A medicinal plaster for treating dermatophytosis based on a traditional Chinese medicine composition and a preparation method thereof

Abstract

The present application belongs to the technical field of pharmacy, and mainly relates to a kind of ointment for treating dermatophytosis based on traditional Chinese medicine composition and a preparation method thereof.In the present application, the traditional Chinese medicinal materials such as biofermented sophora, belamcanda, bauhinia, gallnut, cnidium, aloe, vitex, kochia, oak, stemona, saposhnikovia, gleditsia, and smilax can inhibit fungal growth, and can also relieve itching and swelling.The present application has less side effects.In the present application, Ti / ZIF-8 is further added, and the bacteriostatic effect is more obvious.Acute and chronic dermatitis, eczema, beriberi, foot rot, tinea, foot sweat, fungal infection, skin itching, papules, maculopapular rash, erythroderma, stubborn skin eczema, and redness and itching caused by mosquito bites can be inhibited, and white candida and staphylococcus aureus can be inhibited.In general, the present application has very wide application prospects in the field.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and mainly relates to an ointment for treating fungal infections of dermatitis based on a traditional Chinese medicine composition and its preparation method. Background Technology

[0002] Fungal skin infections such as dermatitis, tinea, and eczema are common skin lesions caused by fungi (such as Candida albicans and Staphylococcus aureus) or mixed infections. These diseases are characterized by high incidence, easy recurrence, and long treatment cycles. Clinical manifestations include skin itching, erythema, and desquamation, which seriously affect patients' quality of life. Currently, clinical treatment mainly relies on chemically synthesized antifungal drugs such as azoles (such as clotrimazole) and allylamines (such as terbinafine), but long-term use can easily lead to drug resistance, skin irritation, and systemic adverse reactions. Although traditional Chinese medicine preparations have a relatively high safety profile, they generally suffer from slow onset of action and insufficient inhibitory effect on stubborn fungi.

[0003] In existing technologies, conventional traditional Chinese medicine preparations (such as CN114392310A) are prepared through water decoction extraction, resulting in insufficient release of active ingredients and difficulty in effectively inhibiting deep fungal infections. While compound preparations of chemical drugs (such as CN115590908A) improve the antibacterial effect, they introduce the risk of side effects associated with chemical drugs. Neither of these methods can solve the dual dilemma of low utilization rate of active ingredients in traditional Chinese medicine and poor safety of chemical drugs.

[0004] This invention innovatively combines bio-fermentation technology using specific bacterial strains (Lactobacillus plantarum CICC 25125 and Bifidobacterium adolescentis CICC6178) with Ti / ZIF-8@Mullite porous microsphere antibacterial agent. This not only significantly improves the dissolution rate and bioavailability of active ingredients in traditional Chinese medicine, but also specifically disrupts fungal cell structure through a dual mechanism of physical adsorption and chemical action. This comprehensive solution overcomes the shortcomings of traditional Chinese medicine preparations, such as slow onset of action and insufficient efficacy, while avoiding the potential side effects of chemical drugs, achieving a balance between high efficiency and safety. Summary of the Invention

[0005] To address the above problems, this invention provides a medicated ointment based on a traditional Chinese medicine composition for treating fungal infections of dermatitis and its preparation method.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In one aspect, the present invention provides a method for preparing an ointment based on a traditional Chinese medicine composition for treating fungal infections of dermatitis, the specific steps of which are as follows:

[0008] S1. *Lactobacillus plantarum* (CICC 25125) and *Bifidobacterium adolescentis* (CICC 6178) were activated. The activated *Lactobacillus plantarum* and *Bifidobacterium adolescentis* were then transferred to shake flasks containing 800 ml of MRS liquid medium and TPY liquid medium, respectively, and fermented for 18-24 hours to obtain a bacterial concentration of 1.5 × 10⁻⁶. 8 -3.5×10 8 cfu•ml -1 The concentration of the *Lactobacillus plantarum* fermentation broth and strain was 2.0 × 10⁻⁶. 8 -8×10 8 cfu•ml -1 Bifidobacterium adolescentis fermentation liquid;

[0009] The MRS liquid culture medium consisted of: 1L distilled water-soluble peptone: 10g, beef extract powder: 10g, yeast extract: 5g, glucose: 20g, dipotassium hydrogen phosphate: 2g, diammonium citrate: 2g, sodium acetate: 5g, Tween 80: 1ml, magnesium sulfate: 0.5g, and manganese sulfate: 0.25g. The medium was autoclaved at 121℃ for 15 minutes and set aside for later use.

[0010] TPY liquid culture medium: Dissolve 26.4g of TPY liquid culture medium (solid powder, catalog number HB8570-1) in 1000ml of distilled water by heating, then autoclave at 121℃ for 15 minutes. TPY liquid culture medium, catalog number HB8570-1, Haibo Biotechnology Co., Ltd.

[0011] S2. Add 5-6g of Sophora flavescens, 3-5g of Belamcanda chinensis, 2-3g of Dictamnus dasycarpus root bark, 1-3g of Galla chinensis, 2-3g of Cnidium monnieri, 1-3g of Aloe vera, 1-1.8g of Pseudolarix amabilis root bark, 2-3g of Kochia scoparia fruit, 2-3g of Phellodendron chinense bark, 1-1.9g of Arisaema heterophyllum, 0.9-1.6g of Stemona japonica, 1.8-2.5g of Hedyotis diffusa, 2.3-3.5g of Gleditsia sinensis thorns, and 2-3.1g of Smilax glabra to a fermentation tank. Then add 10-20ml of Lactobacillus plantarum fermentation liquid and 10-20ml of Bifidobacterium adolescentis fermentation liquid. Perform anaerobic fermentation at 37℃ for 4-5 hours in the fermentation tank. Sophora flavescens, Belamcanda chinensis, Dictamnus dasycarpus root bark, Galla chinensis, Cnidium monnieri, Aloe vera, Pseudolarix amabilis root bark, Kochia scoparia fruit, Phellodendron chinense bark, Arisaema heterophyllum, Stemona japonica, Hedyotis diffusa, Gleditsia sinensis thorns, and Smilax glabra are all dried and pulverized into 100-mesh particles. In this step, the fermented Sophora flavescens, Belamcanda chinensis, Dictamnus dasycarpus, and Galla chinensis not only have a good effect on inhibiting fungal growth, but also relieve itching and reduce swelling.

[0012] S3. Place the fermented Sophora flavescens, Belamcanda chinensis, Dictamnus dasycarpus, Galla chinensis, Cnidium monnieri, Aloe vera, Pseudolarix amabilis, Kochia scoparia, Phellodendron chinense, Arisaema heterophyllum, Stemona japonica, Hedyotis diffusa, Gleditsia sinensis, and Smilax glabra into an enamel container and soak them in water to a depth of about 2-3 cm above the herbs. Soak at room temperature for 8-10 hours.

[0013] S4. Simmer the decoction and dregs prepared in S3 in a clay pot, then add 0.1-0.3g of deer antler, 0.2-0.3g of Panax notoginseng, 0.1-0.3g of ginseng, and 0.05-0.1g of saffron. Add 500-600ml of water, simmer at 90-100℃ for 1-1.5 hours. After simmering, pour out the decoction. Add another 300-400ml of water and simmer again at 90-100℃ for 0.5-1 hours. After simmering, combine the two decoctions and press the dregs multiple times in a press. Collect the decoction and combine all the decoctions. Place the mixture in an enamel container to settle and cool for 10-12 hours. Then, take the supernatant and pass it through an 80-mesh sieve to obtain the decocted decoction for later use.

[0014] S5. Preparation of Ti / ZIF-8@Mullite powder: Dissolve 0.3-0.4g zinc chloride, 0.05-0.08g titanium tetrafluoride, and 1.6-1.7g 2-methylimidazole in 50-60ml methanol to obtain a clear solution. Then, first soak 3-5g of porous mullite beads in the clear solution and circulate it using a peristaltic pump for 2 hours to ensure that the pores of the mullite beads are filled with the clear solution. Then, remove the porous mullite beads soaked in the clear solution and place them in 20-30ml of 0.1% sodium bis(trifluoromethanesulfonyl)imide solution (NaNTf2, solvent: DMSO) and stir for 3-4 hours. Wash the obtained porous mullite ceramic beads with methanol and dry them overnight at 55℃. Repeat the above steps (impregnation with Zn-containing...) 2+ and Ti 4+ The Ti / ZIF-8@Mullite beads were obtained by washing and drying three times with a clear solution of NaNTf2 solution. The Ti / ZIF-8@Mullite beads were then ground into powder and passed through an 80-mesh sieve for later use.

[0015] S6. Mix the decoction obtained in step S4 with 10-20g of turtle shell glue, heat it in a medicine pot, bring it to a boil over high heat, then reduce to low heat to concentrate. During the concentration process, a lot of foam will often be produced when the decoction is boiling. The foam should be skimmed off continuously. When the decoction is concentrated to a paste with a relative density of 1.2 (60℃), cool it down to 55℃ and add 0.1-1g of peppermint essential oil, 0.05-0.1g of nicotinamide and 0.3-0.5g of Ti / ZIF-8 powder obtained in step S5. Stir well.

[0016] S7. Take the ointment prepared in S6 and place it in a cooling room for 12-15 hours. Once the ointment has completely cooled to room temperature, a traditional Chinese medicine composition ointment for treating fungal dermatitis is obtained.

[0017] Preferably, the activated Lactobacillus plantarum and Bifidobacterium adolescentis obtained in step S1 are transferred to shake flasks containing 800 ml of MRS liquid culture medium and TPY liquid culture medium, respectively, and fermented for 21 h.

[0018] Preferably, in step S2, the fermentation broth of Lactobacillus plantarum and the fermentation broth of Bifidobacterium adolescentis are subjected to anaerobic fermentation for 4 hours.

[0019] Preferably, in step S3, the fermented Sophora flavescens, Belamcanda chinensis, Dictamnus dasycarpus, Galla chinensis, Cnidium monnieri, Aloe vera, Pseudolarix amabilis bark, Kochia scoparia, Phellodendron chinense, Arisaema heterophyllum, Stemona japonica, Hedyotis diffusa, Gleditsia sinensis thorns, and Smilax glabra are placed in an enamel container and soaked at room temperature for 9 hours.

[0020] Preferably, in step S4, the medicinal liquid is collected and all the medicinal liquids are combined, and then placed in an enamel vessel to settle and cool for 12 hours.

[0021] Preferably, 0.07g of titanium tetrafluoride is added in step S5.

[0022] Preferably, 0.3g of Ti / ZIF-8 powder is added in step S6.

[0023] On the other hand, the present invention provides an ointment prepared by the above method.

[0024] The beneficial effects of the present invention are as follows: 1. The traditional Chinese medicinal materials such as Sophora flavescens, Belamcanda chinensis, Dictamnus dasycarpus, and Galla chinensis after biological fermentation in the present invention not only have good inhibitory effects on fungal growth, but also relieve itching and reduce swelling.

[0025] 2. The present invention adds the prepared modified Ti / ZIF-8 as an antibacterial agent to the ointment, which has excellent antibacterial and anti-inflammatory effects. Attached Figure Description

[0026] Figure 1 This is a histogram showing the anti-Staphylococcus aureus effect of the traditional Chinese medicine ointment in this invention.

[0027] Figure 2 This is a histogram showing the effect of the traditional Chinese medicine ointment in this invention on inflammatory factors in LPS-induced macrophages.

[0028] Figure 3 This is a product image of the ointment obtained in Example 1 of the present invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] In this invention, both *Lactobacillus plantarum* (accession number CICC 25125) and *Lactobacillus plantarum* (accession number CICC10481) were purchased from the China Industrial Microbial Culture Collection Center.

[0031] Bifidobacterium adolescentis (accession number CICC 6178), China Industrial Microbial Culture Collection Center;

[0032] Porous mullite ceramic beads, purchased from Pingxiang Keyuan Environmental Protection Equipment Packing Co., Ltd.

[0033] Sodium bis(trifluoromethanesulfonyl)imide, CAS No.: 91742-21-1, purchased from Zhengzhou Huiju Chemical Co., Ltd.

[0034] Example 1

[0035] S1. *Lactobacillus plantarum* (CICC 25125) and *Bifidobacterium adolescentis* (CICC 6178) were activated. The activated *Lactobacillus plantarum* and *Bifidobacterium adolescentis* were then transferred to shake flasks containing 800 ml of MRS liquid medium and TPY liquid medium, respectively, and fermented for 21 h to obtain a bacterial concentration of 2.0 × 10⁻⁶. 8 cfu•ml -1 The concentration of the *Lactobacillus plantarum* fermentation broth and strain was 5.0 × 10⁻⁶. 8 cfu•ml -1 Bifidobacterium adolescentis fermentation liquid;

[0036] S2. Add 5g of Sophora flavescens, 3g of Belamcanda chinensis, 2g of Dictamnus dasycarpus root bark, 1g of Galla chinensis, 2g of Cnidium monnieri, 1g of Aloe vera, 1g of Pseudolarix amabilis root bark, 2g of Kochia scoparia fruit, 2g of Phellodendron chinense bark, 1g of Arisaema heterophyllum, 0.9g of Stemona japonica, 1.8g of Hedyotis diffusa, 2.3g of Gleditsia sinensis thorns, and 2g of Smilax glabra to a fermentation tank, then add 10ml of Lactobacillus plantarum fermentation liquid and 10ml of Bifidobacterium adolescentis fermentation liquid, and carry out anaerobic fermentation at 37℃ for 4 hours in the fermentation tank;

[0037] S3. Place the fermented Sophora flavescens, Belamcanda chinensis, Dictamnus dasycarpus, Galla chinensis, Cnidium monnieri, Aloe vera, Pseudolarix amabilis, Kochia scoparia, Phellodendron chinense, Arisaema heterophyllum, Stemona japonica, Hedyotis diffusa, Gleditsia sinensis, and Smilax glabra into an enamel container and soak them in water to a depth of about 2 cm above the herbs. Soak at room temperature for 8 hours.

[0038] S4. Simmer the decoction and dregs prepared in S3 in a clay pot, then add 0.2g of deer antler, 0.25g of Panax notoginseng, 0.2g of ginseng, and 0.075g of saffron. Add 500ml of water and simmer at 95℃ for 1.5 hours. After simmering, pour out the decoction. Add another 300ml of water and simmer again at 95℃ for 1 hour. After simmering, combine the two decoctions and press the dregs multiple times in a press. Collect the decoction and combine all the decoctions. Place the mixture in an enamel container to settle and cool for 10 hours. Then, take the supernatant and pass it through an 80-mesh sieve to obtain the decocted decoction for later use.

[0039] S5. Dissolve 0.3g zinc chloride, 0.05g titanium tetrafluoride, and 1.6g 2-methylimidazole in 50ml of methanol to obtain a clear solution. Then, first soak 3g of porous mullite beads in the above-mentioned Zn-containing solution. 2+ and Ti 4+ In a clear solution, the mixture was circulated using a peristaltic pump for 2 hours to ensure that the pores of the mullite beads were filled with the Zn-containing solution. 2+ and Ti 4+ The clear solution was filled; then the porous mullite beads soaked in the clear solution were taken out and placed in 20 ml of 0.1% sodium bis(trifluoromethanesulfonyl)imide in DMSO solution and stirred for 3 h; the obtained porous mullite ceramic beads were washed with methanol and dried at 55 °C overnight. The above steps were repeated three times to obtain Ti / ZIF-8@Mullite beads. The Ti / ZIF-8@Mullite beads were ground into powder for later use.

[0040] S6. Mix the decoction obtained in step S4 with 10g of turtle shell glue, heat it in a medicine pot, bring it to a boil over high heat, then reduce to low heat to concentrate. During the concentration process, a lot of foam will often be produced when the decoction is boiling. The foam should be skimmed off continuously. When the decoction is concentrated to a paste with a relative density of 1.2 (60℃), cool it down to 55℃ and add 0.1g of peppermint essential oil, 0.05g of nicotinamide and 0.3g of Ti / ZIF-8 powder obtained in step S5. Stir well.

[0041] S7. Take the ointment obtained in S6 and place it in a cooling room for 12 hours until it has completely cooled to room temperature. This yields an ointment for treating fungal dermatitis. (See product image below.) Figure 3 As shown.

[0042] Example 2

[0043] In this embodiment: In step S1, the activated *Lactobacillus plantarum* and *Bifidobacterium adolescentis* were respectively transferred to shake flasks containing 800 ml of MRS liquid medium and TPY liquid medium for fermentation for 18 hours, resulting in a bacterial concentration of 1.5 × 10⁻⁶. 8 cfu•ml -1 The concentration of the *Lactobacillus plantarum* fermentation broth and strain was 2.0 × 10⁻⁶.8 cfu•ml -1 The fermentation broth of *Bifidobacterium adolescentis* was prepared; the herbal formula for step S2 was: 6g *Sophora flavescens*, 3g *Belamcanda chinensis*, 3g *Dictamnus dasycarpus*, 1g *Rhus chinensis*, 3g *Cnidium monnieri*, 1g *Aloe vera*, 1.8g *Pseudolarix amabilis*, 2g *Kochia scoparia*, 3g *Phellodendron chinense*, 1g *Arisaema heterophyllum*, 1.6g *Stemona japonica*, 1.8g *Hedyotis diffusa*, 3.5g *Gleditsia sinensis* thorns, and 2g *Smilax glabra*. All other steps were the same as in Example 1.

[0044] Example 3

[0045] In this embodiment: In step S1, the activated *Lactobacillus plantarum* and *Bifidobacterium adolescentis* were respectively transferred to shake flasks containing 800 ml of MRS liquid medium and TPY liquid medium for fermentation for 24 h, resulting in a bacterial concentration of 3.5 × 10⁻⁶. 8 cfu•ml -1 The concentration of the *Lactobacillus plantarum* fermentation broth and strain was 8 × 10⁻⁶. 8 cfu•ml -1 The fermentation broth of *Bifidobacterium adolescentis* was prepared; the traditional Chinese medicine formula in step S2 was as follows: 5g *Sophora flavescens*, 5g *Belamcanda chinensis*, 2g *Dictamnus dasycarpus*, 3g *Rhus chinensis*, 2g *Cnidium monnieri*, 3g *Aloe vera*, 1g *Pseudolarix amabilis*, 3g *Kochia scoparia*, 2g *Phellodendron chinense*, 1.9g *Arisaema heterophyllum*, 0.9g *Stemona japonica*, 2.5g *Hedyotis diffusa*, 2.3g *Gleditsia sinensis* thorns, and 3.1g *Smilax glabra*. All other steps were the same as in Example 1.

[0046] Comparative Example 1

[0047] In this comparative example, except for step S2 where Sophora flavescens, Belamcanda chinensis, Dictamnus dasycarpus, Galla chinensis, Cnidium monnieri, Aloe vera, Pseudolarix amabilis bark, Kochia scoparia, Phellodendron chinense, Arisaema heterophyllum, Stemona japonica, Hedyotis diffusa, Gleditsia sinensis thorns, and Smilax glabra are not fermented with Lactobacillus plantarum (accession number CICC 25125) and Bifidobacterium adolescentis (accession number CICC 6178), all other steps are the same as in Example 1.

[0048] Comparative Example 2

[0049] In this comparative example: except for step S2, where Sophora flavescens, Belamcanda chinensis, Dictamnus dasycarpus, Galla chinensis, Cnidium monnieri, Aloe vera, Pseudolarix amabilis, Kochia scoparia, Phellodendron chinense, Arisaema heterophyllum, Stemona japonica, Hedyotis diffusa, Gleditsia sinensis, and Smilax glabra are not fermented with Lactobacillus plantarum (accession number CICC 25125) and Bifidobacterium adolescentis (accession number CICC 6178), but with Lactobacillus plantarum CICC 10481 and Bifidobacterium adolescentis CICC 6178, all other steps are the same as in Example 1.

[0050] Comparative Example 3

[0051] In this comparative example, except for step S2 where Sophora flavescens, Belamcanda chinensis, Dictamnus dasycarpus, Galla chinensis, Cnidium monnieri, Aloe vera, Pseudolarix amabilis, Kochia scoparia, Phellodendron chinense, Arisaema heterophyllum, Stemona japonica, Hedyotis diffusa, Gleditsia sinensis, and Smilax glabra are not fermented with Lactobacillus plantarum (preservation number CICC 25125) (i.e., only Bifidobacterium adolescentis CICC 6178 is used for fermentation), all other steps are the same as in Example 1.

[0052] Comparative Example 4

[0053] In this comparative example, except for step S2 where Sophora flavescens, Belamcanda chinensis, Dictamnus dasycarpus, Galla chinensis, Cnidium monnieri, Aloe vera, Pseudolarix amabilis bark, Kochia scoparia, Phellodendron chinense, Arisaema heterophyllum, Stemona japonica, Hedyotis diffusa, Gleditsia sinensis thorns, and Smilax glabra are not fermented with Bifidobacterium adolescentis (accession number CICC 6178) (i.e., only Lactobacillus plantarum CICC 25125 is used), all other steps are the same as in Example 1.

[0054] Comparative Example 5

[0055] In this comparative example, except that 0.3g of the Ti / ZIF-8 powder obtained in step S5 is not added in step S6, all other steps are the same as in Example 1.

[0056] Comparative Example 6

[0057] In this comparative example, except for step S2 where Belamcanda chinensis is not added for bio-fermentation, all other steps are the same as in Example 1.

[0058] Comparative Example 7

[0059] In this comparative example, except for step S6 where turtle shell glue is not added, all other steps are the same as in Example 1.

[0060] I. Research on antibacterial effects against dermatophytes

[0061] 1. Research on antifungal properties

[0062] (1) Source of strain

[0063] Trichophyton rubrum, product number: HZB129765, purchased from Gray Algae Biotechnology Co., Ltd.

[0064] Candida albicans, product number: HZB141142, purchased from Gray Algae Biotechnology Company.

[0065] (2) Preparation of bacterial culture

[0066] Activate the bacterial strain according to the instructions. Inoculate the activated bacterial solution into fresh RPMI-1640 culture medium at a 1% inoculum volume and incubate normally for 24 hours to allow it to enter the fermentation phase. Count the cells under a microscope using a hemocytometer, and adjust the bacterial concentration to 2 × 10⁻⁶ cells / mL with RPMI-1640 culture medium. 6CFU / ml.

[0067] (3) Experimental grouping

[0068] The experiment was divided into a negative control group, Examples 1-3 groups, and Comparative Examples 1-7 groups. The stock solution of the negative control group was sterile distilled water, while the stock solutions (640 mg / mL) of Examples 1-3 and Comparative Examples 1-7 groups were prepared with the corresponding Chinese herbal ointments (sterile distilled water).

[0069] (4) Determination of minimum inhibitory concentration (MIC)

[0070] Trichophyton rubrum: A sterile 96-well plate was used. 100 μl of RPMI-1640 liquid culture medium was added to well 1 in each row as a blank control. 100 μl of freshly prepared bacterial suspension was added to wells 3-11. Well 2 was added with 195 μl of bacterial suspension and 5 μl of the drug. Well 12 contained no drug and only 100 μl of bacterial suspension as a negative control. The drug in wells 2-11 was serially diluted (the drug was the herbal ointment from Examples 1-3 and Comparative Examples 1-7) to achieve final drug concentrations of 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg / ml, respectively. The plates were incubated at 35°C for 48 hours. The inhibitory effect of the drug on the growth of the tested bacteria was observed and compared with the control group. The experiment was repeated three times. The results are shown in Table 1.

[0071] Candida albicans: A sterile 96-well plate was used. 100 μl of RPMI-1640 liquid medium was added to well 1 in each row as a blank control. 100 μl of freshly prepared bacterial suspension was added to wells 3-11. Well 2 was added with 195 μl of bacterial suspension and 5 μl of the drug. Well 12 was a negative control with only 100 μl of bacterial suspension. The drug in wells 2-11 was serially diluted (the drug was the herbal ointment from Examples 1-3 and Comparative Examples 1-7) to achieve final drug concentrations of 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg / ml, respectively. The plates were incubated at 35°C for 48 hours. The inhibitory effect of the drug on the growth of the tested bacteria was observed and compared with the control group. The experiment was repeated three times. The results are shown in Table 1.

[0072] Result interpretation: Visually observe the growth status of the test strains on the plate. Compared with the growth control wells without drugs, MIC refers to the minimum drug concentration that completely inhibits the growth of the test strain. At this time, the solution in the experimental wells is clear and transparent with no visible turbidity / precipitate, and the negative control wells (untreated bacterial solution) show obvious turbidity due to bacterial growth.

[0073] Table 1:

[0074] Grouping Trichophyton rubrum MIC Candida albicans MIC negative control group >32 >32 Example 1 2 0.5 Example 2 4 1 Example 3 4 1 Comparative Example 1 16 4 Comparative Example 2 8 2 Comparative Example 3 8 2 Comparative Example 4 8 2 Comparative Example 5 16 4 Comparative Example 6 8 2 Comparative Example 7 8 2

[0075] As shown in Table 1, the antibacterial effect of Example 1 was significantly higher than that of the other groups, demonstrating its superior antibacterial effect. Comparative Examples 1-4 indicate that the herbal ointment needs to be fermented with *Lactobacillus plantarum* (accession number CICC 25125) and *Bifidobacterium adolescentis* (accession number CICC 6178) to achieve its optimal antibacterial level. Comparative Examples 5-7 indicate that Ti / ZIF-8 powder, bio-fermented *Belamcanda chinensis*, and turtle shell glue are also key components for achieving the best antibacterial effect of the herbal ointment.

[0076] 2. Research on antibacterial agents

[0077] (1) Source of strain

[0078] Staphylococcus aureus ATCC 25923 was purchased from the China Industrial Microbial Culture Collection Center.

[0079] (2) Preparation of Staphylococcus aureus bacterial culture

[0080] First, prepare sterile LB medium (ILLB medium contains 10g trypsin, 5g yeast extract, 10g sodium chloride, and the remainder is made up with distilled water). Take 2ml of the medium and add it to the purchased strain bottle to dissolve. Pour the dissolved bacterial solution into a test tube containing 8ml of LB medium and incubate at 36℃ for 24 hours at 100rpm / min. On the second day, take 1ml of the medium and add it to a test tube containing 9ml of fresh LB medium. Incubate at 36℃ at 100rpm / min until OD560=1. Place the bacterial solution in a 4℃ refrigerator for later use.

[0081] (3) Preparation of bacterial plates

[0082] In a clean bench, melted (microwaved on high) solid culture medium (ILLB medium containing 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar powder, and made up with distilled water) is poured into sterilized petri dishes. Allow it to cool and solidify, then incubate at 37°C for 24 hours. Once sterility is confirmed, it is ready for use. A suspension of Staphylococcus aureus at a suitable concentration, determined qualitatively, is then spread onto the solid culture medium to prepare bacterial plates.

[0083] (4) Preparation of filter paper containing drug solution

[0084] Take filter paper and prepare small circular paper discs with a diameter of 6 mm. Autoclave and sterilize for later use. Dissolve the herbal ointments from Examples 1-3 and Comparative Examples 1-7 in sterile distilled water; dissolve 50 mg of the herbal ointment in 1 ml of sterile distilled water to obtain the corresponding medicinal solutions. Take 10 μL of each of the medicinal solutions from Examples 1-3 and Comparative Examples 1-7 and drop them onto the small circular paper discs. Allow them to dry to obtain the medicinal solution filter paper.

[0085] (5) Determination of inhibition zone diameter

[0086] The study was divided into 12 groups: blank group, positive control group, Examples 1-3, and control groups 1-7. The blank group plates were coated with sterile PBS without Staphylococcus aureus; the other groups were plates containing bacteria. In the blank group, filter paper discs were not treated with any medication, but with sterile distilled water. The positive control group was treated with penicillin solution (2 mg dissolved in 1 ml of sterile distilled water), and the other groups were treated with their respective medication solutions. 6 mm filter paper discs, after being treated and dried, were placed on the plates containing bacteria and incubated at 37°C for 24 hours. The diameter of the inhibition zone was measured using the cross-hatching method, and the average value was recorded. Six plates were used for each group. The results are shown in Table 2 and [Table data would be inserted here]. Figure 1 As shown.

[0087] Table 2:

[0088]

[0089] Table 2 shows that the antibacterial effect of Example 1 is much higher than that of other groups, proving the superior antibacterial effect of the traditional Chinese medicine ointment. Comparative Examples 1-4 indicate that traditional Chinese medicine needs to be fermented with *Lactobacillus plantarum* (accession number CICC 25125) and *Bifidobacterium adolescentis* (accession number CICC 6178) to achieve the best antibacterial effect. Comparative Examples 5-7 indicate that Ti / ZIF-8 powder, bio-fermented *Belamcanda chinensis*, and turtle shell glue are also key components for achieving the best antibacterial effect of the traditional Chinese medicine ointment, with Ti / ZIF-8 powder showing a more significant antibacterial effect.

[0090] II. Anti-inflammatory experiment

[0091] RAW264.7 cells, catalog number: SNL-112, were purchased from Shangen Biotechnology Co., Ltd.

[0092] LPS, product code: HY-D1056, purchased from MCE Biotechnology Co., Ltd.

[0093] 1. Cell Culture: RAW264.7 cells were added to DMEM medium containing 10% fetal bovine serum and cultured in a 37℃, 5% CO2 incubator, passaged every 2 days. RAW264.7 cells were cultured at a density of 5×10⁶ cells / year. 5 0.5 mL per well was inoculated into a 24-well plate.

[0094] 2. Preparation of Chinese herbal ointment solution: Dissolve the ointment in sterile distilled water to prepare a 50mg / mL solution.

[0095] 3. Experiment and Grouping: The cells were divided into a blank group, a model group, Examples 1-3, and Comparative Examples 1-7. The blank group received only 1 mL of DMEM culture medium. The model group received 1 mL of DMEM culture medium containing LPS (1 μg / mL). The drug treatment group received 1 mL of a mixture of LPS (1 μg / mL) and DMEM culture medium containing traditional Chinese medicine ointment. Each group had three replicates. After 24 hours of culture, the cell supernatant was collected, and the levels of IL-6 and TNF-α were detected by ELISA. The levels of IL-6 and TNF-α are shown in Table 3. Figure 2 As shown.

[0096] Table 3:

[0097]

[0098] Table 3 shows that the levels of inflammatory factors in the model group were significantly higher than those in the control group, proving that LPS successfully induced an inflammatory response in RAW264.7 cells. The anti-inflammatory level in Example 1 was significantly higher than in other groups, demonstrating the superior anti-inflammatory effect of the herbal ointment. Comparative Examples 1-4 indicate that the herbal ointment needs to be fermented with *Lactobacillus plantarum* (accession number CICC 25125) and *Bifidobacterium adolescentis* (accession number CICC 6178) to achieve its optimal anti-inflammatory effect. Comparative Examples 5-7 indicate that Ti / ZIF-8 powder, bio-fermented *Belamcanda chinensis*, and turtle shell glue are also key components for achieving the best anti-inflammatory effect of the herbal ointment.

[0099] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a medicated ointment based on a traditional Chinese medicine composition for treating fungal infections of dermatitis, characterized in that, The preparation steps are as follows: S1, The concentration of the prepared strain is 1.5 × 10⁻⁶. 8 -3.5×10 8 cfu•ml -1 The concentration of the *Lactobacillus plantarum* fermentation broth and strain was 2.0 × 10⁻⁶. 8 -8×10 8 cfu•ml -1 Bifidobacterium adolescentis fermentation broth; S2. Add 5-6g of Sophora flavescens, 3-5g of Belamcanda chinensis, 2-3g of Dictamnus dasycarpus root bark, 1-3g of Galla chinensis, 2-3g of Cnidium monnieri, 1-3g of Aloe vera, 1-1.8g of Pseudolarix amabilis root bark, 2-3g of Kochia scoparia fruit, 2-3g of Phellodendron chinense bark, 1-1.9g of Arisaema heterophyllum, 0.9-1.6g of Stemona japonica, 1.8-2.5g of Hedyotis diffusa, 2.3-3.5g of Gleditsia sinensis thorns, and 2-3.1g of Smilax glabra to a fermentation tank, then add 10-20ml of Lactobacillus plantarum fermentation liquid and 10-20ml of Bifidobacterium adolescentis fermentation liquid. Perform anaerobic fermentation at 37℃ for 4-5 hours in the fermentation tank. S3. Soak the fermented Sophora flavescens, Belamcanda chinensis, Dictamnus dasycarpus, Galla chinensis, Cnidium monnieri, Aloe vera, Pseudolarix amabilis bark, Kochia scoparia, Phellodendron chinense, Arisaema heterophyllum, Stemona japonica, Hedyotis diffusa, Gleditsia sinensis thorns, and Smilax glabra in water, with the water level 2-3 cm above the herbs, at room temperature for 8-10 hours. S4. In a clay pot, decoct the medicinal liquid and dregs prepared in S3, then add 0.1-0.3g of deer antler, 0.2-0.3g of Panax notoginseng, 0.1-0.3g of ginseng, and 0.05-0.1g of saffron. Add 500-600ml of water, simmer at 90-100℃ for 1-1.5 hours. After decoction, pour out the liquid. Add another 300-400ml of water and decoct again at 90-100℃ for 0.5-1 hours. After decoction, combine the two liquids and press the dregs multiple times in a press. Collect the liquid and combine all the liquids. Allow it to settle and cool for 10-12 hours. Then, take the supernatant and pass it through an 80-mesh sieve to obtain the decoction for later use. S5. Dissolve 0.3-0.4g zinc chloride, 0.05-0.08g titanium tetrafluoride, and 1.6-1.7g 2-methylimidazole in 50-60ml methanol to obtain a clear solution. Then, soak 3-5g of porous mullite beads in the clear solution and circulate it using a peristaltic pump for 2 hours to ensure that the pores of the mullite beads are filled with the clear solution. Then, remove the porous mullite beads soaked in the clear solution and place them in 20-30ml of 0.1% sodium bis(trifluoromethanesulfonyl)imide in DMSO solution and stir for 3-4 hours. Wash with methanol and dry overnight at 55℃. Repeat the above steps of soaking in the clear solution, circulating with a peristaltic pump, stirring in sodium bis(trifluoromethanesulfonyl)imide in DMSO solution, washing with methanol, and drying overnight at 55℃ three times to obtain Ti / ZIF-8@Mullite beads. Grind the Ti / ZIF-8@Mullite beads into powder for later use. S6. Mix the decoction obtained in step S4 with 10-20g of turtle shell glue. Heat the decoction in a medicine pot. After boiling over high heat, reduce to low heat and concentrate. During the concentration process, foam often forms when the decoction boils. Skim off the foam continuously. When the decoction is concentrated to a paste with a relative density of 1.2 measured at 60℃, cool it to 55℃ and add 0.1-1g of peppermint oil, 0.05-0.1g of nicotinamide and 0.3-0.5g of Ti / ZIF-8 powder prepared in step S5. Stir well. S7. Take the ointment obtained in S6 and place it in the ointment cooling room for 12-15 hours. Once the ointment has completely cooled to room temperature, a medicinal ointment for treating fungal dermatitis is obtained.

2. The method for preparing a medicated ointment for treating fungal dermatitis based on a traditional Chinese medicine composition according to claim 1, characterized in that: The selected Lactobacillus plantarum is Lactobacillus plantarum CICC 25125, and the selected Bifidobacterium adolescentis is Bifidobacterium adolescentis CICC 6178.

3. The method for preparing a medicated ointment for treating fungal dermatitis based on a traditional Chinese medicine composition according to claim 2, characterized in that: Lactobacillus plantarum CICC 25125 and Bifidobacterium adolescentis CICC 6178 were activated separately. The activated Lactobacillus plantarum and Bifidobacterium adolescentis were then transferred to shake flasks containing 800 ml of MRS liquid medium and TPY liquid medium, respectively, and fermented for 18-24 hours to obtain strains with a concentration of 1.5 × 10⁻⁶. 8 -3.5×10 8 cfu•ml -1 The concentration of the *Lactobacillus plantarum* fermentation broth and strain was 2.0 × 10⁻⁶. 8 -8×10 8 cfu•ml -1 Bifidobacterium adolescentis fermentation liquid.

4. The method for preparing a medicated ointment for treating fungal dermatitis based on a traditional Chinese medicine composition according to claim 3, characterized in that: The activated Lactobacillus plantarum and Bifidobacterium adolescentis were transferred to shake flasks containing 800 ml of MRS liquid medium and TPY liquid medium, respectively, and fermented for 21 h.

5. The method for preparing a medicated ointment for treating fungal dermatitis based on a traditional Chinese medicine composition according to claim 4, characterized in that: In step S2, the fermentation broth of Lactobacillus plantarum and the fermentation broth of Bifidobacterium adolescentis are subjected to anaerobic fermentation for 4 hours.

6. The method for preparing a medicated ointment for treating fungal dermatitis based on a traditional Chinese medicine composition according to claim 4, characterized in that: In step S3, the fermented Sophora flavescens, Belamcanda chinensis, Dictamnus dasycarpus, Galla chinensis, Cnidium monnieri, Aloe vera, Pseudolarix amabilis bark, Kochia scoparia, Phellodendron chinense, Arisaema heterophyllum, Stemona japonica, Hedyotis diffusa, Gleditsia sinensis thorns, and Smilax glabra are soaked in water for 9 hours at room temperature.

7. The method for preparing a medicated ointment for treating fungal dermatitis based on a traditional Chinese medicine composition according to claim 6, characterized in that: In step S4, the medicinal liquid is collected and all the medicinal liquids are combined, and the sediment is cooled for 12 hours.

8. The method for preparing a medicated ointment for treating fungal dermatitis based on a traditional Chinese medicine composition according to claim 7, characterized in that: In step S5, 0.07g of titanium tetrafluoride is added.

9. A method for preparing a medicated ointment for treating fungal dermatitis based on a traditional Chinese medicine composition according to claim 8, characterized in that: In step S6, 0.3g of Ti / ZIF-8 powder is added.

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