A bacteriostatic composition and its use

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a bacteriostatic composition and application thereof. The application provides a bacteriostatic composition, which comprises a first component, a second component and a third component. The first component comprises cypressol; the second component comprises mulberry bark extract and / or hemp extract; and the third component comprises hosta plantaginea and / or tiger-lily root extract. The bacteriostatic composition provided by the application has effective inhibiting effects on gram-positive bacteria, gram-negative bacteria and fungi, especially antibiotic-resistant bacteria. The different components have a synergistic effect, the use amount of the extract can be reduced, and thus the product cost is reduced. The bacteriostatic composition provided by the application is derived from widely available plant raw materials, has a simple preparation process, low cost and market-friendly product.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antibacterial composition and its application. Background Technology

[0002] Bacterial infections pose a persistent threat to human health. Since the discovery of penicillin, various antibiotics have been widely used to treat bacterial infections. However, with the widespread use of antimicrobial drugs, the problem of bacterial resistance has become increasingly serious. The World Health Organization has pointed out that drug-resistant bacteria have become a major challenge in the field of public health. This problem cannot be solved by relying solely on existing antibiotics and other methods; there is an urgent need to find new, green antibiotics.

[0003] Plant-derived natural antibacterial agents possess advantages such as good antibacterial activity, safety, high efficiency, and abundant resources, making them suitable as alternatives to traditional antibacterial agents. However, existing technologies for traditional plant-derived antibacterial compositions have some limitations. For example, their antibacterial effects are often relatively singular, failing to effectively cover multiple microorganisms and drug-resistant microorganisms. Furthermore, due to the lack of clarity regarding their antibacterial and antimicrobial active ingredients, effective quality control is difficult, which may affect the stable efficacy of the product. Summary of the Invention

[0004] The purpose of this invention is to provide an antibacterial composition that has broad-spectrum antibacterial properties and stable antibacterial effect.

[0005] This invention provides an antibacterial composition comprising a first component, a second component, and a third component;

[0006] The first component includes juniper alcohol; the second component includes mulberry bark extract and / or hemp extract; the third component includes hosta root extract and / or tiger paw extract.

[0007] Preferably, in the antibacterial composition, the concentration of the first component is ≥32 μg / mL; the concentration of the second component is ≥4 μg / mL; and the concentration of the third component is ≥16 μg / mL.

[0008] Preferably, the total amount of morin, morinone G and morinone H in the mulberry bark extract is 10wt% to 60wt% based on the content of dried product;

[0009] The total amount of cannabidiol and hypocannabidiol in the cloud hemp extract is 10wt% to 60wt%.

[0010] The total amount of (25R)-2α,3β-dihydroxy-5α-spirost-9-en-12-one-3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside in the root extract of the hosta flower is 1wt% to 10wt%.

[0011] The content of secondary saponin CP4 in the tiger paw grass extract is 20wt% to 80wt%.

[0012] Preferably, the preparation method of the mulberry bark extract includes: mixing mulberry bark powder with 80%–95% v / v ethanol solution, soaking for 12–24 hours, extracting by reflux at 80–90°C 2–5 times, each time for 3–5 hours, combining the extracts, filtering under reduced pressure, concentrating, and drying to constant weight to obtain crude mulberry bark extract; purifying the crude extract to obtain mulberry bark extract;

[0013] The preparation method of the cloud hemp extract includes: mixing cloud hemp powder with 80%–95% v / v ethanol solution, soaking for 12–24 hours, extracting by reflux at 80–90°C 2–5 times, each time for 3–5 hours, combining the extracts, filtering under reduced pressure, concentrating, and drying to constant weight to obtain a crude cloud hemp extract; purifying the crude cloud hemp extract to obtain the cloud hemp extract.

[0014] The preparation method of the Hosta plant root extract includes: mixing Hosta plant root powder with 80%–95% v / v ethanol solution, soaking for 12–24 hours, extracting by reflux at 80–90°C 2–5 times, each time for 3–5 hours, combining the extracts, filtering under reduced pressure, concentrating, and drying to constant weight to obtain a crude Hosta plant root extract; purifying the crude Hosta plant extract to obtain the Hosta plant root extract.

[0015] The preparation method of the tiger paw grass extract includes: mixing tiger paw grass powder with 80%–95% v / v ethanol solution, soaking for 12–24 hours, extracting by reflux at 80–90°C 2–5 times, each time for 3–5 hours, combining the extracts, filtering under reduced pressure, concentrating, and drying to constant weight to obtain crude tiger paw grass extract; redissolving the crude tiger paw grass extract in water, filtering, precipitating with hot acetone, and filtering again to obtain the tiger paw grass extract.

[0016] The ratio of mulberry bark powder to 80%–95% v / v ethanol solution is 40–60 g / L; the ratio of hemp fiber powder to 80%–95% v / v ethanol solution is 40–60 g / L; the ratio of hosta root powder to 80%–95% v / v ethanol solution is 40–60 g / L; and the ratio of tiger paw grass powder to 80%–95% v / v ethanol solution is 40–60 g / L.

[0017] Preferably, the purification includes purification using a macroporous resin.

[0018] The present invention also provides the application of the antibacterial composition described above in the preparation of antibacterial products.

[0019] Preferably, the antibacterial product includes a product that inhibits the activity of pathogens; the pathogens include bacteria and / or fungi, and the bacteria and / or fungi include antibiotic-resistant bacteria and / or fungi.

[0020] Preferably, the dosage form of the product includes one or more of the following: paste, cream, emulsion, gel, spray, aqueous solution, lotion, soap, ointment, suppository, capsule, powder, granule, tablet, and effervescent tablet.

[0021] The present invention also provides an antibacterial cream comprising the following components: 0.02wt%–1wt% of the antibacterial composition described in the above technical solution, 10wt%–30wt% of glycerol, 4wt%–10wt% of beeswax, 4wt%–10wt% of liquid paraffin, 1wt%–10wt% of stearyl alcohol, 0.5wt%–5wt% of cetyl alcohol, 1wt%–10wt% of white petrolatum, 1wt%–8wt% of silicone oil, 0.2wt%–5wt% of glyceryl monostearate, 1wt%–6wt% of propylene glycol, 0.1wt%–2wt% of Tween 80, 0.1wt%–1wt% of triethanolamine, and the balance being water and polyoxyethylene hydrogenated castor oil; wherein the mass ratio of the polyoxyethylene hydrogenated castor oil to the antibacterial composition is 0.5–3:1.

[0022] The present invention also provides an antibacterial ointment comprising the following components: 0.02wt%–1wt% of the antibacterial composition described in the above technical solution, 60wt%–80wt% of white petrolatum, 5wt%–20wt% of glycerol, 1wt%–10wt% of lanolin, 0.5wt%–5wt% of liquid paraffin, 0.2wt%–5wt% of glyceryl monostearate, 1wt%–6wt% of propylene glycol, 0.1wt%–2wt% of Tween 80, 0.0wt%–0.5wt% of borneol, and the balance being water and polyoxyethylene hydrogenated castor oil; wherein the mass ratio of the polyoxyethylene hydrogenated castor oil to the antibacterial composition is 0.5–3:1.

[0023] Beneficial effects:

[0024] The antibacterial composition provided by this invention has an effective inhibitory effect on Gram-positive bacteria, Gram-negative bacteria, and fungi, especially antibiotic-resistant bacteria. Furthermore, some components exhibit synergistic effects, reducing the amount of extract required and thus lowering product costs. The antibacterial composition provided by this invention is derived from widely available plant materials, has a simple preparation process, low cost, and is commercially viable.

[0025] Furthermore, this invention clarifies the active ingredients and their contents in different extracts. These active ingredients can be used as quality markers for quality control of each extract, ensuring the stability of the antibacterial effect of the composition.

[0026] The antibacterial composition provided by this invention has a wide range of applications and can be formulated into various dosage forms such as ointments, emulsions, gels, sprays, and aqueous solutions. Its applications include improving fungal or bacterial infections of the skin, mucous membranes, and body surface, as well as exhibiting antibacterial activity against antibiotic-resistant bacteria. It covers various infections such as those caused by external injuries, surgical wounds, reproductive tract infections, external genitalia infections, oral infections, and hand and foot infections. Specific applications include the prevention and treatment of sores, dermatitis, eczema, psoriasis, rosacea, athlete's foot, onychomycosis, bedsores, burns, scalds, sunburns, trauma, insect bites, skin redness and swelling, stomatitis, vaginitis, and similar diseases of the skin and mucous membranes in animals. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0028] Figure 1 A flowchart illustrating the preparation process of the antibacterial composition provided by this invention;

[0029] Figure 2 Structural diagrams of antibacterial activity markers in each extract;

[0030] Figure 3 The total ion chromatograms of each extract are shown in the LC-MS.

[0031] Figure 4 Antibacterial curves for each extract;

[0032] Figure 5 The antibacterial curve of juniperol;

[0033] Figure 6 The antibacterial curves of composition 1 and composition 2 are shown.

[0034] Figure 7 The antibacterial curves of composition 3 and composition 4 are shown.

[0035] Figure 8 The antibacterial curve of composition 5 is shown.

[0036] Figure 9 The antibacterial curve of composition 6 is shown.

[0037] Figure 10 The antibacterial curve of composition 7 is shown.

[0038] Figure 11 The antibacterial curve of composition 8 is shown. Detailed Implementation

[0039] The present invention provides an antibacterial composition comprising a first component, a second component, and a third component; the first component comprises juniper alcohol; the second component comprises mulberry bark extract and / or hemp extract; and the third component comprises hosta root extract and / or tiger paw extract.

[0040] In this invention, the first component comprises juniperol, more preferably juniperol; the concentration of the first component is preferably ≥32 μg / mL, more preferably ≥128 μg / mL, and even more preferably 1600 μg / mL. Juniperol described in this invention is a highly safe plant component with good antibacterial activity and significant inhibitory effect on Gram-negative bacteria. Its structure is as follows:

[0041]

[0042] In this invention, the second component comprises mulberry bark extract and / or hemp extract, preferably mulberry bark extract and hemp extract or mulberry bark extract or hemp extract, more preferably mulberry bark extract. The concentration of the second component is preferably ≥4 μg / mL, more preferably ≥16 μg / mL, and even more preferably 800 μg / mL; the mulberry bark extract and hemp extract in the second component can exert an anti-Gram-positive bacterial effect.

[0043] In this invention, the third component comprises *Hosta salsa* root extract and / or *Spathiphyllum tigrinum* extract, preferably *Hosta salsa* root extract and *Spathiphyllum tigrinum* extract, or either *Hosta salsa* root extract or *Spathiphyllum tigrinum* extract. The concentration of the third component is preferably ≥16 μg / mL, more preferably ≥64 μg / mL, and even more preferably 800 μg / mL; the *Hosta salsa* root extract and *Spathiphyllum tigrinum* extract in the third component can effectively inhibit the activity of pathogenic fungi.

[0044] In a preferred embodiment of the present invention, the antibacterial composition comprises juniperol, mulberry bark extract, and hosta root extract; in a preferred embodiment of the present invention, the antibacterial composition comprises juniperol, mulberry bark extract, and tiger paw extract; in a preferred embodiment of the present invention, the antibacterial composition comprises juniperol, hemp extract, and hosta root extract; in a preferred embodiment of the present invention, the antibacterial composition comprises juniperol, hemp extract, and tiger paw extract.

[0045] The antibacterial composition provided by this invention has broad-spectrum and highly effective antibacterial efficacy, and can effectively combat Gram-positive bacteria, Gram-negative bacteria and fungi, including antibiotic-resistant bacteria.

[0046] The antibacterial composition provided by the present invention has a synergistic effect, as verified by the examples. Specifically: the combination of mulberry bark extract and hosta root extract showed a synergistic effect against methicillin-resistant Staphylococcus aureus (MRSA). Hosta root extract reduced the minimum inhibitory concentration (MIC) of mulberry bark extract, and the synergistic effect remained even with the addition of juniper alcohol. The combination of mulberry bark extract and tiger paw extract showed a synergistic effect against both MRSA and vancomycin-resistant enterococci (VRE). Tiger paw extract reduced the MIC of mulberry bark extract, and the synergistic effect remained even with the addition of juniper alcohol. The combination of hemp extract and hosta root extract had no effect on MRSA activity but showed an antagonistic effect on VRE. However, the combination of juniper alcohol with hemp extract and hosta root extract showed a synergistic effect against both MRSA and VRE. The combination of hemp extract and tiger paw extract showed an antagonistic effect against both MRSA and VRE, but the combination of juniper alcohol with hemp extract and tiger paw extract showed a synergistic effect against both MRSA and VRE.

[0047] In this invention, the total amount of morin, morinone G and morinone H in the mulberry bark extract, calculated on a dried basis, is preferably 10wt% to 60wt%, more preferably 30wt% to 60wt%, and even more preferably 60wt%. The preferred method for preparing the mulberry bark extract of the present invention includes: mixing mulberry bark powder with 80%–95% v / v ethanol solution, soaking for 12–24 hours, extracting by reflux at 80–90°C 2–5 times, each time for 3–5 hours, combining the extracts, filtering under reduced pressure, concentrating, and drying to constant weight to obtain crude mulberry bark extract; purifying the crude extract to obtain mulberry bark extract; the preferred material-to-liquid ratio of mulberry bark powder to ethanol is 40–60 g / L, more preferably 50 g / L; the purification preferably includes purification using macroporous resin; the macroporous resin is preferably D101 macroporous resin; the purification step using macroporous resin preferably includes: eluting sequentially with pure water, 40% v / v ethanol, and 80% v / v ethanol, using 4–6 column volumes for each eluent, collecting the 80% v / v ethanol eluent, concentrating, and drying to constant weight.

[0048] In this invention, the total amount of cannabidiol and hypocannabidiol in the cloud hemp extract, calculated on a dried basis, is preferably 10wt% to 60wt%, more preferably 40wt% to 60wt%, and even more preferably 60wt%. The preferred method for preparing the hemp extract includes: mixing hemp powder with 80%–95% v / v ethanol solution, soaking for 12–24 hours, extracting by reflux at 80–90°C 2–5 times, each time for 3–5 hours, combining the extracts, filtering under reduced pressure, concentrating, and drying to constant weight to obtain crude hemp extract; purifying the crude extract to obtain hemp extract; the preferred material-to-liquid ratio of hemp powder to ethanol is 40–60 g / L, more preferably 50 g / L; the purification preferably includes purification using macroporous resin; the preferred macroporous resin is D101 macroporous resin; the purification step using macroporous resin preferably includes: eluting sequentially with pure water, 40% v / v ethanol, and 80% v / v ethanol, using 4–6 column volumes for each eluent, collecting the 80% v / v ethanol eluent, concentrating, and drying to constant weight.

[0049] In this invention, the total amount of (25R)-2α,3β-dihydroxy-5α-spirost-9-en-12-one-3-O-β-D-glucopyranoside-(1→2)-[β-D-xylopyranoside-(1→3)]-β-D-glucopyranoside-(1→4)-β-D-galactopyranoside in the *Hosta* root extract is preferably 1% to 10 wt%, more preferably 3 wt% to 10 wt%, and even more preferably 10 wt%. The preferred method for preparing the *Hosta plantago-aquatica* root extract includes: mixing *Hosta plantago-aquatica* root powder with an 80%–95% v / v ethanol solution, soaking for 12–24 hours, and extracting by reflux at 80–90°C 2–5 times, each time for 3–5 hours; combining the extracts, filtering under reduced pressure, concentrating, and drying to constant weight to obtain a crude extract of *Hosta plantago-aquatica* root; purifying the crude extract to obtain the *Hosta plantago-aquatica* root extract; the preferred material-to-liquid ratio of *Hosta plantago-aquatica* root powder to ethanol is 40 g / L–60 g / L, more preferably 50 g / L; the purification preferably includes purification using a macroporous resin; the preferred macroporous resin is D101 macroporous resin; the purification step using a macroporous resin preferably includes: eluting sequentially with pure water, 40% v / v ethanol, and 80% v / v ethanol, using 4–6 column volumes for each eluent, collecting the 80% v / v ethanol eluent, concentrating, and drying to constant weight.

[0050] In this invention, the content of CP4, calculated on a dried basis, in the *Smilax china* extract is preferably 20wt%–80wt%, more preferably 60wt%–80wt%, and even more preferably 80wt%. The preferred method for preparing the *Smilax china* extract includes: mixing *Smilax china* powder with an 80%–95% v / v ethanol solution, soaking for 12–24 hours, extracting by reflux at 80–90°C 2–5 times, each time for 3–5 hours, combining the extracts, filtering under reduced pressure, concentrating, and drying to constant weight to obtain a crude *Smilax china* extract; redissolving the crude extract in water, filtering, precipitating with hot acetone, and filtering again to obtain the *Smilax china* extract; the ratio of *Smilax china* powder to ethanol is preferably 40–60 g / L, more preferably 50 g / L.

[0051] This invention effectively controls the quality of each extract by controlling the content of antibacterial activity markers in each extract, thereby ensuring the stability of the antibacterial effect of the composition.

[0052] The present invention also provides the application of the antibacterial composition described above in the preparation of antibacterial products.

[0053] In this invention, the antibacterial product preferably includes a product that inhibits the activity of pathogenic bacteria; the pathogenic bacteria preferably include bacteria and / or fungi, and the bacteria and / or fungi preferably include antibiotic-resistant bacteria and / or fungi; the bacteria are further preferably antibiotic-resistant bacteria, more preferably one or more of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), drug-resistant Escherichia coli, and drug-resistant Acinetobacter baumannii; the fungi are preferably antibiotic-resistant fungi, more preferably fluconazole-resistant Candida albicans.

[0054] In this invention, the dosage form of the product includes one or more of the following: ointment, cream, emulsion, gel, spray, aqueous solution, lotion, soap, ointment, suppository, capsule, powder, granule, tablet, and effervescent tablet. The antibacterial compositions and products provided by this invention are suitable for the prevention and treatment of skin and mucous membrane infections in humans and animals, covering various infections such as trauma, surgical wounds, reproductive tract infections, external genitalia infections, oral infections, and hand and foot infections. Specific applications include the prevention and treatment of sores, dermatitis, eczema, psoriasis, rosacea, athlete's foot, onychomycosis, bedsores, burns, scalds, sunburn, trauma, insect bites, skin redness and swelling, stomatitis, vaginitis, and similar diseases of the skin and mucous membranes in animals.

[0055] The present invention also provides an antibacterial cream comprising the following components: 0.02wt%–1wt% of the antibacterial composition described in the above technical solution, 10wt%–30wt% of glycerol, 4wt%–10wt% of beeswax, 4wt%–10wt% of liquid paraffin, 1wt%–10wt% of stearyl alcohol, 0.5wt%–5wt% of cetyl alcohol, 1wt%–10wt% of white petrolatum, 1wt%–8wt% of silicone oil, 0.2wt%–5wt% of glyceryl monostearate, 1wt%–6wt% of propylene glycol, 0.1wt%–2wt% of Tween 80, 0.1wt%–1wt% of triethanolamine, and the balance being water and polyoxyethylene hydrogenated castor oil; wherein the mass ratio of the polyoxyethylene hydrogenated castor oil to the antibacterial composition is 0.5–3:1. As a preferred embodiment of the present invention, the antibacterial cream comprises, by weight, the following components: 0.32 wt% antibacterial composition, 16 wt% glycerol, 5 wt% beeswax, 5 wt% liquid paraffin, 4 wt% octadecyl alcohol, 2 wt% hexadecyl alcohol, 3 wt% white petrolatum, 2 wt% silicone oil, 1 wt% glyceryl monostearate, 3 wt% propylene glycol, 0.8 wt% Tween 80, 0.4 wt% triethanolamine, 0.4 wt% polyoxyethylene hydrogenated castor oil, and the balance being water.

[0056] The present invention also provides an antibacterial ointment, which, by weight, comprises the following components: 0.02wt% to 1wt% of the antibacterial composition described in the above technical solution, 60wt% to 80wt% of white petrolatum, 5wt% to 20wt% of glycerol, 1wt% to 10wt% of lanolin, 0.5wt% to 5wt% of liquid paraffin, 0.2wt% to 5wt% of glyceryl monostearate, 1wt% to 6wt% of propylene glycol, 0.1wt% to 2wt% of Tween 80, 0.0wt% to 0.5wt% of borneol, and the balance being water and polyoxyethylene hydrogenated castor oil; wherein the mass ratio of the polyoxyethylene hydrogenated castor oil to the composition is 0.5 to 3:1. As a preferred embodiment of the present invention, the antibacterial ointment comprises the following components by weight: 0.32 wt% antibacterial composition, 74 wt% white petrolatum, 14 wt% glycerol, 4 wt% lanolin, 2 wt% liquid paraffin, 2 wt% glyceryl monostearate, 2.28 wt% propylene glycol, 0.4 wt% polyoxyethylene hydrogenated castor oil, 0.8 wt% Tween 80, and 0.2 wt% borneol.

[0057] The antibacterial cream and antibacterial cream provided by this invention have strong antibacterial activity against antibiotic-resistant bacteria, can be used to inhibit the proliferation of fungi or bacteria, and improve infection symptoms, while being non-irritating to the skin.

[0058] In the embodiments of this invention, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, fluconazole-resistant Candida albicans, drug-resistant Escherichia coli, and drug-resistant Acinetobacter baumannii were used as examples for experiments. However, the composition provided by this invention also has inhibitory and killing effects on other pathogenic bacteria and super-drug-resistant bacteria.

[0059] To further illustrate the present invention, the following detailed description of an antibacterial composition and its application provided by the present invention, in conjunction with the accompanying drawings and embodiments, is provided, but should not be construed as limiting the scope of protection of the present invention.

[0060] It should be emphasized that the following embodiments are merely illustrative examples and do not limit the scope of protection of this invention, which is determined by the claims. Non-essential improvements and adjustments are still within the scope of protection of this invention.

[0061] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field; the technical or scientific terms used should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0062] In this embodiment of the invention, the medicinal materials mulberry bark, hemp, hosta root, and tiger paw grass were purchased from the Kunming medicinal materials market; cypress alcohol was purchased from Tianjin Xiens Biochemical Technology Co., Ltd.

[0063] Example 1

[0064] according to Figure 1 The process was used to prepare mulberry bark extract, hemp extract, hosta root extract, and tiger paw extract.

[0065] 1. Take 60g of dried mulberry bark, crush it into coarse powder, and place it in a round-bottom flask. Add 1200mL of 80% v / v ethanol solution, soak for 12h, then reflux at 80℃ for 3h. Repeat the extraction three times and combine the three extracts. Then filter under reduced pressure, concentrate, and dry to constant weight to obtain 13.5g of crude mulberry bark extract. Further purify the obtained crude mulberry bark extract using D101 macroporous resin: elute sequentially with pure water, 40% v / v ethanol, and 80% v / v ethanol, using 4-6 column volumes for each eluent. Collect the 80% v / v ethanol eluent, concentrate, and dry to constant weight to obtain 2.5g of mulberry bark extract (SBP). The yield is calculated (Table 1).

[0066] 2. Take 60g of dried hemp seeds, crush them into coarse powder, and place them in a round-bottom flask. Add 1200mL of 80% v / v ethanol solution, soak for 12h, then reflux at 80℃ for 3h. Repeat the extraction three times and combine the three extracts. Then filter under reduced pressure, concentrate, and dry to constant weight to obtain 5.6g of crude hemp extract. Further purify the obtained crude hemp extract using D101 macroporous resin: elute sequentially with pure water, 40% v / v ethanol, and 80% v / v ethanol, using 4-6 column volumes for each eluent. Collect the 80% v / v ethanol eluent, concentrate, and dry to constant weight to obtain 1.5g of hemp extract (ym). The yield is calculated (Table 1).

[0067] 3. Take 60g of dried Hosta root, crush it into coarse powder, and place it in a round-bottom flask. Add 1200mL of 80% v / v ethanol solution, soak for 12h, and then reflux extract. Heat and reflux at 80℃ for 3h, repeat the extraction three times, and combine the three extracts. Then filter under reduced pressure, concentrate, and dry to constant weight to obtain 17.4g of crude Hosta root extract. Further purify the obtained crude Hosta root extract using D101 macroporous resin: elute successively with pure water, 40% v / v ethanol, and 80% v / v ethanol. Collect the 80% v / v ethanol eluent, concentrate, and dry to constant weight to obtain 1.4g of Hosta root extract (yzg). The yield is calculated (Table 1).

[0068] 4. Take 60g of dried *Hypericum vulgare*, crush it into coarse powder, and place it in a round-bottom flask. Add 1200mL of 80% v / v ethanol solution, soak for 12h, and then reflux extract. Heat to reflux at 80℃ for 3h, repeat the extraction three times, and combine the three extracts. Then filter under reduced pressure, concentrate, and dry to constant weight to obtain 13.2g of crude *Hypericum vulgare* extract. Redissolve the prepared crude *Hypericum vulgare* extract in water, filter the precipitate; precipitate the precipitate with hot acetone, filter to obtain crude crystals, and obtain 8.8g of the *Hypericum vulgare* extract (hzc). The yield is statistically analyzed (Table 1).

[0069] Table 1. Yield of antibacterial extract (dry weight)

[0070] Extract Name abbreviation crude extract yield Extraction yield Mulberry bark extract sbp 22.5％ 4.2％ Cloud hemp extract ym 9.3％ 2.5％ Hosta root extract yzg 29.0％ 2.3％ Tiger Paw Grass Extract hzc 22.0％ 14.7％

[0071] Example 2

[0072] Detection of active biomarkers in extracts

[0073] The antibacterial activity markers in the four extracts obtained in Example 1 were qualitatively and quantitatively analyzed using LC-MS technology.

[0074] Detection Method: Extract samples and standards were dissolved separately in methanol (mass spectrometry grade, Merck) and filtered through a 0.22 μm organic membrane. Data acquisition was performed using an Agilent Technologies 1290 Infinity II ultra-high performance liquid chromatography-mass spectrometry (UHPLC) system coupled with a G6545B quadrupole-time-of-flight tandem mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Extracts were separated using an Agilent SB C18 reversed-phase column (2.1 mm × 100 mm, 1.8 μm, Agilent Technologies). The column temperature was set at 35 °C, and the mobile phase consisted of 0.1% formic acid in water (phase A) and pure acetonitrile (phase B), with a gradient elution flow rate of 0.15 mL / min. The optimized elution program was as follows: 0–2 min, 20%–40% B; 2–15 min, 40%–60% B; 15–20 min, 60%–70% B; 20–40 min, 70%–90% B; 40–45 min, 90%–95% B. Data were acquired in both ESI positive and negative ion modes.

[0075] Compound identification was performed by comparing the mass spectrometry data of the compounds with those of corresponding standards and mass spectrometry data reported in the literature. The structural formulas of each compound are as follows: Figure 2 As shown in Table 2. The identified compounds were quantitatively analyzed using a standard curve, with diosgenin used as a substitute for the active compounds in *Hosta plantago-aquatica* root. The results are shown in Table 2 and... Figure 3 .

[0076] Table 2. Qualitative and quantitative analysis results of antibacterial activity markers in extracts.

[0077]

[0078]

[0079] The table number corresponds to Figure 2 and Figure 3 The number in the middle.

[0080] From Table 2 and Figure 3It can be seen that the main antibacterial activity markers of each extract are as follows: Mulberry bark extract is mainly composed of flavonoids, specifically represented by Morusin, Kuwanon G, and Kuwanon H; Hemp root extract is mainly composed of cannabinoids, specifically represented by cannabidiol and cannabidivarin; Hosta root extract is mainly composed of saponins, specifically represented by (25R)-2α,3β-dihydroxy-5α-spirost-9-en-12-one-3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (hereinafter referred to as Q-Marker 6); Tiger's paw extract is mainly composed of saponins, specifically represented by prosapogenin CP4. CP4 is the representative.

[0081] Example 3

[0082] Antibacterial activity detection of extracts and their antibacterial activity markers

[0083] The minimum inhibitory concentration (MIC) and inhibition curves were used as the main indicators to detect and evaluate the antibacterial activity of the extract prepared in Example 1 and the active markers detected in Example 2.

[0084] (1) MIC value detection method: Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), fluconazole-resistant Candida albicans, drug-resistant Escherichia coli (E. coli), and drug-resistant Acinetobacter baumannii (A. baumannii) were cultured in trypsin-soybean broth (TSB) to the exponential phase, and the bacterial concentration was adjusted to approximately 5 × 10⁻⁶. 5 Five microbial suspensions were prepared using CFU / mL. Samples or antibiotics were prepared in 96-well plates using a two-fold dilution method, incubated at 37°C for 24 hours, and then OD was measured using a microplate reader. 600 The nm value was used to determine the MIC value. A control group was included in the experiment, and the experiment was repeated three times independently. The test results are shown in Table 3.

[0085] (2) Method for testing the inhibition curve: The above five microbial suspensions were mixed with test samples at concentrations of 1 / 4 and 1 / 2 MIC, respectively, mixed with a positive antibiotic at 1 / 2 MIC, and used as a control group with a blank solvent. The mixtures were incubated at 37°C. The OD values ​​of each sample were measured using an ELISA reader at 0h, 2h, 4h, 6h, 8h, 12h, 16h, 20h, and 24h of incubation. 600nmValues ​​were calculated and independently repeated three times. This was used to demonstrate the effect of the extract on microbial growth. The results are as follows: Figure 4 As shown.

[0086] Table 3. MIC values ​​(μg / mL) of extracts and their biomarkers

[0087]

[0088] Where a represents positive control drug; b represents negative control drug; NT represents not detected. Table 3 shows that the extracts of mulberry bark and hemp seed have significant anti-MRSA and VRE activities, with MICs of 4–8 μg / mL; the extracts of hosta root and tiger's paw have significant anti-C. albicans activities, with a MIC of 16 μg / mL; juniper alcohol has significant anti-C. albicans, E. coli, and A. baumannii activities, with MICs of 4–32 μg / mL; simultaneously, the active markers in the extracts showed good antibacterial activity, with MICs of 2–16 μg / mL, which are the material basis for the antibacterial activity of the extracts.

[0089] Examples 4 to 11

[0090] Preparation of antibacterial compositions and detection of their antibacterial activity

[0091] Prepare antibacterial compositions according to the formulations recorded in Table 4.

[0092] Table 4. Composition details of Examples 4 to 11

[0093]

[0094] The antibacterial activity of the compositions described in the above eight examples was tested according to the method described in Example 3. The minimum inhibitory concentration (MIC) of each composition in the bacterial culture is shown in Table 5. The results are shown in Table 5 and... Figures 4 to 11 As shown.

[0095] Table 5. MIC and interaction relationships of each antibacterial composition.

[0096]

[0097] NT indicates that it was not detected.

[0098] According to the antibacterial test results in Table 5, the compositions of Example 4, Example 5, Example 10, and Example 11 exhibited a synergistic effect on MRSA, while the compositions of Example 5, Example 10, and Example 11 also showed a synergistic effect on VRE. That is, the root extract of *Hosta plantago-aquatica* and the extract of *Gnaphalium affine* enhanced the antibacterial activity of *Morus alba* bark extract against MRSA; simultaneously, the addition of juniper alcohol improved the antibacterial activity of the compositions of Example 6 and Example 7 against both MRSA and VRE.

[0099] Figures 4 to 11 The antibacterial test results showed that the compositions of the present invention have good antibacterial activity. At a concentration of 1 / 2 MIC, the extracts of mulberry bark and hemp seed were superior to positive control antibiotics against MRSA, the root extract of hosta was superior to fluconazole-resistant Candida albicans, and juniper alcohol was superior to positive control antibiotics against drug-resistant Acinetobacter baumannii and drug-resistant Escherichia coli. Specifically, the compositions of Examples 4 to 11 showed superior antibacterial activity against MRSA compared to vancomycin; the compositions of Examples 5, 10, and 11 showed superior antibacterial activity against VRE compared to meropenem; and the compositions of Examples 8, 9, 10, and 11 showed superior antibacterial activity against drug-resistant Acinetobacter baumannii compared to cefoperazone.

[0100] Example 12

[0101] Preparation of antibacterial cream based on Example 8

[0102] The raw materials for making antibacterial cream are shown in Table 6.

[0103] Table 6. Ingredients and Content of Antibacterial Cream

[0104]

[0105] The production steps are as follows:

[0106] (1) Phase A: Weigh each component of Phase A into an emulsifying pot according to the dosages recorded in Table 6. Set the heating temperature to 80°C and stir while heating until the oil phase melts. Then lower the temperature to 60°C and keep it warm for later use.

[0107] (2) Phase B: Weigh each component of composition 5 (mulberry bark extract, hosta root extract, and juniper alcohol) according to the dosages recorded in Table 6, and add polyoxyethylene hydrogenated castor oil. Stir and mix at 50°C until the composition and polyoxyethylene hydrogenated castor oil are fully mixed, then add propylene glycol. Continue stirring until the composition is fully dissolved in propylene glycol, then add Tween 80, stir and mix well, and keep warm for later use.

[0108] (3) Phase C: Weigh glycerol, triethanolamine, benzyl alcohol and purified water, heat to 60°C, stir and mix well, and keep warm for later use.

[0109] (4) Mixing: Slowly pump phase B into the emulsifying tank and stir it evenly with phase A. Then pump phase C into the emulsifying tank and stir it evenly again. If necessary, vacuum degassing can be performed to remove air bubbles. Stop stirring after the mixture temperature drops to room temperature, and then dispense it into containers to obtain the final product.

[0110] Example 13

[0111] Preparation of antibacterial ointment based on Example 9

[0112] The raw materials for making the antibacterial ointment are shown in Table 7.

[0113] Table 7. Ingredients and Content of Antibacterial Cream

[0114]

[0115] The production steps are as follows:

[0116] (1) Phase A: Weigh each component of Phase A into an oil pan according to the dosages recorded in Table 7. Set the heating temperature to 80°C, and stir while heating until the oil phase melts and the matrix becomes clear and transparent. Then lower the temperature to 60°C and keep it warm for later use.

[0117] (2) Phase B: Weigh each component of composition 6 (mulberry bark extract, tiger paw grass extract, and juniper alcohol) according to the dosages recorded in Table 7, and add polyoxyethylene hydrogenated castor oil. Stir and mix thoroughly at 50°C to ensure the composition is fully mixed with the polyoxyethylene hydrogenated castor oil. Then, continue adding propylene glycol and stirring to ensure the composition is fully dissolved in the propylene glycol. Next, add borneol and Tween 80, and continue stirring until the borneol is completely dissolved, ensuring the entire system is uniformly mixed. Maintain the temperature at 50°C for later use.

[0118] (3) Mixing: Slowly pour the well-mixed phase B into phase A and stir until homogeneous. If necessary, vacuum degassing can be performed to remove air bubbles. After homogeneous mixing, maintain the temperature at 60°C for dispensing, and then cool to room temperature to obtain the final product.

[0119] Example 14

[0120] Testing of the bactericidal and bacteriostatic properties of antibacterial creams and antibacterial creams

[0121] The antibacterial cream prepared in Example 12 and the antibacterial cream prepared in Example 13 were used as test samples to test their antibacterial properties against Staphylococcus aureus, Escherichia coli, Candida albicans, methicillin-resistant Staphylococcus aureus, and drug-resistant Acinetobacter baumannii. The test methods were performed according to WS / T 650-2019 "Evaluation Methods for Antibacterial and Antimicrobial Efficacy" 5.2.2.5 and 2.1.1.5; the test results are shown in Tables 8 and 9.

[0122] Table 8. Bactericidal effect of antibacterial cream.

[0123] strains Inhibition rate (%) after 5.0 min of action Mean colony count in positive control Staphylococcus aureus 93.69 <![CDATA[3.71×10 4 ]]> E. coli 94.64 <![CDATA[6.16×10 4 ]]> Candida albicans 90.23 <![CDATA[3.07×10 4 ]]> Methicillin-resistant Staphylococcus aureus 93.82 <![CDATA[3.67×10 4 ]]> Drug-resistant Acinetobacter baumannii 94.04 <![CDATA[3.51×10 4 ]]>

[0124] The inhibition rate was the average of three repeated tests; the negative control showed no bacterial growth.

[0125] Table 9. Antibacterial effect of antibacterial ointment in antibacterial test.

[0126] strains Inhibition rate (%) after 5.0 min of action Mean colony count in positive control Staphylococcus aureus 99.95 <![CDATA[8.37×10 4 ]]> E. coli 99.98 <![CDATA[8.18×10 4 ]]> Candida albicans 99.67 <![CDATA[8.12×10 4 ]]> Methicillin-resistant Staphylococcus aureus 99.97 <![CDATA[7.55×10 4 ]]> Drug-resistant Acinetobacter baumannii 99.96 <![CDATA[8.05×10 4 ]]>

[0127] The inhibition rate was the average of three repeated tests; the negative control showed no bacterial growth.

[0128] As shown in Tables 8 and 9, the antibacterial cream provided in Example 12, after 5.0 minutes of application, exhibited a bactericidal rate >90% against all five types of bacteria, demonstrating antibacterial activity and meeting the requirements of WS / T 650-2019 "Evaluation Method for Antibacterial and Bacteriostatic Effects". The antibacterial cream provided in Example 13, after 5.0 minutes of application, showed an inhibition rate >90% against all five types of bacteria, demonstrating strong antibacterial activity and meeting the requirements of WS / T 650-2019 "Evaluation Method for Antibacterial and Bacteriostatic Effects".

[0129] Example 15

[0130] The antibacterial cream prepared in Example 12 and the antibacterial cream prepared in Example 13 were used as test samples. Multiple complete skin irritation tests were conducted on Japanese rabbits. The test method was based on Section 2.3.3.3.3 of the "Disinfection Technical Specifications" (2002 edition). The test results are shown in Tables 10 and 11.

[0131] Table 10. Scoring results of repeated intact skin irritation reactions to the antibacterial cream in Example 5.

[0132]

[0133]

[0134] Table 11. Results of repeated intact skin irritation reactions to the antibacterial ointment in Example 6.

[0135]

[0136] As shown in Tables 10 and 11, the total average score (irritation index) of the antibacterial cream prepared in Example 12 and the antibacterial cream prepared in Example 13 in multiple complete skin irritation tests was 0.00 (0—<0.5), indicating that the irritation intensity was non-irritating. This meets the requirements of the "Disinfection Technical Specifications" (2002 edition).

[0137] As can be seen from the above embodiments, the antibacterial composition provided by the present invention has good inhibitory effects on methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), fluconazole-resistant Candida albicans, drug-resistant Escherichia coli, and drug-resistant Acinetobacter baumannii. The antibacterial creams and antibacterial ointments prepared based on this antibacterial composition have strong antibacterial activity against antibiotic-resistant bacteria, can be used to inhibit the proliferation of fungi or bacteria, and improve infection symptoms, while being non-irritating to the skin.

[0138] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antibacterial composition, characterized in that, The antibacterial composition is juniper alcohol, hemp extract and hosta root extract; or juniper alcohol, hemp extract and tiger paw extract; In the antibacterial composition, the final concentration of juniper alcohol is 32 μg / mL, the final concentration of hemp extract is 4 μg / mL, the final concentration of hosta root extract is 16 μg / mL, and the final concentration of tiger paw extract is 16 μg / mL. The antibacterial composition inhibits pathogens that are methicillin-resistant Staphylococcus aureus and / or vancomycin-resistant Enterococci. The preparation method of the cloud hemp extract includes: mixing cloud hemp powder with 80%~95% v / v ethanol solution, soaking for 12~24 hours, extracting by reflux at 80~90℃ 2~5 times, each time for 3~5 hours, combining the extracts, filtering under reduced pressure, concentrating, and drying to constant weight to obtain cloud hemp crude extract; purifying the cloud hemp crude extract to obtain cloud hemp extract; The preparation method of the Hosta root extract includes: mixing Hosta root powder with 80%~95% v / v ethanol solution, soaking for 12~24 hours, extracting by reflux at 80~90℃ 2~5 times, 3~5 hours each time, combining the extracts, filtering under reduced pressure, concentrating, and drying to constant weight to obtain a crude Hosta root extract; purifying the crude Hosta root extract to obtain the Hosta root extract. The preparation method of the tiger paw grass extract includes: mixing tiger paw grass powder with 80%~95% v / v ethanol solution, soaking for 12~24 hours, extracting by reflux at 80~90℃ 2~5 times, each time for 3~5 hours, combining the extracts, filtering under reduced pressure, concentrating, and drying to constant weight to obtain crude tiger paw grass extract; redissolving the crude tiger paw grass extract in water, filtering, precipitating with hot acetone, and filtering again to obtain the tiger paw grass extract. The ratio of the hemp powder to the 80%~95% v / v ethanol solution is 40~60 g / L; the ratio of the hosta root powder to the 80%~95% v / v ethanol solution is 40~60 g / L; the ratio of the tiger paw grass powder to the 80%~95% v / v ethanol solution is 40~60 g / L.

2. The antibacterial composition according to claim 1, characterized in that, Based on the content of dried product, the total amount of cannabidiol and hypocannabidiol in the cloud hemp extract is 10wt%~60wt%. The total amount of (25R)-2α,3β-dihydroxy-5α-spirost-9-en-12-one-3-O-β-D-glucopyranoside-(1→2)-[β-D-xylopyranoside-(1→3)]-β-D-glucopyranoside-(1→4)-β-D-galactopyranoside in the *Hosta* root extract is 1 wt% to 10 wt%. The content of secondary saponin CP4 in the tiger paw grass extract is 20wt%~80wt%.

3. The antibacterial composition according to claim 1, characterized in that, The purification process includes purification using macroporous resins.

4. The use of the antibacterial composition according to any one of claims 1 to 3 in the preparation of antibacterial creams and / or antibacterial ointments, characterized in that, The antibacterial cream and / or antibacterial cream inhibit pathogens that are methicillin-resistant Staphylococcus aureus and / or vancomycin-resistant Enterococci.

5. An antibacterial cream, characterized in that, The antibacterial cream is made from the following components: 0.02wt%~1wt% of the antibacterial composition according to any one of claims 1 to 3, 10wt%~30wt% of glycerol, 4wt%~10wt% of beeswax, 4wt%~10wt% of liquid paraffin, 1wt%~10wt% of stearyl alcohol, 0.5wt%~5wt% of cetyl alcohol, 1wt%~10wt% of white petrolatum, 1wt%~8wt% of silicone oil, 0.2wt%~5wt% of glyceryl monostearate, 1wt%~6wt% of propylene glycol, 0.1wt%~2wt% of Tween 80, 0.1wt%~1wt% of triethanolamine, and the balance being water and polyoxyethylene hydrogenated castor oil; the mass ratio of the polyoxyethylene hydrogenated castor oil to the antibacterial composition is 0.5~3:1; The antibacterial cream inhibits pathogens that are methicillin-resistant Staphylococcus aureus and / or vancomycin-resistant Enterococci.

6. An antibacterial ointment, characterized in that, The antibacterial ointment is made from the following components: 0.02wt% to 1wt% of the antibacterial composition according to any one of claims 1 to 3, 60wt% to 80wt% of white petrolatum, 5wt% to 20wt% of glycerol, 1wt% to 10wt% of lanolin, 0.5wt% to 5wt% of liquid paraffin, 0.2wt% to 5wt% of glyceryl monostearate, 1wt% to 6wt% of propylene glycol, 0.1wt% to 2wt% of Tween 80, 0.0wt% to 0.5wt% of borneol, and the balance being water and polyoxyethylene hydrogenated castor oil; The mass ratio of the polyoxyethylene hydrogenated castor oil to the antibacterial composition is 0.5~3:1; The antibacterial ointment inhibits pathogens that are methicillin-resistant Staphylococcus aureus and / or vancomycin-resistant Enterococci.

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