Preparation method of bitter wine soup powder for treating radiation-induced oral mucositis

Abstract

The invention provides a preparation method of bitter wine soup powder for treating radiation-induced oral mucositis. The preparation method comprises the following steps: a, weighing the following raw materials in parts by weight: rhizoma pinelliae preparata, egg white and rice vinegar; b, adding rice vinegar into the rhizoma pinelliae preparata, soaking, then dipping, percolating by adopting a percolation method, and collecting percolate; and c, uniformly mixing the percolate with egg white, filtering, and freeze-drying to obtain freeze-dried powder. Compared with a traditional method, the preparation method has the advantages that effective substances in pinellia ternate are fully dissolved out, and the transfer rate is higher. The preparation method is simpler, and the problems of uncontrollable egg white industrial heating quality, easy solidification and the like are avoided. Compared with a liquid form in an ancient method, the bitter wine soup powder prepared by the method disclosed by the invention is better in taste, free from fishy odor and easy to store. Compared with a bitter wine soup obtained by an ancient method, the bitter wine soup powder disclosed by the invention has a better treatment effect on RTOM.

Description

Technical Field

[0001] This invention relates to a method for preparing a bitter wine powder for treating radiation-induced oral mucositis, belonging to the field of pharmaceuticals. Background Technology

[0002] Radiation-induced oral mucositis (RTOM) is the most common and serious dose-limiting toxicity in radiotherapy for head and neck tumors, with an incidence rate exceeding 80%. Its clinical features include severe pain, dysphagia, and mucosal ulceration, significantly impacting patients' quality of life and often leading to radiotherapy interruption, thus affecting tumor control. Currently, both domestic and international clinical practice agree that there is still a lack of radical and specific drugs for this disease. Existing treatments mainly focus on symptom relief and wound management, and these are generally insufficient. While local analgesics (such as mouthwashes containing lidocaine or morphine) can temporarily relieve pain, they only provide symptomatic relief and cannot fundamentally eliminate inflammation or promote tissue repair; their effects are short-lived and require frequent use. Topical cell growth factor preparations (such as epidermal growth factor gel) can directly stimulate wound regeneration, but their efficacy varies among individuals, and their high cost limits their widespread application. Meanwhile, due to considerations of oral microecological protection and drug resistance control, the prophylactic use of antibiotics is strictly limited. Systemic glucocorticoids have been significantly reduced in clinical treatment due to potential adverse reactions. Existing medications generally face multiple challenges, including limited efficacy (treating symptoms but not the underlying cause), side effects, and financial burden. These issues severely impact patients' treatment and long-term quality of life. Therefore, there is an urgent clinical need to explore novel treatment strategies that can effectively relieve symptoms, promote mucosal repair, and are easy to use.

[0003] Bitter Wine Decoction, originating from the *Treatise on Cold Damage*, is a classic Chinese medicine formula for treating sore throat and ulcers. It demonstrates 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 clear heat, moisten dryness, and promote tissue regeneration. The three herbs work together to promote tissue regeneration, clear heat, and resolve phlegm, effectively relieving mucosal redness, swelling, and pain, and accelerating ulcer healing.

[0004] Huang Yan et al., Clinical observation on the prevention of radiation-induced oral mucositis with bitter wine decoction, Chinese Journal of Emergency Traditional Chinese Medicine, November 2021, Vol. 30, No. 11, published the efficacy of bitter wine decoction in preventing radiation-induced oral mucositis (RTOM). This literature also disclosed that the difficulty in preparing bitter wine decoction lies in its decoction process. According to the annotations, egg white coagulation often occurs during decoction. Therefore, improvements were made to the decoction method without violating the original formula. Meng Xufang et al., Clinical observation on the treatment of radiation-induced pharyngitis with modified bitter wine decoction, Guangming Journal of Traditional Chinese Medicine, April 2018, Vol. 33, No. 7, published the following process: First, take 10 g of raw Pinellia ternata and put it in 1000 ml of water, decoct for 1.5 hours, and when about 150 ml of liquid remains, remove the dregs, add 50 ml of bitter wine, boil three times, let cool, and then add one egg (yolk removed) and stir well. Zhang Jin et al., in their article "Experience in Treating Post-Radiation Oral Ulcers with Bitter Wine Decoction," published in the September 2015 issue of the Journal of Practical Traditional Chinese Medicine (Vol. 31, No. 9), disclosed a slight modification to the decoction method. First, the Pinellia ternata was decocted and the dregs removed. While the Pinellia ternata decoction was still lukewarm, egg white was added while stirring constantly to prevent coagulation. Finally, bitter wine was added. During the decoction process, a large amount of acetic acid (boiling point approximately 118℃) evaporated, reducing the acidity of the decoction. This not only weakened its "healing" effect but also altered the properties of the solvent, potentially affecting the dissolution type and content of the effective components of Pinellia ternata. If the residual heat of the vinegar was not dissipated or the mixture was improperly mixed, irreversible flocculent or lumpy precipitates could form. These precipitates would encapsulate and adsorb the effective components of the drug, severely affecting their release.

[0005] CN202010719207.X, Invention Title: A Bitter Wine Decoction and Its Preparation Method, discloses a bitter wine decoction and its preparation method, wherein the bitter wine decoction is prepared from Pinellia ternata, bitter wine (rice vinegar) and chicken eggs; the preparation method of the bitter wine decoction includes: preparing the raw material bitter wine decoction and making the bitter wine decoction into the target dosage form - ice. The preparation process includes the following steps: Step 1: Select and clean Pinellia ternata to obtain clean Chinese medicinal materials. Slice Pinellia ternata and grind it into powder. Step 2: Pour the egg white from the eggshell into a beaker for later use. Add Pinellia ternata to the eggshell. Use a pipette to add egg white and an appropriate amount of rice vinegar to the eggshell. Mix the three together, heat evenly, boil three times, remove the dregs, and cool to room temperature to obtain the raw material of bitter wine soup. Step 3: Dilute the prepared raw material of bitter wine soup with ultrapure water to the required drug concentration, mix evenly, wash and dry the mold, pour the prepared medicine into the mold until it reaches the surface of the mold, and freeze. Step 4: After freezing and solidification, scrape off the overflow with a knife, refrozen, and after storage, remove the medicine from the mold, package it, and store it in the freezer. The process disclosed in this document relies on the operator's personal experience, and it is difficult to accurately control the heat and the denaturation state of the egg white. The steps are cumbersome and cannot meet the requirements of modern standardized drug production, large-scale preparation and long-term storage. It has obvious shortcomings in terms of clinical convenience and application promotion, and cannot meet the needs of modern pharmaceutical industrialization.

[0006] The common defects of the aforementioned traditional and modern preparation methods, including poor stability, uncontrollable quality, and inconvenience of use, seriously hinder the large-scale production of bitter wine soup and the evaluation of its quality controllability.

[0007] In recent years, the country has vigorously promoted the development of classic prescriptions, and the role of traditional Chinese medicine in supportive treatment of tumor radiotherapy has received increasing attention. This has brought important opportunities for the modernization research of Kujiu Decoction. However, the prescription still faces challenges such as insufficient verification of key information and inconvenience in using traditional dosage forms. Summary of the Invention

[0008] This invention provides a method for preparing a bitter wine decoction powder for treating radiation-induced oral mucositis.

[0009] This invention provides a method for preparing a bitter wine decoction powder for treating radiation-induced oral mucositis, comprising the following steps:

[0010] a. Weigh the ingredients according to the specified weight ratio: Pinellia ternata, egg white, and rice vinegar;

[0011] 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;

[0012] c. Mix the percolate with egg white, filter, and freeze-dry to obtain freeze-dried powder.

[0013] The amount of raw materials used in step a is as follows: for every 4g of Pinellia ternata, there are 12ml of rice vinegar and 35ml of egg white.

[0014] In step a, the Pinellia ternata is pulverized into the coarsest powder, coarse powder, and medium powder.

[0015] 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.

[0016] 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%.

[0017] The freeze-drying conditions described in step c are: pre-freezing at -80℃ for 6 hours, followed by drying at a vacuum of 20 Pa and a cold trap temperature of -50℃ for 24 hours.

[0018] Preferably,

[0019] The prepared Pinellia ternata powder described in step a is pulverized to medium powder;

[0020] 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.

[0021] In step c, a freeze-drying protectant is added, which is mannitol; the amount of freeze-drying protectant is 5%.

[0022] In step c, 1.8%-2.4% of the total freeze-dried powder is added as a flavoring agent.

[0023] The flavoring agent is a composition of peppermint flavoring, orange flavoring, and mogroside, with the following weight ratio:

[0024] Peppermint flavor 0.6-1.4 parts, orange flavor 0.6-1.4 parts, monk fruit glycosides 0.3-0.4 parts.

[0025] Preferably, the flavoring agent is a combination of peppermint flavoring and orange flavoring, with the following weight ratio:

[0026] 1.2 parts peppermint flavoring, 0.8 parts orange flavoring, and 0.3 parts monk fruit glycosides.

[0027] This invention systematically evaluates the therapeutic effects of different preparation processes of Kujiu Decoction on a rat model of radiation-induced oral mucositis (RTOM) from a pharmacodynamic perspective. It focuses on verifying the feasibility of percolation as a substitute for traditional ancient methods and modern decoction methods in terms of efficacy, thereby screening out a process scheme with better efficacy and suitable for modern production. This provides key experimental evidence for the standardization and modernization of the process of this classic prescription. The powder prepared by this invention has stable quality, is convenient to take, and has a good taste.

[0028] The beneficial effects of this invention are:

[0029] 1. Compared with traditional methods, the effective substances in Pinellia ternata are fully dissolved, resulting in a higher transfer rate.

[0030] 2. The preparation method is simpler, avoiding problems such as uncontrollable quality and easy coagulation caused by industrial heating of egg whites.

[0031] 3. Compared to the traditional liquid form, the bitter wine powder prepared by the method of this invention has a better taste, no fishy smell, and is easier to store.

[0032] 4. Compared with the bitter wine decoction obtained by the ancient method, the bitter wine decoction powder of this invention has a better therapeutic effect on RTOM. Attached Figure Description

[0033] Figure 1 Graph showing cumulative weight change in rats;

[0034] Figure 2 Ulcer condition at different time points in each group (A: Ulcer manifestations after drug administration at different time points in each group; B: Ulcer scores after drug administration in each group at different time points).

[0035] Figure 3 Tongue histopathological sections and mucosal thickness images (A: Histopathological sections of the dorsal and ventral sides of the tongue in each group of rats; B: Quantitative analysis of the thickness of the dorsal tongue mucosa in each group; C: Quantitative analysis of the thickness of the ventral tongue mucosa in each group).

[0036] Figure 4 The effect of different particle sizes on extraction (A: The effect of different particle sizes on extract yield, total alkaloids and comprehensive evaluation indicators; B: The effect of different particle sizes on extract yield and total alkaloids).

[0037] Figure 5 Effects of different soaking times on extraction (A: Effects of different soaking times on extract yield, total alkaloids and comprehensive evaluation indicators; B: Effects of different soaking times on extract yield and total alkaloids).

[0038] Figure 6 Effects of different percolation rates on extraction (A: Effects of different percolation rates on extract yield, total alkaloids and comprehensive evaluation indicators; B: Effects of different percolation rates on extract yield and total alkaloids).

[0039] Figure 7 The effects of different freeze-drying protectants on the freeze-drying process (A: Violin plots of different freeze-drying protectants on appearance evaluation, freeze-dried powder yield, and lysozyme content; B: Significance plots of different freeze-drying protectants on appearance evaluation, freeze-dried powder yield, and lysozyme content).

[0040] Figure 8 The effect of different amounts of mannitol on the freeze-drying process;

[0041] Figure 9 Sensory evaluation of different flavoring formulations (wherein, (A) sensory evaluation of each flavoring formulation; (B) odor sensory score; (C) taste sensory score; (D) aftertaste sensory score; (E) overall sensory score). Detailed Implementation

[0042] Example 1: Preparation method of bitter wine soup powder

[0043] Take 4g of Pinellia ternata, 12ml of rice vinegar, and 35ml of egg white;

[0044] 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).

[0045] Example 2: Screening Test of the Efficacy of Different Preparation Processes of Bitter Wine Decoction

[0046] 1. Experimental Materials and Instruments

[0047] 1.1 Laboratory Animals

[0048] 75 male SPF-grade SD rats, 8 weeks old and weighing 180-200g, were housed in an SPF-grade animal room with constant temperature (22±2℃), constant humidity (55±5%), and a 12-hour light-dark cycle, with free access to food and water.

[0049] 1.2 Experimental Materials and Reagents

[0050] Pinellia ternata, rice vinegar (food grade, total acidity ≥5.0g / mL) / fresh eggs; sodium pentobarbital, isoflurane, 4% paraformaldehyde fixative, HE staining reagent, 0.9% sterile physiological saline, tissue scissors, forceps, animal-specific mouth opener, sterile cotton swabs.

[0051] 1.3 Instruments and Equipment

[0052] X-RAY Small Animal Precision Irradiation System (Brand / Model: [ ]), Isoflurane Evaporation Canister and Induction Box (Brand / Model: [ ]), Small Animal Ventilator (Brand / Model: [ ]), Custom Lead Shielding Box (Lead Plate Thickness 5mm), 33℃ Constant Temperature Incubator Paraffin Embedding Machine (Brand / Model: [ ]), Tissue Slicer (Brand / Model: [ ]), Slice Spreader (Brand / Model: [ ]), Oven (Brand / Model: [ ]), Optical Microscope and Image Acquisition System (Brand / Model: [ ]), Electronic Balance (Brand / Model: [ ]), Timer.

[0053] 2. Experiment Content

[0054] 2.1 Methods for establishing RTOM rat model

[0055] This invention successfully established a rat model of radiation-induced oral mucositis (RTOM). The modeling process was conducted according to standard procedures to ensure the consistency and reliability of the model. After acclimatization in an SPF environment for 3 days, all animals except the blank control group were anesthetized by intraperitoneal injection of 3% sodium pentobarbital. After anesthesia, the rats were fixed in a supine position in a specially designed lead-shielded box, with the head and neck exposed to the irradiation area, while the trunk and limbs, and other non-target tissues, were shielded with 5 mm thick lead plates to achieve localized and precise irradiation.

[0056] This experiment employed a single high-dose localized X-ray irradiation. Rats, along with their lead boxes, were placed in a small animal precision irradiation system. The irradiation parameters were set as follows: voltage 160 kV, current 20 mA, total absorbed dose 25 Gy, dose rate 1.903 Gy / min, and system dose uniformity controlled within ±3%. Immediately after irradiation, the rats were transferred to a 33℃ constant-temperature incubator until fully recovered (approximately 2 hours) to prevent hypothermia. For the first 24 hours post-surgery, 1 mL of 0.9% sterile saline was subcutaneously injected every 6 hours to maintain fluid balance and physiological homeostasis.

[0057] 2.2 Dosing regimen and drug preparation

[0058] 2.2.1 Dosing regimen

[0059] On day 5 post-irradiation, once typical symptoms of RTOM had fully appeared, drug intervention was initiated. Rats were induced and maintained under light anesthesia by isoflurane inhalation, and were fixed with an oral gag to fully expose the dorsum of the tongue mucosa. The operator used a sterile cotton swab to apply a measured amount of drug solution (treatment group) or an equal volume of sterile saline (blank control group and model control group) evenly to the ulcers on the tongue surface and the surrounding congested and edematous mucosa. Each operation lasted about 3 minutes to ensure sufficient contact between the drug and the lesion.

[0060] The medication was administered once every two days for a total of six times to assess the cumulative therapeutic effect. Patients were instructed to abstain from food and water for one hour after each administration, followed by resumption of free food and water intake to ensure adequate local drug absorption.

[0061] 2.2.2 Drug preparation method

[0062] Traditional method: Take the prescribed amount of Pinellia ternata, rice vinegar and egg white, put them together into an eggshell, heat with an alcohol lamp until boiling, then remove the heat source, repeat the heating-stopping operation 3 times, then filter and freeze dry to obtain freeze-dried powder.

[0063] Decoction method: Take the prescribed amount of Pinellia ternata, add the prescribed amount of rice vinegar and decoct for 1 hour. After the decoction cools, add the prescribed amount of egg white and mix thoroughly. Filter and freeze-dry to obtain freeze-dried powder.

[0064] Percolation method: Take the prescribed amount of Pinellia ternata, soak it in rice vinegar 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 it and freeze dry to obtain freeze-dried powder.

[0065] 2.3 Experimental Grouping

[0066] Based on the optimal dose determined in the previous dose screening experiment, this experiment compared the efficacy of different preparation processes. Seventy-five rats were randomly divided into five groups of 15 each (n=15).

[0067] (1) Blank control group: No radiation was received and the animals were fed normally.

[0068] (2) Model control group: received radiation treatment but no drugs were given, only an equal volume of sterile saline was applied.

[0069] (3) Process A: Receive radiation and apply medicine prepared by ancient methods.

[0070] (4) Process B: The drug prepared by percolation is subjected to radiation and then coated.

[0071] (5) Process C group: The drug prepared by decoction is subjected to radiation and then coated.

[0072] 2.4 Observation Indicators and Detection Methods

[0073] 2.4.1 General condition and weight of rats

[0074] Observe the rats' mental state, activity, food intake, and fur condition daily. Weigh and record the rats on days 1, 3, 5, 7, 9, and 11 after irradiation.

[0075] 2.4.2 RTOM Severity Assessment

[0076] This invention employs a standardized macroscopic scoring system, in which three researchers, uniformly trained and unaware of their experimental group assignments, independently and blindly assess the severity of tongue mucosal damage. The scoring is based on macroscopic morphological changes such as mucosal erythema, bleeding, epithelial ulceration, and abscesses, using a modified 0-5 point scale.

[0077] 0 points: The mucosa has normal color, a smooth and intact surface, and no visible damage.

[0078] 1 point: Mild erythema is visible on the mucosa, but there is no epithelial damage or ulceration.

[0079] 2 points: Severe erythema appears, accompanied by pinpoint erosions or scattered bleeding points.

[0080] 3 points: Focal epithelial desquamation occurs, forming a clear ulcer, but its cumulative area accounts for less than 25% of the observed area.

[0081] 4 points: Ulcers merge or enlarge, with the cumulative area accounting for 25% to 50% of the observed area.

[0082] 5 points: Extensive and severe ulcers appear, with a cumulative area greater than 50%, or accompanied by deep tissue necrosis.

[0083] The arithmetic mean of the independent scores from the three evaluators was used as the final score for each rat at that time point. Scoring was performed before each administration (days 1, 3, 5, 7, 9, and 11) to dynamically monitor mucosal damage and treatment efficacy.

[0084] 2.4.3 Sample Collection and Processing

[0085] 2.4.3.1 Sample Collection

[0086] Twenty-four hours after the last drug administration intervention, all rats were euthanized. Rats were deeply anesthetized via intraperitoneal injection of sodium pentobarbital, and cervical dislocation was performed after anesthesia was lost. The tongue tissue was then rapidly dissected and rinsed in pre-cooled 0.9% sterile saline to remove oral secretions and uneaten food. The tissue collection process was performed on ice to minimize tissue autolysis.

[0087] 2.4.3.2 Preparation of Paraffin Sections

[0088] Fresh tongue tissue was immediately immersed in sufficient 4% paraformaldehyde phosphate buffer (pH 7.4) and fixed at 4°C for 24–48 h. After fixation, the tissue was rinsed with running water and then subjected to a series of ethanol dehydration processes (70%, 80%, 90%, 95%, 100% I, 100% II), xylene clearing (I, II), and paraffin infiltration and embedding (melting point 56–58°C) to prepare paraffin blocks. Sections were continuously prepared using a rotary microtome to a thickness of 5 μm. The sections were horizontally spread on poly-L-lysine pretreated slides and dried overnight in a 60°C oven for later use.

[0089] 2.4.3.3 Hematoxylin-eosin (H&E) staining

[0090] Sections were dewaxed with xylene, hydrated with graded ethanol, and then stained with Hematoxylin and Escherichia coli (H&E). The procedure was as follows: Harris hematoxylin staining of the nuclei for 5–8 min, rinsing with running water, differentiation with 1% hydrochloric acid-ethanol for a few seconds, and blueing with running water for 15 min; 0.5% eosin staining of the cytoplasm for 1–3 min. After staining, the sections were graded dehydrated, cleared with xylene, mounted on neutral resin, and air-dried for later use.

[0091] 2.4.3.4 Pathological observation and evaluation

[0092] (1) H&E sections were observed by pathology professionals in a single-blind manner, with standardized microscope parameters and digital acquisition of typical fields of view. The assessment included: integrity of mucosal epithelium (continuity, thickness, keratinization and exfoliation), inflammatory response (density and distribution of inflammatory cells infiltrating the lamina propria and muscularis propria), and dynamics of tissue damage and repair (edema, hyperemia, necrosis and granulation tissue morphology).

[0093] (2) Thickness of the tongue dorsal and ventral mucosa: The thickness of the tongue dorsal and ventral mucosa was quantitatively analyzed using Image-Pro Plus 9.0 software.

[0094] 3. Experimental Results

[0095] 3.1 Effects on the general condition and body weight of rats

[0096] The cumulative weight loss in rats showed that the control group exhibited a natural weight gain trend during the experiment, with a cumulative weight loss of 56.18 g, significantly higher than other groups. The radiation-only group showed a highly significant weight loss, with a cumulative weight loss of -89.72 g, suggesting that this model leads to severe weight loss in rats. After interventions using percolation, traditional methods, and decoction, the weight loss in rats was significantly alleviated. The percolation group showed the smallest cumulative weight loss among the three intervention groups (-47.63 g), followed by the decoction group (-57.32 g) and the traditional method group (-66.35 g). Statistical analysis showed highly significant differences between the control group and all other groups, and between the radiation group and each intervention group. Statistically, statistical differences were also observed between the percolation group and the traditional method group, and between the decoction group and the intervention group. These results indicate that all three intervention methods effectively alleviate radiation-induced weight loss, with percolation showing a relatively more significant protective effect (see...). Figure 1 ).

[0097] 3.2 Effect on macroscopic damage score of RTOM in rats

[0098] This invention establishes a rat model of radiation-induced oral mucositis and systematically evaluates the intervention effects of different drug preparation processes (percolation, traditional method, and decoction). Rats were randomly divided into four groups of six after radiotherapy, and administered the drug six times consecutively. Mucosal damage scores were recorded before each administration. Results showed significant differences in the degree of mucosal damage among the different process groups. The percolation group maintained a low score at all time points, slowly increasing from 0.17 points at the first administration to 1.83 points at the fourth administration, and then gradually decreasing to 1.17 points at the sixth administration. The tongue surface showed only mild erythema without obvious ulceration, indicating that this process has good mucosal protection and repair effects. The scoring trends of the traditional group and the decoction group were similar, with both showing a peak in damage during the middle of the experiment (third to fourth administration), reaching scores of 2.42 and 2.33 respectively. The tongue surface exhibited characteristics of "severe erythema with punctate erosions," subsequently gradually recovering, decreasing to 1.5 and 1.33 respectively before the sixth administration. This suggests that while both methods could control damage progression, the repair process was relatively slow. The radiation group showed the most severe damage, with the score continuously rising from 1.00 at the first administration to 2.67 at the fifth administration. Although it slightly decreased to 2.50 at the sixth administration, it remained at a high level, corresponding to focal ulceration on the tongue surface, with no obvious repair trend observed.

[0099] In summary, the results of this invention demonstrate that, in a radiation-induced oral mucositis model, the percolation process exhibits significant advantages in reducing mucosal damage and promoting early repair. Traditional and decoction processes also provide some protection, while the radiation process failed to effectively control damage progression. Therefore, in the preparation of drugs for the prevention and treatment of radiation-induced oral mucositis, the percolation process should be given priority, and further process optimization research should be conducted (see...). Figure 2 ).

[0100] 3.3 Pathological observation results of tongue tissue

[0101] As shown in Figure 3A, the pathological histological observation of the dorsum and ventral mucosa of the tongue in the model group exhibited typical characteristics of acute radiation-induced oral mucositis, with severe damage and discontinuity of the epithelial structure, extensive infiltration of inflammatory cells in the lamina propria, and significant widening and edema in the submucosal interstitial space. In the traditional intervention group, although the epithelial layer showed some repair, some defects remained. Inflammatory cell infiltration in the lamina propria was less severe than in the model group, and submucosal edema was alleviated, indicating that this intervention had a certain repair effect on mucosal damage, but the effect was limited. In the decoction intervention group, the epithelial layer continuity was basically restored and the thickness was close to normal. Inflammatory cells in the lamina propria were significantly reduced, and submucosal edema was basically subsided, indicating that this intervention could effectively promote epithelial repair and inhibit the inflammatory response, with a repair effect superior to the traditional group. In the percolation intervention group, the epithelial layer was intact, of uniform thickness, and with regular cell arrangement. Inflammatory cell infiltration in the lamina propria was basically eliminated, and there was no significant submucosal edema, showing that it had the best repair effect on radiation-induced mucositis, maximally restoring the normal morphology and structure of the mucosa.

[0102] according to Figure 3Analysis of the tongue dorsum and ventral mucosa thickness data in AC showed that the mucosa in the model group was almost completely sloughed off, with a thickness approaching zero, indicating that radiation exposure had caused severe structural atrophy and tissue damage to the oral mucosa. In the traditional method group, the tongue dorsum and ventral mucosa thicknesses were approximately 109.25 μm and 75.03 μm, respectively, both the lowest values ​​among all intervention groups, suggesting that this method has relatively limited therapeutic effect on radiation-induced oral mucositis. In contrast, after the decoction method intervention, the tongue dorsum mucosa thickness in rats recovered to approximately 197.92 μm, and the ventral mucosa thickness recovered to approximately 118.11 μm, showing a certain degree of repair. The percolation method intervention group showed a more significant repair effect, with a tongue dorsum mucosa thickness of 208.56 μm and a ventral mucosa thickness of 118.64 μm. These values ​​are close to the normal physiological mucosal thickness of the blank group (tongue dorsum and ventral mucosa thicknesses of 214.76 μm and 123.02 μm, respectively), and both groups' data were significantly better than the model group and the traditional method group. Statistical analysis results showed that, in terms of repairing the dorsum of the tongue mucosa, the percolation method was significantly more effective than other intervention methods; while in terms of the ventral mucosa, both the percolation method and the decoction method were significantly better than the traditional method. In conclusion, all three intervention methods could alleviate radiation-induced mucosal atrophy to varying degrees, with the percolation method showing the most significant repair effect. Its mucosal morphology and thickness were closer to the normal physiological state, demonstrating its clear therapeutic effect on radiation-induced oral mucositis.

[0103] 4. Summary and Discussion

[0104] This invention compares the therapeutic effects of three different preparation processes of bitter wine decoction on rats with RTOM (Related Temporal Osteoarthritis). The results show that all three methods effectively alleviated the clinical symptoms of RTOM and promoted mucosal repair, confirming the effectiveness of bitter wine decoction in traditional Chinese medicine for "fire-toxin erosion sores." The results suggest that differences in drug component dissolution caused by different processes may be key to the differences in efficacy. Traditional methods may denature egg white protein and cause loss of some volatile components; decoction methods may have limited extraction efficiency; while percolation, as a dynamic, room-temperature percolation process, may be more conducive to the full dissolution of effective components such as alkaloids and organic acids, while better preserving the active protein components in egg white, thus showing advantages in anti-inflammation and promoting epithelial regeneration. The results of this invention provide preliminary experimental basis for optimizing the modern preparation process of bitter wine decoction; its specific chemical composition differences and mechanisms of action require further in-depth research.

[0105] Under the experimental conditions, the bitter wine decoction prepared by all three processes showed a clear therapeutic effect on radiation-induced oral mucositis, improving the general condition of the model rats, reducing mucosal damage, and promoting tissue repair. Considering both macroscopic scoring and microscopic pathological results, the bitter wine decoction prepared by percolation exhibited the relatively optimal efficacy and can serve as a preferred process reference for the subsequent development and application of this formula.

[0106] Example 3: Optimization of the preparation process of bitter wine soup powder

[0107] 1. Experimental Instruments and Materials

[0108] instrument

[0109] Table 1 Instrument Information Instrument Name / Model freeze dryer Electronic analytical balance UV-Vis spectrophotometer pH meter Percolation device (homemade)

[0110] 1.2 Materials

[0111] Pinellia ternata (batch number: 240802, place of origin: Sichuan), rice vinegar (Haitian rice vinegar, acidity ≥5.0 g / 100 mL), ephedrine hydrochloride, bromothymol blue, citrate-sodium citrate buffer (pH=6), chloroform, ammonia, mannitol, sucrose, glucose, trehalose, peppermint flavor, lemon flavor, peach flavor, orange flavor, monk fruit glycosides, etc.

[0112] 2. Experiment Content

[0113] 2.1 Optimization of Percolation Process

[0114] 2.1.1 Degree of powdering of medicinal materials

[0115] Weigh out 12 g each of the coarsest, coarse, and medium-coarse powders of Pinellia ternata, and divide into three parallel portions. Add 1.5 times the amount of rice vinegar to each portion, soak for 1 hour, then pack into a column. Add 3 times the amount of rice vinegar to each portion and soak for 12 hours. Control the percolation flow rate at 2 mL / min·kg, collect the percolate, filter, and set aside to determine the optimal degree of powdering of the medicinal material.

[0116] 2.1.2 Investigation of soaking time

[0117] Weigh out 12 g of Pinellia ternata and divide it into three equal portions. Soak the portions for 0.5, 1, 1.5, and 2 hours respectively, observing the degree of wetting to determine the optimal soaking time.

[0118] 2.1.3 Immersion Time

[0119] Weigh 12 g of Pinellia ternata and divide into three parallel portions. Add 1.5 times the amount of rice vinegar to each portion and soak for 1 hour. Then pack the mixture into a column and add 3 times the amount of rice vinegar. Soak for 6, 12, 18, 24, and 36 hours respectively, controlling the percolation flow rate at 2 mL / min*kg. Collect the percolate, filter, and set aside for later use to determine the optimal soaking time.

[0120] 2.1.4 Percolation rate

[0121] Weigh 12 g of Pinellia ternata and divide it into three parallel portions. Add 1.5 times the amount of rice vinegar to each portion and soak for 1 hour. Then pack the mixture into a column and add 3 times the amount of rice vinegar. Soak for 18 hours. The percolation flow rates are 1 mL / min*kg, 1.5 mL / min*kg, 2 mL / min*kg, 2.5 mL / min*kg, and 3 mL / min*kg, respectively. Collect the percolate, filter it, and set it aside to determine the optimal percolation rate.

[0122] 2.1.5 Evaluation Indicators

[0123] 2.1.5.1 Method for determination of total alkaloids

[0124] (1) Determination of reference solution

[0125] Add 10 mL of chloroform, 1 mL of bromothymol blue indicator, and 10 mL of pH 6.0 citrate buffer solution to the standard solution sequentially. Mix thoroughly and let stand for 60 min. After separation, collect the organic phase, evaporate to dryness in a water bath, redissolve in chloroform, and bring the volume to 10 mL. Measure the absorbance A at 416 nm.

[0126] (2) Determination of the test solution

[0127] Accurately pipette 1 ml of the percolate, dilute it 3 times with water, adjust the pH to 12 with an appropriate amount of ammonia, add 10 mL of pH 6.0 citrate-sodium citrate buffer and 1 ml of bromothymol blue solution, shake and let stand for 60 min, take the chloroform layer, evaporate to dryness, and make up to 10 mL with chloroform. Using chloroform as a blank, measure A at 416 nm and calculate the alkaloid content.

[0128] 2.1.5.2 Determination of extract yield

[0129] Take 20 mL of percolate into a constant-weight evaporating dish, evaporate to dryness in a water bath, dry at 105 ℃ for 3 h, cool in a desiccator for 30 min, weigh quickly, and calculate its extract yield.

[0130] Extract yield (%) =

[0131] 2.1.5.3 Comprehensive Evaluation Indicators

[0132] To comprehensively evaluate the quality of the extraction process, two key indicators were selected: extract yield (E) and total alkaloid content (T). First, each indicator was normalized to eliminate the influence of dimensions and ensure comparability. The normalization formula is as follows:

[0133]

[0134]

[0135] in, and These are the minimum values ​​of the corresponding indicators. and These represent the range of the indicator.

[0136] Furthermore, a comprehensive evaluation index (S) was constructed by assigning corresponding weights to each indicator based on their importance in the extraction process. Given that the total alkaloid content has a more significant impact on the extraction effect, it was given a higher weight (0.6), while the extract yield had a weight of 0.4. The formula for calculating the comprehensive index is as follows:

[0137]

[0138] 2.1.6 Methodological Examination

[0139] 2.1.6.1 Preparation of the reference standard stock solution

[0140] Accurately weigh 2.17 mg of ephedrine hydrochloride, dilute to 10 mL with pure water in a volumetric flask, and shake well to obtain a 217 μg / mL reference solution.

[0141] 2.1.6.2 Preparation of test solution

[0142] Accurately weigh an appropriate amount of Pinellia ternata powder, add an appropriate amount of rice vinegar, and perform percolation extraction for 18 hours at a percolation rate of 2.0 mL / min*kg. Filter to obtain the percolation. Take 1 mL of the percolation and dilute it with water to 3 mL to obtain the test solution.

[0143] 2.1.6.3 Examination of Linear Relationships

[0144] Accurately pipette an appropriate amount of the reference solution and prepare a series of reference solutions with different mass concentrations for determination. Prepare a series of standard solutions with concentrations of 1.085, 5.425, 27.125, 43.400, and 54.250 μg / mL.

[0145] 2.1.6.4 Precision Examination

[0146] The same test solution was used for content determination, and the determination was performed in parallel six times.

[0147] 2.1.6.5 Repeatability Test

[0148] Take six parallel portions of the same batch of Pinellia ternata powder, perform percolation extraction, and prepare the solution according to the method for preparing the test sample solution for content determination.

[0149] 2.1.6.6 Content Determination

[0150] Take an appropriate amount of Pinellia ternata powder and prepare it into a test solution according to the method under the test sample preparation section. Determine the content using the method under section "2.1.5.1" in triplicate.

[0151] 2.1.7 Orthogonal Experiment

[0152] The results of the single-factor experiment were used to select three levels of factors—grinding degree, soaking time, and percolation rate—for orthogonal design to observe their effects on the total alkaloid content and extract yield in the percolation process, and to find the optimal percolation conditions.

[0153] 2.2 Screening of freeze-drying protectants

[0154] 2.2.1 Preparation of bitter wine solution

[0155] Take 12 mL of the clear Pinellia ternata percolate, add 35 mL of egg white mixture, stir at 500 r / min for 5 min, mix evenly and filter to obtain the bitter wine soup solution for later use.

[0156] 2.2.2 Types of freeze-drying protectants

[0157] Take 10 mL of the bitter liquor solution prepared by the optimized process, and add 5% (w / v) mannitol, sucrose, glucose, and trehalose respectively, stirring until completely dissolved. Pre-freeze at -80℃ for 6 h, and then dry in a freeze dryer (vacuum degree 20 Pa, cold trap temperature -50℃) for 24 h to obtain lyophilized powder. The optimal lyophilization protectant was screened based on appearance evaluation (shape, color, reconstitution) and lysozyme content.

[0158] 2.2.3 Dosage of freeze-drying protectant

[0159] Take 10 mL of the bitter liquor solution prepared by the optimized process, and add 2.5%, 5%, 7.5%, and 10% (w / v) mannitol respectively, stirring until completely dissolved. Pre-freeze at -80℃ for 6 h, and then dry in a freeze dryer (vacuum degree 20 Pa, cold trap temperature -50℃) for 24 h to obtain lyophilized powder. The optimal amount of lyophilizing protectant was screened based on appearance evaluation (shape, color, reconstitution) and lysozyme content.

[0160] 2.2.4 Evaluation Indicators

[0161] 2.2.4.1 Appearance Evaluation

[0162] (1) Appearance: It is best to maintain the original volume, not collapse, not shrink, and have a smooth surface, but not break apart.

[0163] (2) Color: The best color is uniform, without spots, and with a fine texture.

[0164] (3) Reconstitution: Take each lyophilized powder and add 5 mL of water to determine the time required for reconstitution.

[0165] Each indicator is scored on a scale of 10, and the scoring criteria are shown in Table 2.

[0166] Table 2 Appearance Evaluation Table of Freeze-Dried Powder

[0167] score shape Color Reconstitution time 0-2 <![CDATA[Atrophy +++ > Layering, obvious color difference between the top and bottom. ＞120s 3-5 <![CDATA[Atrophy ++ > Layered, with obvious color difference between the top and bottom. 90-120s 6-8 <![CDATA[Atrophy + > Layered, with obvious color difference between the top and bottom* 60-90s 9-10 full Uniform, with no color difference ＜90s

[0168] Layering, with obvious color difference between the top and bottom: The resulting freeze-dried powder has a rough texture, many spots, and a very obvious color difference between the top and bottom;

[0169] Layering and noticeable color difference between the top and bottom: The resulting freeze-dried powder has a relatively coarse texture, occasionally with mottled patterns, and a noticeable color difference between the top and bottom.

[0170] Layering, with obvious color difference between the top and bottom*: The resulting freeze-dried powder has a good texture, no spots, and no obvious color difference between the top and bottom;

[0171] Uniform and without color difference: The obtained freeze-dried powder has a uniform color, no color difference, no spots, and a delicate texture.

[0172] 2.2.4.2 Yield of freeze-dried powder

[0173] Accurately pipette 10 mL of the liquid before freeze-drying, freeze-dry it, and weigh the mass (m) of the freeze-dried powder. Calculate the yield using the following formula.

[0174]

[0175] 2.2.4.3 Lysozyme content

[0176] Dissolve 0.5 g of bitter wine powder in 10 mL of 0.9% sodium chloride solution. Dilute 1 mL of the solution 50 times with 0.9% sodium chloride solution. Adjust the pH to 4.5 with acetic acid. Incubate at 77 ℃ for 10 min, then let stand for 1 h. Centrifuge at 12000 r / min for 10 min, collect the supernatant, and measure its absorbance at 281 nm. Calculate the lysozyme content in the bitter wine sample using the standard curve regression equation.

[0177] 2.2.4.4 Comprehensive Evaluation Indicators

[0178] To comprehensively evaluate the quality of the extraction process, appearance evaluation (A), freeze-dried powder yield (B), and lysozyme content (C) were selected as indicators. First, each indicator was normalized to eliminate the influence of dimensions and ensure comparability. The normalization formula is as follows:

[0179]

[0180]

[0181]

[0182] in, , and These are the minimum values ​​of the corresponding indicators. , and These represent the range of the indicator.

[0183] Furthermore, a comprehensive evaluation index (Z) was constructed by assigning corresponding weights to each indicator based on their importance in the extraction process. Given that lysozyme content has a more significant impact on freeze-drying efficiency, it was given a higher weight (0.4), while appearance evaluation and freeze-dried powder yield were each weighted at 0.3. The formula for calculating the comprehensive index is as follows:

[0184]

[0185] 2.2.5 Methodological Examination

[0186] 2.2.5.1 Preparation of the reference standard stock solution

[0187] Accurately weigh 12.78 mg of lysozyme and dilute to a 25 mL volumetric flask with 0.9% sodium chloride solution. Shake well to obtain a 511.2 μg / mL reference solution.

[0188] 2.2.5.2 Preparation of test solution

[0189] Accurately weigh an appropriate amount of the lyophilized bitter wine powder, add an appropriate amount of 0.9% sodium chloride solution to dissolve it, and this is the test solution.

[0190] 2.2.5.3 Examination of Linear Relationships

[0191] Accurately pipette an appropriate amount of the reference solution and prepare a series of reference solutions with different mass concentrations for determination.

[0192] 2.2.5.4 Precision Examination

[0193] The absorbance of the same test solution was measured in six parallel measurements.

[0194] 2.2.5.5 Repeatability Test

[0195] Take six parallel portions of the same batch of bitter wine powder and prepare them according to the method for preparing the test solution for content determination.

[0196] 2.2.2.6 Content Determination

[0197] Take an appropriate amount of bitter wine powder and prepare it into a test solution according to the method under the test sample preparation section. Determine the content using the method under section "2.2.4.3" in triplicate.

[0198] 2.3 Screening of Flavoring Agents

[0199] 2.3.1 Sensory evaluation

[0200] 2.3.1.1 Selection of evaluators

[0201] Twelve healthy volunteers were selected based on the following criteria: ① Aged between 20 and 40 years old and passed a taste perception test; ② Not prone to allergies and had not taken any medications recently; ③ Had not consumed alcohol, smoked, chewed gum, or eaten foods that strongly stimulated the taste buds within 4 hours prior to the evaluation.

[0202] 2.3.1.2 Classic Population Taste Evaluation Method

[0203] Powder samples were placed in sealed plastic bags for odor evaluation. Then, 10 mL of sample solution was placed in a tasting cup and held in the mouth by volunteers. During this time, the volunteers rinsed their mouths for about 15 seconds to allow their tongues to fully experience the taste of the drug. Afterward, the liquid was spat out, and the volunteers rinsed their mouths with purified water until there was no lingering taste before proceeding to the next sample test (the experiment was conducted using a randomized, single-blind method).

[0204] 2.3.1.3 Establishment of Evaluation Criteria

[0205] The evaluation indicators were odor (sour, fishy), taste (sour, fishy), aftertaste, and overall mouthfeel. The evaluation results were scored from 1 to 10. After tasting the samples, volunteers gave their evaluation results and scored them according to the taste evaluation criteria in Table 3.

[0206] Table 3 Taste Evaluation Criteria

[0207] Fraction Odor (sour, fishy) Taste (sour, fishy) Aftertaste Overall evaluation 0-2 The smell is strange and unacceptable. Too sweet or too sour, or with a fishy smell is unacceptable. Strong and persistent residue Unacceptable 2-4 The smell is unpleasant and difficult to mask. The sweet and sour flavors are mismatched, and the fishy smell is obvious. Obvious residue, strong discomfort Unacceptable 4-6 The smell is so-so, it partially masks the odor. The sweet and sour flavors are generally balanced, but a fishy smell is noticeable. There may be residue, but it will fade. reluctantly accepted 6-8 The smell is good and it basically masks the odor. The sweet and sour flavors are well-balanced, with a slight fishy smell. Slight residue, fades quickly Willing to accept 8-10 Pleasant scent, completely masking The sweet and sour flavors are well-balanced, with no unpleasant tastes. No odor residue, comfortable and refreshing Very accepting

[0208] 2.3.2 AHP (Analog-Philosophical Hierarchy Process)

[0209] The Analytic Hierarchy Process (AHP) is a multi-criteria decision-making method that decomposes complex problems into multiple levels, calculates the weights of each factor by constructing a judgment matrix, and finally obtains a comprehensive evaluation result. This invention applies AHP to assign weights to sensory evaluation indicators to improve the scientific rigor and objectivity of the evaluation.

[0210] 2.3.2.1 Establishment of Hierarchical Structure

[0211] The hierarchical structure is generally divided into three layers, from top to bottom: the target layer, the criteria layer, and the scheme layer, with each layer influenced by the layers below. Based on the characteristics of bitter wine powder after adding flavoring agents, four evaluation indicators—odor, mouthfeel, aftertaste, and overall evaluation—were selected from the factors affecting taste to form the criteria layer. The comprehensive evaluation index was used as the target layer, and the prescriptions with different flavoring ratios were used as the scheme layer, thus constructing a multi-level structural analysis model. .

[0212] 2.3.2.2 Constructing the judgment matrix

[0213] Based on the 1-9 scale method, the relative importance of indicators at the same level is determined, and a judgment matrix A is constructed (see Table 4) to determine the weight coefficients of the indicators.

[0214] The geometric mean of each row vector of the judgment matrix A is used to calculate the initial weight coefficient Wi according to formula (1), and then the normalized weight coefficient Wj is calculated according to formula (2). Finally, the weight coefficients of the four indicators are obtained, namely odor 0.3042, taste 0.2386, aftertaste 0.0762, and overall evaluation 0.3811.

[0215] (1)

[0216] (2)

[0217] Table 4. Priority Matrix for Comparing Indicator Components

[0218] A odor Aftertaste taste Overall evaluation odor 1 4 1 1 Aftertaste 1 / 4 1 1 / / 3 1 / 5 taste 1 3 1 1 / 2 Overall evaluation 1 5 2 1

[0219] 2.3.2.3 Consistency Check

[0220] The judgment matrix is ​​constructed by the decision-maker's subjective consciousness, and there may be contradictions in the importance of various indicators. In order to verify the rationality of the weight coefficients, it is essential to perform a consistency test on the judgment matrix. Among them, the consistency ratio CR is an important basis for measuring the rationality of the weight coefficients, and RI is the random consistency index. When CR < 0.1 (CR = CI / RI), the judgment matrix is ​​considered to have satisfactory consistency. First, the maximum eigenvalue λmax is calculated according to formula (3), and then the consistency index CI is calculated according to formula (4), CI = 0.0693. Finally, the CR value is 0.0779, which is less than 0.1, indicating that the matrix has consistency.

[0221]

[0222] 2.3.2.4 Comprehensive Indicators

[0223] Given the weight values ​​of the four indicators, multiply the corresponding scores by their respective weights and sum them up to obtain the comprehensive indicator for each product. Where Yi is the score and Wj is the weight. .

[0224] 3. Experimental Results

[0225] 3.1 Optimization of Percolation Process

[0226] The effects of the degree of powdering of medicinal materials, soaking time, maceration time and percolation rate on the percolation effect were investigated through single-factor experiments. The optimal percolation process conditions were determined by using the total alkaloid content and extract rate as comprehensive evaluation indicators.

[0227] 3.1.1 The degree of powdering of medicinal materials affects

[0228] This invention investigated the effects of three different particle sizes (coarseest, coarse, and medium) of Pinellia ternata powder on the percolation extraction efficiency. As shown in Figure 4A, the medium-powder group had the highest mean comprehensive index, reaching 0.8970, significantly better than the coarse powder group (0.2097) and the coarsest powder group (0.0596). Combined with... Figure 4 The test results for group B show that the medium-sized powder exhibits the best performance in both extract yield (13.8%) and total alkaloid content (approximately 340 mg / L). The coarse powder group has lower extract yield (12.4%) and total alkaloid content (approximately 280 mg / L) than the medium-sized powder group, indicating a slightly lower extraction effect. The coarsest powder group has the lowest extract yield (12.2%) and total alkaloid content (approximately 260 mg / L) among the three groups, with an overall extraction effect significantly inferior to the other two groups. The medium-sized powder ensures both a high extraction rate and maximizes the retention of effective components (total alkaloids). In summary, based on the multi-dimensional evaluation results of extract yield, total alkaloid content, and comprehensive indicators, medium-sized powder is determined to be the optimal particle size for the percolation extraction process of Pinellia ternata.

[0229] 3.1.2 Effect of soaking time

[0230] The effect of soaking time (0.5, 1, 1.5, 2 h) on percolation was investigated. The results showed that the medicinal materials had a hard core after soaking for 0.5 h, and percolation was incomplete; after soaking for 1 h, the medicinal materials were fully moistened, so the optimal soaking time was determined to be 1 h.

[0231] 3.1.3 Effect of immersion time

[0232] This invention investigated the effects of five different soaking times (6 h, 12 h, 18 h, 24 h, and 36 h) on the percolation extraction efficiency of Pinellia ternata. As shown in Figure 5A, the 18 h group had the highest mean comprehensive index, reaching 0.9428, significantly better than the other time groups (approximately 0.27 for the 6 h group, 0.57 for the 12 h group, 0.84 for the 24 h group, and 0.25 for the 36 h group). Combined with the results in Figure 5B, the 18 h group showed the best performance in both extract yield (13.2%) and total alkaloid content (approximately 330). The 24 h group had lower extract yield (12.7%) and total alkaloid content (approximately 310) than the 18 h group, indicating a slightly lower extraction efficiency. The 6 h and 36 h groups had the lowest extract yield and total alkaloid content among all groups, showing significantly inferior overall extraction efficiency compared to the other time groups. In summary, based on the multi-dimensional evaluation results of extract yield, total alkaloid content, and comprehensive indicators, 18 hours was determined to be the optimal soaking time for the percolation extraction process of Pinellia ternata.

[0233] 3.1.4 Effect of percolation rate

[0234] This invention investigated the effects of five different percolation flow rates (1.0, 1.5, 2.0, 2.5, and 3.0 mL / min·kg) on ​​the percolation extraction efficiency of Pinellia ternata. As shown in Figure 6A, the average value of the 2.0 mL / min·kg group was the highest, reaching 0.8917, significantly better than the other flow rate groups (approximately 0.27 for the 1.0 mL / min·kg group, approximately 0.47 for the 1.5 mL / min·kg group, approximately 0.20 for the 2.5 mL / min·kg group, and approximately 0.40 for the 3.0 mL / min·kg group). As shown in Figure 6B, the 2.0 mL / min·kg group exhibited the best performance in both extract yield (13.0%) and total alkaloid content (approximately 340 mg / kg). The 1.5 mL / min·kg and 3.0 mL / min·kg groups had lower extract yields and total alkaloid contents than the 2.0 mL / min·kg group, indicating slightly inferior extraction efficiency. The 1.0 mL / min·kg and 2.5 mL / min·kg groups had the lowest extract yields and total alkaloid contents among all groups, showing significantly worse overall extraction efficiency than the other flow rate groups. In conclusion, based on the multi-dimensional evaluation results considering extract yield, total alkaloid content, and other comprehensive indicators, 2.0 mL / min·kg was determined to be the optimal percolation rate for the percolation extraction of Pinellia ternata.

[0235] 3.1.5 Methodological Examination

[0236] 3.1.5.1 Examination of Linear Relationships

[0237] The absorbance of the reference standard in each sample was calculated, and the results are shown in Figure 1-1. The regression equation for the total alkaloid absorbance (Y) versus concentration (X) is Y = 0.0118X + 0.2659, R0. 2 =0.9965. The results indicate that the total alkaloids exhibit a good linear relationship in the range of 1.085–54.25 μg / mL.

[0238] 3.1.5.2 Precision Examination

[0239] Record the absorbance of the reference standard in each sample. The results show that the RSD value of the absorbance of ephedrine hydrochloride is 0.750%, indicating that the instrument has good precision.

[0240] 3.1.5.3 Repeatability Test

[0241] The total alkaloid content in the six parallel samples was calculated, and the RSD value was 1.79%, indicating that the method has good repeatability.

[0242] 3.1.6 Results of Orthogonal Experiment

[0243] Based on the single-factor experiment, the degree of powdering of medicinal materials (A), soaking time (B), and percolation speed (C) were selected as the factors to be investigated. Three levels were selected for each factor. The total alkaloid content and extract rate were used as evaluation indicators. An L9(33) orthogonal experimental table was designed to optimize the percolation process parameters.

[0244] 3.1.6.1 Factor Levels in Orthogonal Experiments

[0245] The factors and their levels are shown in Table 5.

[0246] Table 5 Factor Level Table

[0247]

[0248] 3.1.6.2 Orthogonal Design Results and Analysis

[0249] The orthogonal experimental design and results are shown in Table 6. Using the comprehensive score as the evaluation index, intuitive analysis shows that the order of influence of each factor on the extraction effect is: B (impregnation time) > A (grinding degree) > C (percolation flow rate). Based on the K value, the optimal combination of factors is A3B2C2, i.e., medium grinding degree, impregnation time of 18 h, and percolation flow rate of 2.0 ml / min·kg.

[0250] Table 6 Orthogonal Experimental Design and Results

[0251]

[0252] 3.1.6.3 Analysis of Variance

[0253] To further determine the significance of the effects of each factor, an analysis of variance was performed on the orthogonal experimental results, and the results are shown in Table 7. The F-values ​​indicate that factors B (impregnation time) and A (degree of pulverization) have significant effects on the extraction effect (p < 0.05), while factor C (percolation flow rate) has no significant effect (p > 0.05), consistent with the range ranking results in the intuitive analysis.

[0254] In summary, orthogonal experimental analysis revealed the optimal parameter combination for the percolation process to be A3B2C2, namely: medium-fine powder of the medicinal material, 18 h of soaking time, and a percolation flow rate of 2.0 ml / min·kg. Under these conditions, the extraction effect is optimal, providing a basis for subsequent process validation.

[0255] Table 7 Results of Analysis of Variance

[0256]

[0257] 3.2 Screening of freeze-drying protectants

[0258] 3.2.1 Methodological Examination

[0259] 3.2.1.1 Examination of Linear Relationships

[0260] The absorbance of the reference standard in each sample was calculated, and the results are shown in Figure 1-1. The regression equation for the absorbance (Y) and concentration (X) of lysozyme was obtained as Y = 0.002X + 0.0146, R0. 2 =0.9994. The results indicate that lysozyme exhibits good linearity in the range of 81.792–408.96 μg / mL.

[0261] 3.2.1.2 Precision test

[0262] Record the absorbance of the reference standard in each sample. The results show that the RSD of the lysozyme absorbance is 0.54%, indicating that the instrument has good precision.

[0263] 3.2.1.3 Repeatability Test

[0264] The total alkaloid content in the six parallel samples was calculated, and the RSD value was 0.68%, indicating that the method has good repeatability.

[0265] 3.2.2 Types of lyophilization protectants

[0266] This invention systematically investigated the effects of four preservatives—glucose, mannitol, sucrose, and trehalose—on the freeze-drying effect of bitter liquor. As shown in Figure 7A, the mannitol group exhibited the highest normalized values ​​for appearance evaluation, freeze-drying yield, and lysozyme content, while the trehalose group performed poorly in all normalized indicators. Further combined with... Figure 7 Analysis of the bar chart results (B) shows that, in terms of appearance, the mannitol group scored the highest and significantly higher than the trehalose group, while the glucose and sucrose groups scored in the middle. Regarding freeze-drying yield, the mannitol group had the highest yield (15.82%), followed by the sucrose and trehalose groups, while the glucose group had the lowest yield and differed from the other groups. In terms of lysozyme activity, the mannitol group had the highest activity (334.70 mg / g), followed by the glucose and sucrose groups, while the trehalose group had the lowest activity, with the mannitol group showing significantly higher activity than the other groups. The protective mechanism of mannitol may be related to the glassy structure formed at low temperatures, which can stabilize the hydrogen bond network of enzyme molecules to reduce denaturation. The poor performance of trehalose in this system suggests that its protective effect may be related to the surface charge characteristics and binding affinity of the enzyme molecules. In summary, based on the multi-dimensional evaluation results of appearance, yield, and activity, mannitol was determined to be the optimal protectant for the freeze-drying process of bitter liquor.

[0267] 3.2.3 Dosage of lyophilization protectant

[0268] Based on the determination that mannitol is a suitable preservative, the effects of different concentrations (2.5%, 5%, 7.5%, 10%, w / v) on the quality of the bitter soup freeze-dried powder were further investigated. The study found that there was no significant difference in the appearance of the freeze-dried powder with increasing mannitol concentration; therefore, lysozyme content was used as the core indicator. Results are as follows... Figure 8 As shown, the 7.5% group had the optimal lysozyme content, reaching 334.43 mg / g. However, when the concentration increased to 10%, the lysozyme content decreased significantly (259.867 mg / g). This indicates that excessively high concentrations of mannitol may interfere with the native conformation of proteins or form an overly dense glassy structure during freeze-drying, thus adversely affecting the stability of lysozyme. Further comparison revealed no significant difference in lysozyme content between the 5% and 7.5% mannitol groups. Considering both process cost and excipient usage, and balancing protective effect with economy, 5% mannitol was ultimately selected as the protective agent for the bitter soup freeze-drying process.

[0269] 3.3 Screening of Flavoring Agents

[0270] 3.3.1 Sample preparation and formulation design

[0271] 3.3.3.1 Preparation of Sample Solution

[0272] Powder: Take the bitter wine soup powder prepared by the optimal percolation process combined with the best freeze-drying protectant, add the flavoring agent in the formula of Table 3-8, and vortex shake until evenly mixed to obtain the powder.

[0273] Solution: Accurately weigh 5 g of the above bitter wine powder, add 30 mL of purified water, and stir magnetically to dissolve completely.

[0274] 3.3.3.2 Flavoring Formula Design

[0275] Based on the preliminary experimental results, this experiment designed nine different flavoring agent formulations to investigate the effects of the type, ratio, and dosage of flavoring agents on the taste of bitter wine soup. The specific design is shown in Table 8.

[0276] Table 8. Formulation design of bitter wine powder with different flavoring agents

[0277] NO. Flavoring agent 1 Dosage / % Flavoring agent 2 Dosage / % 1 Peppermint flavoring 1.5 Monk fruit glycosides 0.3 2 Orange flavoring 2 Monk fruit glycosides 0.3 3 Peppermint flavoring 1.5 Sucralose 0.3 4 Orange flavoring 2 Sucralose 0.3 5 Mint and orange flavoring 1：1 Monk fruit glycosides 0.3 6 Mint and orange flavoring 1.4：0.6 Monk fruit glycosides 0.3 7 Mint and orange flavoring 0.6：1.4 Monk fruit glycosides 0.3 8 Mint and orange flavoring 1.2：0.8 Monk fruit glycosides 0.3 9 Mint and orange flavoring 1.2：0.8 Monk fruit glycosides 0.4

[0278] 3.3.2 Sensory evaluation results of volunteers

[0279] Twelve qualified volunteers conducted a systematic evaluation of all nine sample groups based on the criteria in Table 3. The scores for each indicator were the arithmetic mean of the scores given by the twelve volunteers, and the comprehensive index (Zi) was calculated according to the method described in Section 2.3.2.4. The complete evaluation results are shown in Table 9.

[0280] Table 9. Results of Volunteers' Sensory Evaluation

[0281] Serial Number odor taste Aftertaste Overall score Final result 1 8.0417 4.1667 6.0833 3.9583 5.4125 2 6.0833 5.1667 4.0417 5.1250 5.3444 3 8.0000 2.1667 7.0417 4.0417 5.0274 4 6.0833 2.1667 4.0000 4.9167 4.7648 5 8.0417 5.0833 6.9583 5.9583 6.4601 6 8.9167 3.0417 6.7917 6.0833 6.2741 7 6.8333 6.0000 6.0417 6.8750 6.5907 8 8.7500 7.9167 7.0417 7.8333 8.0725 9 8.8750 6.0417 7.0833 6.0833 6.9994

[0282] According to Table 9 and Figure 9 The analysis results show that sample 8 (overall score Z8=8.0725) performed significantly better than other samples and was the formula with the best overall flavor correction effect; the overall scores of samples 5, 7 and 9 were all above 6.4, which is above average; while the overall scores of samples 1–4 were all below 5.5, and the overall flavor correction effect was not good.

[0283] Analyzing from four dimensions—aroma, taste, aftertaste, and overall evaluation—sample 8 scored highest in taste (7.9167) and overall evaluation (7.8333), while also ranking highly in aroma (8.7500) and aftertaste (7.0417), demonstrating well-balanced and excellent sensory characteristics. There may be synergistic effects among the flavoring components; for example, peppermint flavoring may effectively mask the eggy taste of the bitter soup, orange flavoring helps improve the aftertaste, and monk fruit glycosides can harmonize the sweet and sour ratio of the medicinal liquid.

[0284] In terms of formula optimization, the overall performance of compound flavor formulations (samples 5–9) was better than that of single flavor formulations (samples 1–4), indicating that flavor compounding has a synergistic effect. Among them, the best effect was achieved when peppermint and orange flavor were compounded in a 6:4 ratio (sample 8), which maintained the cooling sensation and odor masking effect of peppermint while improving the aftertaste with the fruity aroma of orange. In addition, increasing the amount of mogroside from 0.3% to 0.4% did not improve the overall score, indicating that 0.3% was sufficient to achieve a suitable sweet and sour balance, and excessive use may lead to a cloying sweetness and affect the harmony of the taste.

[0285] Based on the combined results of analytic hierarchy process (AHP) and sensory evaluation, sample 8 was determined to be the optimal flavoring agent for bitter wine soup. Its composition is: a peppermint-orange blended flavoring (6:4 ratio, 2% total) and 0.3% monk fruit glycosides. This formula, through scientific proportioning, achieves comprehensive optimization in masking odor, harmonizing taste, and improving aftertaste, providing a basis for the clinical application and taste enhancement of bitter wine soup.

[0286] 4. Summary and Discussion

[0287] This chapter focuses on the development of the classic formula Kujiu Tang into powder form, and systematically conducts research on the optimization of the entire process chain from "extraction-forming-flavoring", aiming to establish a scientific, stable and industrially suitable preparation process.

[0288] In the extraction stage, a combination of single-factor and orthogonal experimental design was used to construct a comprehensive evaluation index (S-value) with total alkaloid content and extract yield as the core. The optimal extraction process parameters were determined to be: Pinellia ternata powdered to medium powder, soaked for 1 h, then soaked in rice vinegar for 18 h, with a percolation rate controlled at 2.0 mL / (min·kg). In the freeze-drying stage, the appearance of the formulation, freeze-drying yield, and lysozyme activity were used as evaluation indicators, and 5% mannitol was selected as the best freeze-drying protectant. To address the key issue of improving the formulation's taste, an analytic hierarchy process (AHP) was introduced to quantify and objectify subjective sensory evaluations, constructing a comprehensive taste evaluation system with clear weights. This upgraded the selection of flavoring agents from traditional experience-based judgment to scientific quantitative decision-making, ultimately determining Formula 8 as the optimal flavoring process, with the optimal ratio of 1.2% peppermint flavoring, 0.8% orange flavoring, and 0.3% mogroside.

[0289] The optimized percolation process (medium powder, 18-hour impregnation) may achieve selective enrichment of target components (alkaloids) by controlling the solute diffusion rate and dissolution balance. Mannitol was chosen as a protective agent not only because of its inertness and non-hygroscopic properties, but also because it can form a glassy protective layer around proteins through a "water displacement" mechanism, effectively maintaining the activity of macromolecules such as lysozyme. Taste improvement studies have shown that a "flavor-oriented and modified" strategy can systematically improve the inherent taste of traditional Chinese medicine: diverting attention through olfactory intervention with flavorings, modifying the aftertaste with pleasant flavors, and then balancing the overall taste profile with natural sweeteners. This multi-target, multi-layered taste improvement strategy provides insights into the regulation of the taste of traditional Chinese medicine.

Claims

1. A method for preparing a bitter wine decoction powder for treating radiation-induced oral mucositis, characterized in that: It includes the following steps: a. Weigh the ingredients according to the specified weight ratio: Pinellia ternata, egg white, and rice vinegar; 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, and freeze-dry to obtain freeze-dried powder.

2. The method for preparing the bitter wine decoction powder for treating radiation-induced oral mucositis according to claim 1, characterized in that: The ingredient amounts mentioned in step a are as follows: for every 4g of Pinellia ternata, there are 12ml of rice vinegar and 35ml of egg white.

3. The method for preparing the bitter wine decoction powder for treating radiation-induced oral mucositis according to claim 1 or 2, characterized in that: Step a describes pulverizing Pinellia ternata into the coarsest, coarse, and medium powders; 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 hours, followed by drying at a vacuum of 20 Pa and a cold trap temperature of -50℃ for 24 hours.

4. The method for preparing the bitter wine decoction powder for treating radiation-induced oral mucositis according to claim 3, characterized in that: 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%.

5. The method for preparing the bitter wine decoction powder for treating radiation-induced oral mucositis according to any one of claims 1-4, characterized in that: In step c, 1.8%-2.4% of the total freeze-dried powder is added as a flavoring agent.

6. The method for preparing the bitter wine decoction powder for treating radiation-induced oral mucositis according to claim 5, characterized in that: The flavoring agent is a composition of peppermint flavoring, orange flavoring, and mogroside, with the following weight ratio: Peppermint flavor 0.6-1.4 parts, orange flavor 0.6-1.4 parts, monk fruit glycosides 0.3-0.4 parts.

7. The method for preparing the bitter wine decoction powder for treating radiation-induced oral mucositis according to claim 6, characterized in that: The flavoring agent is a combination of peppermint flavoring and orange flavoring, in the following weight ratio: 1.2 parts peppermint flavoring, 0.8 parts orange flavoring, and 0.3 parts monk fruit glycosides.

Images