraction of 50%, a shear rate of 13000 rpm / min, and a shear time of 2 min, single-factor experiments were conducted using the emulsion height and ESI of the Pickering emulsion after centrifugation as evaluation indicators. The effects of the addition amount (2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25 μg / mL), oil phase volume fraction (45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%), shear rate (5000, 7000, 9000, 11000, 13000, 15000 rpm / min), and shear time (1, 2, 3, 4 min) on the Pickering emulsion were investigated. The experiments were repeated three times.

[0143] 1.2.3 Pickering emulsion characterization

[0144] 1.2.3.1 Staining Phenomena

[0145] Apply an appropriate amount of the Sudan III and methylene blue staining emulsion to a glass slide, making a certain angle between the coverslip and the slide. Slowly lower the coverslip to avoid air bubbles. Fix the slide on the microscope stage, adjust the aperture and focus, and observe the staining of the modified amber particle-stabilized Pickering emulsion under a 10x objective lens.

[0146] 1.2.3.2 Particle size and particle size distribution

[0147] Using a Microtrac S35000 laser particle size analyzer, the particle size and distribution of Pickering emulsion were measured under the wet method based on the principle of static laser scattering. The measurements were performed in three parallel steps.

[0148] 1.2.3.3 Near-infrared spectroscopy

[0149] Appropriate amounts of Acorus calamus oil, modified amber particle suspension, and Pickering emulsion were placed in a quartz sample cup for near-infrared spectroscopy acquisition. The acquisition workflow was the solid powder average spectrum workflow, and the acquisition range was 4000–10000 cm⁻¹. -1 The acquisition temperature was room temperature, the number of scans was 32, and the resolution was 8cm. -1 The gain was 4x. Spectra were collected with air as a reference and background subtraction subtracted. Each sample was collected three times to obtain the average NIRS spectrum.

[0150] 1.2.4 Study on the thermal stability of Pickering emulsion stabilized by modified amber particles

[0151] 1.2.4.1 Retention Rate Measurement

[0152] Appropriate amounts of Acorus tatarinowii volatile oil, a physical mixture (Acorus tatarinowii volatile oil volume ratio of 65%, modified amber particle addition amount of 0.0075 g / mL), and Pickering emulsion were placed in 100 mL evaporating dishes and placed in an electric heating blast oven at 40℃ for 1, 3, and 8 hours. After removal, the volume of Acorus tatarinowii volatile oil was recorded. The physical mixture and Pickering emulsion were distilled using steam distillation for 8 hours to separate the oil phase. Three sets of experiments were performed in parallel, and the oil phase volume was recorded.

[0153] 1.2.4.2 Determination of Peroxides

[0154] Pickering emulsion, Acorus calamus oil, and the physical mixture were subjected to thermal stability treatment as described above. Acorus calamus oil was stored at 4°C for later use. Pickering emulsion and the physical mixture were centrifuged at high speed (10000 rpm / min, 30 min), and the upper oil layer was collected for later use. 500 μL of each of the three volatile oil samples were placed into a stoppered conical flask, 10 mL of a chloroform-glacial acetic acid mixture (4:6) was added, followed by 1 mL of saturated potassium iodide solution. The flask was sealed tightly, shaken for 0.5 min, and placed in the dark for 3 min. Then, 30 mL of water and 1 mL of 1% starch indicator were added. Titration was performed with 0.1 mol / L sodium thiosulfate standard solution (26 g of sodium thiosulfate and 0.20 g of anhydrous sodium carbonate were dissolved in 1000 mL of water, slowly boiled for 10 min, cooled, and then diluted) until the blue color of the solution disappeared. The volume of sodium thiosulfate standard solution consumed was recorded, and the peroxide value was calculated. Take the same volume of chloroform-glacial acetic acid solution, saturated potassium iodide solution, distilled water, and 1% starch indicator as described above, and perform a blank test using the same method.

[0155] 1.2.4.3 Malondialdehyde Determination

[0156] (1) Plotting the standard curve:

[0157] Accurately weigh an appropriate amount of 1,1,3,3-tetraethoxypropane and dilute to 500 mL to obtain a malondialdehyde standard solution. Accurately transfer 0, 1, 3, 5, 10, 20, 30, and 40 μL of the malondialdehyde standard solution into 10 mL volumetric flasks, and dilute to 10.00 mL with distilled water to obtain a series of malondialdehyde standard solutions with mass concentrations of 0, 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, and 0.4 μg / mL, respectively. Take 5.00 mL of each of the above standard solutions, add 5 mL of TBA solution (0.02 mol / L), mix well, heat in a 90℃ water bath for 40 min, remove, cool in the dark for 1 h, add 5.00 mL of chloroform, shake well, let stand for 1 h, take the supernatant and measure the absorbance value at 532 nm. Plot a standard curve with the mass concentration of the standard series solutions as the abscissa (X) and absorbance as the ordinate (Y), and perform linear regression to obtain the regression equation Y = 1.0797X - 0.0042(R²). 2 =0.9993).

[0158] (2) Sample preparation and determination:

[0159] Take 500 μL of each group of volatile oil samples from the "1.2.4.2 Peroxide Determination" section into a 10 mL volumetric flask, add a trichloroacetic acid mixed solution (37.5 g trichloroacetic acid and 0.50 g sodium ethylenediaminetetraacetate dissolved in water and diluted to 500 mL), and dilute to the mark. Sonicate at 40℃ for 30 min, remove, cool to room temperature, filter, and take 5.00 mL of the filtrate into a stoppered conical flask. Add 5.00 mL of TBA solution, heat at 90℃ for 40 min, remove, cool in the dark for 1 h, add 5.00 mL of chloroform, shake thoroughly, and let stand for 1 h. Take the supernatant, measure its absorbance at 532 nm, and calculate the malondialdehyde content.

[0160] 1.2.4.4 Determination of volatile oil components by GC-MS

[0161] (1) GC-MS detection conditions:

[0162] Chromatographic conditions: HP-5MS quartz capillary column (30m×0.25mm, 0.25μm) was used; helium (purity 99.999%) was used as the carrier gas; flow rate was 1mL / min; injection volume was 1μL; split ratio was 10:1; injection port temperature was 230℃; temperature program: 50℃ held for 2min, increased to 110℃ at 5℃ / min, held for 2min, increased to 120℃ at 2℃ / min, held for 5min, increased to 125℃ at 0.5℃ / min, held for 10min, increased to 200℃ at 4℃ / min, held for 2min, increased to 250℃ at 10℃ / min, held for 2min.

[0163] Mass spectrometry conditions: ionization mode EI, ion mode ESI, electron energy 70 eV, quadrupole temperature 150 ℃, ion source temperature 230 ℃, scan range m / z 35~500, solvent delay 3 min.

[0164] (2) Preparation of sample solution

[0165] Accurately measure 100 μL of each group of volatile oil samples and n-dodecane internal standard solution (10 mg / mL) under "1.2.4.2 Determination of Peroxides" into a 10 mL brown volumetric flask. Add n-hexane to dilute to the mark, add an appropriate amount of anhydrous sodium sulfate for dehydration, shake to mix, filter the sample solution through a 0.22 μm microporous membrane into a vial, and perform GC-MS detection according to the GC-MS detection conditions under "1.2.4.4 GC-MS Determination of Volatile Oil Components".

[0166] 1.2.5 Screening and Analysis of Differential Components in Volatile Oils

[0167] After GC-MS data acquisition, Data Analysis software was used to access the W11N17main.L database. After automatic integration, data from each group was read, and the peak area and CAS number of volatile components in each group were selected based on a matching degree ≥60. The relative content of volatile components in each group was calculated using the internal standard peak area. Based on this, RStudio software was used with packages such as Pheatmap, Limma, ggbiplot, ggplot2, and dplyr for data analysis and plotting. A thermographic analysis was performed on the overall relative content of components in the Pickering emulsion group, the Acorus calamus oil group, and the physical mixture group under different heat treatment times. Volcano plots were used with LogFC ≥1.5 and P < 0.05 as indicators to screen for differentially expressed components between Acorus calamus oil treated for 1, 3, and 8 hours and untreated Acorus calamus oil. Principal component analysis was performed using the relative content information of differentially expressed components in each group, and line graphs showing the change of relative content of differentially expressed components with heat treatment time were plotted, further illustrating the protective effect of Pickering emulsion on Acorus calamus oil at the volatile component level. The screening of qualitative change components (disappearing components and newly generated components) and related analyses were performed using OmicShare, a free online data analysis platform (https: / / www.omicshare.com / tools).

[0168] 1.3 Data Processing

[0169] All experiments were repeated three times, and the results are expressed as follows: One-way ANOVA was performed on the three groups of data using SPSS 26. LSD (homogeneity of variance) and Tamhany T2(M) (unequal variance) were used for multiple tests to compare the significance of the differences among the three groups. The significance level was P < 0.05.

[0170] 2 Results and Analysis

[0171] 2.1 Results of Single-Factor Investigation of Pickering Milk Preparation Process

[0172] 2.1.1 Consideration of Addition Amount

[0173] The dosage was evaluated using a comprehensive score (comprehensive score = ESI / maximum ESI × 50% + emulsion height / maximum emulsion height × 50%). Results showed that the comprehensive score increased with increasing dosage, making it impossible to determine the optimal dosage. Furthermore, observation of the emulsion morphology after centrifugation revealed that at dosages of 2.5 and 5 μg / mL, the emulsion was not dense enough and contained gaps; however, at dosages of 7.5 μg / mL and above, the emulsion was dense. Considering the principles of excipient usage, the optimal dosage was determined to be 7.5 μg / mL.

[0174] 2.1.2 Investigation of oil phase volume fraction

[0175] The oil phase volume fraction was evaluated using a comprehensive score (comprehensive score = maximum ESI / maximum ESI × 50% + emulsion height / maximum emulsion height × 50%). When the oil phase volume fraction was 80%, the prepared Pickering emulsion exhibited a complex emulsion type, containing both W / O and O / W emulsions; when the oil phase volume fraction was 85%, the Pickering emulsion completely inverted to the W / O type. Based on the comprehensive score results, an oil phase volume fraction of 65% was determined.

[0176] 2.1.3 Shear Rate Examination

[0177] The shear rate was evaluated using a comprehensive score (comprehensive score = ESI / maximum value of ESI × 50% + emulsion height / maximum value of emulsion height × 50%). Based on the comprehensive score results, the shear rate was determined to be 11000 rpm / min.

[0178] 2.1.4 Examination of shearing time

[0179] The shearing time was evaluated using a comprehensive score (comprehensive score = ESI / maximum ESI × 50% + emulsion height / maximum emulsion height × 50%). Based on the comprehensive score results, the shearing time was determined to be 2 minutes. (See...) Figure 11 )

[0180] 2.2 Pickering breast characterization

[0181] 2.2.1 Staining Phenomena

[0182] Depend on Figure 12As shown in AB, the outer phase of the Pickering emulsion was stained blue after methylene blue staining, while the inner phase was stained red after Sudan III staining. The microscopic staining results further indicate that the Pickering emulsion stabilized by modified amber particles is of the O / W type.

[0183] 2.2.2 Particle size and particle size distribution

[0184] The particle size of the Pickering emulsion was determined using a Microtrac S35000 laser particle size analyzer. The emulsion particle size of the modified amber particle-stabilized Pickering emulsion was 36.20 ± 0.77 μm.

[0185] 2.2.3 Near-infrared spectroscopy

[0186] Near-infrared spectroscopy showed that the average near-infrared spectrum of the Pickering emulsion stabilized with modified amber particles was basically consistent with that of the modified amber particle suspension, except for the difference in the 5000–6000 cm⁻¹ range. -1 The presence of a faint characteristic peak of Acorus calamus oil indicates that in Pickering emulsion, modified amber particles are coated on the outer layer of Acorus calamus oil droplets.

[0187] 2.3 Study on the thermal stability of Pickering emulsion

[0188] 2.3.1 Ownership Rate Assessment

[0189] Depend on Figure 13 As shown in Figure A, under conditions of 3 hours and 8 hours at 40℃, the retention rate of volatile oil in the Pickering emulsion group was higher than that in the Acorus calamus volatile oil group, with a statistically significant difference. However, there was no significant difference in the retention rate between the physical mixture group and the Acorus calamus volatile oil group, indicating that the Pickering emulsion has an enhancing effect on the retention rate of Acorus calamus oil under thermal conditions.

[0190] 2.3.2 Investigation of Peroxides

[0191] Depend on Figure 13 As shown in section B, under conditions of 40℃ for 1, 3, and 8 hours, the peroxide content in the Pickering emulsion group was lower than that in the Acorus calamus oil group, with highly significant or extremely significant differences. The peroxide content in the physical mixture group and the Acorus calamus oil group showed significant differences after heat treatment for 3 and 8 hours. Throughout the entire heat treatment process, the peroxide content in the Pickering emulsion group < the physical mixture group < the Acorus calamus oil group, indicating that the Pickering emulsion can delay the oxidation of Acorus calamus oil under a hot environment.

[0192] 2.3.3 Malondialdehyde (MDA) Investigation

[0193] Depend on Figure 13As shown in D, under the conditions of 1, 3, and 8 hours of heat treatment at 40℃, the malondialdehyde (MDA) content in the Pickering emulsion group was significantly lower than that in the Acorus calamus oil group. However, the MDA content in the physical mixture group and the Acorus calamus oil group only showed a significant difference after 8 hours of heat treatment. Throughout the entire heat treatment process, the MDA content in the Pickering emulsion group < the physical mixture group < the Acorus calamus oil group, indicating that the Pickering emulsion can delay the formation of MDA in Acorus calamus oil under heat conditions.

[0194] 2.4 Screening and Analysis of Differential Components

[0195] Data Analysis software was used to automatically search and match the data in the W11N17main.L database, and the data for each group were analyzed. The GC-MS total ion chromatogram of Acorus tatarinowii oil is shown below. Figure 14 The chromatograms showed good separation, with symmetrical peaks, no overlap, and flat baselines, meeting the measurement requirements. The analytical results of Acorus calamus oil, physical mixtures, and Pickering emulsion at different heat treatment time points were compiled, yielding a total of 72 volatile components and their relative contents.

[0196] 2.4.1 Overall Composition Heatmap

[0197] In RStudio software, the Pheatmap package was used to perform heatmap analysis on the average content of three groups of overall components under different heat treatment times. Figure 15 It is evident that the overall average content of components in each group falls into two major clusters. ① The overall average content of components in the *Acorus calamus* oil and physical mixture group after 1 hour of heat treatment, as well as the Pickering emulsion group after 1, 3, and 8 hours of heat treatment, clusters into one large cluster, indicating that the types and contents of volatile components in the Pickering emulsion are largely consistent with those in the *Acorus calamus* oil and physical mixture group after 1 hour of heat treatment at 1, 3, and 8 hours. ② The overall average content of components in the *Acorus calamus* oil and physical mixture group after 3 and 8 hours of heat treatment clusters into one large cluster, with the *Acorus calamus* oil and physical mixture group after 3 and 8 hours respectively clustering into smaller clusters. These results indicate that high temperature affects the types and contents of volatile components in *Acorus calamus* oil, and that the Pickering emulsion has a certain protective effect on the volatile components in *Acorus calamus* oil, maintaining a volatile component type and content closer to that of crude oil. This result helps us better understand the protective effect of the Pickering emulsion on *Acorus calamus* oil during heat treatment and its application potential.

[0198] 2.4.2 Screening for Differential Components using Volcano Plots

[0199] In Rstudio, the Limma package was used to compare the relative contents of volatile components between Acorus calamus oil and untreated Acorus calamus crude oil after heat treatment for 1, 3, and 8 hours using volcano plots. Differential components were selected with LogFC≥1.5 and P<0.05 as the cutoff. 9, 28, and 35 differential components were obtained after heat treatment for 1, 3, and 8 hours, respectively. After summarizing and deduplication, 35 differential components were selected. They are (+)-Cedrol(000077-53-2), Geraniol(000106-24-1), (+)-delta-Cadinene(000483-76-1), (R)-Citronello l(001117-61-9), Tricyclo[4.4.0.02,7]dec-3-ene,1,3-dimethyl-8-(1-methylethyl)-,stereoisomer(014912 -44-8), 1-methyl-4-[(1R)-1,2,2-trimethylcyclopentyl]benzene(016982-00-6), alpha-Calacorene(021391-99-1), alpha-Cadinene(024406-05-1), (4E)-1-methyl-4-(6-meth ylhept-5-en-2-ylidene)cyclohexene(053585-13-0), 4a,8-Dimethyl-2-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,7-octahydrona Phthalene(103827-22-1), α-Selinene(000473-13-2), (1R,4S,4aR,8aR)-4-Isopropyl-1,6-Dimethyl-1,2,3,4,4A,7,8,8A-Oc tahydro-1-Naphthalenol(000481-34-5), α-muurolene(010208-80-7), beta-Selinene(017066-67-0), Hedycaryol(021657-90 -9), 1-methyl-4-(1,2,2-trimethylcyclopentyl)cyclohexa-1,3-diene(029621-78-1), 4,8,11,11-tetramethylbicyclo[7.2.0] undec-3-en-5-ol(913176-41-7), Italiancene(997220-41-0), α-Phellandrene(000099-83-2), p-Cymene(000099-87-6), Citronellal(000106-23-0), Myrcene(000123-35-3), Beta-Pinene(000 127-91-3), Cineole(000470-82-6), Cyclene(000508-32-7), Terpinolene(000586-62-9), 3-Cyclohexen-1-ol,1-methyl-4-(1-methylethyl)-(000586-82-3), Sabinene(003387 -41-5), Bicyclo[2.2.1]heptane (005794-04-7), (+)-4-Carene (029050-33-7), alpha-Terpinene (000099-86-5), 2-Bornene (000464-17-5), 1,4-Cineole (000470-67-7), (1S)-(-)-alpha-Pinene (007785-26-4), (1R)-(+)-alpha-Pinene (007785-70-8). The relative contents of these differentially expressed components were significantly different from those of untreated Acorus calamus oil after heat treatment for 1, 3, and 8 hours. Further analysis of the performance of these differentially expressed components in Pickering emulsion, Acorus calamus oil, and physical mixture groups can help us understand the protective effect of Pickering emulsion on volatile oils (see...). Figure 16 ).

[0200] 2.4.3 PCA Analysis of Differential Components

[0201] Principal component analysis was performed using the relative content information of the three sets of differential components. Figure 17 AC results showed that the three Pickering milk samples exhibited a clear aggregation trend after heat treatment for 1, 3, and 8 hours, and were significantly different from the Acorus calamus oil and physical mixture groups. Figure 17D indicates that the sample distribution of all samples in the Acorus calamus oil and physical mixture groups basically overlaps, indicating that the relative content of the aforementioned differential components is basically consistent in the two groups. However, the sample distribution in the Pickering milk group does not overlap with that in the Acorus calamus oil and physical mixture groups. The contribution values ​​of PC1 and PC2 are 64.8% and 21.0%, respectively. The above results indicate that the differential components in the Pickering milk group show significant differences from those in the Acorus calamus oil and physical mixture groups after heat treatment for 1, 3, and 8 hours.

[0202] 2.4.4 Linear Analysis of Differential Components

[0203] To further investigate the changing trends of the relative contents of the differential components in each group with heat treatment time, a comparison of the linear graphs of the differential components in the three groups shows that the following components are present in the Acorus tatarinowii oil and physical mixture groups: 000077-53-2, 000106-24-1, 000473-13-2, 000481-34-5, 000483-76-1, 001117-61-9, 010208-80-7, 014912-44-8, 016982-00-6, 017066-67-0, and 021391-99- 1. The relative contents of 021657-90-9, 024406-05-1, 029621-78-1, 053585-13-0, 103827-22-1, and 997220-41-0 increased continuously with the extension of heat treatment time. However, in the Pickering milk group, the relative contents showed a trend of first increasing and then decreasing or increasing more slowly with the extension of heat treatment time, and the overall contents were relatively stable. This indicates that Pickering milk can slow down the increasing trend of the above 17 components during heat treatment. In the Acorus calamus oil and physical mixture groups, 00... The content of 0099-83-2, 000099-86-5, 000099-87-6, 00123-35-3, 000127-91-3, 000464-17-5, 000470-67-7, 000470-82-6, 000508-32-7, 000586-62-9, 003387-41-5, 005794-04-7, 007785-26-4, 007785-70-8, and 029050-33-7 decreases continuously with increasing heat treatment time. The relative content of the volatile components decreased to zero, but the trend of decreasing relative content was slowed down in the Pickering milk group. Furthermore, the relative content of these components in the Pickering milk was generally higher than that in the Acorus calamus oil and physical mixture groups, indicating that the Pickering milk can slow down the volatilization or degradation of the above 15 components during heat treatment. The content decrease or increase trends of 000106-23-0, 000586-82-3, and 913176-41-7 with heat treatment time were relatively similar in the three groups, and their change trend was larger in the Pickering milk. In summary, the content of volatile components in Acorus calamus oil showed an increasing and decreasing behavior with prolonged heat treatment time. The Pickering milk exhibited excellent protective effect on Acorus calamus oil during heat treatment, slowing down the volatilization and increase of most volatile components with heat treatment time, while having a smaller impact on other components (see...). Figure 18 ).

[0204] 2.4.5 Analysis of the physicochemical properties of the differential components

[0205] By analyzing the changes in the content of 35 differential components in the *Acorus calamus* oil group with heat treatment time, two main trends were observed: increasing and decreasing content with heat treatment time. Physicochemical properties, including relative molecular mass, density, boiling point, flash point, and LogP, were retrieved for the differential components with increasing content (UP group) and decreasing content (DOWN group). Numerical normalization was performed on different physicochemical properties, and the average values ​​of each physicochemical parameter for the differential components in the UP and DOWN groups were calculated. The petal plots of the physicochemical properties of the two groups show that the differential components in the UP group have higher relative molecular mass, boiling point, and flash point; while the differential components in the DOWN group have higher density and LogP. Further comparison of the physicochemical properties between the two groups using radar charts... Figure 19 As shown in Figure C, the relative molecular mass, density, boiling point, flash point, and LogP of the UP group are significantly higher than those of the DOWN group. The PCA plots of the physicochemical properties of the differential components in the UP and DOWN groups indicate that the differential components are well distinguished in both groups, showing significant differences, suggesting a clear distinction in the physicochemical properties of the differential components between the two groups.

[0206] Further investigation was conducted based on different physicochemical parameters to explore the principal components influencing the changes in volatile component content under thermal conditions. A total of five principal components were obtained. The first two principal components had relatively large variance contribution rates, with a cumulative variance contribution rate of 74.60%, and can be considered as the principal components influencing the changes in volatile component content under thermal conditions. Specifically: PC1 = -0.58 × relative molecular mass - 0.17 × density - 0.56 × boiling point - 0.38 × flash point - 0.42 × LogP, with a variance contribution rate of 53.4%; PC2 = 0.09 × relative molecular mass + 0.88 × density + 0.02 × boiling point - 0.06 × flash point - 0.45 × LogP, with a variance contribution rate of 21.2%. Figure 19As shown in D, the differences between the UP and DOWN groups are clearly distinguished in PC1. Combined with the principal component composition, PC1 is a principal component with a negative correlation between relative molecular mass, density, boiling point, flash point, and LogP. This means that components with high relative molecular mass, high boiling point, high flash point, high density, and high LogP are more likely to increase in content under thermal conditions; while components with low relative molecular mass, low boiling point, low flash point, low density, and low LogP are more volatile. This is consistent with the findings of Jahangir Farahbakhsh et al., whose low-boiling-point compounds significantly decreased during storage. These results indicate that the physicochemical properties of volatile components themselves affect the behavior of volatile oil components under thermal conditions, suggesting that we should pay attention to the key physicochemical properties of volatile components in volatile oils, such as relative molecular mass, boiling point, and flash point, as these may be related to the instability of volatile components. Pickering emulsions, with solid microparticles coating the surface of oil droplets, possess strong stability and protect the oil droplets from external influences. They can mitigate the increase or decrease in the content of these "thermally different components" with heat treatment time, and reduce the impact of the physicochemical properties of volatile components themselves.

[0207] 2.4.6 Qualitative Factor Analysis

[0208] After being placed at 40℃ for 8 hours, the GC-MS stacking diagrams of Acorus calamus oil, physical mixture, and Pickering emulsion showed that there were significant differences in the composition and content of volatile components in the three groups in the first 15 minutes. Figure 18 Similarly, it was shown that the content of some differentially expressed components decreased to zero with prolonged heat treatment time. The content of some differentially expressed components was zero at 1 and 3 hours of heat treatment, but became prominent with further heat treatment, indicating that the content of some components underwent qualitative changes during heat treatment, manifested as volatile components changing from present to absent or from absent to present. Therefore, it is necessary to further explore the protective effect of Pickering emulsion technology on *Acorus calamus* oil from the perspective of component qualitative changes. By comparing the relative content of volatile components in untreated *Acorus calamus* oil with that in *Acorus calamus* oil treated for 1, 3, and 8 hours using upset plots, "qualitatively changing components" were selected. Figure 20 As can be seen from B, during the heat treatment process, 16 components in Acorus tatarinowii oil disappeared with the extension of heat treatment time, while 5 components were newly formed during the heat treatment process. Furthermore, the total number of volatile components in Acorus tatarinowii oil continuously decreased with the extension of heat treatment time. From... Figure 20As shown in Figure C, compared to crude oil, 16 components disappeared from the *Acorus calamus* oil and the physical mixture after 8 hours of heat treatment, while Pickering emulsion retained 14 of these components. A total of 5 new components were formed from the *Acorus calamus* oil, the physical mixture, and the Pickering emulsion, with 2 of these new components unique to the Pickering emulsion. These results indicate that high temperature has a significant impact on the composition of volatile oils, and the modified amber-particle-stabilized Pickering emulsion can protect the volatile components in *Acorus calamus* oil from volatilization and maintain their original composition.

[0209] Further analysis was conducted on the changes in the content of the aforementioned qualitative components in Acorus tatarinowii oil, physical mixtures, and Pickering emulsions with heat treatment time. Figure 21 The results showed that the contents of 007785-26-4, 005794-04-7, 003387-41-5, 000127-91-3, 000500-00-5, 00123-35-3, 000099-83-2, 007785-70-8, 000470-67-7, 029050-33-7, 000099-87-6, 000470-82-6, and 000586-67-4 in the Acorus calamus oil and physical mixture group decreased continuously with the extension of heat treatment time, and the contents were completely reduced to zero after 3 or 8 hours of heat treatment (a qualitative change occurred). However, the Pickering milk group still retained the above components after 8 hours of heat treatment, and its contents were significantly higher than those in the Acorus calamus oil and physical mixture group at all time points. In all three groups, the contents of 000110-93-0 and 007786-67-6 were completely reduced to zero after 3 or 8 hours of heat treatment. 000124-18-5 was present in high amounts in untreated Acorus calamus oil, but its content was zero in the three groups treated for 1 and 3 hours, as well as in the Acorus calamus oil and physical mixture group treated for 8 hours. However, it suddenly appeared in the Pickering emulsion group after 8 hours of heat treatment, the possible reasons for which require further discussion. With the extension of heat treatment time, seven new components were generated, indicating that volatile components react to produce new components under heat. Among them, 000481-34-5, 997220-41-0, 003856-25-5, and 110983-38-5 had a slower formation rate and lower content in the Pickering emulsion group; 913176-41-7 had a faster formation rate and the highest content in the Pickering emulsion group. Among them, 000768-91-2 and 997332-93-7 are two newly formed components unique to Pickering milk. By analyzing the disappearing and newly formed qualitative changes, it was found that Pickering milk can slow down the loss of volatile components under high temperature conditions and reduce the formation rate of some new components.

Claims

1. A Pickering emulsion containing Acorus calamus oil, characterized in that: It contains modified amber particles, calamus oil, and water, and is an O / W type Pickering emulsion prepared using a high-speed shearing method. The content of modified amber particles and calamus oil is as follows: Modified amber particles 2.5-25 μg / mL, oil phase volume fraction 45-85%; The modified amber particles are prepared by adding fine amber powder to molten PEG6000, followed by melting and drying. The weight ratio of PEG6000 to amber is as follows: PEG6000 1-4 parts, amber 4 parts.

2. The Pickering emulsion containing Acorus calamus oil according to claim 1, characterized in that: The weight ratio of PEG6000 and amber is as follows: PEG6000 2 parts, amber 4 parts.

3. The Pickering emulsion containing Acorus calamus oil according to claim 1 or 2, characterized in that: The modification conditions are as follows: Place PEG6000 in an evaporating dish, transfer it to a heating mantle, set the voltage of the heating mantle to 100-150 volts, preheat the heating mantle for 10 minutes, heat and melt PEG6000, add amber powder, stir rapidly with a glass rod for 5 minutes to melt, remove the evaporating dish, dry at room temperature for 24 hours, and grind to obtain the product. The moisture content of the amber powder is ≤5%.

4. The Pickering emulsion containing Acorus calamus oil according to claim 3, characterized in that: The contact angle of the modified particles is 72-74°; under scanning electron microscopy (SEM), the surface of the modified amber particles is covered in clusters.

5. The Pickering milk according to claim 1, characterized in that: The modified amber particles and Acorus calamus oil content are as follows: modified amber particles 7.5 μg / mL, and oil phase volume fraction 65%.

6. The Pickering milk according to claim 1, characterized in that: The conditions for the high-speed shearing method are: shearing speed 5000-15000 rpm / min and shearing time 1-4 min.

7. The Pickering milk according to claim 6, characterized in that: The conditions for the high-speed shearing method are: shearing speed 11000 rpm / min and shearing time 2 min.

8. A preparation of Lingzhu powder, characterized in that: The raw materials are in the following weight proportions: 100-200 parts antelope horn, 100-200 parts pearl, 250-400 parts bezoar, 250-400 parts silkworm, 100-200 parts cinnabar, 250-350 parts amber, 550-650 parts Arisaema cum Bile, 20-40 parts borneol, and 10-30 parts Acorus tatarinowii oil. The amber and Acorus tatarinowii oil are further prepared into the Pickering emulsion containing Acorus tatarinowii oil as described in any one of claims 1-7.

9. The Lingzhu powder preparation according to claim 8, characterized in that: The weight parts of the raw materials are: 150 parts antelope horn, 150 parts pearl, 300 parts bezoar, 300 parts silkworm, 150 parts cinnabar, 300 parts amber, 600 parts bile arisaema, 30 parts borneol, and 20 parts calamus oil.

Images