Qingjiang aurantii extract with soothing and repairing effects on atopic dermatitis and application thereof

Abstract

The application discloses a Qingjiang Aurantii Fructus extract with soothing and repairing effects on atopic dermatitis and application thereof, and a preparation method of the Qingjiang Aurantii Fructus extract comprises the following steps: A1, crushing Qingjiang Aurantii Fructus, adding water to soak, and then sterilizing; A2, inoculating probiotics in the solution after sterilization in step A1 to perform fermentation; A3, filtering the obtained fermentation liquor after sterilization, and purifying to obtain the Qingjiang Aurantii Fructus extract; wherein the probiotics are at least one selected from lactic acid bacteria, bacillus and yeast fungi. The Qingjiang Aurantii Fructus extract obtained by fermenting Qingjiang Aurantii Fructus with specific probiotics has significant soothing, repairing and anti-inflammatory effects, and has the soothing and repairing effects on atopic dermatitis in particular.

Description

Technical Field

[0001] This invention belongs to the field of daily chemical product technology, specifically relating to a method for preparing and applying a Qingjiang Zhi Ke extract with soothing and repairing effects, and particularly to a Qingjiang Zhi Ke extract with soothing and repairing effects on atopic dermatitis and its application. Background Technology

[0002] Atopic dermatitis (AD) is a chronic, relapsing, inflammatory skin disease clinically characterized by rashes on the face, extremities, eyelids, and backs of the hands and feet, accompanied by intense itching. The etiology of atopic dermatitis is unclear, but it may be related to genetics, immunity, environmental factors, and abnormal skin barrier function. Common clinical manifestations include dry skin, eczematous rashes, and intense itching. Especially for moderate to severe cases, the condition is protracted and difficult to cure, causing long-term suffering from intense itching, severe skin lesions, insomnia, and other symptoms, significantly impacting quality of life. The pathogenesis of atopic dermatitis (AD) is a complex vicious cycle network of "barrier-inflammation-itch". Impaired barrier function leads to the invasion of allergens and bacteria, which activates the immune system and then triggers inflammation and itching (mast cell degranulation and histamine release). Itching prompts scratching, which further damages the barrier, forming a vicious cycle of "barrier-inflammation-itch". Therefore, the core of treatment is to break this cycle, which requires both repairing the barrier (repair function) and blocking key signaling pathways (secretion of inflammatory factors such as TLSP, IL-8, IL-6, and TNF-α).

[0003] Traditional Chinese medicine (TCM) has a long history, outstanding efficacy, and few adverse reactions. External TCM treatments also show clear clinical effectiveness in treating Alzheimer's disease (AD). In the acute phase of AD, wet compresses and medicated baths are commonly used, while gels, ointments, and creams are frequently used in the chronic phase. These treatments are safe, effective, and easily accepted by patients, demonstrating significant advantages in AD treatment. CN119633052A discloses a TCM wash for treating infantile eczema, composed of honeysuckle, dictamnus root bark, cicada slough, raw peppermint, scutellaria root, fleeceflower stem, forsythia, stir-fried immature bitter orange peel, dandelion, and raw licorice, along with its preparation method. This wash possesses the effects of dispelling wind and clearing heat, removing dampness, and relieving itching. CN109464369A discloses an anti-dermatitis bath liquid containing extracts of traditional Chinese medicine made from Cnidium monnieri, safflower, yam, seahorse, Lobelia chinensis, Saposhnikovia divaricata, Coptis chinensis, Poria cocos, Prunella vulgaris, Citrus aurantium, Curculigo orchioides, and gypsum. It utilizes warm bath water to promote drug penetration into the skin, solving the problem of significant side effects from long-term use in existing anti-dermatitis treatments, and achieving significant improvement in symptoms and quality of life for neurogenic and atopic dermatitis. CN112546108A discloses a compound traditional Chinese medicine eczema ointment composed of Schizonepeta tenuifolia, Saposhnikovia divaricata, Cicadae periostracum, Bombyx mori, Forsythia suspensa, Poria cocos, Citrus aurantium, Paeonia lactiflora, Kochia scoparia, Vigna angularis, oil phase, and water phase, and its preparation method. This solves the problem of inconvenient ointment use, preserves the efficacy of the medicine, and allows for rapid skin absorption, providing convenient anti-allergic and antipruritic treatment effects.

[0004] Currently, most Chinese herbal extracts consist of multiple herbs with complex chemical compositions, and may contain ineffective or even harmful substances, posing risks to product use. Therefore, it is highly significant to identify plant-based ingredients that truly deliver soothing, repairing, anti-inflammatory, and anti-itch benefits. Plants, after fermentation, produce active substances with significant skincare benefits, and numerous studies have reported obtaining cosmetic raw materials with significant skincare effects through the microbial fermentation of Chinese herbs.

[0005] Zhangshu City (formerly Qingjiang County), Jiangxi Province, formerly known as "Shangzhou," has long been renowned as the "Hometown of Bitter Orange Peel." The famous Ming Dynasty physician Li Shizhen recorded in his *Compendium of Materia Medica* that "Bitter orange peel...from Shangzhou is the best." Qingjiang bitter orange peel, a locally produced medicinal material from Zhangshu, Jiangxi, is characterized by its green skin, thick flesh, firm texture, and strong aroma. It is described as having "thick flesh, turning outwards like an inverted bowl, with numerous segments," and its medicinal active ingredients are superior to other varieties. The chemical components of bitter orange peel include flavonoids, coumarins, and volatile oils, with flavonoids being the main chemical components and active ingredients. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a Qingjiang Zhi Ke extract with soothing and repairing effects and its application, especially a Qingjiang Zhi Ke extract with soothing and repairing effects on atopic dermatitis and its application.

[0007] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for preparing an extract of Citrus aurantium, comprising the following steps: A1. After crushing the Qingjiang bitter orange peel, soak it in water and then sterilize it; A2. Inoculate probiotics into the sterilized solution in step A1 for fermentation; A3. After sterilizing the fermentation broth obtained in step A2, filter it. The filtrate is then purified using resin to obtain the Qingjiang Zhi Ke extract. The probiotics are selected from at least one of lactic acid bacteria (including at least one of Lactobacillus acidophilus and Lactobacillus bulgaricus), Bacillus (including at least one of Bacillus licheniformis, Bacillus subtilis, and Bacillus cereus), and yeast fungi (including at least one of Saccharomyces cerevisiae and Saccharomyces petroleum).

[0008] As a preferred embodiment, in step A1, the amount of water added is 15-20 times the amount of water, that is, 1g of Qingjiang Zhi Ke is added to 15-20 ml of water.

[0009] As a preferred embodiment, in step A1, after the Qingjiang citrus peel is crushed, it also includes the step of passing it through a 40-60 mesh sieve.

[0010] As a preferred embodiment, in step A2, the probiotics are selected from at least one of Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus bulgaricus.

[0011] As a preferred embodiment, the probiotic is Bacillus subtilis or a combination of Bacillus subtilis and Saccharomyces cerevisiae.

[0012] As a preferred embodiment, when the probiotic is a compound of Bacillus subtilis and Saccharomyces cerevisiae, the inoculation ratio of Bacillus subtilis to Saccharomyces cerevisiae is 1-2:1. More preferably, the inoculation ratio of Bacillus subtilis to Saccharomyces cerevisiae is 1:1.

[0013] As a preferred embodiment, step A2 further includes activation and expansion culture steps before inoculating the probiotics.

[0014] As a further preferred embodiment, the activation and expansion culture steps are as follows: the probiotics are inoculated onto an activation medium for activation, and then the activated probiotics are inoculated onto a seed medium for expansion culture.

[0015] As a preferred embodiment, the bacterial concentration of the probiotic seed liquid obtained after expanded culture is 10. 6 -10 8 CFU / mL.

[0016] As a preferred embodiment, the ratio of the mass of *Citrus aurantium* to the inoculation amount of probiotics is 20 g: 1.0-5.0 × 10⁻⁶. 9 CFU.

[0017] As a further preferred embodiment, the ratio of the mass of *Citrus aurantium* to the inoculation amount of probiotics is 20 g: 1.0-3.0 × 10⁻⁶. 9 CFU. The optimal ratio is 20 g: 3.0 × 10⁻⁶. 9 CFU.

[0018] As a preferred embodiment, in step A2, the fermentation conditions are: fermentation at 37℃ and 200r / min for 48-96h.

[0019] As a further preferred option, the fermentation time is 48-72 hours.

[0020] As a preferred embodiment, in step A3, the resin type is Seplite LX-94.

[0021] Secondly, the present invention provides a Qingjiang Zhi Ke extract prepared according to the aforementioned method.

[0022] Thirdly, the present invention provides the application of the Qingjiang Zhi Ke extract prepared according to the aforementioned method or the Qingjiang Zhi Ke extract according to claim 8 in the preparation of cosmetics or pharmaceuticals with soothing and repairing effects.

[0023] As a preferred embodiment, the application includes the preparation of cosmetics or pharmaceuticals that have a soothing and repairing effect on atopic dermatitis.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention utilizes specific types of probiotics to ferment *Citrus aurantium* (Qingjiang Citrus aurantium), resulting in an extract that exhibits significant soothing, repairing, and anti-inflammatory effects, particularly for atopic dermatitis. Specifically, the *Citrus aurantium* extract prepared using *Bacillus subtilis* fermentation showed a healing rate exceeding 54.81%, a mast cell degranulation rate below 35.00%, and IL-6 and TNF-α secretion levels below (834.82±2.14) pg / mL and (18800.32±336.97) pg / mL, respectively, while IL-8 and TLSP secretion levels were below (877.29±9.49) pg / mL and (11.61±0.72) pg / mL, respectively. Furthermore, human efficacy evaluation experiments have demonstrated the soothing effects of the *Citrus aurantium* extract prepared using *Bacillus subtilis* fermentation.

[0025] 2. Furthermore, this invention utilizes a specific ratio of probiotics, Bacillus subtilis and Saccharomyces cerevisiae, to ferment and prepare the Qingjiang Citrus aurantium extract, achieving a healing rate of 81.52% and obtaining better repair effects. Attached Figure Description

[0026] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 A typical example of the redness and swelling reduction effect of Antera3D photography on subjects; Figure 2 Typical example two of the redness and swelling reduction effects of Antera3D photography on subjects. Detailed Implementation

[0027] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0028] The terms "preferred," "more preferably," and "more suitable" used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0029] It should be understood that, except in any operational instance or otherwise indicated, all figures representing the amounts of ingredients used, for example, in the specification and claims, should be understood to be modified in all cases by the term "about". Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximations varying with the desired performance to be obtained according to the invention. It is not at all an attempt to limit the application of the doctrine of equivalents to the scope of the claims; each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques.

[0030] Although the numerical ranges and parameters illustrating the broad scope of the invention are approximate, the values ​​listed in the specific examples are reported as precisely as possible. However, any numerical value inherently contains some error that is necessarily caused by the standard deviation found in their respective test measurements.

[0031] In a specific embodiment of the present invention, a Qingjiang Zhi Ke extract with soothing and repairing effects is obtained by using plant microbial fermentation technology to extract Qingjiang Zhi Ke.

[0032] The plant microbial fermentation used in this invention is not only an extraction technology, but also a biotransformation technology. It transforms plant resources into high-value products through microbial processing.

[0033] In the extraction process of Citrus aurantium from Qingjiang in this invention, the addition of microbial fermentation generates catalytic enzymes. On one hand, the fermenting bacteria release various hydrolytic enzymes, effectively disrupting the cell wall structure and releasing the active ingredient, Citrus aurantium flavonoids, from the cell wall. On the other hand, it degrades large flavonoid glycosides into flavonoid aglycones; these lower molecular weight forms generally have higher bioavailability and stronger pharmacological activity.

[0034] This application provides a general and / or specific description of the materials and experimental methods used in the experiments.

[0035] In the following examples, all raw materials and reagents used were commercially available products or prepared by existing methods, and the present invention does not impose any particular limitations. For example, Bacillus subtilis (strain BNCC339991), Saccharomyces cerevisiae (strain BNCC362974), Lactobacillus bulgaricus (strain BNCC186626), and Lactobacillus casei (strain BNCC186562) were all purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0036] Example 1 This embodiment provides a method for obtaining Qingjiang Citrus aurantium extract by fermentation with Bacillus subtilis, which is mainly carried out according to the following steps: (1) Pulverizing medicinal materials: Select the bitter orange peel that meets the requirements of the Jiangxi Provincial Local Standard DB36 / T 1467-2021 Geographical Indication Product Qingjiang Zhi Ke, pulverize it, and pass it through a 40-mesh sieve; (2) Seed culture preparation: Colonies were picked from the Bacillus subtilis preservation slant and inoculated onto the activation medium (nutrient agar medium) for activation to obtain the activated strain; the activated strain was inoculated into the seed medium (LB liquid medium) for expansion culture to obtain Bacillus subtilis seed culture; (3) Plant fermentation: Weigh 20g of crushed and sieved bitter orange peel powder, add 370ml of water, soak for 10 minutes, and then sterilize at 121℃ for 15 minutes; after cooling, inoculate 30ml of Bacillus subtilis seed solution (1.0×10⁻⁶) in a clean bench. 8 The fermentation was carried out at 37℃ and 200r / min for 72h (CFU / mL). After fermentation, the mixture was sterilized and filtered through a 0.22μm filter membrane. The filtrate was the fermentation filtrate of Citrus aurantium.

[0037] (4) Resin purification: Take 20g of pre-treated resin (seplite LX-94, Xi'an Lanxiao Technology New Material Co., Ltd.) and fill it into a resin column (column volume BV is about 30ml); take the fermentation filtrate of Citrus aurantium obtained in step (3) and load it at a flow rate of 1.0 BV / h, collect the eluent, and obtain the extract of Citrus aurantium from Qingjiang.

[0038] Example 2 This embodiment provides a method for obtaining Qingjiang Zhi Ke extract by fermentation with Bacillus subtilis. The operation steps are basically the same as in Example 1, except that in step (3), 390 ml of water is added and 10 ml of Bacillus subtilis seed solution (1.0 × 10⁻⁶) is inoculated. 8 (CFU / mL).

[0039] Example 3 This embodiment provides a method for obtaining Qingjiang Zhi Ke extract by fermentation with Bacillus subtilis. The operation steps are basically the same as in Example 1, except that in step (3), 350 ml of water is added and 50 ml of Bacillus subtilis seed solution (1.0 × 10⁻⁶) is inoculated. 8 (CFU / mL).

[0040] Example 4 This embodiment provides a method for obtaining Qingjiang Zhi Ke extract by fermentation with Bacillus subtilis. The operation steps are basically the same as those in Embodiment 2, except that the fermentation time in step (3) is 48h.

[0041] Example 5 This embodiment provides a method for obtaining Qingjiang Zhi Ke extract by fermentation with Bacillus subtilis. The operation steps are basically the same as those in Example 3, except that the fermentation time in step (3) is 96 hours.

[0042] Example 6 This embodiment provides a method for obtaining Qingjiang Zhi Ke extract using brewer's yeast fermentation, which mainly involves the following steps: (1) Pulverizing medicinal materials: Select the bitter orange peel that meets the requirements of the Jiangxi Provincial Local Standard DB36 / T 1467-2021 Geographical Indication Product Qingjiang Zhi Ke, pulverize it, and pass it through a 40-mesh sieve; (2) Seed culture preparation: Colonies were picked from the preserved slant of Saccharomyces cerevisiae and inoculated onto the activation medium (YPD solid medium) for activation to obtain the activated strain; the activated strain was inoculated into the seed medium (YPD medium) for expansion culture to obtain the Saccharomyces cerevisiae seed culture; (3) Plant fermentation: Weigh 20g of crushed and sieved bitter orange peel powder, add 370ml of water, soak for 10 minutes, and then sterilize at 121℃ for 15 minutes; after cooling, inoculate 30ml of brewer's yeast seed liquid (1.0×10⁻⁶) in a clean bench. 8 The fermentation was carried out at 30℃ and 200r / min for 72h (CFU / mL). After fermentation, the mixture was sterilized and filtered through a 0.22μm filter membrane. The filtrate was the fermentation filtrate of Citrus aurantium.

[0043] (4) Resin purification: Take 20g of pre-treated resin (seplite LX-94, Xi'an Lanxiao Technology New Material Co., Ltd.) and fill it into a resin column (column volume BV is about 30ml); take the fermentation filtrate of Citrus aurantium obtained in step (3) and load it at a flow rate of 1.0 BV / h, collect the eluent, and obtain the extract of Citrus aurantium from Qingjiang.

[0044] Example 7 This embodiment provides a method for obtaining Qingjiang bitter orange extract by fermentation with Saccharomyces cerevisiae. The operation steps are basically the same as those in Example 6, except that in step (3), 390 ml of water is added and 10 ml of Saccharomyces cerevisiae seed liquid (1.0 × 10⁻⁶) is inoculated. 8 (CFU / mL), fermented for 48 hours.

[0045] Example 8 This embodiment provides a method for obtaining Qingjiang bitter orange extract by fermentation with Saccharomyces cerevisiae. The operation steps are basically the same as those in Example 6, except that in step (3), 350 ml of water is added and 50 ml of Saccharomyces cerevisiae seed liquid (1.0 × 10⁻⁶) is inoculated. 8 CFU / mL), fermented for 96 hours.

[0046] Example 9 This embodiment provides a method for obtaining Qingjiang Zhi Ke extract by fermentation with Lactobacillus bulgaricus, which is mainly carried out according to the following steps: (1) Pulverizing medicinal materials: Select the bitter orange peel that meets the requirements of the Jiangxi Provincial Local Standard DB36 / T 1467-2021 Geographical Indication Product Qingjiang Zhi Ke, pulverize it, and pass it through a 40-mesh sieve; (2) Seed culture preparation: Colonies were picked from the preserved slant of Lactobacillus bulgaricus and inoculated onto the activation medium (MRS solid medium) for activation to obtain the activated strain; the activated strain was inoculated into the seed medium (MRS broth medium) for expansion culture to obtain Lactobacillus bulgaricus seed culture; (3) Plant fermentation: Weigh 20g of crushed and sieved bitter orange peel powder, add 390ml of water, soak for 10 minutes, and then sterilize at 121℃ for 15 minutes; after cooling, inoculate 10ml of Lactobacillus bulgaricus seed solution (1.0×10⁻⁶) in a clean bench. 8 The fermentation was carried out at 37℃ and 200r / min for 48h (CFU / mL). After fermentation, the mixture was sterilized and filtered through a 0.22μm filter membrane. The filtrate was the fermentation filtrate of Citrus aurantium.

[0047] (4) Resin purification: Take 20g of pre-treated resin (seplite LX-94, Xi'an Lanxiao Technology New Material Co., Ltd.) and fill it into a resin column (column volume BV is about 30ml); take the fermentation filtrate of Citrus aurantium obtained in step (3) and load it at a flow rate of 1.0 BV / h, collect the eluent, and obtain the Citrus aurantium extract of Qingjiang described in this invention.

[0048] Example 10 This embodiment provides a method for obtaining Qingjiang Zhi Ke extract by fermentation with Lactobacillus bulgaricus. The operation steps are basically the same as those in Example 9, except that in step (3), 350 ml of water is added and 50 ml of Lactobacillus bulgaricus seed solution (1.0 × 10⁻⁶) is inoculated. 8 CFU / mL), fermented for 96 hours.

[0049] Example 11 This embodiment provides a method for obtaining Qingjiang Zhi Ke extract using Lactobacillus casei fermentation, which mainly involves the following steps: (1) Pulverizing medicinal materials: Select the bitter orange peel that meets the requirements of the Jiangxi Provincial Local Standard DB36 / T 1467-2021 Geographical Indication Product Qingjiang Zhi Ke, pulverize it, and pass it through a 40-mesh sieve; (2) Seed culture preparation: Colonies were picked from the preserved Lactobacillus casei slant and inoculated onto the activation medium (MRS solid medium) for activation to obtain the activated strain; the activated strain was inoculated into the seed medium (MRS broth medium) for expansion culture to obtain Lactobacillus casei seed culture; (3) Plant fermentation: Weigh 20g of crushed and sieved bitter orange peel powder, add 370ml of water, soak for 10 minutes, and then sterilize at 121℃ for 15 minutes; after cooling, inoculate 30ml of Lactobacillus casei seed solution (1.0×10⁻⁶) in a clean bench. 8 The fermentation was carried out at 37℃ and 200r / min for 72h (CFU / mL). After fermentation, the mixture was sterilized and filtered through a 0.22μm filter membrane. The filtrate was the fermentation filtrate of Citrus aurantium.

[0050] (4) Resin purification: Take 20g of pre-treated resin (seplite LX-94, Xi'an Lanxiao Technology New Material Co., Ltd.) and fill it into a resin column (column volume BV is about 30ml); take the fermentation filtrate of Citrus aurantium obtained in step (3) and load it at a flow rate of 1.0 BV / h, collect the eluent, and obtain the Citrus aurantium extract of Qingjiang described in this invention.

[0051] Example 12 This embodiment provides a method for obtaining Qingjiang Citrus aurantium extract by fermentation using a combination of Bacillus subtilis and Saccharomyces cerevisiae, which mainly involves the following steps: (1) Pulverizing medicinal materials: Select the bitter orange peel that meets the requirements of the Jiangxi Provincial Local Standard DB36 / T 1467-2021 Geographical Indication Product Qingjiang Zhi Ke, pulverize it, and pass it through a 40-mesh sieve; (2) Seed culture preparation: Colonies were picked from the Bacillus subtilis preservation slant and inoculated onto the activation medium (nutrient agar medium) for activation to obtain the activated strain; the activated strain was inoculated into the seed medium (LB liquid medium) for expansion culture to obtain Bacillus subtilis seed culture; colonies were picked from the Saccharomyces cerevisiae preservation slant and inoculated onto the activation medium (YPD solid medium) for activation to obtain the activated strain; the activated strain was inoculated into the seed medium (YPD medium) for expansion culture to obtain Saccharomyces cerevisiae seed culture; (3) Plant fermentation: Weigh 20g of crushed and sieved bitter orange peel powder, add 370ml of water, soak for 10 minutes, and then sterilize at 121℃ for 15 minutes; after cooling, inoculate 15ml of Bacillus subtilis seed solution (1.0×10⁻⁶) in a clean bench. 8 CFU / mL) and 15 ml of Saccharomyces cerevisiae seed culture (1.0 × 10⁻⁶ CFU / mL) 8 The fermentation was carried out at 37℃ and 200r / min for 72h (CFU / mL). After fermentation, the mixture was sterilized and filtered through a 0.22μm filter membrane. The filtrate was the fermentation filtrate of Citrus aurantium.

[0052] (4) Resin purification: Take 20g of pre-treated resin (seplite LX-94, Xi'an Lanxiao Technology New Material Co., Ltd.) and fill it into a resin column (column volume BV is about 30ml); take the fermentation filtrate of Citrus aurantium obtained in step (3) and load it at a flow rate of 1.0 BV / h, collect the eluent, and obtain the extract of Citrus aurantium from Qingjiang.

[0053] Example 13 This embodiment provides a method for obtaining Qingjiang citrus extract by fermentation with Saccharomyces cerevisiae. The operation steps are basically the same as those in Example 12, except that in step (3), 20 ml of Bacillus subtilis seed solution (1.0 × 10⁻⁶) is inoculated. 8 CFU / mL) and 10 ml of Saccharomyces cerevisiae seed culture (1.0 × 10⁻⁶ CFU / mL) 8 (CFU / mL).

[0054] Example 14 This embodiment provides a method for obtaining Qingjiang Zhi Ke extract by fermentation with Saccharomyces cerevisiae. The operation steps are basically the same as those in Example 12, except that in step (3), 10 ml of Bacillus subtilis seed solution (1.0 × 10⁻⁶) is inoculated. 8 CFU / mL) and 20 ml of Saccharomyces cerevisiae seed culture (1.0 × 10⁻⁶ CFU / mL) 8 (CFU / mL).

[0055] Example 15 This embodiment provides a method for obtaining Qingjiang Citrus aurantium extract by fermentation using a combination of Bacillus subtilis and Lactobacillus bulgaricus, which is mainly carried out according to the following steps: (1) Pulverizing medicinal materials: Select the bitter orange peel that meets the requirements of the Jiangxi Provincial Local Standard DB36 / T 1467-2021 Geographical Indication Product Qingjiang Zhi Ke, pulverize it, and pass it through a 40-mesh sieve; (2) Seed culture preparation: Colonies were picked from the Bacillus subtilis preservation slant and inoculated onto the activation medium (nutrient agar medium) for activation to obtain the activated strain; the activated strain was inoculated into the seed medium (LB liquid medium) for expansion culture to obtain Bacillus subtilis seed culture; colonies were picked from the Lactobacillus bulgaricus preservation slant and inoculated onto the activation medium (MRS solid medium) for activation to obtain the activated strain; the activated strain was inoculated into the seed medium (MRS broth medium) for expansion culture to obtain Lactobacillus bulgaricus seed culture; (3) Plant fermentation: Weigh 20g of crushed and sieved bitter orange peel powder, add 370ml of water, soak for 10 minutes, and then sterilize at 121℃ for 15 minutes; after cooling, inoculate 15ml of Bacillus subtilis seed solution (1.0×10⁻⁶) in a clean bench. 8 CFU / mL) and 15 ml of Lactobacillus bulgaricus seed culture (1.0 × 10⁻⁶ CFU / mL) 8 The fermentation was carried out at 37℃ and 200r / min for 72h (CFU / mL). After fermentation, the mixture was sterilized and filtered through a 0.22μm filter membrane. The filtrate was the fermentation filtrate of Citrus aurantium.

[0056] (4) Resin purification: Take 20g of pre-treated resin (seplite LX-94, Xi'an Lanxiao Technology New Material Co., Ltd.) and fill it into a resin column (column volume BV is about 30ml); take the fermentation filtrate of Citrus aurantium obtained in step (3) and load it at a flow rate of 1.0 BV / h, collect the eluent, and obtain the extract of Citrus aurantium from Qingjiang.

[0057] Example 16 This embodiment provides a method for obtaining Qingjiang Citrus aurantium extract by fermentation using a combination of Saccharomyces cerevisiae and Lactobacillus bulgaricus, which mainly involves the following steps: (1) Pulverizing medicinal materials: Select the bitter orange peel that meets the requirements of the Jiangxi Provincial Local Standard DB36 / T 1467-2021 Geographical Indication Product Qingjiang Zhi Ke, pulverize it, and pass it through a 40-mesh sieve; (2) Seed culture preparation: Colonies were picked from the preserved slant of Saccharomyces cerevisiae and inoculated onto activation medium (YPD solid medium) for activation to obtain activated strains; the activated strains were inoculated into seed medium (YPD medium) for expansion culture to obtain Saccharomyces cerevisiae seed culture; colonies were picked from the preserved slant of Lactobacillus bulgaricus and inoculated onto activation medium (MRS solid medium) for activation to obtain activated strains; the activated strains were inoculated into seed medium (MRS broth medium) for expansion culture to obtain Lactobacillus bulgaricus seed culture; (3) Plant fermentation: Weigh 20g of crushed and sieved bitter orange peel powder, add 370ml of water, soak for 10 minutes, and then sterilize at 121℃ for 15 minutes; after cooling, inoculate 15ml of Bacillus subtilis seed solution (1.0×10⁻⁶) in a clean bench. 8 CFU / mL) and 15 ml of Lactobacillus bulgaricus seed culture (1.0 × 10⁻⁶ CFU / mL) 8 The fermentation was carried out at 37℃ and 200r / min for 72h (CFU / mL). After fermentation, the mixture was sterilized and filtered through a 0.22μm filter membrane. The filtrate was the fermentation filtrate of Citrus aurantium.

[0058] (4) Resin purification: Take 20g of pre-treated resin (seplite LX-94, Xi'an Lanxiao Technology New Material Co., Ltd.) and fill it into a resin column (column volume BV is about 30ml); take the fermentation filtrate of Citrus aurantium obtained in step (3) and load it at a flow rate of 1.0 BV / h, collect the eluent, and obtain the extract of Citrus aurantium from Qingjiang.

[0059] Comparative Example 1 This comparative example provides a method for obtaining Qingjiang Zhi Ke extract, which mainly involves the following steps: (1) Pulverizing medicinal materials: Select the bitter orange peel that meets the requirements of the Jiangxi Provincial Local Standard DB36 / T 1467-2021 Geographical Indication Product Qingjiang Zhi Ke, pulverize it, and pass it through a 40-mesh sieve; (3) Blank fermentation: Weigh 20g of crushed and sieved bitter orange powder, add 400ml of water, soak and sterilize at 121℃ for 15min; then ferment blank at 37℃ and 200r / min for 72h; after fermentation, sterilize and filter with a 0.22μm filter membrane, the filtrate is bitter orange fermentation filtrate.

[0060] (4) Resin purification: Take 20g of pre-treated resin (seplite LX-94, Xi'an Lanxiao Technology New Material Co., Ltd.) and fill it into a resin column (column volume BV is about 30ml); take the fermentation filtrate of Citrus aurantium obtained in step (3) and load it at a flow rate of 1.0 BV / h, collect the eluent, and obtain the extract of Citrus aurantium from Qingjiang.

[0061] The fermentation process parameters used in the above embodiments and comparative examples are shown in Table 1 below.

[0062] Table 1 Efficacy verification: Verification Experiment Example 1: Evaluation Experiment of the Repairing Efficacy of Extracts The extracts of Citrus aurantium prepared using Examples 1-16 and Comparative Example 1 were used as test samples to test the healing properties of Hacat keratinocytes (purchased from Beina Chuanglian Biotechnology Co., Ltd.). The specific test method was as follows: 1) Collect cells in the logarithmic growth phase, at a cell density of 2 × 10⁻⁶. 5 Inoculate one well per well into a 24-well culture plate.

[0063] 2) After culturing in an incubator (37℃, 5% CO2) for 24-48 hours, treat the cells with mitomycin C (10 μg / mL) for 1 hour. Then, use a 200 μL pipette tip to draw "damage" on a 24-well plate. Wash the cells with PBS 1-3 times to remove the drawn cells.

[0064] 3) According to the experimental groups, 1 ml of FBS-free medium was added to the negative control group, 1 ml of medium containing 10% FBS was added to the positive control group, and 900 μl of FBS-free medium and 100 μl of each test sample (the Qingjiang Zhi Ke extract prepared in Examples 1-16 and Comparative Example 1 was diluted to 0.5% with PBS solution) were added to each sample group. The samples were incubated in an incubator (37℃, 5% CO2) for 24 h, with 3 replicates per group.

[0065] 4) Photograph each group of migrating cells using an inverted microscope, and calculate the average scratch area using Image Pro Plus software. Healing rate % = (initial scratch area - current scratch area) / initial scratch area × 100%.

[0066] 5) Experimental results: The average healing rate of HaCaT cells in the negative control group was 25.92%±0.52%, and the average healing rate of HaCaT cells in the positive control group was 82.36%±0.91%. The healing rate of HaCaT cells by Qingjiang Zhi Ke extract compared with the negative control is shown in Table 2 below.

[0067] Table 2 As shown in Table 2, the average healing rate of the *Citrus aurantium* extract prepared in each example ranged from 31.83% to 81.52%, which was significantly higher than that of the negative control group, indicating that the *Citrus aurantium* extract prepared in each example had a repairing effect. In contrast, the average healing rate of the *Citrus aurantium* extract prepared in Comparative Example 1 was only 28.68%, a significant difference from the other examples, indicating that fermentation with probiotics can significantly improve the repairing effect of the *Citrus aurantium* extract. Furthermore, a comparison of the results of Examples 1-5 and Examples 6-11 shows that the healing rate of the *Citrus aurantium* extract prepared by fermentation with *Bacillus subtilis* reached over 54.81%, and its repairing effect was significantly better than that of the *Citrus aurantium* extract prepared by fermentation with *Saccharomyces cerevisiae*, *Lactobacillus bulgaricus*, or *Lactobacillus casei*. Further comparisons of the results from Examples 1 and 2-3, Examples 2 and 4, and Examples 3 and 5 show that with 20g of Citrus aurantium and 30ml of seed culture (i.e., a ratio of Citrus aurantium to inoculum of 20g: 3.0×10⁻⁶), it is found that... 9 When CFU is used, the healing rate can be significantly improved and better repair effects can be obtained; when the fermentation time is 72h, the healing rate can be significantly improved and better repair effects can be obtained.

[0068] Further comparison of the results from Examples 12 and 15-16 shows that the healing rate of the *Citrus aurantium* extract prepared by combined fermentation with *Bacillus subtilis* and *Saccharomyces cerevisiae* is significantly higher than that prepared by combined fermentation with *Bacillus subtilis* and *Lactobacillus bulgaricus* or by combined fermentation with *Saccharomyces cerevisiae* and *Lactobacillus bulgaricus*. Comparison of the results from Examples 1 and 12-14 shows that the healing rate of the *Citrus aurantium* extract prepared by combined fermentation with *Bacillus subtilis* and *Saccharomyces cerevisiae* in a specific ratio of 1-2:1 can reach 71.09-81.52%, which is better than the *Citrus aurantium* extract prepared by fermentation with *Bacillus subtilis* alone in Example 1; while the healing rate of the *Citrus aurantium* extract prepared by combined fermentation with *Bacillus subtilis* and *Saccharomyces cerevisiae* in a ratio of 1:2 is worse than that in Example 1. This indicates that the present invention, by further employing a specific ratio of combined fermentation with *Bacillus subtilis* and *Saccharomyces cerevisiae*, can further improve the healing rate of the *Citrus aurantium* extract and obtain better repair efficacy.

[0069] Verification Experiment Example 2: Evaluation Experiment of the Antipruritic Efficacy of the Extract The extracts of Citrus aurantium prepared in Examples 1-11 and Comparative Example 1 were used as test samples to test the degranulation of mast cell P815 (purchased from Beina Chuanglian Biotechnology Co., Ltd.). The specific test method is as follows: 1) Cell seeding and preparation: 1×10 5 Cells were seeded at a density of cells per well into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2).

[0070] 2) Solution preparation: Add the Qingjiang Zhi Ke extract prepared in Examples 1-11 and Comparative Example 1 to PBS solution to prepare a test sample solution containing 0.5% test sample.

[0071] 3) Drug administration: When the cell deposition rate in the 24-well plate reaches 40%–50%, drugs are administered to the following groups, with three replicates per group. The blank control group and negative control group are each given 100 μl of PBS solution, the positive control group is given 100 μl of sodium cromoglycate solution (pre-diluted with PBS to 10 mg / ml), and the sample group is given 100 μl of the test sample solution. After drug administration, the 24-well plate is incubated in an incubator (37℃, 5% CO2) for 24 h.

[0072] 4) C48 / 80 stimulation: After incubation for 24 hours, the sample solution was discarded. According to the experimental groups, the blank control group was given 1 mL of serum-free high-glucose DMEM culture medium, the negative control group was given 990 μL of serum-free high-glucose DMEM culture medium and 10 μL of solution containing C48 / 80 (C48 / 80 was purchased from Sigma-Aldrich, USA, and prepared in advance with serum-free high-glucose DMEM culture medium to 100 μg / mL), the positive control group was given 890 μL of serum-free high-glucose DMEM culture medium, 100 μL of sodium cromoglycate solution and 10 μL of solution containing C48 / 80, and the sample group was given 890 μL of serum-free high-glucose DMEM culture medium, 100 μL of the test sample solution and 10 μL of solution containing C48 / 80. After the administration, C48 / 80 stimulation was performed for 45 min, and the reaction was terminated by ice bath at 0℃ for 10 min.

[0073] 5) Cell morphology observation: After culture, the degranulation of cells in each group was observed under an inverted microscope and photographed. The photos were analyzed and the degranulation rate was calculated using IPP software.

[0074] Degranulation rate (%) = Total number of degranulated cells / (Total number of degranulated cells + Total number of non-degranulated cells) 100% 6) Experimental Results: In the blank control group, mast cells were round and translucent, exhibiting normal cell morphology; in the negative control group, most mast cells were shriveled and dull, showing a degranulated morphology, and the degranulation rate was 91.97%±1.74% compared with the blank control group, significantly higher. p <0.01), indicating that the degranulation model was successfully established in this experiment; the positive control group was 14.04%±1.31%.

[0075] The mast cell degranulation rate of the extracts prepared from *Citrus aurantium* in each example (average values) is shown in Table 3. Compared with the negative control group, all of them significantly reduced the mast cell degranulation rate. pThe result <0.01 indicates that the *Citrus aurantium* extract prepared in each embodiment has antipruritic effects. While the mast cell degranulation rate of the *Citrus aurantium* extract prepared in Comparative Example 1 was significantly lower than that of the negative control group, it was significantly higher than that of the *Citrus aurantium* extracts prepared in each embodiment. Furthermore, a comparison of the results of Examples 1-5 and Examples 6-11 shows that the mast cell degranulation rate of the *Citrus aurantium* extract prepared by fermentation with *Bacillus subtilis* was only below 35.00%, and its antipruritic effect was significantly better than that of the *Citrus aurantium* extract prepared by fermentation with *Saccharomyces cerevisiae*, *Lactobacillus bulgaricus*, or *Lactobacillus casei*. Further comparison of the results of Examples 1 and Examples 2-3 shows that with a *Citrus aurantium* dosage of 20g and a seed culture volume of 30ml (i.e., a ratio of *Citrus aurantium* dosage to inoculum volume of 20g: 3.0×10⁻⁶), the *Citrus aurantium* dosage was significantly lower than that of the negative control group, but still significantly higher than that of the *Citrus aurantium* extracts prepared by fermentation in each embodiment. 9 When CFU is used, it can significantly reduce the degranulation rate of mast cells and achieve better antipruritic effects.

[0076] Table 3 Validation Experiment Example 3: Evaluation Experiment of the Anti-inflammatory (Macrophage) Efficacy of the Extract The extracts of Citrus aurantium prepared using Examples 1-11 and Comparative Example 1 were used to test the secretion of LPS (lipopolysaccharide)-induced inflammatory factors IL-6 and TNF-α in RAW264.7 cells. The specific test methods were as follows: 1) Cell seeding and preparation: Macrophages (purchased from Beina Chuanglian Biotechnology Co., Ltd.) were seeded at a rate of 1×10⁶ cells / year. 5 Cells were seeded at a density of cells per well into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2).

[0077] 2) Solution preparation: Add the Qingjiang Zhi Ke extract prepared in Examples 1-11 and Comparative Example 1 to PBS solution to prepare a test sample solution containing 0.5% test sample.

[0078] 3) Drug administration: According to the experimental groups, when the cell deposition rate in the 24-well plates reached 40%~50%, the drugs were administered to the groups. Each group had 3 replicates. 100 μl of PBS solution was added to the blank control group and the negative control group, 100 μl of dexamethasone solution (pre-diluted with PBS solution to 1 mg / ml) was added to the positive control group, and 100 μl of the sample solution to be tested was added to the sample group. After drug administration, the 24-well plates were placed in an incubator (37℃, 5% CO2) and incubated for 2 h.

[0079] 4) Stimulation: After the samples were cultured for 2 h after drug addition, 10 μl of LPS working solution (pre-diluted to 100 μg / ml with serum-containing high-glucose DMEM culture medium) was added to the well plates of the negative control group, positive control group and sample group according to the experimental design. The well plates were shaken left and right to mix the drug in the well plates. The final concentration of LPS was 1 μg / mL. The plates were then incubated in an incubator (37℃, 5% CO2) for 22 h.

[0080] 5) Sample collection: After incubation, collect the cell culture supernatant into EP tubes (Note: Determine the amount of sample to be collected according to the detection indicators). After collection, freeze the sample in a -80℃ freezer.

[0081] 6) Perform the test according to the ELISA kit's instruction manual.

[0082] 7) Experimental Results: In the blank control group, the secretion levels of inflammatory factors IL-6 and TNF-α were (71.54±0.84) pg / mL and (411.84±2.58) pg / mL, respectively; in the negative control group, the secretion levels of inflammatory factors IL-6 and TNF-α were (2858.32±9.80) pg / mL and (26378.52±143.28) pg / mL, respectively, which were significantly higher than those in the blank control group. p The value <0.01 indicates that the inflammation model was successfully established in this experiment. The secretion levels of inflammatory factors IL-6 and TNF-α in the positive control group were (1609.30±2.14) pg / mL and (14532.21±50.24) pg / mL, respectively.

[0083] The results (average values) of the Qingjiang Zhi Ke extract prepared in each example on the secretion of cellular inflammatory factors IL-6 and TNF-α are shown in Table 4. Compared with the negative control group, they were significantly reduced. p <0.05), indicating that the sample has anti-inflammatory effects. The Qingjiang Citrus aurantium extract prepared in Comparative Example 1 showed significantly lower levels of IL-6 and TNF-α secretion compared to the negative control group, but significantly higher levels compared to the Qingjiang Citrus aurantium extracts prepared in each example. Furthermore, a comparison of the results of Examples 1-5 and Examples 6-11 shows that the Qingjiang Citrus aurantium extract prepared by Bacillus subtilis fermentation had IL-6 and TNF-α secretion levels below (834.82±2.14) pg / mL and (18800.32±336.97) pg / mL, respectively, demonstrating significantly better anti-inflammatory effects than the Qingjiang Citrus aurantium extract prepared by fermentation with Saccharomyces cerevisiae, Lactobacillus bulgaricus, or Lactobacillus casei. Further comparison of the results of Examples 1 and Examples 2-3 shows that with 20g of Citrus aurantium and 10-30ml of seed culture (i.e., a ratio of Citrus aurantium to inoculum of 20g: 1.0-3.0×10⁻⁶), the anti-inflammatory effects were significantly better. 9When CFU is used, it can significantly reduce the secretion of IL-6 and TNF-α, thus achieving better anti-inflammatory effects.

[0084] Table 4 Validation Experiment Example 4: Evaluation Experiment of the Anti-inflammatory (Keratinocyte) Efficacy of the Extract The extracts of Citrus aurantium prepared using Examples 1-11 and Comparative Example 1 were used to test the secretion levels of IL-8 and TLSP, which are related to TNF-α and IFN-γ co-stimulation of keratinocytes (HaCaT). The specific test methods are as follows: 1) Cell seeding and preparation: Keratinocytes were seeded at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of cells per well into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2).

[0085] 2) Solution preparation: Add the Qingjiang Zhi Ke extract prepared in Examples 1-11 and Comparative Example 1 to PBS solution to prepare a test sample solution containing 1% test sample.

[0086] 3) Stimulation: According to the experimental design, when the cell deposition rate in the 24-well plate reached 40%~50%, 10 μl of TNF-α+IFN-γ working solution (pre-diluted to 2 mg / ml with serum-containing high-glucose DMEM culture medium) was added to the wells of the negative control group, positive control group, and sample group respectively to stimulate the model. The wells were shaken left and right to mix the drug in the wells. The final concentration of TNF-α+IFN-γ was 20 ng / mL. The wells were then incubated in an incubator (37℃, 5% CO2) for 2 h.

[0087] 4) Drug administration: Two hours after the stimulant was applied, according to the experimental groups, 100 μl of PBS solution was added to the blank control group and the negative control group, 100 μl of dexamethasone solution (pre-diluted with PBS solution to 1 mg / ml) was added to the positive control group, and 100 μl of the test sample solution was added to the sample group. After drug administration, the 24-well plate was placed in an incubator (37℃, 5% CO2) and incubated for 16 hours.

[0088] 5) Sample collection: After incubation, collect the cell culture supernatant into EP tubes (Note: Determine the amount of sample to be collected according to the detection indicators). After collection, freeze the sample in a -80℃ freezer.

[0089] 6) Perform the test according to the ELISA kit's instruction manual.

[0090] 7) Experimental Results: In the blank control group, the secretion levels of inflammatory factors IL-8 and TLSP were (176.32±0.42) pg / mL and (18.03±0.35) pg / mL, respectively; in the negative control group, the secretion levels of inflammatory factors IL-8 and TLSP were (930.45±1.08) pg / mL and (14.37±1.09) pg / mL, respectively, which were significantly higher than those in the blank control group. p The value <0.01 indicates that the inflammation model was successfully established in this experiment. The secretion levels of inflammatory factors IL-8 and TLSP in the positive control group were (822.46±2.10) pg / mL and (11.00%±0.71%) pg / mL, respectively.

[0091] The results (average values) of the Qingjiang Zhi Ke extract prepared in each example (1-10) on the secretion of cellular inflammatory factors IL-8 and TLSP are shown in Table 5. Compared with the negative control group, they were significantly reduced. p <0.05), indicating that the sample has anti-inflammatory effects. The Qingjiang Citrus aurantium extract prepared in Comparative Example 1 showed similar levels of IL-8 and TLSP secretion to the negative control group, and was higher than the Qingjiang Citrus aurantium extracts prepared in each example. The Qingjiang Citrus aurantium extract prepared in Example 11 showed a certain decrease in IL-8 and TLSP secretion compared to the negative control group, but the effect was significantly worse than in Example 1. Furthermore, a comparison of the results of Examples 1-5 and Examples 6-10 shows that the Qingjiang Citrus aurantium extract prepared by Bacillus subtilis fermentation had IL-8 and TLSP secretion levels below (877.29±9.49) pg / mL and (11.61±0.72) pg / mL, respectively, with significantly better anti-inflammatory effects than the Qingjiang Citrus aurantium extract prepared by Saccharomyces cerevisiae and Lactobacillus bulgaricus fermentation. Further comparing the results of Example 1 with those of Examples 2-3, when the dosage of Citrus aurantium was 20g, the seed culture volume was 30ml (i.e., the ratio of Citrus aurantium dosage to inoculum volume was 20g: 3.0×10). 9 When CFU is used, it can further reduce the secretion of IL-8 and TLSP, thus achieving better anti-inflammatory effects.

[0092] Table 5 Validation Experiment Example 5: Human Efficacy Evaluation Experiment of Extracts The soothing efficacy of the Qingjiang Citrus aurantium extract prepared in Example 1 was tested on human subjects. The specific test method was as follows: 1) Sample preparation: The Qingjiang Zhi Ke extract prepared in Example 1 was diluted with purified water to 1% to obtain the test group sample; and purified water was used as the control group sample.

[0093] 2) Two 3 cm × 3 cm areas of skin were randomly marked on the inner forearm of the subject as test areas, designated as the control group and the experimental group, respectively, with an interval of at least 2 cm. Transparent tape was used to peel off the test areas in both the experimental and negative control groups, repeating the peeling process 10 times for each area. After peeling, a 3 cm × 3 cm piece of non-woven fabric was soaked in a 10 mg / mL histamine solution in both test areas. Once completely saturated, the fabric was applied to both areas, left for 10 minutes, and then removed. Subsequent testing began after the surface was dry and free of solution residue. Photos were taken to assess various skin parameters in each area, serving as baseline values ​​for the modeled results.

[0094] 3) Test samples were used within the test area of ​​the experimental group, with a sample volume of 2 mg / cm³. 2 Purified water was used in the test area of ​​the negative control group. Antera 3D images were taken at 30 min, 60 min, 80 min, 100 min, and 120 min after the subjects applied the samples to assess the redness reduction and swelling reduction effect.

[0095] Examples of redness and swelling reduction effects in Antera3D photography from two subjects are shown below. Figure 1 and Figure 2 As shown. By Figure 1 and Figure 2 It can be seen that after using the test sample containing the Qingjiang Zhi Ke extract of Example 1, the subjects in the experimental group experienced a shorter time to reduce redness and swelling compared to the control group. Furthermore, the subjects in the sample group also experienced faster and more significant relief from itching compared to the control group, indicating that the Qingjiang Zhi Ke extract prepared in this invention has a soothing effect.

[0096] In summary, the results show that the *Citrus aurantium* extract obtained using the method of this invention has significant repairing, antipruritic, and anti-inflammatory effects. The IL-8 and TLSP tested in the above-mentioned validation experiments are associated with atopic dermatitis. International clinical trials using the tape peeling method to measure IL-8 reflect the response of AD patients to topical corticosteroid treatment have found that changes in IL-8 concentration are correlated with visual improvement of symptoms. IL-8 is a valuable biomarker for monitoring the treatment effect in AD patients (Susumu Murata, Sakae Kaneko, Eishin Morita. Interleukin-8 Levels in the Stratum Corneum as a Biomarker for Monitoring Therapeutic Effect in Atopic Dermatitis Patients. Int Arch Allergy Immunol, 2021,182:592–606). TLSP is also a targeted target of atopic dermatitis drugs (such as Tezepelumab). Therefore, the *Citrus aurantium* extract of this invention has significant soothing and repairing effects on atopic dermatitis.

[0097] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. A method for preparing an extract of Citrus aurantium, characterized in that, Includes the following steps: A1. After crushing the Qingjiang bitter orange peel, soak it in water and then sterilize it; A2. Inoculate probiotics into the sterilized solution in step A1 for fermentation; A3. After sterilizing the fermentation broth obtained in step A2, filter it. The filtrate is then purified using resin to obtain the Qingjiang Zhi Ke extract. The probiotics are selected from at least one of lactic acid bacteria, Bacillus, and yeast fungi.

2. The method for preparing the extract of *Citrus aurantium* according to claim 1, characterized in that, In step A1, the amount of water added is 15-20 times the normal amount.

3. The method for preparing the extract of *Citrus aurantium* according to claim 1, characterized in that, In step A2, the probiotics are selected from at least one of Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus bulgaricus.

4. The method for preparing the extract of *Citrus aurantium* according to claim 3, characterized in that, The probiotics are Bacillus subtilis or a combination of Bacillus subtilis and Saccharomyces cerevisiae.

5. The method for preparing the extract of *Citrus aurantium* according to claim 4, characterized in that, When the probiotic is a compound of Bacillus subtilis and Saccharomyces cerevisiae, the inoculation ratio of Bacillus subtilis to Saccharomyces cerevisiae is 1-2:

1.

6. The method for preparing the extract of *Citrus aurantium* according to claim 1, characterized in that, The ratio of the weight of *Citrus aurantium* (Qingjiang) to the inoculation amount of probiotics was 20 g: 1.0-5.0 × 10⁻⁶. 9 CFU.

7. The method for preparing the extract of *Citrus aurantium* according to claim 1, characterized in that, In step A2, before the probiotics are inoculated, the steps of activation and expansion culture are also included; In step A2, the fermentation conditions are: fermentation at 37℃ and 200r / min for 48-96h; In step A3, the resin type is Seplite LX-94.

8. A *Citrus aurantium* extract prepared by the method according to any one of claims 1-7.

9. The use of a Qingjiang Zhi Ke extract prepared by any one of claims 1-7 or the Qingjiang Zhi Ke extract according to claim 8 in the preparation of cosmetics or pharmaceuticals with soothing and repairing effects.

10. The application according to claim 9, characterized in that, The applications include the preparation of cosmetics or pharmaceuticals that have soothing and repairing effects on atopic dermatitis.

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