Soothing agent of bitter wine soup powder as well as preparation method and simulation degree evaluation method of soothing agent

Abstract

The invention provides a placebo of bitter wine soup powder, which is prepared from the following raw materials in parts by weight: 2,080 to 3,120 parts of filling agent, 60 to 90 parts of turbid agent, 0.0704 to 0.1056 part of pigment, 40 to 60 parts of tackifier, 107.2 to 160.8 part of taste auxiliary material, 44.2 to 66.3 parts of smell auxiliary material and 67.84 to 101.76 parts of foaming agent. The invention also provides a preparation method of the placebo of the bitter wine soup powder and a simulation degree evaluation method. A percolation process is screened as an optimal extraction process through radioactive oral mucosa animal experiments; further systematic optimization is carried out on an extraction-freeze-drying-taste modifying process, so that'ready-to-use and ready-to-prepare 'type bitter wine soup powder is successfully prepared; the high-simulation placebo is developed on the basis of multi-dimensional sensory characteristics of bitter wine soup powder in shape, color, smell, taste and the like. According to the evaluation method, raw medicine similarity evaluation is carried out on the powder, the dissolution behavior and the solution appearance of the placebo respectively, the three states are simulated synchronously, and in order to solve the problem that the solubility of the raw medicine powder and the placebo is poor, improvement is carried out through a freeze-drying protective agent and micronization, so that the raw medicine powder and the placebo are similar; in addition, the appearance and physicochemical properties of a solution obtained after the raw medicine is dissolved are special, and simulation optimization is carried out.

Description

Technical Field

[0001] This invention relates to a placebo of bitter wine powder, its preparation method, and a method for evaluating the degree of simulation. Background Technology

[0002] Kujiu Decoction, originating from the *Shanghan Lun* (Treatise on Cold Damage), is a classic formula in Traditional Chinese Medicine (TCM) for treating sore throat and ulcers. It has demonstrated unique advantages in treating radiation-induced oral mucositis. The formula combines bitter wine (rice vinegar) to astringe and reduce swelling, pinellia to resolve phlegm and dissipate nodules, and egg to moisten dryness and promote tissue regeneration. The three herbs work synergistically to astringe sores, promote tissue regeneration, clear heat, and resolve phlegm, effectively relieving mucosal redness, swelling, and pain, and promoting ulcer healing. In recent years, the country has vigorously promoted the development of classic formulas, and the role of TCM in supportive treatment of tumor radiotherapy has received increasing attention, presenting a significant opportunity for the modernization of Kujiu Decoction research. However, this formula currently faces challenges such as insufficient verification of key information, inconvenience in using traditional dosage forms, and the lack of a highly realistic placebo, which restricts its clinical scientific evaluation. Systematic research is urgently needed to promote its standardization, dosage form improvement, and the construction of an evaluation system.

[0003] Traditional Chinese medicine decoctions possess unique colors, aromas, and tastes. Without a highly similar placebo, subjects and researchers are highly susceptible to unblinding, leading to expectation effects and observer bias, which seriously affect the scientific validity and reliability of trial results (Shao Jingyuan, et al., Research Progress on the Preparation Technology and Evaluation Methods of Placebos in Traditional Chinese Medicine, Drug Evaluation Research, Vol. 46, No. 5, May 2023). As a traditional formula, Kujiu Decoction has distinct appearance, color, and aroma characteristics. Preparing a sensorily consistent placebo is a necessary condition for achieving a randomized, double-blind, placebo-controlled (RCT) design. Summary of the Invention

[0004] This invention provides a placebo of bitter wine soup powder, its preparation method, and a method for evaluating the degree of simulation.

[0005] This invention provides a placebo of bitter wine powder, which is prepared from the following raw materials in the indicated weight ratios:

[0006] 2,080 to 3,120 parts filler, 60 to 90 parts turbidity agent, 0.0704 to 0.1056 parts pigment, 40 to 60 parts thickener, 107.2 to 160.8 parts flavoring agent, 44.2 to 66.3 parts odor agent, and 67.84 to 101.76 parts foaming agent;

[0007] The filler includes one or a mixture of mannitol and maltodextrin; the turbidity agent includes soybean lecithin; the pigment includes one or a mixture of tartrazine and sunset yellow; the thickener includes gum arabic; the flavoring agent includes one or a mixture of salt, monosodium glutamate, acetic acid powder, and mogroside; the aroma agent includes one or a mixture of acetic acid, peppermint flavoring, and orange flavoring; and the foaming agent includes Tween 80.

[0008] Preferably, it is prepared from raw materials in the following weight ratio:

[0009] 2600 parts filler, 75 parts turbidity agent, 0.088 parts pigment, 50 parts thickener, 134 parts flavoring agent, 55.25 parts odor agent, and 84.8 parts foaming agent;

[0010] The filler includes a mixture of mannitol and maltodextrin; the turbidity agent includes soybean lecithin; the pigment includes a mixture of tartrazine and sunset yellow; the thickener includes gum arabic; the flavoring agent includes a mixture of salt, monosodium glutamate, acetic acid powder, and mogroside; the aroma agent includes a mixture of acetic acid, peppermint flavoring, and orange flavoring; and the foaming agent includes Tween 80.

[0011] More preferably, it is prepared from raw materials in the following weight ratio:

[0012] Tween 84.8 parts, Mannitol 1040.3448 parts, Maltodextrin 1560.5172 parts, Salt 54 mg parts, Acetic acid powder 66 parts, Monosodium glutamate 9 mg parts, Lemon yellow pigment 0.07 parts, Sunset yellow pigment 0.018 parts, Soy lecithin 75 parts, Gum arabic 50 parts, Peppermint flavor 30 parts, Orange flavor 20 parts, Monk fruit glycoside 5 parts, Acetic acid 5.25 parts.

[0013] The preparation method of the bitter wine powder is as follows:

[0014] a. Weigh the ingredients according to the specified weight ratio: for every 4g of Pinellia ternata, use 12ml of rice vinegar and 35ml of egg white;

[0015] b. Add rice vinegar to the prepared Pinellia ternata, soak it first, then steep it, and then use the percolation method to percolate and collect the percolate;

[0016] c. Mix the percolate with egg white, filter, freeze dry to obtain freeze-dried powder; then add 1.8%-2.4% of flavoring agent to the total amount of freeze-dried powder.

[0017] Preferably, in the preparation method of the bitter wine powder,

[0018] Step a describes pulverizing Pinellia ternata into the coarsest, coarse, and medium powders;

[0019] The soaking time in step b is 0.5-2.0 h; the immersion time is 6-36 h; and the percolation flow rate is 1 mL / min·kg-3 mL / min·kg.

[0020] In step c, a freeze-drying protectant is added, which may include mannitol, sucrose, glucose, or trehalose; the amount of freeze-drying protectant used is 2.5-10%.

[0021] The freeze-drying conditions described in step c are: pre-freezing at -80℃ for 6 h, followed by drying at a vacuum of 20 Pa and a cold trap temperature of -50℃ for 24 h.

[0022] The flavoring agent mentioned in step c is a combination of peppermint flavoring, orange flavoring, and mogroside, with the following weight ratio:

[0023] Peppermint flavor 0.6-1.4 parts, orange flavor 0.6-1.4 parts, monk fruit glycosides 0.3-0.4 parts.

[0024] Preferably, the flavoring agent is a combination of peppermint flavoring and orange flavoring, with the following weight ratio:

[0025] 1.2 parts peppermint flavoring, 0.8 parts orange flavoring, and 0.3 parts monk fruit glycosides.

[0026] In the preparation method of the bitter wine powder,

[0027] The prepared Pinellia ternata powder described in step a is pulverized to medium powder;

[0028] The soaking time in step b is 1 hour; the immersion time is 18 hours; and the percolation flow rate is 2.0 mL / min·kg.

[0029] In step c, a freeze-drying protectant is added, which is mannitol; the amount of freeze-drying protectant is 5%.

[0030] The present invention also provides a method for preparing a placebo of the aforementioned bitter wine powder, comprising the following steps:

[0031] Weigh the filler and solution turbidity agent, place them in a mortar and grind them evenly; add the pigment, flavor excipient and odor excipient solution, and continue mixing; finally add the thickener and foaming agent, grind evenly, so that all the drug passes through a No. 5 sieve (80 mesh, pore size 180 micrometers), and at least 95% of the powder can pass through a No. 6 sieve, and the drug is obtained.

[0032] Specifically, it includes the following steps:

[0033] Take the prescribed amount of Tween 80, add it to a mortar and grind it evenly. Add the prescribed amounts of mannitol, salt, acetic acid, monosodium glutamate, and mogroside and grind evenly. Then add the prescribed amounts of lemon yellow pigment and sunset yellow pigment to the mortar and grind evenly. Add the prescribed amounts of soybean lecithin, gum arabic, and maltodextrin to the mortar and grind evenly. Dry at 105℃ for 30 minutes, let cool, add the prescribed amounts of acetic acid, peppermint flavor, and orange flavor to the mortar and grind evenly. Pass through a No. 6 sieve to obtain placebo powder.

[0034] This invention also provides a method for evaluating the placebo simulation of bitter wine soup powder. It uses headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS), electronic tongue, colorimeter, powder property analyzer and viscometer to analyze the "shape, color, aroma and taste" of bitter wine soup powder in multiple dimensions.

[0035] Specifically, it includes the following steps:

[0036] a. Volunteer evaluation was conducted using a Likert scale. Evaluation indicators included: powder color, powder odor; solution color, solution odor, clarity, and solution taste. The judgment criteria were: similarity threshold: a score > 6 indicates high similarity; PyCharm 2022.2 software was used to calculate the cosine of the angle between the two sides. The closer the cosine value is to 1, the higher the similarity.

[0037] b. Instrument evaluation: including

[0038] Similarity of formulation characteristics: Texture characteristics include angle of repose, dispersibility, clarity, viscosity, and foaming properties;

[0039] Odor similarity: The odor of samples in powder form is evaluated and distinguished using an electronic nose;

[0040] Taste similarity: The taste of samples in solution is evaluated and distinguished using an electronic tongue;

[0041] Color similarity: The color difference meter similarity evaluation method was used;

[0042] c. Compare the placebo with the bitter wine powder standard to obtain the simulation results.

[0043] The evaluation method of this invention evaluated the similarity of the placebo powder, dissolution behavior, and solution appearance to the original drug, and simultaneously simulated these three states. The main technical challenge lies in the poor solubility of the original drug powder and the placebo, which was improved through lyophilization protectants and micronization to achieve similarity. Furthermore, the appearance and physicochemical properties of the solution after dissolution of the original drug are quite unique, and simulation optimization was performed.

[0044] This invention screened percolation as the optimal extraction process through animal experiments on radioactive oral mucosa; further, it systematically optimized the extraction-freeze-drying-flavoring process, successfully preparing a "prepared on demand" bitter wine soup powder; finally, based on the multi-dimensional sensory characteristics of the bitter wine soup powder in terms of shape, color, aroma, and taste, a highly realistic placebo was developed, providing reliable support for subsequent blinded clinical studies.

[0045] This invention proposes a "powder-solution" dual-phase synergistic imitation strategy, conducting systematic imitation research focusing on the complex properties of the powder and reconstituted form, such as characteristic foam, odor, and color. Using modern analytical methods, the physical properties and solution characteristics of the original bitter wine decoction were comprehensively characterized, and based on this, a compound excipient system was screened and constructed. This system uses mannitol and maltodextrin (2:3) as the filling matrix, Tween 80 to reproduce foaming characteristics, soybean lecithin to simulate turbidity, gum arabic to adjust viscosity, tartrazine and sunset yellow to regulate color, acetic acid, peppermint flavor, and orange flavor to reproduce odor, and acetic acid powder, salt, monosodium glutamate, and monk fruit glycosides to synergistically reproduce flavor. A combination of volunteer sensory evaluation and objective instrumental detection was used to evaluate the system similarity. The results showed that the prepared placebo, in both powder and reconstituted states, highly closely resembled the original drug in terms of color, odor, taste, clarity, flowability, viscosity, and foaming properties, effectively meeting the requirements of blinded clinical trial design and providing a reliable placebo-controlled solution for the clinical research of this classic formula. Attached Figure Description

[0046] Figure 1. Powder properties data of bitter wine soup powder;

[0047] Figure 2 Data on the properties of bitter wine solution;

[0048] Figure 3 Color distribution of ten batches of bitter wine powder;

[0049] Figure 4 Color distribution of ten batches of bitter wine powder;

[0050] Figure 5 The taste of bitter wine soup powder;

[0051] Figure 6 Results of placebo odor and taste screening for bitter wine powder;

[0052] Figure 7 Results of placebo color prescription screening for bitter wine decoction powder;

[0053] Figure 8 Similarity evaluation results between volunteers taking bitter wine powder and those taking placebo;

[0054] Figure 9. PCA diagram of bitter wine powder measured by electronic nose;

[0055] Figure 10. PCA diagram of the electronic tongue determination of bitter wine powder solution. Detailed Implementation

[0056] Example 1: Method for preparing the placebo of the present invention

[0057] Take 80 μL of Tween and place it in a mortar. Grind it evenly. Add 1 g of mannitol, 54 mg of salt, 66 mg of acetic acid, and 9 mg of monosodium glutamate to the mortar and grind it evenly. Then add 70 μL of lemon yellow pigment solution (1 mg / mL) and 18 μL of sunset yellow pigment solution (1 mg / mL) to the mortar and grind it evenly. Then add 75 mg of soybean lecithin, 50 mg of gum arabic, and 1.5 g of maltodextrin to the mortar and grind it evenly. Dry it at 105℃ for 30 min, let it cool, add 5 μL of acetic acid, 30 mg of peppermint flavoring, and 20 mg of orange flavoring to the mortar and grind it evenly. Pass it through a No. 6 sieve to obtain placebo powder.

[0058] Example 2: Screening test of the preparation process conditions for the placebo of the bitter wine powder of the present invention.

[0059] 1. Instruments and Materials

[0060] 1.1 Instruments

[0061] Magnetic stirrer (model), HS-SPME-GC-MS, electronic tongue, turbidimeter, intelligent rheometer, laser particle size analyzer, rotational viscometer, multiple light scattering instrument

[0062] 1.2 Reagents

[0063] Drug: Bitter Wine Powder (batch number: manufacturer), Bitter Wine Powder Placebo (batch number:), manufactured by Chengdu University of Traditional Chinese Medicine. Pigments: Lemon Yellow, Sunset Yellow. Filler excipients: Mannitol, Glucose, Sodium Alginate. Pharmaceutical excipients: Soy lecithin, Soy protein powder, Xanthan gum, Sodium caseinate, Hydroxypropyl methylcellulose, Mannitol, Tween 80. Flavoring agents: Acetic acid, Acetic acid, Monk fruit extract. Flavorings: Peppermint flavor, Orange flavor.

[0064] 1.3 Data Processing

[0065] Origin, Python, MATLAB 2016b, and SPSS 21.0 software were used.

[0066] 2 Methods

[0067] 2.1 Determination of Key Quality Attribute Parameters of Bitter Wine Powder

[0068] To systematically improve the similarity of placebos, key quality attributes (CQAs) need to be identified from four dimensions: dosage form characteristics, visual characteristics, and odor and taste characteristics. Among these, the color of the formulation and the odor and taste characteristics are the core parameters of sensory CQAs.

[0069] The 2025 edition of the Chinese Pharmacopoeia has specific inspection requirements for particle size, moisture content, and fill weight variation of powders. Furthermore, after reconstitution, the Kujiu Tang powder exhibits certain foaming properties and viscosity. Therefore, parameters such as the angle of repose, bulk density, tap density, and Hausner ratio of the powder, as well as the surface tension, viscosity, clarity, and foaming properties of the solution, need to be included in the dosage form CQAs as the basis for the characteristics of the Kujiu Tang powder.

[0070] 2.1.1 Physicochemical properties

[0071] 2.1.1.1 Powder Characteristic Parameters

[0072] The angle of repose and dispersibility of the samples were determined using a BT-1000 powder property analyzer.

[0073] 2.1.1.2 Solution characteristic parameters

[0074] (1) Clarity: 200 µL of sample and blank solvent were added to a 96-well plate and the optical density OD value was measured at a wavelength of 600 nm.

[0075] (2) Foaming ability: Take a certain volume (Vl) of solution, place it in a stoppered graduated cylinder, and oscillate vertically at a fixed frequency and amplitude for a fixed number of times. Immediately record the foam volume (Vf) to evaluate the foaming ability (FC (%) = Vf / Vl × 100%).

[0076] (3) Viscosity: The apparent viscosity of each solution was measured using a rotational viscometer at 25°C and a shear rate of 60 RMB.

[0077] 2.1.2 Color Measurement

[0078] 2.1.2.1 Preparation of the test sample

[0079] In powder form, the powder is directly measured; in solution form, the powder is prepared into a solution with a mass concentration of 1 g / 6 mL for measurement.

[0080] 2.1.2.2 Colorimeter Technical Parameters

[0081] Illumination: Halogen tungsten lamp used for the instrument; Illumination conditions: 0 / d conditions (vertical illumination, diffuse reflection reception), D65 standard light source; Standard observer: 10-degree field of view. Before use, preheat the instrument for 30 minutes. After preheating, first adjust to black, then adjust to white. After completion, the measurement can be performed. Each sample is measured in triplicate. Record the L*, a*, and b* values.

[0082] 2.1.3 Determination of volatile components

[0083] 2.1.3.1 Gas Chromatography Conditions

[0084] The capillary column used was an SH-Polar Wax (60 mm × 0.25 mm × 0.25 μm), with an injection port temperature of 250℃. The injection method was split injection with a split ratio of 5:1. High-purity helium (mass fraction ≥ 99.999%) was used as the carrier gas. The control mode was constant linear velocity with a linear velocity of 25.50 cm / s. The purge flow rate was set to 3.0 mL / min. The column temperature was programmed, with an initial temperature of 40℃, held for 5 min, and then increased to 250℃ at a rate of 3℃ / min, held for 15 min. The column equilibration time was 3.0 min.

[0085] 2.1.3.2 Mass Spectrometry Conditions

[0086] Ionization was performed using electron impact ionization (EI) at an energy of 70 eV. The ion source temperature was maintained at 200 °C, and the mass spectrometry transmission interface temperature was set to 250 °C. Argon was used as the collision gas. Full scan mode was used for mass spectrometry acquisition, with a scan mass number range of 35–500 amu. The detector voltage was set to +0.10 kV relative to the tuning result, and the solvent delay time was 1 min.

[0087] 2.1.3.3 Headspace-Solid Phase Microextraction Conditions

[0088] Accurately weigh 0.50 g of bitter wine powder and place it in a 20 ml inert headspace vial; add 10 μL each of internal standards 4-bromofluorobenzene, 1,2-dichlorobenzene-d4, and acenaphthene-d10 (10 μg / mL, methanol as solvent). After equilibration at 50 °C for 5 min, the headspace vial containing the sample is transferred to the extraction apparatus. The SPME arrow solid-phase extraction head is inserted into the headspace vial through a polytetrafluoroethylene headspace septum, without contacting the sample. Extraction and adsorption are performed at 50 °C for 15 min. After the autosampler removes the extraction head, it is quickly inserted into the GC-MS / MS injection port, and desorption is performed at 250 °C for 2 min. The solid-phase extraction head is a composite of DVB / CWR / PDMS materials, with a film thickness of 120 μm and a length of 20 mm. The solid-phase extraction head is aged at 250 °C for 3 min before and after sample injection.

[0089] 2.1.3.4 Qualitative and quantitative analysis of volatile components

[0090] Volatile components were identified by comparing the retention time and retention index of odor components with the NIST 23 mass spectrometry library and standard alkanes (C7–C30). Volatile components in *Codonopsis pilosula* were quantitatively analyzed using the internal standard method, with the RRF value determined to be 1 by calculating the relative response factor (RRF) of the three internal standards. Quantitative comparisons were performed using 504 composite standard curves from the Smart Aroma Database built into the Shimadzu TQ8050 reanalysis software. The quantification of volatile components was calculated according to formulas (1) and (2), and the OAV value was calculated according to formula (3).

[0091]

[0092]

[0093] RRF is the relative response factor, C i The value of A represents the mass fraction of the unknown volatile component (μg / kg), C0 represents the internal standard concentration, V0 is the internal standard injection volume (μL), and A represents the internal standard injection volume (μL). i A0 represents the peak area of ​​the unknown volatile component, m represents the measured mass of the sample.

[0094]

[0095] C i Indicates the mass fraction of aroma compounds; OT I It is the odor threshold of aroma components.

[0096]

[0097] In the formula C j and T jThese represent the relative percentage content of each volatile substance and its corresponding olfactory threshold. These represent the relative percentage content of the component that contributes the most to the overall odor of the sample and its corresponding olfactory threshold.

[0098] 2.1.4 Determination of taste parameters

[0099] 2.1.4.1 Sensors

[0100] This instrument can utilize five sensors (C00, AE1, CA0, CT0, and AAE) and two standard electrodes for a food five-taste analysis system; one sensor (GL1) and one standard electrode for a sweetness analysis system; and two sensors (ANO and BTO) and one standard electrode for a drug bitterness analysis system. The sensors are activated and calibrated before sample measurement.

[0101] 2.1.4.2 Test solution

[0102] Reference solution: 30 mM potassium chloride + 0.3 mM tartaric acid; Negative electrode cleaning solution: 100 mM hydrochloric acid + 30% ethanol (volume); Positive electrode cleaning solution: 10 mM potassium hydroxide + 100 mM potassium chloride + 30% ethanol (volume).

[0103] 2.1.4.3 Sample Testing Methods

[0104] The reconstituted solution of bitter wine powder can be directly tested on the instrument.

[0105] Equilibrium: The sensor is first cleaned in the cleaning solution for 90 seconds, then cleaned in the reference solution for 120 seconds, and then cleaned in another reference solution for 120 seconds. The sensor is then zeroed at the equilibrium position for 30 seconds. Testing: The test time is 30 seconds, and the initial taste value is output. Then, the sensor is cleaned in the reference solution for 3 seconds, and the aftertaste is tested in the new reference solution for 30 seconds. The five taste sensors C00, AE1, CA0, CT0, and AAE, as well as the bitter taste sensors ANO and BTO, are tested 4 times. The first cycle is removed, and the average of the last three cycles is taken as the test result. The sweet taste sensor GL1 is tested 5 times. The first and last cycles are removed, and the average of the middle three cycles is taken as the test result.

[0106] 2.2 Screening and Preparation of Placebo Formula for Bitter Wine Decoction Powder

[0107] 2.2.1 Preparation of placebo for bitter wine decoction powder

[0108] 2.2.1.1 Placebo Powder Preparation Process

[0109] Weigh the filler and turbidity agent according to the prescription, place them in a mortar and grind them evenly; add an appropriate amount of pigment and acidulant solution, and continue mixing; finally add the thickener and foaming agent, grind evenly, so that all the medicine passes through a No. 5 sieve (80 mesh, 180 micrometers aperture), and at least 95% of the powder can pass through a No. 6 sieve.

[0110] 2.2.1.2 Preparation of placebo solution

[0111] Weigh 5 g of placebo powder, add 30 mL of water, and stir to dissolve.

[0112] 2.2.2 Turbidity screening of excipients

[0113] Preliminary experiments showed that the bitter wine solution itself has a certain degree of turbidity. During the screening of excipients with turbidity-enhancing effects, it was found that soybean lecithin, soybean protein powder, and microcrystalline cellulose could all affect the turbidity of the solution, but their characteristics and stability differed. While soybean protein powder and microcrystalline cellulose could increase turbidity in the short term after dissolution, they were prone to precipitation after standing, resulting in poor system stability. Soybean lecithin, on the other hand, not only effectively enhanced the turbidity of the sample but also significantly increased its viscosity, exhibiting better dispersion stability. Therefore, this study selected soybean lecithin as a turbidity-enhancing excipient and further systematically investigated its dosage, using turbidity as the core evaluation index to optimize the formulation.

[0114] 2.2.3 Screening of Toughening Agent Excipients

[0115] The viscosity of the bitter liquor solution at a shear rate of 60 RPM (rotor 0) was 2.29 mPa·s, which is relatively low. Considering its pale yellow color and low turbidity, this study selected light-colored excipients such as gum arabic, xanthan gum, and pectin as candidates for thickeners. Xanthan gum exhibits gelling properties, significantly increasing the system viscosity far beyond the base viscosity of the bitter liquor solution. Pectin, on the other hand, tends to clump, and its viscosity is significantly affected by environmental pH, temperature, and its own concentration, exhibiting poor reproducibility and stability. In contrast, gum arabic showed good compatibility, with moderate viscosity and rheological behavior less affected by pH, temperature, and shear conditions, which is beneficial for maintaining consistency with the bitter liquor solution during preparation and storage. Therefore, gum arabic was selected as the thickener excipient, and viscosity was used as the evaluation index to optimize the formulation.

[0116] 2.2.4 Screening of foaming agent excipients

[0117] The original bitter wine soup contains egg white protein, which generates bubbles when stirred. Initial attempts to simulate its foaming properties using sodium caseinate, a protein-containing excipient, revealed a precipitation reaction with the flavoring agent, acetic acid powder. Therefore, this study shifted to using excipients that reduce surface tension to simulate its foaming performance. When examining commonly used foaming agents such as monoglycerides, polyglycerol fatty acid esters, and sucrose fatty acid esters, it was found that they all had low solubility in water and poor foaming ability. Ultimately, Tween 80 was selected as the foaming agent, and its dosage was investigated, with foaming ability serving as the screening criterion.

[0118] 2.2.5 Screening of Odor and Taste Additives

[0119] 2.2.5.1 Volunteer Sensory Evaluation Method

[0120] The specific methods for volunteer selection are as described in sections 2.3.1.1-2.3.1.2. The sensory evaluation method is as follows: Similarity scores were calculated for the original drug and placebo prescriptions. The similarity scoring criteria used the internationally recognized Likert scale: 0-2 points indicate complete inconsistency, 2-4 points indicate uncertainty, 4-6 points indicate close similarity, 6-8 points indicate very close similarity, and 8-10 points indicate almost perfect similarity. The simulated subjects and the placebo were evaluated based on two sensory CQAs parameters: powder odor and solution taste.

[0121] 2.2.5.2 Odor and Taste Prescription Screening

[0122] The results of aroma and taste characterization of the original herb, Kujiu Tang, showed that it has aromas such as sourness and fishiness, as well as taste characteristics such as sourness, saltiness, and umami. Based on previous research on flavoring processes, acetic acid, peppermint flavoring, and orange flavoring were selected as aroma simulating additives, while salt, monosodium glutamate, acetic acid powder, and mogroside were selected as taste simulating additives. Different prescriptions were screened and optimized through sensory evaluation by volunteers.

[0123] 2.2.6 Color Accessory Screening

[0124] Both the powder and solution of the bitter wine decoction are pale yellow, with the solution being turbid and having some translucency. Based on its color characteristics, this study selected lemon yellow and sunset yellow as color simulation excipients. While directly adding powdered pigments can effectively simulate the powder color, it results in a significantly darker solution color compared to the original. Therefore, solutions of both pigments were prepared at a concentration of 1 mg / mL, and their dosage was investigated using these as a baseline. The color difference value (ΔE) was used as the evaluation index; a smaller ΔE value indicates a closer similarity between the simulated color and the original drug color.

[0125] 2.2.7 Screening of filler excipients

[0126] The original bitter wine decoction is pale yellow with low turbidity. To maintain the uniformity of the formulation's appearance, a colorless filler excipient was selected. After preliminary screening, mannitol, maltodextrin, and microcrystalline cellulose were identified as candidate excipients. Preliminary experiments showed that microcrystalline cellulose had poor water solubility and was therefore excluded.

[0127] Considering that fillers account for over 80% of the formulation, their powder properties have a decisive influence on the final powder characteristics. Therefore, it is necessary to select excipients that match the characteristics of the active pharmaceutical ingredient powder. Mannitol has good solubility but poor flowability; maltodextrin, on the other hand, has better flowability, and its aqueous solution is colorless and transparent, with solubility between that of mannitol and microcrystalline cellulose. To balance solubility and flowability, subsequent experiments used a compound of mannitol and maltodextrin to leverage their synergistic and complementary effects, and systematically investigated these effects using powder angle of repose and dispersibility as key evaluation indicators.

[0128] 2.3 Similarity Evaluation Methods

[0129] There are two common methods for evaluating the similarity of traditional Chinese medicine placebos: the healthy volunteer evaluation method and the objective evaluation method using sensory and physical property measuring instruments. The volunteer evaluation method includes manual scoring and manual judgment of the similarity between preparations, evaluating individual characteristic parameters or the entire preparation. The objective evaluation method includes the measurement of physical property parameters, the use of biomimetic devices to measure taste and odor, and the use of colorimeters or image evaluation methods to measure color.

[0130] 2.3.1 Volunteer Evaluation

[0131] The specific methods for volunteer selection are as described in Sections 2.3.1.1-2.3.1.2 of Chapter 3. The comparative evaluation is implemented as follows: A simulated subject is used as the reference drug. A placebo and the simulated subject are randomly packaged into drug A and drug B, respectively. Drug A and drug B are compared with the reference drug using a similarity scoring standard based on the internationally accepted Likert scale: 0-2 points indicate complete inconsistency, 2-4 points indicate uncertainty, 4-6 points indicate close similarity, 6-8 points indicate very close similarity, and 8-10 points indicate almost perfect similarity. The simulated subject and placebo are evaluated using six sensory CQAs parameters: powder color, powder odor; solution color, solution odor, clarity, and solution taste. A score greater than 6 indicates high similarity between the two subjects. The cosine of the angle between the simulated subject and the placebo is calculated using PyCharm 2022.2 software. A cosine value closer to 1 indicates higher similarity.

[0132] 2.3.2 Instrument Evaluation

[0133] 2.3.2.1 Similarity of formulation characteristics

[0134] The texture characteristics of bitter wine powder and solution, including angle of repose, dispersibility, clarity, viscosity, and foaming properties, need to be simulated using suitable matrix materials. These were determined according to the methods described in section "2.1.1 Physicochemical Properties". The cosine of the angle between the parameters was calculated using PyCharm 2022.2 software and used as an indicator to evaluate the similarity between the placebo and bitter wine powder.

[0135] 2.3.2.2 Odor Similarity

[0136] An electronic nose was used to evaluate and differentiate the odor of samples in powder form.

[0137] (1) Sample preparation: KJT bitter wine powder, KJT placebo, KJT placebo (tasteless simulated excipient), KJT placebo (odorless simulated excipient), KJT placebo (tasteless and odorless simulated excipient), and blank group were prepared.

[0138] (2) Methodological investigation of the measurement conditions: Record the response values ​​of 10 electrodes during the measurement of the test sample and calculate the RSD. (3) Measurement conditions and data acquisition: The electronic nose electrodes are W1C, W5S, W3C, W6S, W5C, W1S, W1W, W2S, W2W, and W3S, a total of 10 electrodes. The sampling time is 1 s / group, the sensor self-cleaning time is 100 s, the sensor zeroing time is 5 s, the sample preparation time is 5 s, the injection flow rate is 400 mL / min, and the analysis sampling time is 100 s.

[0139] (4) Data analysis: Based on the raw response data collected by the sensor, PCA was performed using Origin 2022 software. The difference in odor between samples was determined by the distance between samples on the PCA graph. The smaller the distance, the smaller the difference and the higher the sample similarity.

[0140] 2.3.2.3 Taste Similarity

[0141] An electronic tongue is used to evaluate and differentiate the taste of samples in solution.

[0142] (1) Preparation of sample solutions: Weigh the powder, add water to dissolve and filter. KJT, KJT placebo, KJT placebo (odorless simulated excipient), KJT placebo (odorless simulated excipient), KJT placebo (odorless and tasteless simulated excipient) and pure water were prepared in this way.

[0143] (2) Methodological investigation of determination conditions: including investigation of mass concentration and precision. Sample solutions of 1.0 and 0.1 mg / mL were prepared for determination, and the concentration with better electronic tongue discrimination ability was selected as the determination concentration of the sample. At the mass concentration level of 0.1 mg / mL, the response value of the electrode was recorded and the RSD was calculated.

[0144] (3) Measurement conditions and data acquisition: The electronic tongue electrodes are ZZ, JE, BB, CA, GA, DA and JB, a total of 7 electrodes. The acquisition time for each sample was set to 120 s, with one acquisition per second. The average response value of each sample at 100 and 120 s was used as the measurement data and included in the calculation. After each measurement, the sample was automatically cleaned for 10 s. Each sample was measured 10 times, and the last 3 data points with stable response values ​​were selected for calculation.

[0145] (4) Data analysis: Based on the raw response data collected by the sensor, principal component analysis (PCA) was performed using the Alphasoft 14.0 software that comes with the system. The difference in taste between samples was determined by the distance between samples on the PCA graph. The smaller the distance, the smaller the difference and the higher the similarity of the samples.

[0146] 2.3.2.4 Color Similarity

[0147] In visual characteristic attributes, color similarity is a key focus and challenge in the preparation of powder placebos because similarity requirements must be met in both powder and solution states. While manual evaluation is commonly used for color assessment, the application of colorimeters for color measurement is also gradually increasing. Therefore, this study will use the colorimeter similarity evaluation method.

[0148] Colorimeter measurement is a simple and rapid method for sample pretreatment, and it can determine L*, a*, and b* color space parameters, generally used for rapid screening of color simulation prescriptions. In this study, the L*, a*, and b* color spaces of bitter wine powder and its placebo in powder and solution states were determined using a colorimeter. The determination was performed according to the method under "2.1.2 Color Measurement". The L*, a*, b*, and ΔE* values ​​obtained by the colorimeter can be used to determine the color similarity between two samples. The formula for calculating ΔE* is shown below.

[0149] ΔE *=(L *2+a *2+b *2) 1 / 2. The ΔE value is calculated using the CIEDE2000 color difference formula. ΔE<3 is defined as "visually indistinguishable", 3≤ΔE<6 as "slight difference", and ΔE≥6 as "significant difference".

[0150] 3 Results

[0151] 3.1 Extraction of key parameters for bitter wine powder

[0152] 3.1.1 Pharmaceutical Characteristics

[0153] 3.1.1.1 Powder

[0154] The powder flowability and dispersibility of 10 batches of samples were tested. The results showed that the angle of repose ranged from 38.33° to 40.33°, with an average of 39.54° ± 0.73°; ​​the dispersibility ranged from 23.47% to 26.92%, with an average of 24.99% ± 1.06%. The bar chart showed some fluctuations among different batches. Batch 250802 had the lowest angle of repose (38.33°), indicating the best powder flowability; while batches 250601 and 2408050 had the highest angles of repose (both 40.33°), indicating relatively poor flowability. Regarding dispersibility, batch 250305 exhibited the best dispersibility (26.92%), while batch 240802 had the lowest dispersibility (23.47%).

[0155] Further analysis using violin plots and box plots showed that the angle of repose data were relatively uniformly distributed with no obvious outliers, indicating good reproducibility of the preparation process. The dispersibility data also showed good consistency, with the median and mean close, indicating that the dispersion performance of each batch of samples was relatively stable (e.g., ...). Figure 1 (As shown).

[0156] 3.1.1.2 Solution

[0157] The clarity, foaming ability, and viscosity of 10 batches of samples were tested. The results showed that the clarity ranged from 0.53 to 0.59, with an average value of 0.56 ± 0.02; the foaming ability ranged from 1.7 to 1.8, with an average value of 1.76 ± 0.04; and the viscosity ranged from 2.11 to 2.20, with an average value of 2.15 ± 0.03.

[0158] The bar chart shows some fluctuations in data between different batches. Batch 250504 had the lowest clarity (0.53), while batches 250601 and 250802 had the highest clarity (both 0.59). In terms of foaming ability, batches 240108 and 250504 performed best (both 1.8), while batches 250601 and 250802 had the lowest (both 1.7). In viscosity testing, batch 240108 had the lowest viscosity (2.11) and the best flowability; batch 250504 had the highest viscosity (2.20) and relatively poor flowability.

[0159] Further analysis using violin plots and box plots revealed that the clarity data were relatively concentrated with no significant outliers, indicating good control over solution transparency across different batches. The foaming ability and viscosity data also showed good consistency, with the median and mean values ​​close, reflecting relative stability in these two physical properties across batches. Figure 2 (As shown).

[0160] 3.1.2 Color Parameters

[0161] 3.1.2.1 Powder

[0162] This study used a colorimeter to characterize and visualize the color characteristics of the bitter wine powder. The results of the three-dimensional scatter plot (Figure 3A) and the two-dimensional projection plots (Figure 3B–D) of a*-b*, L*-a*, and L*-b* show that all samples exhibit high brightness characteristics, with L values ​​stable in the range of 94.04–95.41 and small brightness fluctuations. In terms of hue, a values ​​are distributed between -0.17 and 0.37, with no significant red-green bias, while b values ​​are concentrated between 12.31 and 15.47, exhibiting an overall low-saturation light beige tone. Further analysis of the sample distribution patterns in the two-dimensional projection plots reveals that the color differences between samples mainly stem from subtle fluctuations in b values ​​(yellow hue concentration) and slight shifts in a values, while the brightness remains largely consistent. Combined with the actual color bars of the samples (…),… Figure 3 E) indicates that all batches exhibited a uniform light yellow hue, demonstrating high color consistency. These results indicate that the bitter wine powder exhibits good color stability and controllability during preparation, providing a quantitative basis for subsequent color quality control of the bitter wine powder placebo.

[0163] 3.1.2.2 Solution

[0164] To prepare a highly realistic placebo of bitter wine, accurate characterization and simulation of the color of its reconstituted solution are also required. The color characterization results of the reconstituted solution are as follows: Figure 4 As shown, a three-dimensional scatter plot ( Figure 4 A) and two-dimensional projection diagrams of a*-b*, L*-a*, and L*-b* (A) Figure 4 (BD) The results show that the L values ​​of all samples are stable within the range of 79.64 to 80.71, indicating high overall brightness with minimal fluctuations; the a values ​​range from -3.33 to -3.18, indicating a slight greenish tint to the solution; the b values ​​are concentrated in the range of 29.39 to 29.65, showing a clear yellowish tendency. Both these factors indicate that the reconstituted solution generally presents a low-saturation, light yellowish-green color. Further analysis of the distribution patterns of the sample points in the projection diagram reveals that the color differences between samples mainly originate from subtle variations in the a values ​​(green saturation) and b values ​​(yellow saturation), while the lightness L values ​​remain largely consistent. Combined with... Figure 4The actual color bands of E show that the color of each sample is a highly consistent light yellow-green, providing a reliable quantitative basis for the subsequent preparation of placebos.

[0165] 3.1.3 HS-SPME-GC-MS Analysis Results of Odor of Bitter Wine Powder

[0166] Odor analysis was performed on the bitter wine powder samples. A total of 55 volatile components were detected in the powder samples, with alcohols, acids, and heterocyclic compounds being the most prevalent categories. Specifically, among the 55 components, there were 14 alcohols (25.45%), 10 acids (18.18%), 10 heterocyclic compounds (18.18%), 6 esters (10.91%), 4 aldehydes (7.27%), 4 ketones (7.27%), 4 phenols (7.27%), 2 sulfides (3.64%), and 1 ether (1.82%). The relative odor activity value (ROAV) was used to analyze the overall contribution of each compound to the sample. The "olfactory threshold" refers to the minimum concentration or dilution factor at which a substance can be perceived by the human olfactory system. Under certain conditions, the lower the olfactory threshold and the higher the concentration, the easier the odor is to be perceived and the greater its contribution to the overall odor. The key volatile aroma compounds in bitter liquor powder were determined using the ROAV method. First, the odor activity value (OAV) method was used to identify the volatile components that contribute most to the flavor of the sample. The odor activity values ​​of each substance were calculated, and the components that contribute most to the aroma of the sample were defined. The ROAV values ​​of other volatile components were calculated. All components met the condition 0 ≤ ROAV ≤ 100, and the larger the ROAV, the greater the contribution of the component to the overall odor of the sample. Components with ROAV ≥ 1 are generally considered key odor substances in the sample, while components with ROAV ≤ 0.1 < 1 have a modifying effect on the overall odor. In this sample, acetic acid (ROAV = 100) was identified as the key component with the largest contribution to the overall odor. Four key odor substances with ROAV ≥ 1 (acetic acid, isovaleric acid, n-valeric acid, and hexanoic acid) and seven odor-modifying components with ROAV ≤ 0.1 < 1 (butyric acid, n-hexanal, p-cresol, and isoamyl alcohol) were screened. These substances together constitute the unique complex odor characteristics of this sample: "strong rancidity, cheese fermentation, fatty and slightly burnt." Acetic acid, as the most significant odor contributor, establishes the basic sour tone of the sample, while the fecal and cheese-like odors brought by isovaleric acid and n-valeric acid, and the barn fermentation aroma presented by hexanoic acid, further enrich the odor layers of the sample, collectively forming the characteristic flavor profile.

[0167] Table 1. Analysis results of odor substances in bitter wine powder.

[0168] Chinese name English name ROAV Scent description Acetic acid Acetic acid 100 acid, cheese, fruit, pungent, sour Isovalerate Isovaleric acid 29.64681139 acid, cashew, cheese, fat, fecal valeric acid Valeric acid 1.931595929 acid, cheese, fecal, fruit, pungent hexanoic acid Capronic acid 1.544726834 acid, barnyard, cheese, fat, fermented butyric acid Butyric acid 0.750716889 acid, butter, cheese, must, rancid hexanol Hexanal 0.552526835 apple, beans, cut grass, fat, fish p-Cresol p-Cresol 0.277613027 medicine, phenol, smoke Isoamyl alcohol Isoamyl alcohol 0.259828134 alcohol, balsamic, banana, burnt, cheese 2,6-Dimethylpyrazine 2,6-Dimethylpyrazine 0.195851004 burnt, cocoa, coffee, fried, fried potato Isobutyric acid Isobutyric acid 0.138304609 acid, burnt, butter, cheese, cooked 3-Hydroxy-2-Butanone Acetoin 0.115088108 butter, cream, fat, green pepper, rancid

[0169] 3.1.4 Flavor Parameters

[0170] This study comprehensively analyzed the taste characteristics of bitter wine soup samples using a combination of electronic tongue detection and human sensory evaluation. The results showed that the sample's flavor profile exhibited distinct structural features. Objective detection data showed that the saltiness intensity value was 4.45, significantly higher than its tastelessness point (-6), indicating a clear perception of saltiness; the umami value was 2.86, also significantly higher than the tastelessness point (0), showing a pronounced umami characteristic; and the richness value was 3.46, higher than the tastelessness point (0), suggesting that the sample possesses certain umami and saltiness, which together constitute the base of its overall flavor.

[0171] In the sourness dimension, there was a certain discrepancy between instrumental detection results and sensory evaluation. The electronic tongue measured a sourness value of -14.35, which is lower than the tasteless point (-13) based on tartaric acid, and from an instrumental perspective, it did not reach the significant sourness detection threshold. However, human sensory evaluation consistently confirmed that the sample had a distinct sourness characteristic. Analysis suggests that this discrepancy may be related to the insufficient sensitivity of the electronic tongue's sourness sensor to the actual acetic acid content in the sample. Therefore, based on a comprehensive assessment, "sourness" is considered one of its fundamental flavor attributes.

[0172] Among the other taste indicators, the astringency value was 0.95, which is higher than the tasteless point (0), and the bitterness value was 0.28, which is only slightly higher than the tasteless point (0), indicating that the astringency and bitterness were relatively weak. The two aftertaste indicators (Aftertaste-B was 0.36 and Aftertaste-A was -0.44) were close to or lower than the tasteless point, indicating that the sample had a weak persistence in the aftertaste stage.

[0173] In summary, the overall flavor characteristics of bitter wine soup are characterized by a base of sourness, supplemented by significantly detectable saltiness and umami, exhibiting clear flavor layers and a distinct structure (e.g., Figure 5 (As shown).

[0174] 3.2 Screening Results of Placebo Preparation Process for Bitter Wine Powder

[0175] 3.2.1 Screening results of turbidity agent excipients

[0176] The results are shown in Table 2. As the dosage of soybean lecithin increased from 1.60% to 3.40%, the turbidity of the solution system showed a significant upward trend, reaching 0.996, 1.342, and 1.863 respectively. This indicates that soybean lecithin has a significant enhancing effect on the turbidity of the solution. When the dosage was 2.50%, the turbidity of the system (1.342) was closest to that of the original bitter wine decoction (1.343). To further quantify the similarity between each formulation and the original solution, the multidimensional characteristic cosine similarity between each formulation and the original solution was calculated. The results showed that the similarity was highest at a dosage of 2.50%, reaching 99.99994%, indicating that this dosage most effectively simulated the turbidity of the original solution.

[0177] Table 2. Similarity results of different dosages of soybean lecithin

[0178]

[0179] 3.2.2 Screening Results of Toughener Excipients

[0180] The results are shown in Table 3. As the amount of gum arabic increased from 0.8% to 3.2%, the viscosity of the solution gradually increased, corresponding to 1.977, 2.23, and 2.53, respectively. When the amount was 1.6%, the viscosity of the system (2.23) was closest to that of the original bitter liquor solution (2.29). To further quantify the similarity between each formulation and the original solution, the multidimensional characteristic cosine similarity between each formulation and the original solution was calculated. The results showed that the similarity was highest at 1.6%, reaching 99.9985%, indicating that this addition amount most effectively simulated the viscosity of the original solution.

[0181] Table 3. Similarity results of different dosages of gum arabic

[0182]

[0183] 3.2.3 Screening results of foaming excipients

[0184] Table 4 shows that the foaming ability of the drug solution increased dose-dependently as the dosage of Tween 80 increased from 40 μL to 100 μL, with corresponding values ​​of 0.68, 1.05, 1.46, and 1.82, respectively. Similarity algorithms were used to quantify the similarity between each formulation and the stock solution. The results showed that the 100 μL dosage group had the highest similarity (99.9999%), while the 80 μL dosage group also had a similarity of 99.9996%, suggesting that higher dosages of Tween 80 more accurately simulate the foaming characteristics of the stock solution. However, considering the sensory requirements of the formulation, excessive Tween 80 dosage would introduce a noticeable odor. Therefore, considering both the matching degree of foaming characteristics and the influence of odor, 80 μL was ultimately determined to be the optimal dosage of Tween 80.

[0185] Table 4. Similarity results of different dosages of Tween 80

[0186]

[0187] 3.2.4 Results of screening excipients for odor and taste

[0188] Based on the sensory evaluations of volunteers, the similarity of each placebo prescription to the original drug in terms of smell and taste was compared. The results showed that prescription 2 was the most similar to the original drug in both smell and taste dimensions. Its smell similarity score was 8.00, and its taste similarity score was 8.08, with the least fluctuation in both data sets. Prescription 5 also performed well in smell similarity, scoring 7.29, but its taste similarity score was only 3.17, indicating a significant inconsistency between the smell and taste of this prescription. In contrast, prescription 3 performed the weakest in both evaluations, with smell and taste scores of only 3.13 and 3.00 respectively, indicating a low overall sensory match. Prescription 1 performed reasonably well in taste similarity, scoring 6.21, but its smell similarity score was relatively low at 4.33. Prescription 4 was at a moderate level, with smell and taste scores of 6.33 and 4.50 respectively. In conclusion, prescription 2 has the highest sensory match with the original drug and can be considered the optimal prescription for placebo smell and taste (e.g., ...). Figure 6 (As shown).

[0189] Table 5. Placebo Odor and Taste Prescription Screening

[0190] prescription Peppermint flavoring (mg) Orange flavoring (mg) Acetic acid (μL) Acetic acid powder (mg) Salt (mg) MSG (mg) Monk fruit glycosides (mg) 1 36 24 8 100 110 15 5 2 30 20 5 90 110 15 5 3 24 16 3 90 120 20 8 4 30 20 5 110 110 20 5 5 30 20 5 90 120 15 5

[0191] Table 6 Placebo Odor and Taste Prescription Screening

[0192] prescription odor smell 1 4.33±0.44 6.21±0.58 2 8.00±0.48 8.08±0.19 3 3.13±0.31 3.00±0.30 4 6.33±0.49 4.50±0.52 5 7.29±0.45 3.17±0.33

[0193] 3.2.5 Results of Color Accessory Screening

[0194] A systematic analysis of the placebo color formulations revealed significant differences in total color difference (ΔE) between the different formulations in solution and powder states. In the solution system, formulation 4 had the lowest ΔE value (0.70), indicating the best color matching, while formulation 1 had the highest ΔE value (3.29), showing the most significant deviation from the target color. Overall, the ΔE value decreased significantly from formulation 1 to formulation 4, then slightly increased again with formulation 6. In the powder system, formulation 4 had the lowest ΔE value (1.31), indicating the most stable color, while formulation 1 still had the highest ΔE value (2.77), showing a similar overall trend to the solution system. Except for formulation 1, the ΔE values ​​of all other formulations were lower in solution than in powder, indicating that the solution system generally had better color stability. Formulation 4 maintained a low ΔE level in both states, demonstrating the most balanced overall performance. In the color space dimension, the powder brightness of all formulations was significantly higher than that of the solution, and both exhibited a yellowish tint. However, the solution showed higher yellow saturation and a more pronounced green tendency. Formulation 4 showed the best color deviation ratio in both the a and b dimensions, with good color balance. In summary, formulation 4 demonstrated the best color stability and is suitable for subsequent placebo preparation (e.g., ...). Figure 7 (As shown).

[0195] Table 7 Color Accessory Prescription Table

[0196] Lemon yellow (μL) solution Sunset Yellow (μL) Prescription 1 60 10 Prescription 2 65 15 Prescription 3 70 15 Prescription 4 70 18 Prescription 5 72 18 Prescription 6 75 18

[0197] Table 8. Screening of prescriptions for bitter wine decoction powder and solution color.

[0198]

[0199] 3.2.6 Screening of filler excipients

[0200] Based on the angle of repose and similarity test results, the similarity between each placebo formulation and the original drug in terms of flowability and overall matching degree was compared. The results showed that formulation 1 (40% mannitol + 60% maltodextrin) was most similar to the original drug in both the angle of repose and similarity dimensions. Its angle of repose was 41°, which was closest to the original powder's 40°, indicating a high degree of consistency in flowability with the original drug, with a similarity of 99.9999%, the highest among all formulations. Formulation 2 (50% mannitol + 50% maltodextrin) had a similarity of 99.9924%, which was at a moderate level, but its angle of repose was 37°, showing a significant difference in flowability from the original powder. In contrast, formulation 3 (60% mannitol + 60% maltodextrin) performed the weakest in both evaluations, with an angle of repose of only 33°, showing the largest difference in flowability from the original powder, and also the lowest similarity at only 99.9913%. In summary, prescription 1 has the highest degree of matching with the original drug in terms of flowability and overall compatibility, and can be regarded as the optimal prescription in placebo powder form.

[0201] Table 9 Screening of fillers and excipients

[0202]

[0203] 3.3 Results of placebo similarity evaluation of bitter wine soup powder

[0204] 3.3.1 Volunteer Evaluation Results

[0205] The sensory evaluation results showed that the similarity scores of the bitter wine powder and the placebo were all above 8 points in all dimensions of the study, indicating that the two had a high degree of consistency in the sensory evaluation of volunteers. This can effectively reduce the possibility that subjects will identify the investigational drug due to sensory differences, thus meeting the basic requirements of a double-blind trial.

[0206] Further analysis showed that the sample scored higher in color, clarity, and taste in the solution state than in the powder state. The solution clarity (8.83 points) and solution color (8.75 points) showed the most significant similarity, indicating that the solution state sample had better visual consistency with the placebo. In contrast, the powder odor and solution taste scores were both 8.17 points, the lowest among all dimensions.

[0207] Regarding the stability of the ratings, the standard deviations for powder odor, solution taste, and solution clarity were all 0.39, reflecting a high degree of consistency in the volunteers' evaluations of these three indicators; while the standard deviation for powder color was 0.49, suggesting that different subjects had some differences in their judgments of the similarity of this indicator.

[0208] In summary, the bitter wine powder and the placebo exhibit good consistency in sensory characteristics, providing effective support for the successful implementation of double-blind trials and helping to reduce trial bias caused by sensory differences (such as...). Figure 8 (As shown).

[0209] 3.3.2 Instrument Evaluation Results

[0210] 3.3.2.1 Similarity of formulation characteristics

[0211] Table 10 evaluates the formulation characteristics of the drug and placebo from multiple dimensions, covering four core indicators: viscosity, turbidity, foaming ability, and angle of repose. Looking at individual indicators, the viscosities of the drug and placebo are 2.28 and 2.23, respectively, and the turbidities are 1.35 and 1.34, respectively, showing a high degree of overlap. While there is a slight difference in foaming ability (1.77 for the drug and 1.48 for the placebo), the overall levels remain similar. For the angle of repose, a core indicator of flowability, the drug is 39.33° and the placebo is 40.67°, a difference of only 1.34, indicating no significant difference in flowability. The overall matching degree is quantified by multidimensional feature similarity measurement. The results show a similarity of 99.9957%, indicating that the prepared placebo is highly similar to the drug in formulation characteristics, effectively mimicking the physicochemical characteristics of the drug and meeting the requirements of a double-blind clinical trial.

[0212] Table 10. Similarity of Formulation Characteristics

[0213]

[0214] 3.3.2.2 Odor Similarity

[0215] Principal component analysis of flavor characteristics was performed on each group of samples, and the results are as follows: Figure 9 As shown, the cumulative contribution rate of PC1 and PC2 is 82.39% (PC1: 65.71%, PC2: 16.68%), which can effectively represent most of the original flavor information of the sample. From the PCA score plot, it can be seen that the distribution of KJT raw powder and the tasteless group is very similar in the PC1 dimension, while the odorless and tasteless excipient group is significantly different from KJT raw powder in the PC1 direction, suggesting that the odor-simulating excipient is the main contributor to the flavor of KJT raw powder. The distribution of KJT placebo and KJT raw powder in the PC1 direction highly overlaps, indicating that its flavor simulation prescription has good similarity to the simulated object; at the same time, the blank group and the odorless excipient group highly overlap in both PC1 and PC2 dimensions, further confirming the core role of excipients in flavor simulation. In summary, KJT raw powder and KJT placebo have a high flavor similarity, which is consistent with the conclusion of human evaluation, indicating that the simulated prescription can effectively reproduce the flavor characteristics of KJT raw powder.

[0216] 3.3.3 Flavor Similarity

[0217] Principal component analysis (PCA) of flavor characteristics was performed on each group of samples, and the results are as follows: Figure 10 As shown, the cumulative contribution rate of PC1 and PC2 is 84.25% (PC1: 75.27%, PC2: 8.98%), which can effectively represent most of the original flavor information of the samples. From the PCA score map, it can be seen that KJT solution is distributed in the upper left area of ​​the figure, while KJT placebo is distributed in the middle-left area. The coordinate intervals of the two in the PC1 dimension are relatively close, and the distance between the groups is much smaller than the distance between them and other excipient groups and blank groups, indicating that the flavor characteristics of the two are highly similar. The blank excipient is completely independently distributed in the lower left area of ​​the figure, and it is significantly separated from all other groups in the PC1 and PC2 dimensions, with extremely obvious feature differences. The tasteless excipient, odorless and tasteless excipient and blank group are highly clustered in the right side of the figure, and the sample points of the three groups almost completely overlap, suggesting that their flavor characteristics are highly similar. In summary, KJT solution and KJT placebo have certain similarities in flavor characteristics, while the blank excipient has the most significant feature differences from all other groups. This result can provide clear data support for subsequent placebo preparation.

[0218] 3.4.4 Color Similarity

[0219] Table 11 evaluates the color similarity between the original Kujiu Tang powder and the placebo in both powder and solution forms. The results show that the L*, a*, and b* color parameters of both are highly similar in both states. The color difference ΔE of the powder group is 1.31, and that of the solution group is 0.70, both of which are below the color difference threshold that the human eye can distinguish. This indicates that the two have good color similarity, which can effectively reduce the risk of subjects judging grouping by color, meet the masking requirements of double-blind trials, and the color matching degree in the solution state is better.

[0220] Table 11. Evaluation results of color similarity between the original drug and placebo powders and solutions of Kujiu Decoction.

[0221]

[0222] 4. Summary and Discussion

[0223] This chapter addresses the core bottleneck of insufficient placebo similarity in traditional Chinese medicine (TCM), which limits the scientific evaluation of clinical trials. It focuses on the preparation and evaluation of a placebo for Kujiu Tang powder, conducting a systematic study and constructing a "powder-solution" dual-phase synergistic generic research strategy to specifically solve the key problems of low placebo simulation and difficulty in meeting the requirements of blinded trials. By systematically characterizing the physical properties, sensory attributes, and solution characteristics of the original drug in powder and reconstituted states, a highly realistic placebo formulation system based on precise compatibility of functional excipients was constructed. The results show that the prepared placebo exhibits high consistency with the original drug in key evaluation indicators such as color, odor, taste, clarity, flowability, viscosity, and foaming properties. This was verified by both volunteer sensory evaluation and objective instrument evaluation. The placebo effectively avoids subjective judgment bias between subjects and researchers, meeting the implementation standards of blinded clinical trials. This study not only provides an effective placebo control scheme for the standardized clinical evaluation of Kujiu Tang but also offers a referable technical path and evaluation method for the development of placebos for other complex TCM preparations, promoting the scientific rigor and reliability of TCM clinical trial design.

[0224] Example 3: Preparation method of bitter wine soup powder

[0225] Take 4g of Pinellia ternata, 12ml of rice vinegar, and 35ml of egg white;

[0226] Take the prescribed amount of Pinellia ternata, soak for 1 hour, add rice vinegar and soak for 18 hours, percolate at a rate of 2.0 mL / (min・kg), collect the percolate and mix it with the prescribed amount of egg white, filter, and freeze-dry to obtain freeze-dried powder. The freeze-drying conditions are: pre-freezing at -80℃ for 6 hours, then drying at a vacuum of 20 Pa and a cold trap temperature of -50℃ for 24 hours. Add 2.3% flavoring agent (1.2 parts peppermint flavoring, 0.8 parts orange flavoring, and 0.3 parts monk fruit glycoside).

Claims

1. A placebo of a bitter wine powder, characterized in that: It is prepared from the following raw materials in the following weight ratio: 2,080 to 3,120 parts filler, 60 to 90 parts turbidity agent, 0.0704 to 0.1056 parts pigment, 40 to 60 parts thickener, 107.2 to 160.8 parts flavoring agent, 44.2 to 66.3 parts odor agent, and 67.84 to 101.76 parts foaming agent; The filler includes one or a mixture of mannitol and maltodextrin; the turbidity agent includes soybean lecithin; the pigment includes one or a mixture of tartrazine and sunset yellow; the thickener includes gum arabic; the flavoring agent includes one or a mixture of salt, monosodium glutamate, acetic acid powder, and mogroside; the aroma agent includes one or a mixture of acetic acid, peppermint flavoring, and orange flavoring; and the foaming agent includes Tween 80.

2. The placebo of the bitter wine powder according to claim 1, characterized in that: It is prepared from the following raw materials in the following weight ratio: 2600 parts filler, 75 parts turbidity agent, 0.088 parts pigment, 50 parts thickener, 134 parts flavoring agent, 55.25 parts odor agent, and 84.8 parts foaming agent; The filler includes a mixture of mannitol and maltodextrin; the turbidity agent includes soybean lecithin; the pigment includes a mixture of tartrazine and sunset yellow; the thickener includes gum arabic; the flavoring agent includes a mixture of salt, monosodium glutamate, acetic acid powder, and mogroside; the aroma agent includes a mixture of acetic acid, peppermint flavoring, and orange flavoring; and the foaming agent includes Tween 80.

3. The placebo of the bitter wine powder according to claim 2, characterized in that: It is prepared from the following raw materials in the following weight ratio: Tween 84.8 parts, Mannitol 1040.3448 parts, Maltodextrin 1560.5172 parts, Salt 54 mg parts, Acetic acid powder 66 parts, Monosodium glutamate 9 mg parts, Lemon yellow pigment 0.07 parts, Sunset yellow pigment 0.018 parts, Soy lecithin 75 parts, Gum arabic 50 parts, Peppermint flavor 30 parts, Orange flavor 20 parts, Monk fruit glycoside 5 parts, Acetic acid 5.25 parts.

4. The placebo of the bitter wine powder according to any one of claims 1-3, characterized in that: The preparation method of the aforementioned bitter wine powder is as follows: a. Weigh the ingredients according to the specified weight ratio: for every 4g of Pinellia ternata, use 12ml of rice vinegar and 35ml of egg white; b. Add rice vinegar to the prepared Pinellia ternata, soak it first, then steep it, and then use the percolation method to percolate and collect the percolate; c. Mix the percolate with egg white, filter, freeze dry to obtain freeze-dried powder; then add 1.8%-2.4% of flavoring agent to the total amount of freeze-dried powder.

5. The placebo of the bitter wine powder according to claim 4, characterized in that: In the preparation method of the bitter wine powder, Step a describes grinding Pinellia ternata into coarse powder, coarse powder, and medium powder; The soaking time in step b is 0.5-2.0 h; the immersion time is 6-36 h; and the percolation flow rate is 1 mL / min·kg-3 mL / min·kg. In step c, a freeze-drying protectant is added, which may include mannitol, sucrose, glucose, or trehalose; the amount of freeze-drying protectant used is 2.5-10%. The freeze-drying conditions described in step c are: pre-freezing at -80℃ for 6 h, followed by drying at a vacuum of 20 Pa and a cold trap temperature of -50℃ for 24 h. The flavoring agent mentioned in step c is a combination of peppermint flavoring, orange flavoring, and mogroside, with the following weight ratio: Peppermint flavoring 0.6-1.4 parts, orange flavoring 0.6-1.4 parts, monk fruit extract 0.3-0.4 parts; Preferably, the flavoring agent is a combination of peppermint flavoring and orange flavoring, with the following weight ratio: 1.2 parts peppermint flavoring, 0.8 parts orange flavoring, and 0.3 parts monk fruit glycosides.

6. The placebo of the bitter wine powder according to claim 5, characterized in that: In the preparation method of the bitter wine powder, The prepared Pinellia ternata powder described in step a is pulverized to medium powder; The soaking time in step b is 1 hour; the immersion time is 18 hours; and the percolation flow rate is 2.0 mL / min·kg. In step c, a freeze-drying protectant is added, which is mannitol; the amount of freeze-drying protectant is 5%.

7. A method for preparing a placebo of the bitter wine powder according to any one of claims 1-5, characterized in that: It includes the following steps: Weigh the filler and solution turbidity agent, place them in a mortar and grind them evenly; add the pigment, flavor excipient and odor excipient solution, and continue mixing; finally add the thickener and foaming agent, grind evenly, so that all the drug passes through a No. 5 sieve (80 mesh, pore size 180 micrometers), and at least 95% of the powder can pass through a No. 6 sieve, and the drug is obtained.

8. The method for preparing the placebo of the bitter wine powder according to claim 6, characterized in that: It includes the following steps: Take the prescribed amount of Tween 80, add it to a mortar and grind it evenly. Add the prescribed amounts of mannitol, salt, acetic acid, monosodium glutamate, and mogroside and grind evenly. Then add the prescribed amounts of lemon yellow pigment and sunset yellow pigment to the mortar and grind evenly. Add the prescribed amounts of soybean lecithin, gum arabic, and maltodextrin to the mortar and grind evenly. Dry at 105℃ for 30 minutes, let cool, add the prescribed amounts of acetic acid, peppermint flavor, and orange flavor to the mortar and grind evenly. Pass through a No. 6 sieve to obtain placebo powder.

9. A method for evaluating the placebo simulation of a bitter wine powder, characterized in that: It employs headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS), electronic tongue, colorimeter, powder property analyzer, and viscometer to perform multi-dimensional characteristic analysis of the "shape, color, aroma, and properties" of bitter wine soup powder.

10. The method for evaluating the placebo simulation of the bitter wine powder according to claim 8, characterized in that: It includes the following steps: a. Volunteer evaluation was conducted using a Likert scale. Evaluation indicators included: powder color, powder odor; solution color, solution odor, clarity, and solution taste. The judgment criteria were: similarity threshold: a score > 6 indicates high similarity; PyCharm 2022.2 software was used to calculate the cosine of the angle between the two sides. The closer the cosine value is to 1, the higher the similarity. b. Instrument evaluation: including Similarity of formulation characteristics: Texture characteristics include angle of repose, dispersibility, clarity, viscosity, and foaming properties; Odor similarity: The odor of samples in powder form is evaluated and distinguished using an electronic nose; Taste similarity: The taste of samples in solution is evaluated and distinguished using an electronic tongue; Color similarity: The color difference meter similarity evaluation method was used; c. Compare the placebo with the bitter wine powder standard to obtain the simulation results.

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