Anoectochilus formosanus oral ulcer treatment formula, preparation thereof and anoectochilus formosanus aerosol

Abstract

According to the anoectochilus formosanus oral ulcer treatment formula and preparation thereof and the anoectochilus formosanus aerosol provided by the invention, when the anoectochilus formosanus aerosol is used for treating rats with immunocompromised oral ulcer, the wound healing of the oral ulcer can be promoted, the effect of relieving inflammation symptoms of body ulcer tissues is achieved, and meanwhile, the immune system function of the body can be improved; the spleen coefficient of an organism is obviously increased, and the expression levels of cell factors such as IL-2, IL-10 and TNF-alpha can also be improved. From the perspective of the healing effect of the oral ulcer, the anoectochilus roxburghii aerosol has an obvious effect, the treatment effect on the oral ulcer is not obviously different from that of a mirabilitum praeparatum spray under the condition of low immunity, but the treatment effect on the low immunity is relatively weak compared with that of Yupingfeng. The effect of the anoectochilus formosanus aerosol in the aspect of improving immunity needs to be further studied.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a formula for treating oral ulcers using *Anoectochilus roxburghii* and its preparation, as well as an aerosol form of *Anoectochilus roxburghii* and its application. Background Technology

[0002] *Anoectochilus roxburghii*, a species of orchid in the Orchidaceae family, is a precious and rare traditional Chinese medicine, renowned as the "Southern Cordyceps" and the "King of Herbs." The whole plant is used medicinally, and it is associated with the lung, liver, kidney, and bladder meridians. It has the effects of clearing heat and cooling the blood, dispelling wind and dampness, strengthening the heart and promoting diuresis, detoxifying, tonifying the kidneys, and calming the liver. The main active ingredient in *Anoectochilus roxburghii* is flavonoids, which have a certain therapeutic effect on oral ulcers in rats.

[0003] Recurrent aphthous ulcers, also known as recurrent aphthous ulcers, are the most common oral mucosal disease. Many factors can contribute to its development, including depression, indigestion, and the common cold, making its etiology complex. Based on clinical characteristics, recurrent aphthous ulcers can be classified into three types: mild recurrent aphthous ulcers, severe recurrent aphthous ulcers, and herpetic recurrent aphthous ulcers. These three types share the characteristic of recurrent flare-ups, with everyday activities easily irritating the lesions, causing burning and pain, such as eating and speaking, severely disrupting daily life. Therefore, many research teams both domestically and internationally are studying the etiology and pathogenesis of this disease, aiming to completely cure it from its root cause.

[0004] Recurrent aphthous ulcers are now a common condition of the oral mucosa, and research on them has made significant progress both domestically and internationally. However, it is undeniable that there is still no cure, and the specific pathogenic mechanism remains difficult to diagnose. Nevertheless, efforts are being made both domestically and internationally to make treatments for recurrent aphthous ulcers more convenient and efficient. This is reflected in many aspects, such as the choice of dosage forms. From the earliest common oral liquids and powders, more convenient dosage forms such as sprays have gradually been developed, as well as more efficient forms like liniments and thermosensitive gels to overcome inconvenience. In recent years, laser therapy has been an innovative treatment method in Western medicine, with significant therapeutic effects and no toxic side effects. Traditional Chinese medicine has also drawn inspiration from its traditions, and acupuncture has been incorporated into the treatment of recurrent aphthous ulcers. This technique involves inserting needles into specific acupoints to achieve a curative effect on the specific disease. The biggest advantage of acupuncture is its low toxicity, high efficiency, and safety in treating recurrent oral ulcers. However, the biggest challenge is that more samples are needed for research and verification, so further confirmation of its efficacy is required.

[0005] In summary, there are countless reasons that can lead to recurrent aphthous ulcers, and the mechanisms underlying their development are extremely complex and difficult to summarize. The occurrence of recurrent aphthous ulcers is the result of the combined effects of multiple factors. Thoroughly understanding the mechanisms and causes of recurrent aphthous ulcers, both in depth and breadth, is crucial in clinical practice for finding an effective treatment that can fundamentally inhibit the recurrence of aphthous ulcers. Summary of the Invention

[0006] To address the technical problems existing in the prior art, the present invention provides the following technical solution: On the one hand, a formula for treating oral ulcers using *Anoectochilus roxburghii* is provided, the formula comprising the following components by weight percentage: The contents of the product were: 1.0% flavonoids from Anoectochilus roxburghii, 0.15% Tween-20, 0.5% propylene glycol, and 0.002% benzalkonium chloride.

[0007] On the other hand, a *Anoectochilus roxburghii* aerosol is provided, which contains the above-described *Anoectochilus roxburghii* oral ulcer treatment formula.

[0008] On the other hand, a method for preparing a *Anoectochilus roxburghii* aerosol is also provided, the method comprising the following steps: Weigh out 8.0-12.0 g of Anoectochilus roxburghii flavonoids and dissolve them in 200 mL of pure water at 50 °C. Stir until completely dissolved, then add 0.018-0.022 g of benzalkonium chloride and stir until dissolved. Then add Tween-20 (1.4-1.6 g), stir until homogeneous, form a solution, and add propylene glycol (4.8-5.2 mL) and mix well. Finally, add pure water to 1000 mL, filter and sterilize using a 0.22 μm microporous membrane, fill the liquid into an aerosol can, seal it, and fill it with propellant to obtain the Anoectochilus roxburghii aerosol.

[0009] On the other hand, a formula for treating oral ulcers using *Anoectochilus roxburghii* or a method for treating oral ulcers using *Anoectochilus roxburghii* aerosol is also provided.

[0010] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following: The experimental data provided by this invention indicate that *Anoectochilus roxburghii* aerosol can promote the healing of oral ulcers in immunocompromised rats, effectively alleviate inflammatory symptoms in ulcer tissue, and enhance the body's immune system function, significantly increasing the spleen coefficient and the expression levels of cytokines such as IL-2, IL-10, and TNF-α. From the perspective of oral ulcer healing efficacy, *Anoectochilus roxburghii* aerosol is significantly effective. Under immunocompromised conditions, its therapeutic effect on oral ulcers is not significantly different from that of watermelon frost spray. However, its therapeutic effect on immunocompromised rats is relatively weaker than that of *Yu Ping Feng* spray. Further research is needed to investigate the role of *Anoectochilus roxburghii* aerosol in enhancing immunity. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 This is a standard curve of rutin content provided in an embodiment of the present invention; Figure 2 This is a comparative curve of the number of oral ulcers that have healed, provided by an embodiment of the present invention. Figure 3 This is a comparative curve of the change in the area of ​​a cavity ulcer provided in an embodiment of the present invention; Figure 4 This is a microscopic schematic diagram of the microscopic histopathological results (hematoxylin-eosin staining) of rat mucosal ulcers provided in the embodiments of the present invention (ABCDE represent blank group, model group, immunodeficient Anoectochilus roxburghii intervention group, immunodeficient Pteris vittata intervention group, and immunodeficient Watermelon Frost intervention group, respectively). Figure 5 This is a statistical chart of plasma cytokine levels provided in an embodiment of the present invention; Figure 6 This is a spleen coefficient recording diagram provided in an embodiment of the present invention. Detailed Implementation

[0013] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0014] In embodiments of the present invention, words such as "exemplarily," "for example," etc., are used to indicate that something is an example, illustration, or description. Any embodiment or design described as "exemplary" in the present invention should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of the word "exemplary" is intended to present the concept in a concrete manner. Furthermore, in embodiments of the present invention, the meaning expressed by "and / or" can be both, or either one.

[0015] In the embodiments of this invention, the terms "image" and "picture" may sometimes be used interchangeably. It should be noted that, without emphasizing the distinction between them, they convey the same meaning. Similarly, the terms "of," "corresponding (relevant)," and "corresponding" may sometimes be used interchangeably. It should be noted that, without emphasizing the distinction between them, they convey the same meaning.

[0016] In this embodiment of the invention, sometimes a subscript such as W1 may be mistakenly written as a non-subscript form such as W1. When the difference is not emphasized, the meaning they express is the same.

[0017] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0018] The experimental reagents and equipment required in this embodiment can all be provided by the laboratory or purchased from the market.

[0019] The experimental data and clinical treatment mechanism of the present invention will be described below.

[0020] I. Research Objectives To investigate the effects of *Anoectochilus roxburghii* on the treatment of oral ulcers, this study screened prescriptions and prepared an aerosol that is easy to use daily. An immunodeficiency model was established by intraperitoneal injection of cyclophosphamide (150 mg / kg). Then, an oral ulcer model was established by chemical cauterization of the oral mucosa of rats with NaOH crystals. The efficacy of this aerosol on oral ulcers in the immunodeficiency model was investigated.

[0021] 1. Research Methods: An appropriate amount of *Anoectochilus roxburghii* powder was extracted ultrasonically for 58 minutes at 45℃, with a material-to-liquid ratio of 1:30 and an ethanol concentration of 80%. The total flavonoid content of the filtrate was determined. Based on the physicochemical properties and commonly used dosages of the main drug and excipients, and combined with preliminary experiments, nine prescriptions were designed. The optimal prescription was selected mainly based on sensory evaluation, and the homogeneity and clarity of the drug solution were examined. According to the National Pharmacopoeia standard, nitrogen gas was selected as the propellant, with a gas-to-liquid ratio of 4:6. Seventy-two healthy male Wistar rats were purchased and randomly divided into six groups of 12 rats each after one week of acclimatization: normal group, *Anoectochilus roxburghii* intervention group without immunodeficiency, positive drug intervention group for immunodeficiency (watermelon frost spray), positive drug intervention group for immunodeficiency (Yu Ping Feng San), *Anoectochilus roxburghii* intervention group with immunodeficiency, and model group. Immunodeficiency models were established in all immunodeficiency groups by intraperitoneal injection of cyclophosphamide (CTX, 150 mg / kg / d), while the non-immunodeficiency group received physiological saline as a control. The following day, oral ulcer animal models were established in the immunocompromised groups using chemical cauterization. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital. After anesthesia, filter paper (3 mm in diameter) soaked in NaOH crystals was placed on the lower lip near the corner of the mouth and cauterized for 5-8 seconds. The following groups were treated: no immunocompromised *Anoectochilus roxburghii* intervention group, immunocompromised positive drug intervention group (watermelon frost spray), immunocompromised positive drug intervention group (jade screen powder), and immunocompromised *Anoectochilus roxburghii* intervention group. The *Anoectochilus roxburghii* and watermelon frost intervention groups had their drugs sprayed evenly onto the ulcer surface, while the jade screen powder group received the drug via gavage. All groups received the same medication three times daily. The normal and model groups received sterile water. Twelve rats in each group were sacrificed on day 12. Ulcer tissue was excised, cleaned, and observed. The spleen was removed from the abdominal cavity, blood was removed, and the tissue was dried with filter paper. The spleen was weighed and its coefficient was recorded. Serum and ulcer tissue were collected. Serum levels of TNF-α, IL-2, and IL-10 were detected using an ELISA kit, and ulcer tissue was used for HE staining.

[0022] 2. Research instruments and materials 2.1 Laboratory Animals Seventy-two male Wistar rats, weighing 200-240g, were selected.

[0023] 2.2 Main Materials and Reagents All materials and reagents can be provided by the laboratory or purchased from the market. For example, the medicinal material of Anoectochilus roxburghii was purchased from Jinhua Central Hospital.

[0024] 2.3 Main Instruments and Equipment All instruments and equipment required for the research can be provided by the laboratory or purchased from the market. For example, the electric thermostatic water bath (model: HWS24) was purchased from Shanghai Qixin Scientific Instruments Co., Ltd.

[0025] II. Preparation of Anoectochilus roxburghii aerosol (I) Preparation of Golden Thread Orchid Solution 1. Extraction of total flavonoids from Anoectochilus roxburghii This experiment plans to use two extraction methods and select the one with higher extraction efficiency. Method A is heating and reflux extraction for 2 hours; Method B is ultrasonic extraction for 58 minutes, with 30 times the amount of 80% ethanol added and extracted at 45°C.

[0026] 2. Determination of total flavonoid content in Anoectochilus roxburghii Accurately weigh 5.0 mg of dried rutin standard to constant weight, and add 60% ethanol until the concentration of the rutin standard solution is 0.1 mg / mL. Then, using a pipette, take 0.0, 0.5, 1.0, 1.5, 2.0, and 3.0 mL of the rutin standard solution and pour them into 10 mL volumetric flasks. Add 2 mL of 60% ethanol solution and 0.3 mL of 5% sodium nitrite solution respectively. After standing for 6 min, add 0.3 mL of 10% aluminum nitrate solution. After standing for 6 min, add 4 mL of 1 mol / L sodium hydroxide solution. Finally, add 60% ethanol to bring the volume to 10.0 mL and let stand for 15 min. Measure the absorbance at wavelengths of 350 nm, 450 nm, and 510 nm. Plot a rutin standard curve with absorbance (A) on the ordinate and rutin concentration (mg / mL) on the abscissa.

[0027] like Figure 1 As shown, 1.0 mL of each of the two extraction methods was precisely transferred into a 10 mL volumetric flask and processed using the same method. The group without rutin was used as a blank control group. The absorbance was measured at wavelengths of 350 nm, 450 nm, and 510 nm. By comparing the absorbance at the three wavelengths, the 510 nm group, which exhibited the best wavelength linearity, was selected as the optimal choice.

[0028] The following are the extraction rates for the two methods:

[0029] Table 1. Extraction Rate After comparison, ultrasonic extraction was chosen to extract Anoectochilus roxburghii.

[0030] 3. Screening of Golden Thread Lotus Aerosol Formulation Prescription Based on the physicochemical properties and common dosages of the main and excipient drugs, and combined with preliminary experiments, nine prescriptions were designed as shown in Table 2 below.

[0031]

[0032] Table 2. Prescription Screening Preparation steps of prescription A1: Weigh 8.0-12.0 g of Anoectochilus roxburghii flavonoids and dissolve them in 200 mL of pure water at 50℃. Stir until completely dissolved, then add benzalkonium chloride (0.018-0.022 g) and stir to dissolve. Then add Tween-20 (1.4-1.6 g) and stir to form a homogeneous solution. Add propylene glycol (4.8-5.2 mL) and mix well. Finally, add pure water to 1000 mL, filter and sterilize using a 0.22 μm microporous membrane, fill the solution into an aerosol can, seal it, and fill it with propellant to obtain the finished product.

[0033] Preparation steps of A2 prescription: Weigh 8.0-12.0 g of Anoectochilus roxburghii flavonoids and dissolve them in 200 mL of pure water at 50℃. Stir until completely dissolved, then add benzalkonium chloride (0.018-0.022 g) and stir to dissolve. Then add Tween-40 (1.4-1.6 g) and stir to form a homogeneous solution. Add propylene glycol (4.8-5.2 mL) and mix well. Finally, add pure water to 1000 mL, filter and sterilize using a 0.22 μm microporous membrane, fill the solution into an aerosol can, seal it, and fill it with propellant to obtain the finished product.

[0034] Preparation steps of A3 prescription: Weigh 8.0-12.0 g of Anoectochilus roxburghii flavonoids and dissolve them in 200 mL of pure water at 50℃. Stir until completely dissolved, then add benzalkonium chloride (0.018-0.022 g) and stir to dissolve. Then add Tween-80 (1.4-1.6 g) and stir to form a homogeneous solution. Add propylene glycol (4.8-5.2 mL) and mix well. Finally, add pure water to 1000 mL, filter and sterilize using a 0.22 μm microporous membrane, fill the solution into an aerosol can, seal it, and fill it with propellant to obtain the finished product.

[0035] Preparation steps of prescription B1: Weigh 8.0-12.0 g of Anoectochilus roxburghii flavonoids and dissolve them in 200 mL of pure water at 50℃. Stir until completely dissolved, then add benzalkonium chloride (0.018-0.022 g) and stir to dissolve. Then add Tween-20 (2.4-2.6 g) and stir to form a homogeneous solution. Add propylene glycol (9.6-10.4 mL) and mix well. Finally, add pure water to 1000 mL, filter and sterilize using a 0.22 μm microporous membrane, fill the solution into an aerosol can, seal it, and fill it with propellant to obtain the finished product.

[0036] Preparation steps of prescription B2: Weigh 8.0-12.0 g of Anoectochilus roxburghii flavonoids and dissolve them in 200 mL of pure water at 50℃. Stir until completely dissolved, then add benzalkonium chloride (0.018-0.022 g) and stir to dissolve. Then add Tween-40 (2.4-2.6 g) and stir to form a homogeneous solution. Add propylene glycol (9.6-10.4 mL) and mix well. Finally, add pure water to 1000 mL, filter and sterilize using a 0.22 μm microporous membrane, fill the solution into an aerosol can, seal it, and fill it with propellant to obtain the finished product.

[0037] Preparation steps of B3 formulation: Weigh 8.0-12.0 g of Anoectochilus roxburghii flavonoids and dissolve them in 200 mL of pure water at 50℃. Stir until completely dissolved, then add benzalkonium chloride (0.018-0.022 g) and stir to dissolve. Then add Tween-80 (2.4-2.6 g) and stir to form a homogeneous solution. Add propylene glycol (9.6-10.4 mL) and mix well. Finally, add pure water to 1000 mL, filter and sterilize using a 0.22 μm microporous membrane, fill the solution into an aerosol can, seal it, and fill it with propellant to obtain the finished product.

[0038] C1 formulation preparation steps: Weigh 8.0-12.0 g of Anoectochilus roxburghii flavonoids and dissolve them in 200 mL of pure water at 50℃. Stir until completely dissolved, then add benzalkonium chloride (0.018-0.022 g) and stir to dissolve. Subsequently, add Tween-20 (3.4-3.6 g) and stir to form a homogeneous solution. Add propylene glycol (14.4-15.6 mL) and mix well. Finally, add pure water to 1000 mL, filter and sterilize using a 0.22 μm microporous membrane, fill the solution into an aerosol can, seal it, and fill it with propellant to obtain the finished product.

[0039] C2 formulation preparation steps: Weigh 8.0-12.0 g of Anoectochilus roxburghii flavonoids and dissolve them in 200 mL of pure water at 50℃. Stir until completely dissolved, then add benzalkonium chloride (0.018-0.022 g) and stir to dissolve. Subsequently, add Tween-40 (3.4-3.6 g) and stir to form a homogeneous solution. Add propylene glycol (14.4-15.6 mL) and mix well. Finally, add pure water to 1000 mL, filter and sterilize using a 0.22 μm microporous membrane, fill the solution into an aerosol can, seal it, and fill it with propellant to obtain the finished product.

[0040] C3 formulation preparation steps: Weigh 8.0-12.0 g of Anoectochilus roxburghii flavonoids and dissolve them in 200 mL of pure water at 50℃. Stir until completely dissolved, then add benzalkonium chloride (0.018-0.022 g) and stir to dissolve. Subsequently, add Tween-80 (3.4-3.6 g) and stir to form a homogeneous solution. Add propylene glycol (14.4-15.6 mL) and mix well. Finally, add pure water to 1000 mL, filter and sterilize using a 0.22 μm microporous membrane, fill the solution into an aerosol can, seal it, and fill it with propellant to obtain the finished product.

[0041] (II) Prescription Screening The above nine formulations were selected based on sensory evaluation, with the group exhibiting the best uniformity and clarity. The main observations included chemical stability, solubility, impurity levels, and spray uniformity. The screening results are shown in Table 3 below.

[0042] Table 3. Prescription Screening 1. Chemical stability test Test method: Each group of samples (A1-C3) was placed under accelerated test conditions (40±2℃, RH 75±5%), and samples were taken and tested at 0, 1, 2 and 3 months respectively.

[0043] The flavonoid content of *Anoectochilus roxburghii* was determined by HPLC, and the retention rate (%) was calculated. pH changes were monitored simultaneously.

[0044] Acceptable standards: Flavonoid content decreases by ≤5%, pH change is ≤±0.5.

[0045] Data records (groups A1-C3):

[0046] Table 4. Chemical stability test data 2. Solubility Test Test method: Place each group of samples (A1-C3) at a constant temperature of 25℃ for 24 hours. Observe the clarity of the solution (visual method) and record whether there is precipitation or turbidity. Take 10 mL of sample and centrifuge (3000 rpm, 15 min), and weigh the mass of the precipitate (mg).

[0047] Acceptable standard: No visible precipitate, and the amount of precipitate after centrifugation is ≤0.1%.

[0048] Data records (groups A1-C3):

[0049] Table 5. Solubility Test Data 3. Impurity detection Test method: Degradation products (such as flavonoid aglycones) in each group of samples (A1-C3) were detected by HPLC. The percentage of the total impurity peak area to the main peak area was calculated.

[0050] Acceptance standard: Total impurities ≤ 0.5%.

[0051] Data records (groups A1-C3):

[0052] Table 6. Impurity Detection Data 4. Spray uniformity test Test method: Each sample group (A1-C3) was sprayed 10 times consecutively, and the spray volume (mg / spray) was recorded after each spray. The relative standard deviation (RSD%) was calculated to assess uniformity.

[0053] Acceptable standard: RSD ≤ 5%.

[0054] Data records (groups A1-C3):

[0055] Table 7. Test data on spray uniformity Comprehensive analysis conclusion: Group A1 performed best in chemical stability (98.2% retention), solubility (no precipitation), impurities (0.1%), and spray uniformity (RSD 1.2%), meeting all criteria.

[0056] Groups B1 and C1 performed well in some indicators (such as solubility and impurities), but their stability was slightly worse.

[0057] Groups A2, A3, B2, B3, C2, and C3 require further optimization due to issues such as sedimentation, impurities, or uneven spraying.

[0058] Final recommended prescription: A1 (Anoectochilus roxburghii flavonoids 1.0%, Tween-20 0.15%, Propylene glycol 0.5%, Benzalkonium chloride 0.002%).

[0059] III. Aerosol Preparation (I) Selection of Propellant for Anoectochilus roxburghii Aerosol 1. Screening of Propellants for Anoectochilus roxburghii Aerosol Based on the types of propellants currently used in the preparation of aerosols, and taking into account the physicochemical properties of the propellants and the chemical properties of the total flavonoids of Anoectochilus roxburghii, propane, isobutane, dimethyl ether, and nitrogen are proposed.

[0060] 2. Compatibility test between the prepared solution and the propellant Take a pressure-resistant test tube, add 20 mL of Anoectochilus roxburghii solution and 10 mL of propellant to each, shake well and let stand for a period of time, and observe the phenomena of each group.

[0061] The propane-butane and isobutane groups showed clear stratification with the prepared solution, remaining on the upper layer and indicating poor compatibility; therefore, they were not considered. The dimethyl ether group was miscible with the prepared solution, producing a brownish-yellow, relatively turbid mixture with a small amount of precipitate at the bottom of the test tube; considering the characteristics of an aerosol, this group was also not considered. The nitrogen group showed no significant change in the prepared solution after the addition of nitrogen; the color remained pale yellow, and no precipitate formed. Based on these considerations, nitrogen was chosen as the propellant.

[0062] 3. Deformation pressure and burst pressure test of aerosol Chinese national standards stipulate that the deformation pressure of aerosol cans should be greater than or equal to 1.2 MPa, and the burst pressure should be greater than or equal to 1.4 MPa. Three aerosol cans were taken, and their deformation pressure and burst pressure were measured. The results are shown in Table 9 below.

[0063]

[0064] Table 9. Deformation pressure and burst pressure of aluminum cans As can be seen from the table above, the aluminum can for the aerosol meets the requirements.

[0065] 4. Screening of Aerosol Dosage The volume ratio of the liquid to the propellant in the final aerosol product greatly affects the spraying effect. Therefore, three gas-liquid ratio groups of 2:8, 4:6, and 6:4 were proposed, and their spraying rate and total spraying volume were measured based on the 2020 edition of the Chinese Pharmacopoeia General Chapter 0113.

[0066]

[0067] Table 10. Results of Gas-Liquid Ratio Study As can be seen from the results in Table 2 above, the optimal ratio of liquid to propellant is 4:6. Under the given conditions, the liquid spraying rate is good, the total spraying volume meets the national standard of ≥85%, and the atomization effect is good.

[0068] (II) Study on the efficacy of Anoectochilus roxburghii aerosol in treating oral ulcers 1. Experimental Preparation 1.1 Establishing a Model Seventy-two male Wistar rats, weighing 200-240g, were selected and acclimatized for 7 days. They were then randomly divided into a normal group, a group without immunodeficiency treated with *Anoectochilus roxburghii*, a model group, a positive immunodeficiency drug intervention group (watermelon frost spray), a positive immunodeficiency drug intervention group (Yu Ping Feng San), and an immunodeficiency *Anoectochilus roxburghii* intervention group. On the first day after acclimatization, all immunodeficiency groups were intraperitoneally injected with cyclophosphamide (CTX, 150mg / kg) to establish an immunodeficiency model. The group without immunodeficiency received an equal volume of saline as a control. On day 2, an oral ulcer model was established in all immunocompromised groups using chemical cauterization. Rats were first anesthetized via intraperitoneal injection of 10% sodium pentobarbital (30 mg / kg). After the anesthesia wore off, filter paper (approximately 3 mm in diameter) soaked in sodium hydroxide crystals was applied tightly to the lower lip near the oral mucosa and cauterized for 8-10 seconds. After cauterization, the mucosa was rinsed with physiological saline. The oral mucosa was observed 24 hours later to check for ulcer formation. On day 3, the oral mucosa of the immunocompromised group was observed, confirming that there were significantly more obvious oral ulcer lesions compared to the non-immunocompromised group, with similar size and depth, indicating successful model establishment.

[0069] 1.2 Administration The prepared Anoectochilus roxburghii aerosol dispensed approximately 0.12g per spray. To ensure consistency, the Yupingfeng group also administered the drug by dissolving it in water and then filling the same spray bottle.

[0070] The following groups were divided into four intervention groups: non-immunodeficiency *Anoectochilus roxburghii* intervention group, immunodeficiency positive drug intervention group (watermelon frost spray), immunodeficiency positive drug intervention group (Yu Ping Feng powder), and immunodeficiency *Anoectochilus roxburghii* intervention group. Each group received the same medication, sprayed evenly onto the ulcer surface twice daily, three times a day. The normal group and model group received the same amount of sterile water each time. Medication continued for 12 days until the ulcers were completely healed.

[0071] 2. Experimental Results 2.1 Ulcer condition Ulcer area measurement: Every day after the successful establishment of the oral ulcer model, the maximum transverse diameter and maximum longitudinal diameter (D1, D2) of the ulcers in each group of the immunodeficiency group were measured using vernier calipers, and the oral ulcer area was calculated using the following formula: Oral ulcer area = 0.25 × D1 × D2 × Π Continue measuring until the oral ulcer has healed.

[0072] like Figure 2 and Figure 3As shown, on the second day after establishing the oral ulcer model, the area of ​​oral ulcers in the model group did not change significantly, while the area of ​​oral ulcers in the groups given the positive control drug decreased significantly. In the immunodeficient Watermelon Frost intervention group and the immunodeficient Jade Screen Prickly Pear intervention group, some rats recovered by the fourth day after administration, and were basically healed by the ninth day, while only a small portion of the model group recovered. Regarding the effects of the three drugs on the healing of oral ulcer area, there was no significant difference. Comparing the data with the model group on the seventh day, the ulcer area decreased by 63.2% in the immunodeficient Anoectochilus roxburghii group, 54.5% in the immunodeficient Jade Screen Prickly Pear group, and 50.2% in the immunodeficient Watermelon Frost group. Comparing the ulcer area before administration to the seventh day after administration, the ulcer area on the seventh day was 26.9% of the pre-administration area in the model group, 11.5% in the immunodeficient Anoectochilus roxburghii intervention group, 13.1% in the immunodeficient Jade Screen Prickly Prickly Pear group, and 16.8% in the immunodeficient Watermelon Frost intervention group. The *Anoectochilus roxburghii* group showed the best healing speed for ulcers, followed by the *Phyllostachys nigra* group, and lastly the *Watermelon Frost* group. However, when the ulcer area was small, the *Watermelon Frost* group had a significant advantage in efficacy. 2.2 Histopathological Analysis Two rats were randomly selected from each group on days 2, 4, and 6 after drug administration, and on the day the ulcers were completely healed as observed by the naked eye. The selected rats were euthanized by cervical dislocation, and the ulcer tissue was excised, with an area of ​​0.5cm*0.5cm*0.1cm. After excising, the oral ulcer tissue was immersed in phosphate-buffered saline (PBS) for washing, and after cleaning, it was fixed in 4% paraformaldehyde. After 24 hours, the ulcer tissue was stained with hematoxylin and eosin (HE).

[0073] like Figure 4 As shown in the HE-stained sections of the ulcer tissue above, from left to right and top to bottom, are the blank group, model group, immunocompromised *Anoectochilus roxburghii* intervention group, immunocompromised *Gynostemma pentaphyllum* intervention group, and immunocompromised *Watermelon Frost* intervention group. The oral epithelial tissue in the non-intervention group still showed cracks, indicating that the epithelial tissue was damaged and had not yet healed. This phenomenon proves that the ulcer formation was successful and the model was successfully established. Compared with the non-intervention group, the epithelial cells in the *Anoectochilus roxburghii* intervention group and the *Watermelon Frost* intervention group showed significant healing, and the surface of the epithelial tissue was covered with newly formed granulation tissue. Furthermore, all three treatment groups showed neutrophil infiltration, consistent with inflammatory characteristics.

[0074] 2.3 ELISA Results Twelve rats were used in each group. Four were randomly selected for HE staining, and the rest were used for ELISA (Enzyme-Linked Immunosorbent Assay) testing on day 9. After fasting for one day prior, rats were anesthetized on the second day by intraperitoneal injection of sodium pentobarbital at a dose of 3 mg / 100g. Once the rats were unresponsive, they were placed supine on a flat plate, and their limbs were secured to their four ends with rubber bands. The abdomen was opened with a scalpel, and the organs and internal fat were moved to one side. Blood vessels were located near the spine; the darker-colored vessels were the inferior vena cava, and the lighter-colored vessels were the abdominal aorta. The translucent fascia attached to the upper side was separated with forceps, and a vacuum blood collection tube was inserted into the abdominal aorta to collect blood from the rats. After blood is collected, the blood sample is left to stand at room temperature for 2 hours, then heated in a water bath at 37°C for 30 minutes. The supernatant is then centrifuged at 3000 rpm for 10 minutes, and then the supernatant is centrifuged again at the same speed for 5 minutes. The pale yellow liquid obtained after centrifugation is serum.

[0075] The levels of TNF-α, IL-2, and IL-10 in rat serum were determined using ELISA, strictly following the instructions of the kit.

[0076] like Figure 5 As shown, the TNF-α levels on day 9 in the no-intervention group, *Anoectochilus roxburghii* intervention group, *Gynostemma pentaphyllum* intervention group, *Watermelon Frost* intervention group, and blank group were 345.54±27.04 (pg / mL), 234.03±25.73 (pg / mL), 253.51±22.07 (pg / mL), 253.27±25.65 (pg / mL), and 167.25±16.96 (pg / mL), respectively; the IL-2 levels on day 9 in the no-intervention group, *Anoectochilus roxburghii* intervention group, *Gynostemma pentaphyllum* intervention group, *Watermelon Frost* intervention group, and blank group were 285.64±15.83 (pg / mL), 220... The IL-10 levels on day 9 were 229.94±20.18 (pg / mL), 229.27±18.98 (pg / mL), and 171.56±23.51 (pg / mL), respectively. The IL-10 levels on day 9 in the no-intervention group, the *Anoectochilus roxburghii* intervention group, the *Gynostemma pentaphyllum* intervention group, the *Watermelon Frost* intervention group, and the blank group were 51.90±4.00 (pg / mL), 42.03±3.71 (pg / mL), 38.90±2.98 (pg / mL), 40.06±3.39 (pg / mL), and 23.72±2.90 (pg / mL), respectively.

[0077] The levels of TNF-α, IL-2, and IL-10 in the no-intervention group were significantly higher than those in the control group, demonstrating that inflammation occurred in the mice after the onset of oral ulcers, indicating successful modeling. The IL-2 levels in the *Anoectochilus roxburghii*, *Gynostemma pentaphyllum*, and *Watermelon Frost* intervention groups were higher than those in the no-intervention group, indicating that the drugs effectively activated immune cells and stimulated an immune response. The IL-10 levels in these groups were significantly higher than those in the control group but lower than those in the no-intervention group, suggesting that IL-10 plays a negative regulatory role in the body and was significantly lower than in the no-intervention group, implying a stronger immune response compared to the no-intervention group. The TNF-α levels in these groups were significantly higher than those in the control group and slightly higher than those in the no-intervention group, indicating that these drugs enhanced anti-inflammatory effects.

[0078] 2.4 Immune Organ Index After the rats' blood was completely drawn, the rats were euthanized, the abdominal cavity was opened, the spleen was located and removed from the right posterior side of the abdominal cavity, the blood was removed and the spleen was wiped dry with filter paper, the weight was weighed and recorded, and the spleen coefficient was calculated using the following formula.

[0079] Spleen coefficient = spleen mass (mg) / body mass (g).

[0080] As shown in the figure, based on the spleen coefficient during the ulceration period, the spleen coefficient of the model group was significantly lower than that of the non-immune hypoxia group, indicating that the immunodeficiency model was successfully established. Under immunodeficiency conditions, the spleen coefficient of the immunodeficiency *Anoectochilus roxburghii* intervention group was higher than that of the model group, indicating that *Anoectochilus roxburghii* aerosol has an effect on improving immunity in immunodeficient rats. However, compared with the immunodeficiency *Yu Ping Feng* intervention group and the immunodeficiency *Xi Gua Shuang* intervention group, the increase in spleen coefficient was lower, indicating that the ability of *Anoectochilus roxburghii* aerosol to improve immunity is relatively limited compared to existing drugs on the market.

[0081] After complete healing of oral ulcers, the immunocompromised group generally showed an increased spleen coefficient, with the group taking medication showing a greater increase. This indicates splenomegaly in the immunocompromised group, suggesting that the immune system is playing an anti-inflammatory role. The increased spleen coefficient in the medication group was greater than in the no-intervention group, demonstrating that *Anoectochilus roxburghii* aerosol, *Yu Ping Feng San*, and *Watermelon Frost* spray all contribute to improving the body's immunity. The increase in spleen coefficient after healing with *Watermelon Frost* was relatively limited, suggesting that this drug may be more effective in improving immunity under immunocompromised conditions. The comparison shows that the spleen coefficient was significantly increased in the *Anoectochilus roxburghii* group under immunocompromised conditions, possibly indicating that the main function of the components in *Anoectochilus roxburghii* is to treat immunocompromise.

[0082] In summary, although recurrent aphthous ulcers are now a common condition of the oral mucosa, and research on them has made significant progress both domestically and internationally, it is undeniable that there is still no cure, and the specific pathogenic mechanism remains difficult to diagnose. However, efforts are being made both domestically and internationally to make treatments for recurrent aphthous ulcers more convenient and efficient. This is reflected in many aspects, such as the choice of dosage forms. From the earliest common oral liquids and powders, more convenient dosage forms such as sprays have gradually been developed, as well as more efficient forms such as liniments and thermosensitive gels to overcome inconvenience. In recent years, laser therapy has become an innovative treatment method in Western medicine, with significant therapeutic effects and no toxic side effects. Traditional Chinese medicine has also drawn inspiration from its traditional medical practices. In the treatment of recurrent aphthous ulcers, acupuncture has been introduced into TCM techniques. This technique involves inserting needles into specific acupoints to achieve a curative effect on the specific disease. The biggest advantage of this acupuncture technique is that it has low toxicity and side effects, high efficiency, and safety when treating recurrent oral ulcers. However, the biggest problem is that more samples are still needed for research and verification, so further confirmation of its efficacy is required.

[0083] In summary, numerous factors contribute to the formation of recurrent aphthous ulcers, and the underlying mechanisms are extremely complex and difficult to summarize. The occurrence of recurrent aphthous ulcers is the result of a combination of factors. Thoroughly understanding the mechanisms and causes of recurrent aphthous ulcers, both in depth and breadth, is crucial for finding an effective treatment that can fundamentally inhibit recurrence. Currently, mainstream academic research focuses primarily on the immune system, and a large body of literature supports the crucial role of the immune system in the development of recurrent aphthous ulcers. Within the immune system, the inflammatory cytokines most closely associated with RUA formation are IL-2, IL-10, and TNF-α. IL-2, or interleukin-2, is one of the most important cytokines in the immune system and plays a crucial role in the immune response, typically activating T cells. IL-10, also known as interleukin-10, is a negative regulator of the immune system, negatively modulating the immune response; increased levels of anti-inflammatory factor IL-10 often indicate a decrease in certain immune products. TNF-α, or tumor necrosis factor, produced by macrophages, possesses strong immunomodulatory properties and inhibits tumor growth. In the final ELISA assay, *Anoectochilus roxburghii* showed higher levels of positively regulating cytokines like IL-2 and TNF-α compared to the control group, while lower levels of negatively regulating cytokines like IL-10 were observed. This suggests that *Anoectochilus roxburghii* has a positive effect on the body's immune response, which may explain its ability to promote oral ulcers. Furthermore, the group that took *Anoectochilus roxburghii* showed a significantly increased spleen coefficient, and its effects under immunodeficiency conditions were similar to and even surpassed those of mainstream immunomodulatory drugs for treating oral ulcers.

[0084] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A formula for treating oral ulcers using *Anoectochilus roxburghii*, characterized in that, The formulation comprises the following components by weight percentage: The contents of the product were: 1.0% flavonoids from Anoectochilus roxburghii, 0.15% Tween-20, 0.5% propylene glycol, and 0.002% benzalkonium chloride.

2. A Golden Thread Lotus aerosol, characterized in that, The *Anoectochilus roxburghii* aerosol contains the *Anoectochilus roxburghii* oral ulcer treatment formula as described in claim 1.

3. A method for preparing a *Anoectochilus roxburghii* aerosol, characterized in that, The method includes the following steps: Weigh out 8.0-12.0 g of Anoectochilus roxburghii flavonoids and dissolve them in 200 mL of pure water at 50 °C. Stir until completely dissolved, then add 0.018-0.022 g of benzalkonium chloride and stir until dissolved. Then add Tween-20 (1.4-1.6 g), stir until homogeneous, form a solution, and add propylene glycol (4.8-5.2 mL) and mix well. Finally, add pure water to 1000 mL, filter and sterilize using a 0.22 μm microporous membrane, fill the liquid into an aerosol can, seal it, and fill it with propellant to obtain the Anoectochilus roxburghii aerosol.

4. A method for treating oral ulcers with the *Anoectochilus roxburghii* oral ulcer treatment formula according to claim 1, or the *Anoectochilus roxburghii* aerosol prepared according to claim 2 or claim 3.

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