Preparation method of composition of artemisia apiacea fermentation extract and dogwood extract and application of composition in medicines and cosmetics for treating acne

Abstract

The invention discloses a preparation method of an artemisia apiacea fermentation extract and dogwood extract composition and application of the artemisia apiacea fermentation extract and dogwood extract composition in acne treatment medicines and cosmetics, belongs to the technical field of traditional Chinese medicine and microorganism crossing, and relates to the preparation method and application of the artemisia apiacea fermentation extract and dogwood extract composition. The preparation method comprises the following steps of: 1, obtaining a lactobacillus casei paracasei enlarged culture solution and a phytobacterium plantarum enlarged culture solution; 2, obtaining double-bacterium fermented artemisia apiacea extract powder; 3, obtaining freeze-dried powder of the double-bacterium fermented artemisia apiacea extract; 4, obtaining dogwood extract freeze-dried powder; and 5, mixing the double-bacterium fermented artemisia apiacea extract freeze-dried powder with the dogwood extract freeze-dried powder to obtain the product. The artemisia apiacea fermentation extract is used as an active ingredient in the medicine and cosmetics for treating acne. The invention provides the application of the double-bacterium fermentation artemisia apiacea extract in intervention of acne caused by propionibacterium acnes for the first time, and the double-bacterium fermentation artemisia apiacea extract is strong in antibacterial and anti-inflammatory activity, high in safety and not easy to generate drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of interdisciplinary technology of traditional Chinese medicine and microbiology, and relates to the preparation method and application of a combination of Artemisia annua fermentation extract and Cornus officinalis extract. Background Technology

[0002] Acne vulgaris is a chronic inflammatory skin disease of the pilosebaceous unit, prevalent among adolescents, with a persistently high global incidence. Patients often experience lesions such as comedones, papules, and pustules, which easily lead to scarring and affect appearance. It can also trigger psychological problems such as anxiety, negatively impacting social and mental health. Traditionally, acne is believed to be related to excessive sebum secretion, abnormal keratinization of the pilosebaceous duct, skin microecological imbalance, and immune inflammatory responses, with Staphylococcus aureus and Propionibacterium acnes being the main pathogens. Topical antibiotics are commonly used clinically, but long-term use can easily lead to adverse reactions and drug resistance.

[0003] As the outermost organ of the human body, the skin is the first line of defense against invading pathogens. Numerous microorganisms, including bacteria, fungi, and viruses, reside on the skin's surface, collectively forming the skin's microbiota and microecology. This skin microecology acts as an "invisible" barrier protecting the skin. The dynamic balance between different microbial communities and between the host and the microbiota is fundamental to maintaining skin microecological homeostasis and skin health. Healthy microbial homeostasis helps prevent harmful pathogens from colonizing the skin surface. The skin microbiota is mainly divided into two types: resident microorganisms and transient microorganisms. Resident microorganisms are non-invasive and non-pathogenic, not only having a symbiotic relationship with the host but also stimulating the skin's immune response to resist pathogen invasion, thus helping to maintain skin health and protect the skin barrier. In contrast, transient microorganisms can be the main pathogens causing skin infections. Skin health is closely related to imbalances and changes in the diversity of the skin microecology; any imbalance caused by exogenous or endogenous factors can trigger skin diseases. In cases like acne, the relative abundance of Propionibacterium acnes in acne lesions can reach as high as 91%. This bacteria proliferate rapidly, metabolizing sebum to produce free fatty acids and activating the innate immune system (such as the NLRP3 inflammasome), triggering a strong inflammatory response that leads to redness, swelling, and suppuration. Restoring skin microbiome diversity, inhibiting pathogenic bacteria, promoting the growth of beneficial bacteria, and rebuilding the skin barrier and immune balance represent a new direction for acne treatment. Summary of the Invention

[0004] The purpose of this invention is to solve the technical problem that long-term use of topical antibiotics for treating acne can easily lead to adverse reactions and drug resistance. This invention provides a method for preparing a composition of Artemisia annua fermentation extract and Cornus officinalis extract and its application in acne treatment drugs.

[0005] The preparation method of the combination of Artemisia annua fermentation extract and Cornus officinalis extract is as follows:

[0006] 1. Inoculate *Lactobacillus casei* and *Lactobacillus plantarum* at a 10% inoculation rate into 10 mL of autoclaved MRS liquid medium and incubate at 37°C for 24 h. Subculture this way twice to obtain two activated strains. Inoculate each of the two activated strains at a 5% inoculation rate into 50 mL of MRS liquid medium and incubate at 37°C for 24 h to obtain *Lactobacillus casei* and *Lactobacillus plantarum* expanded culture broths, respectively.

[0007] 2. Prepare a suspension by mixing Artemisia annua powder with purified water at a ratio of 30mg:70mL, autoclave at 121℃ for 15min, cool to room temperature, and inoculate with a mixed culture of Lactobacillus casei and Lactobacillus plantarum at an inoculation rate of 9%. Place in a constant temperature incubator at 37℃ for 7 days for fermentation. After fermentation, dry at 40℃ to obtain Artemisia annua extract powder fermented by two bacteria.

[0008] The volume ratio of *Lactobacillus casei* and *Lactobacillus plantarum* in the mixed bacteria is 2:1.

[0009] 3. Artemisia annua fermentation powder and 95% ethanol at a volume concentration of 1g:10mL were mixed and refluxed for 10h and 6h respectively. The two extracts were combined and freeze-dried to obtain the freeze-dried powder of Artemisia annua extract fermented by two bacteria.

[0010] 4. Weigh the dried Cornus officinalis powder and place it in an Erlenmeyer flask. Add 60% ethanol solution (volume concentration) at a liquid-to-solid ratio of 1g:15mL, 90% ethanol solution (volume concentration) at a liquid-to-solid ratio of 1g:15mL, or 60% ethanol solution (volume concentration) at a liquid-to-solid ratio of 1g:25mL. Then, heat and reflux at 50℃ for 2 hours, filter under reduced pressure, collect the filtrates twice, concentrate, and freeze-dry to obtain the freeze-dried Cornus officinalis extract powder.

[0011] 5. The freeze-dried powder of Artemisia annua extract fermented by double bacteria and the freeze-dried powder of Cornus officinalis extract are mixed at a mass ratio of 1:1 to obtain the composition of Artemisia annua fermented extract and Cornus officinalis extract.

[0012] The Lactobacillus casei was obtained from the China Industrial Microbial Culture Collection Center (CICC), with accession number 20990.

[0013] The *Lactobacillus plantarum* was obtained from the China General Microbiological Culture Collection Center (CGMCC), with accession number 1.557.

[0014] Application of a combination of Artemisia annua fermented extract and Cornus officinalis extract in acne treatment drugs and cosmetics, wherein the Artemisia annua fermented extract is used as an active ingredient in acne treatment drugs and cosmetics.

[0015] The acne treatment medication is an acne lotion, acne gel, acne cream, acne lotion, or acne serum.

[0016] The cosmetics mentioned include cleansing cream, facial cleanser, bath gel, face cream, toner, and face mask.

[0017] Artemisia annua L., the dried aerial part of a plant in the Asteraceae family, contains various components such as sesquiterpenes and flavonoids. It possesses antibacterial and anti-inflammatory pharmacological activities and shows significant potential in the treatment of acne. However, the poor solubility and low bioavailability of some active ingredients limit its application. Microbial fermentation technology can enhance the antibacterial ability of traditional Chinese medicine. Compound microbial fermentation, through the synergistic metabolic effects of microbial communities, can construct a more complex biotransformation network.

[0018] Cornus officinalis (Cornus fructus) is the dried, ripe pulp of the plant Cornus officinalis Sieb. et Zucc., which was first recorded in the Shennong's Classic of Materia Medica. The main bioactive chemical components in Cornus officinalis are iridoids, triterpenoids, flavonoids, tannins, and organic acids, which have various pharmacological activities such as anti-inflammatory, anti-diabetic, antioxidant, antitumor, antibacterial, neuroprotective, and hepatoprotective effects.

[0019] This invention utilizes bio-fermentation technology, employing *Lactobacillus casei* and *Lactobacillus plantarum* for co-fermentation of *Artemisia annua*. The antibacterial activity of *Artemisia annua* is enhanced after dual-strain fermentation. Inhibition zone measurements show that the extract of *Artemisia annua* exhibits improved antibacterial efficacy against acne-causing bacteria after fermentation. This invention provides the first application of dual-strain fermented *Artemisia annua* extract in intervening in acne caused by *Propionibacterium acnes*, opening up a new use for *Artemisia annua*. Furthermore, due to its strong antibacterial and anti-inflammatory activity, high safety, and low likelihood of inducing drug resistance, it shows great promise in the field of acne treatment. Attached Figure Description

[0020] Figure 1 This is a comparison diagram of the Staphylococcus aureus antibacterial experiment in Experiment 1;

[0021] Figure 2 This is a comparison diagram of the antibacterial experiment of Propionibacterium acnes in Experiment 1;

[0022] Figure 3 This is a comparison chart of the DPPH removal capabilities of the samples in Experiment 2;

[0023] Figure 4 This is a diagram of the mouse acne model from Experiment 3;

[0024] Figure 5 These are comparison images of animal experiments on days 3 and 7 of Experiment 3.

[0025] Figure 6 This is a comparative graph showing the effect of Artemisia annua extract on IL-6 levels in the dorsal skin of acne-prone mice in Experiment 3. Note: ***P<0.001, **P<0.01, *P<0.05. ns P>0.05;

[0026] Figure 7 This is a comparative graph showing the effect of Artemisia annua extract on the TNF-α content in the dorsal skin of acne-prone mice in Experiment 3. Note: ***P<0.001, **P<0.01, *P<0.05. ns P>0.05;

[0027] Figure 8 This is a bar chart of species abundance at the phylum level in Experiment 3;

[0028] Figure 9 This is a bar chart of species abundance at the genus level in Experiment 3. Specific Implementation

[0029] Example 1

[0030] The preparation method of Lactobacillus casei fermentation extract is as follows:

[0031] Artemisia annua was pulverized using a multi-functional Chinese medicine pulverizer and passed through an 80-mesh sieve. The resulting Artemisia annua powder was dried and sealed for storage. Artemisia annua powder and purified water were mixed at a ratio of 30 mg:70 mL to form a solid-liquid suspension. The suspension was autoclaved at 121°C for 15 minutes, cooled to room temperature, and inoculated with Lactobacillus casei culture at a concentration of 9%. The suspension was then fermented in a 37°C incubator for 7 days. After fermentation, the suspension was dried at 40°C to obtain the fermented Artemisia annua product.

[0032] Artemisia annua fermentation powder was mixed with 95% ethanol at a ratio of 1g:10mL and refluxed for 10h and 6h respectively. The two extracts were combined and freeze-dried to obtain the freeze-dried powder of Lactobacillus casei fermentation extract.

[0033] Example 2

[0034] The preparation method of plant lactobacillus fermentation extract is as follows:

[0035] Artemisia annua was pulverized using a multi-functional Chinese medicine pulverizer and passed through an 80-mesh sieve. The resulting Artemisia annua powder was dried and sealed for storage. Artemisia annua powder and purified water were mixed at a ratio of 30 mg:70 mL to form a solid-liquid suspension. The suspension was autoclaved at 121°C for 15 minutes, cooled to room temperature, and inoculated with *Lactobacillus plantarum* culture medium at a concentration of 9%. The suspension was then fermented in a 37°C incubator for 7 days. After fermentation, the suspension was dried at 40°C to obtain the fermented Artemisia annua product.

[0036] Artemisia annua fermentation powder was mixed with 95% ethanol at a ratio of 1g:10mL and refluxed for 10h and 6h respectively. The two extracts were combined and freeze-dried to obtain the freeze-dried powder of Bacillus plantarum fermentation extract.

[0037] Example 3

[0038] The preparation method of the dual-strain fermentation extract is as follows:

[0039] Artemisia annua was pulverized using a multi-functional Chinese medicine pulverizer, passed through an 80-mesh sieve, and the resulting Artemisia annua powder was dried and sealed for storage.

[0040] Artemisia annua powder and purified water were mixed in a ratio of 30 mg: 70 mL to form a suspension. The suspension was autoclaved at 121 °C for 15 min. After cooling to room temperature, a mixed culture consisting of Lactobacillus casei culture medium and Lactobacillus plantarum culture medium was inoculated at a rate of 9%. The mixture was placed in a constant temperature incubator at 37 °C for 7 days. After fermentation, the mixture was dried at 40 °C to obtain Artemisia annua fermentation powder.

[0041] The mixed bacteria contained 2 mL of Lactobacillus casei and 1 mL of Lactobacillus plantarum.

[0042] Artemisia annua fermentation powder was mixed with 95% ethanol at a ratio of 1g:10mL and refluxed for 10h and 6h respectively. The two extracts were combined and freeze-dried to obtain the freeze-dried powder of Artemisia annua fermentation extract.

[0043] Example 4

[0044] Preparation method of freeze-dried powder of Cornus officinalis extract:

[0045] Weigh out the dried Cornus officinalis powder and place it in an Erlenmeyer flask. Add 60% ethanol solution at a liquid-to-solid ratio of 1g:15mL. Heat and reflux at 50℃ for 2 hours. Filter under reduced pressure. Collect the filtrates twice, concentrate, and freeze-dry to obtain the freeze-dried Cornus officinalis extract powder.

[0046] Example 5

[0047] Preparation method of freeze-dried powder of Cornus officinalis extract:

[0048] Weigh out the dried Cornus officinalis powder and place it in an Erlenmeyer flask. Add 90% ethanol solution at a liquid-to-solid ratio of 1g:15mL. Heat and reflux at 50℃ for 2 hours. Filter under reduced pressure. Collect the filtrates twice, concentrate, and freeze-dry to obtain the freeze-dried Cornus officinalis extract powder.

[0049] Example 6

[0050] Preparation method of freeze-dried powder of Cornus officinalis extract:

[0051] Weigh out the dried Cornus officinalis powder and place it in an Erlenmeyer flask. Add 60% ethanol solution at a liquid-to-solid ratio of 1g:25mL. Heat and reflux at 50℃ for 2 hours. Filter under reduced pressure. Collect the filtrates twice, concentrate, and freeze-dry to obtain the freeze-dried Cornus officinalis extract powder.

[0052] Example 7

[0053] The preparation method of the combination of Artemisia annua fermentation extract and Cornus officinalis extract is as follows:

[0054] 1. Inoculate *Lactobacillus casei* and *Lactobacillus plantarum* at a 10% inoculation rate into 10 mL of autoclaved MRS liquid medium and incubate at 37°C for 24 h. Subculture this way twice to obtain two activated strains. Inoculate each of the two activated strains at a 5% inoculation rate into 50 mL of MRS liquid medium and incubate at 37°C for 24 h to obtain *Lactobacillus casei* and *Lactobacillus plantarum* expanded culture broths, respectively.

[0055] 2. Prepare a suspension by mixing Artemisia annua powder with purified water at a ratio of 30mg:70mL, autoclave at 121℃ for 15min, cool to room temperature, and inoculate with a mixed culture of Lactobacillus casei and Lactobacillus plantarum at an inoculation rate of 9%. Place in a constant temperature incubator at 37℃ for 7 days for fermentation. After fermentation, dry at 40℃ to obtain Artemisia annua extract powder fermented by two bacteria.

[0056] The mixed bacteria contained 2 mL of Lactobacillus casei and 1 mL of Lactobacillus plantarum.

[0057] 3. Artemisia annua fermentation powder and 95% ethanol at a volume concentration of 1g:10mL were mixed and refluxed for 10h and 6h respectively. The two extracts were combined and freeze-dried to obtain the freeze-dried powder of Artemisia annua extract fermented by two bacteria.

[0058] 4. Weigh the dried Cornus officinalis powder and place it in an Erlenmeyer flask. Add 60% ethanol solution at a liquid-to-solid ratio of 1g:15mL. Then, heat and reflux at 50℃ for 2 hours. Filter under reduced pressure, collect the filtrates twice, concentrate and freeze dry to obtain the freeze-dried Cornus officinalis extract powder.

[0059] 5. The freeze-dried powder of Artemisia annua extract fermented by double bacteria and the freeze-dried powder of Cornus officinalis extract are mixed at a mass ratio of 1:1 to obtain the composition of Artemisia annua fermented extract and Cornus officinalis extract.

[0060] Prescription for 100g acne gel

[0061]

[0062] Acne cream (100g)

[0063]

[0064] Group C (11g-12g) was treated with an ultrasonic method (ultrasonic power 300w, ultrasonic frequency 40kHZ) to completely dissolve the combination of Artemisia annua fermentation extract and Cornus officinalis extract.

[0065] Heat component A (18g) to 70-80℃ and component B (70.1g) to 70-80℃ until completely dissolved. Add component A to component B (except triethanolamine), cool to 60℃ and add triethanolamine. Cool to 50℃ and add component C, stirring until homogeneous.

[0066] The following experiments were used to verify the effectiveness of the invention:

[0067] Experiment 1:

[0068] Antibacterial test (agar perforation method)

[0069] In this experiment, the diameter of the inhibition zone was measured using the agar perforation method recommended by the National Committee for Clinical Laboratory Standards (NCCLS 2020). Under aseptic conditions, 150 μL of the prepared suspensions of the two bacteria (Staphylococcus aureus and Propionibacterium acnes) were evenly spread onto the corresponding agar plates. 60 µL of *Lactobacillus casei* fermentation extract, *Lactobacillus plantarum* fermentation extract, and *Artemisia annua* fermentation extract (final concentration 320 mg / mL), along with the positive control metronidazole (final concentration 2.5 mg / mL), were added to the perforated agar wells, and the plates were capped. *Propionibacterium acnes* was incubated in a 35 ℃ anaerobic glove box for 96 h, and the results were observed afterward. *Staphylococcus aureus* was incubated in a 37 ℃ incubator for 24 h, and the results were observed afterward. Each sample was tested in triplicate, and the diameter of the inhibition zone was measured and the average value was taken.

[0070] Experimental results:

[0071] Antibacterial experiments using the agar perforation method showed that the Artemisia annua extract fermented with two strains had better antibacterial effects against Propionibacterium acnes and Staphylococcus aureus. The results are shown in Table 1.

[0072] Table 1. Inhibition diameter of fermented Artemisia annua extract against the two strains

[0073]

[0074] Note: Inhibition diameter <10 mm is low sensitivity; 10 mm ≤ inhibition diameter ≤ 15 mm is moderate sensitivity; 15 mm ≤ inhibition diameter ≤ 20 mm is high sensitivity.

[0075] As can be seen from this experiment, dual-strain fermentation (Example 3) has a better antibacterial effect. Therefore, dual-strain fermentation is preferred as the fermentation method in this invention.

[0076] Experiment 2:

[0077] Antioxidant comparison experiment:

[0078] Artemisia annua extract fermented by two bacteria (Example 3) was used as a comparative example.

[0079] Conclusion: Ethanol extraction of Cornus officinalis primarily yields phenolic acids, which possess excellent antioxidant properties. Antioxidant experiments confirmed that the antioxidant activity of Artemisia annua ferment was relatively poor.

[0080] Experiment 3:

[0081] The preparation method of the combination of Artemisia annua fermentation extract and Cornus officinalis extract is as follows:

[0082] 1. Inoculate *Lactobacillus casei* and *Lactobacillus plantarum* at a 10% inoculation rate into 10 mL of autoclaved MRS liquid medium and incubate at 37°C for 24 h. Subculture this way twice to obtain two activated strains. Inoculate each of the two activated strains at a 5% inoculation rate into 50 mL of MRS liquid medium and incubate at 37°C for 24 h to obtain *Lactobacillus casei* and *Lactobacillus plantarum* expanded culture broths, respectively.

[0083] 2. Prepare a suspension by mixing Artemisia annua powder with purified water at a ratio of 30mg:70mL, autoclave at 121℃ for 15min, cool to room temperature, and inoculate with a mixed culture of Lactobacillus casei and Lactobacillus plantarum at an inoculation rate of 9%. Place in a constant temperature incubator at 37℃ for 7 days for fermentation. After fermentation, dry at 40℃ to obtain Artemisia annua extract powder fermented by two bacteria.

[0084] The mixed bacteria contained 2 mL of Lactobacillus casei and 1 mL of Lactobacillus plantarum.

[0085] 3. Artemisia annua fermentation powder and 95% ethanol at a volume concentration of 1g:10mL were mixed and refluxed for 10h and 6h respectively. The two extracts were combined and freeze-dried to obtain the freeze-dried powder of Artemisia annua extract fermented by two bacteria.

[0086] 4. Weigh the dried Cornus officinalis powder and place it in an Erlenmeyer flask. Add 60% ethanol solution at a liquid-to-solid ratio of 1g:15mL. Then, heat and reflux at 50℃ for 2 hours. Filter under reduced pressure, collect the filtrates twice, concentrate and freeze dry to obtain the freeze-dried Cornus officinalis extract powder.

[0087] 5. The freeze-dried powder of Artemisia annua extract fermented by double bacteria and the freeze-dried powder of Cornus officinalis extract are mixed at a mass ratio of 1:1 to obtain the composition of Artemisia annua fermented extract and Cornus officinalis extract.

[0088] Animal experiments:

[0089] Sample preparation:

[0090] The drug was administered via gel, using 1% carbomer as a blank matrix. Dosage was determined according to the following dosage forms: high dose 8 mg / mL of Artemisia annua fermented extract and Cornus officinalis extract, medium dose 4 mg / mL, low dose 2 mg / mL, metronidazole 2 mg / mL, and Artemisia annua fermented extract 4 mg / mL (without Cornus officinalis extract).

[0091] Animal grouping:

[0092] Mice were divided into nine groups: blank, model, blank matrix, high, medium, and low composition, unfermented Artemisia annua, double-fermented Artemisia annua extract (without Cornus officinalis), and metronidazole, with 10 mice in each group.

[0093] Modeling method: 50 μL of Propionibacterium acnes solution was injected intradermally into the back of mice. The injection site was observed the next day to see if there was obvious redness or pus. If no pus appeared on the back, the injection site was re-injected.

[0094] Administration: Except for the control group, each group received 0.1 mL daily for 7 consecutive days. After sacrifice, a 1cm × 1cm piece of skin tissue was collected.

[0095] Figure 4 This is an image of a mouse acne model; obvious pustules are visible to the naked eye.

[0096] Based on the appearance of the experimental results ( Figure 5 As can be seen, the abscesses in the model group and the blank matrix group did not rupture or disappear, showing no significant improvement. In the high-dose group, ulceration occurred on day 3, but no redness or swelling appeared. By day 7, the surface showed signs of healing, the abscesses disappeared, and no hyperplasia occurred. In the medium-dose and low-dose groups, although the skin showed signs of healing on day 7, the abscesses had not completely disappeared. In the double-fermented Artemisia annua group, the degree of abscess healing on day 3 was less than that in the low-dose group but much stronger than that in the unfermented Artemisia annua group. On day 7, although the abscesses on the backs of mice in the double-fermented Artemisia annua group had not completely disappeared, the ruptured parts began to heal, while in the unfermented Artemisia annua group, the ruptured abscesses were still in a state of redness and swelling and had not begun to heal. Based on morphological observation, the effect of the high-dose group was approximately equal to that of the positive control group (metronidazole group), which was superior to the medium-dose and low-dose groups.

[0097] Measurement of inflammatory factors:

[0098] Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), two key pro-inflammatory cytokines, are commonly abnormally expressed in acne patients and are widely believed to play important roles in the inflammatory pathogenesis of acne.

[0099] Skin tissue samples were taken and the levels of IL-6 and TNF-α were determined using a kit.

[0100] ELISA test results show ( Figure 6 , Figure 7 Compared with the control group, the levels of IL-6 and TNF-α in the skin tissue of the model group mice were significantly increased (all P < 0.001). There was no statistically significant difference in IL-6 and TNF-α levels between the control group and the model group (all P > 0.05). Compared with unfermented Artemisia annua extract, the levels of IL-6 and TNF-α in the skin tissue of mice treated with fermented Artemisia annua extract were significantly decreased (P < 0.001). Compared with the model group, the high, medium, and low doses of the combination and the fermented and unfermented Artemisia annua extracts all resulted in varying degrees of decrease in IL-6 and TNF-α levels in the mouse skin tissue. However, the combination significantly reduced IL-6 and TNF-α levels (P < 0.001), and compared with the positive control drug metronidazole, the high-dose combination showed almost the same therapeutic effect as metronidazole.

[0101] 16S microbial community sequencing analysis was performed on the blank group, blank matrix group, model group, high-dose combination group, fermented Artemisia annua group, unfermented Artemisia annua group, and positive control group (metronidazole).

[0102] like Figure 8 As shown, based on species composition analysis, the proportion of species composition varied among the mouse groups at the phylum level. However, Pseudomonas, Bacillota, Actinomycetota, and Bacteroidota were the major bacterial groups with relatively high abundance in each group. The phylum abundance composition of the blank matrix group was similar to that of the model group. Compared with the model group, the abundance of Pseudomonas, Bacillota, and Bacteroidota increased to varying degrees after drug administration, while the abundance of Actinomycetota decreased. The high-dose combination group showed the greatest upregulation of Pseudomonas abundance. The colony composition of the unfermented Artemisia annua group and the positive control group was similar, but the changes in the bacterial community were smaller compared to the high-dose combination group.

[0103] like Figure 9As shown, analysis at the genus level revealed that, compared to the model group, the blank group and the high-dose combination group contained no *Propionibacterium*, and their bacterial compositions were most similar. The colony composition of the blank matrix group was similar to that of the model group, with a slightly lower *Propionibacterium* abundance than the model group, but still higher than other treatment groups. The abundance of *Propionibacterium* decreased significantly in all treatment groups, with *Propionibacterium* disappearing entirely in the high-dose combination group. While *Propionibacterium* did not disappear in the fermented *Artemisia annua* extract group, the unfermented *Artemisia annua* extract group, and the positive control group, its abundance was significantly reduced. This indicates that the combination group had a stronger inhibitory effect on *Propionibacterium* than the simple *Artemisia annua* fermentation group.

[0104] *Propionibacterium acnes* belongs to the genus *Propionibacterium*. An animal model was established using *Propionibacterium acnes*. Based on the aforementioned microbial changes, the combination of *Artemisia annua* fermented extract and *Cornus officinalis* extract, from a skin microbiome perspective, has a regulatory effect on the skin microbiome, enriching and regulating its structure and diversity while inhibiting the excessive proliferation of potentially pathogenic bacteria (*Propionibacterium acnes*). This change helps restore a healthy skin microbiome structure. Furthermore, comparing the results with previous antibacterial experiments, it indicates that the combination of *Artemisia annua* fermented extract and *Cornus officinalis* extract exerts antibacterial and anti-inflammatory effects by inhibiting the over-colonization of *Propionibacterium acnes*.

Claims

1. A method for preparing a composition of Artemisia annua fermentation extract and Cornus officinalis extract, characterized in that... The preparation method of the Artemisia annua fermentation extract and Cornus officinalis extract composition is as follows:

1. Inoculate *Lactobacillus casei* and *Lactobacillus plantarum* at a 10% inoculation rate into 10 mL of autoclaved MRS liquid medium and incubate at 37°C for 24 h. Subculture this way twice to obtain two activated strains. Inoculate each of the two activated strains at a 5% inoculation rate into 50 mL of MRS liquid medium and incubate at 37°C for 24 h to obtain *Lactobacillus casei* and *Lactobacillus plantarum* expanded culture broths, respectively.

2. Prepare a suspension by mixing Artemisia annua powder with purified water at a ratio of 30mg:70mL, autoclave at 121℃ for 15min, cool to room temperature, and inoculate with a mixed culture of Lactobacillus casei and Lactobacillus plantarum at an inoculation rate of 9%. Ferment in a constant temperature incubator at 37℃ for 7 days. After fermentation, dry at 40℃ to obtain Artemisia annua extract powder fermented by two bacteria. The volume ratio of *Lactobacillus casei* and *Lactobacillus plantarum* in the mixed bacteria is 2:

1.

3. Artemisia annua fermentation powder and 95% ethanol at a volume concentration of 1g:10mL were mixed and refluxed for 10h and 6h respectively. The two extracts were combined and freeze-dried to obtain the freeze-dried powder of Artemisia annua extract fermented by two bacteria.

4. Weigh the dried Cornus officinalis powder and place it in an Erlenmeyer flask. Add 60% ethanol solution (volume concentration) at a liquid-to-solid ratio of 1g:15mL, 90% ethanol solution (volume concentration) at a liquid-to-solid ratio of 1g:15mL, or 60% ethanol solution (volume concentration) at a liquid-to-solid ratio of 1g:25mL. Then, heat and reflux at 50℃ for 2 hours, filter under reduced pressure, collect the filtrates twice, concentrate, and freeze-dry to obtain the freeze-dried Cornus officinalis extract powder.

5. The freeze-dried powder of Artemisia annua extract fermented by double bacteria and the freeze-dried powder of Cornus officinalis extract are mixed at a mass ratio of 1:1 to obtain the composition of Artemisia annua fermented extract and Cornus officinalis extract.

2. As described in claim 1, characterized in that The Lactobacillus casei was obtained from the China Industrial Microbial Culture Collection Center (CICC), with accession number 20990.

3. As described in claim 1, characterized in that The *Lactobacillus plantarum* was obtained from the China General Microbiological Culture Collection Center (CGMCC), with accession number 1.

557.

4. The application of the combination of Artemisia annua fermented extract and Cornus officinalis extract prepared according to claim 1 in acne treatment drugs and cosmetics, characterized in that, The artemisia annua fermentation extract is used as an active ingredient in acne treatment drugs and cosmetics.

5. The application according to claim 4, characterized in that, The acne treatment medication is an acne lotion, acne gel, acne cream, acne lotion, or acne serum.

6. The application according to claim 4, characterized in that, The cosmetics mentioned include cleansing cream, facial cleanser, bath gel, face cream, toner, and face mask.

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