Application of *Ulmus pumila* extract in the preparation of antibacterial drugs

Abstract

This invention discloses the application of *Ulva prostrata* extract in the preparation of preventive and therapeutic antibacterial drugs. The antibacterial components of *Ulva prostrata* are extracted by ethanol extraction using a reflux extraction method. The extract shows significant antibacterial effects against *Staphylococcus aureus*, *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, and *Micrococcus luteus*. The minimum inhibitory concentrations (MICs) are 3.125 mg / mL, 12.5 mg / mL, 6.25 mg / mL, and 3.125 mg / mL, respectively. The extract exhibits good stability to heat treatment and ultraviolet light, and shows good antibacterial effects in neutral and slightly alkaline environments. Metal ions affect the antibacterial effect of the extract, but the diameter of the inhibition zone is greater than 7 mm.

Description

Technical Field

[0001] This invention relates to novel uses of *Ulva prostrata* and its extracts, specifically to the use of *Ulva prostrata* extracts in the preparation of preventive and therapeutic antibacterial drugs. Background Technology

[0002] Pathogenic microorganisms are microorganisms that can invade the human body, causing infection and even infectious diseases; they are also called pathogens. Among pathogens, bacteria and viruses are the most harmful. Pathogenic microorganisms include prions, fungi, bacteria, spirochetes, mycoplasma, rickettsiae, chlamydia, and viruses. For example, Staphylococcus aureus (S. aureus), also known as "Staphylococcus aureus," belongs to the genus Staphylococcus and is a representative Gram-positive bacterium. It is a common foodborne pathogen that can produce enterotoxins, causing food poisoning. Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa, is an opportunistic pathogen that impairs the immune system. It generally affects the lungs and urinary tract, or causes burns, wounds, and other blood infections, such as sepsis. Although rare, Pseudomonas aeruginosa can also cause pneumonia. Inadequate cleaning of contact lenses can also lead to corneal infections. Many studies on ventilator-associated pneumonia have indicated that Pseudomonas aeruginosa is one of the bacteria that needs to be isolated. Acinetobacter baumannii is a non-fermenting Gram-negative bacillus widely distributed in nature and is considered an opportunistic pathogen. It is an important pathogen causing hospital-acquired infections, primarily respiratory tract infections, but can also lead to bacteremia, urinary tract infections, secondary meningitis, surgical site infections, and ventilator-associated pneumonia. Its resistance to commonly used antibiotics has been increasing year by year, causing serious concern among clinicians and microbiologists. Micrococcus luteus belongs to the Micrococcidae family and the Micrococcus genus. It is a Gram-positive genus of bacteria, and can be aerobic or inter-anaerobic. Micrococcus luteus is distributed in air, water, soil, and on the surfaces of plants and animals. This bacterium is generally non-pathogenic, but can be an opportunistic pathogen, causing local tissue infections such as wounds, and can also cause serious infections such as endocarditis.

[0003] Currently, antibiotics are the main treatment for pathogenic bacteria. However, the extensive use of antibiotics can easily lead to drug resistance, and some patients may also be allergic to antibiotics. Traditional Chinese medicine preparations are less likely to cause drug resistance or allergies, and therefore have promising development prospects.

[0004] *Lysimachia paridiformis* Franch. var. *stenophylla* Franch. and *Lysimachia paridiformis* Franch. (both belonging to the Primulaceae family) are dried whole herbs. They possess properties of dispelling wind, promoting blood circulation, and relieving pain. They are used for rheumatic pain, limb contractures, hemiplegia, injuries from falls, swelling and pain due to blood stasis, and infantile convulsions. They are distributed in southwestern my country. Currently, there are no reports of antibacterial effects of *Lysimachia paridiformis* or its extracts. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems, the present invention aims to provide the application of *Ulva prostrata* extract in the preparation of preventive and therapeutic antibacterial drugs.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] Application of *Ulva prostrata* extract in the preparation of preventive and therapeutic antibacterial drugs.

[0008] The aforementioned application of the extract of *Ulva prostrata* in the preparation of preventive and therapeutic antibacterial drugs is described in that the extract of *Ulva prostrata* has antibacterial effects against *Staphylococcus aureus*, *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, and *Micrococcus luteus*.

[0009] The aforementioned application of *Ulva prostrata* extract in the preparation of preventive and therapeutic antibacterial drugs, wherein the *Ulva prostrata* extract is prepared according to the following steps:

[0010] (1) Take 0.5g of dried Chasing Wind Umbrella into a 250mL round-bottom flask, add 50-55% ethanol, weigh it, the ratio of Chasing Wind Umbrella to 50-55% ethanol is 1g:25-35mL, place it in a 65-70℃ constant temperature water bath for 200-250min, then make up the weight loss with 50-55% ethanol, shake well, filter, take the filtrate, and obtain the initial extract of Chasing Wind Umbrella.

[0011] (2) Take the initial extract of the wind-chasing umbrella, concentrate it under reduced pressure, evaporate it to dryness in a water bath, and then fully dissolve it in sterile water to obtain a solution with a concentration of 90-110 mg / mL. Filter it through a 0.22 μm filter membrane to remove bacteria, and the wind-chasing umbrella extract is obtained.

[0012] Specifically, the aforementioned *Ulva prostrata* extract is used in the preparation of preventive and therapeutic antibacterial drugs, wherein the *Ulva prostrata* extract is prepared according to the following steps:

[0013] (1) Take 0.5g of dried Chasing Wind Umbrella into a 250mL round-bottom flask, add 53% ethanol, weigh it, the ratio of Chasing Wind Umbrella to 53% ethanol is 1g:31mL, place it in a 67℃ constant temperature water bath for 237min, then make up the weight loss with 53% ethanol, shake well, filter, take the filtrate, and obtain the initial extract of Chasing Wind Umbrella.

[0014] (2) Take the initial extract of the wind-chasing umbrella, concentrate it under reduced pressure, evaporate it to dryness in a water bath, and then fully dissolve it in sterile water to obtain a solution with a concentration of 100 mg / mL. Filter it through a 0.22 μm filter membrane to remove bacteria, and the wind-chasing umbrella extract is obtained.

[0015] Application of the Wind Chaser Umbrella in the preparation of preventive and therapeutic antibacterial drugs.

[0016] A preventive and therapeutic antibacterial drug, characterized in that: the drug includes an extract of *Ulva prostrata* or *Ulva prostrata*.

[0017] A drug for prevention, treatment, and antibacterial action, characterized in that: the main active ingredient in the drug is *Ulva prostrata* extract or *Ulva prostrata*.

[0018] The beneficial effects of the present invention are:

[0019] The extract of *Ulva chamaejasminoides* showed significant antibacterial effects against *Staphylococcus aureus*, *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, and *Micrococcus luteus*, with minimum inhibitory concentrations (MICs) of 3.125 mg / mL, 12.5 mg / mL, 6.25 mg / mL, and 3.125 mg / mL, respectively. Stability was studied using *Staphylococcus aureus* and *Acinetobacter baumannii* as test strains. Results showed that the extract exhibited good stability to heat treatment and ultraviolet radiation, and good antibacterial effects in neutral and slightly alkaline environments. Metal ions affected the antibacterial effect, but the diameter of the inhibition zone was greater than 7 mm in all cases. Attached Figure Description

[0020] Figure 1 Effect of temperature on the antibacterial activity of *Ulmus parvifolia* extract;

[0021] Figure 2 The effect of ultraviolet light on the antibacterial activity of *Umbrella charcoalii* extract;

[0022] Figure 3 Effect of pH on the antibacterial activity of *Ulmus parvifolia* extract;

[0023] Figure 4 The effect of metal ions on the antibacterial activity of *Ulmus parvifolia* extract.

[0024] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention. Detailed Implementation

[0025] Example 1:

[0026] Extraction process:

[0027] (1) Take 0.5g of dried Chasing Wind Umbrella into a 250mL round-bottom flask, add 53% ethanol, weigh it, the ratio of Chasing Wind Umbrella to 53% ethanol is 1:31mL, place it in a 67℃ constant temperature water bath for 237min, then make up the weight loss with 53% ethanol, shake well, filter, take the filtrate, and obtain the initial extract of Chasing Wind Umbrella.

[0028] (2) Take the initial extract of the wind-chasing umbrella, concentrate it under reduced pressure, evaporate it to dryness in a water bath, and then fully dissolve it in sterile water to obtain a solution with a concentration of 100 mg / mL. Filter it through a 0.22 μm filter membrane to remove bacteria, and the wind-chasing umbrella extract is obtained.

[0029] Formulation process: Mix the extract of *Ulva chaperone* with 10% starch, granulate with 75% ethanol, dry, granulate, and fill into capsules to obtain the extract of *Ulva chaperone*.

[0030] Efficacy: Treatment of infections caused by Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Micrococcus luteus.

[0031] Dosage and administration: Oral administration, 3-4 capsules each time, 3 times a day.

[0032] Example 2:

[0033] Extraction process:

[0034] (1) Take 0.5g of dried Chasing Wind Umbrella into a 250mL round-bottom flask, add 50% ethanol, weigh it, the ratio of Chasing Wind Umbrella to 50% ethanol is 1g:25mL, place it in a 65℃ constant temperature water bath for 200min, then make up the weight loss with 50% ethanol, shake well, filter, take the filtrate, and obtain the initial extract of Chasing Wind Umbrella.

[0035] (2) Take the initial extract of the wind-chasing umbrella, concentrate it under reduced pressure, evaporate it to dryness in a water bath, and then fully dissolve it in sterile water to obtain a solution with a concentration of 110 mg / mL. Filter it through a 0.22 μm filter membrane to remove bacteria, and the wind-chasing umbrella extract is obtained.

[0036] Formulation process: Take the extract of *Ulva prostrata* and add 10% starch and mix evenly. Use an appropriate amount of starch slurry with a concentration of 10% to make soft material, granulate it through a 16-mesh sieve, dry it at 55℃, add 2% talc powder after granulation, mix evenly, compress into tablets, and coat with film to obtain tablets.

[0037] Efficacy: Treatment of infections caused by Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Micrococcus luteus.

[0038] Dosage and administration: Oral administration, 3-4 tablets each time, 3 times a day.

[0039] Example 3:

[0040] Extraction process:

[0041] (1) Take 0.5g of dried Chasing Wind Umbrella into a 250mL round-bottom flask, add 55% ethanol, weigh it, the ratio of Chasing Wind Umbrella to 55% ethanol is 1g:35mL, place it in a 70℃ constant temperature water bath for 250min, then make up the weight loss with 55% ethanol, shake well, filter, take the filtrate, and obtain the initial extract of Chasing Wind Umbrella.

[0042] (2) Take the initial extract of the Wind Chaser Umbrella, concentrate it under reduced pressure, evaporate it to dryness in a water bath, and then fully dissolve it in sterile water to obtain a solution with a concentration of 90 mg / mL. Filter it through a 0.22 μm filter membrane to remove bacteria, and the Wind Chaser Umbrella extract is obtained.

[0043] Formulation process: Take the extract of *Ulva prostrata* and concentrate it under reduced pressure to a thick paste with a relative density of 1.30-1.35 (65-70℃). Then add 35% dextrin and 45% sucrose by weight, mix well, form into granules, and dry at 55℃ to obtain granules.

[0044] Efficacy: Treatment of infections caused by Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Micrococcus luteus.

[0045] Dosage and administration: Oral administration, 15-20g each time, 3 times a day.

[0046] The inventors conducted numerous experiments, and the following is a study on the antibacterial effect of the *Umbrella chaperone* extract described in this invention:

[0047] 1. Extraction process:

[0048] Reflux method: Take 0.5g of dried *Ulva chaperone* in a 250mL round-bottom flask, add 53% ethanol, weigh it, the ratio of *Ulva chaperone* to 53% ethanol is 1:31mL, reflux in a 67℃ constant temperature water bath for 237min, then make up the weight loss with 53% ethanol, shake well, filter, take the filtrate and concentrate under reduced pressure, evaporate to dryness in a water bath, then fully dissolve in sterile water to 100mg / mL, filter through a 0.22μm filter membrane for sterilization, and obtain *Ulva chaperone* extract.

[0049] 2 Evaluation of antibacterial effect

[0050] 2.1 Determination of Minimum Inhibitory Concentration

[0051] The minimum inhibitory concentration (MIC) of the extract of *Ulva lactuca* was determined in a 96-well microtiter plate using the two-fold dilution method. A bacterial suspension was prepared by shaking the bacterial suspension and broth at a ratio of 10:990 μL. 50 μL of the solution was added to the first well of the microtiter plate and diluted sequentially up to the 10th well. 50 μL of broth was added to wells 2-12, and 50 μL of the bacterial suspension was added to wells 1-11. Well 11 was not treated with the solution, and well 12 contained only broth and no solution, serving as a positive and negative control. The plates were incubated at 37°C for 18-24 h.

[0052] 2.2 Determination of its antibacterial stability using the filter paper disc method

[0053] 2.2.1 Effect of pH on the antibacterial stability of *Ulmus pumila* extract

[0054] The pH of the *Ulva prostrata* extract was adjusted to 3.0, 5.0, 7.0, 9.0, and 11.0 using 1 mol / L HCl and NaOH, respectively. Filter paper discs were then immersed in the solutions to ensure complete absorption. Staphylococcus aureus, *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, and *Micrococcus luteus* were used as test bacteria. The inhibition diameters under various conditions were determined using the filter paper disc method, repeated three times.

[0055] 2.2.2 Effect of temperature on the antibacterial stability of *Ulmus pumila* extract

[0056] Extracts from *Ulmus pumila* were treated for 30 min each under water bath conditions at 40℃, 60℃, 80℃, and 100℃, and under high-pressure humid heat conditions at 121℃. Staphylococcus aureus and Acinetobacter baumannii were used as test bacteria, and the inhibition diameters under each factor were determined using the filter paper disc method, repeated three times.

[0057] 2.2.3 Effect of UV irradiation on the antibacterial stability of *Ulva prostrata* extract

[0058] Extracts from *Ulva prostrata* were irradiated under ultraviolet light for 10 min, 20 min, 30 min, 40 min, and 60 min, respectively. Staphylococcus aureus and Acinetobacter baumannii were used as test bacteria, and the inhibition diameters under various conditions were determined using the filter paper disc method, repeated three times.

[0059] 2.2.4 Effect of Metal Ions on the Antibacterial Stability of *Ulmus pumila* Extract

[0060] Extracts of *Ulva prostrata* were placed under a UV lamp and equal volumes of 0.05 mol / L AlCl3, CaCl2, FeCl3, and NaCl solutions were added respectively. *Ulva prostrata* extracts of the same concentration served as a blank control group. Staphylococcus aureus and Acinetobacter baumannii were used as test bacteria, and the inhibition diameters under various factors were determined using the filter paper disc method, repeated three times.

[0061] Results and analysis of antibacterial activity of *Ulmus pumila* extract.

[0062] 3.1 Determination of the minimum inhibitory concentration (MIC) of the extract of *Ulmus pumila*

[0063] The results of the minimum inhibitory concentrations (MICs) of the *Ulva chamaejasminoides* extract are shown in Table 1. The extract showed significant antibacterial effects against *Staphylococcus aureus*, *Pseudomonas aeruginosa*, *Acinetobacter baumannii*, and *Micrococcus luteus*, with MICs of 3.125 mg / mL, 12.5 mg / mL, 12.5 mg / mL, 6.25 mg / mL, and 3.125 mg / mL, respectively.

[0064] Table 1. Minimum inhibitory concentration of *Umbrella chameleon* extract.

[0065]

[0066] Note: "-" indicates no sterile growth; "+" indicates low bacterial growth; "++" indicates high bacterial growth; "+++" indicates very high bacterial growth.

[0067] 3.2 Determination of antibacterial effect

[0068] Inhibition zones larger than 7 mm showed antibacterial activity, while those smaller than 7 mm had no antibacterial activity. Larger inhibition zones generally showed better activity. Staphylococcus aureus and Acinetobacter baumannii were selected as the test bacteria.

[0069] The KB method was used to immerse 6 mm drug-sensitive filter paper discs in 100 mg / mL drug solution for 15 min under sterile conditions. 50 mg / mL streptomycin sulfate was used as a positive control. The diameter of the inhibition zone was determined by the cross-hatching method. The determination was repeated three times. The results are shown in Table 2.

[0070] Table 2. Determination of antibacterial activity of *Ulmus pumila* extract.

[0071]

[0072] Note: "-" indicates no antibacterial effect.

[0073] 3.3 Antibacterial stability study

[0074] 3.3.1 Effect of temperature on antibacterial activity

[0075] Depend on Figure 1 It can be seen that the diameter of the inhibition zone of the *Staphylococcus aureus* and *Acinetobacter baumannii* extract first increases and then decreases with increasing temperature, indicating that the *Staphylococcus aureus* extract still has a certain antibacterial effect under high temperature treatment. However, high temperatures above 80℃ may destroy the active ingredients in the *Staphylococcus aureus* extract and significantly weaken its antibacterial effect.

[0076] 3.3.2 Effect of ultraviolet light on antibacterial activity

[0077] Depend on Figure 2It is known that under ultraviolet light irradiation, the inhibitory effect of the extract of *Zhuifengsan* on *Staphylococcus aureus* and *Acinetobacter baumannii* weakens with time. However, the inhibitory effect on *Acinetobacter baumannii* remains relatively stable under ultraviolet irradiation for 10-60 minutes, with no significant change in antibacterial activity. In contrast, the inhibitory effect on *Staphylococcus aureus* significantly decreases after 40 minutes of ultraviolet irradiation, with an inhibition zone diameter of 12.5 mm at 60 minutes. This indicates that the antibacterial active ingredients in *Zhuifengsan* have good stability against ultraviolet light.

[0078] 3.3.3 Effect of pH on antibacterial activity

[0079] Depend on Figure 3 It was observed that the levels of Staphylococcus aureus varied significantly after treatment with different pH values. This is likely because strong acids and alkalis disrupted the structure of the antibacterial substances in the *Ulva lactuca* extract, leading to a significant decrease in its antibacterial effect. At pH values ​​of 7.0-9.0, the diameter of the inhibition zone of *Ulva lactuca* extract against various bacterial species was less affected. Therefore, *Ulva lactuca* extract exhibits better antibacterial effects under neutral and slightly alkaline conditions.

[0080] 3.3.4 Effects of Metal Ions on Antibacterial Activity

[0081] Depend on Figure 4 It can be seen that, under the influence of various metal ions, the antibacterial effect of the *Chuifengsan* extract against *Staphylococcus aureus* and *Acinetobacter baumannii* decreased compared with the control group. The antibacterial effect of *Chuifengsan* extract against *Staphylococcus aureus* and *Acinetobacter baumannii* decreased most significantly after treatment with metal ion K+.

[0082] 4. Conclusion

[0083] Regarding antibacterial activity, the extract showed significant inhibitory effects against Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Micrococcus luteus, with minimum inhibitory concentrations (MICs) of 3.125 mg / mL, 12.5 mg / mL, 12.5 mg / mL, 6.25 mg / mL, and 3.125 mg / mL, respectively. The stability of the extract was studied using Staphylococcus aureus and Acinetobacter baumannii as test strains. The results showed that the extract exhibited good stability to heat treatment and ultraviolet radiation, and good antibacterial effects in neutral and slightly alkaline environments. Metal ions affected the antibacterial effect of the extract, but the diameter of the inhibition zone was greater than 7 mm in all cases.

[0084] 5 Discussions

[0085] This invention employs a heating reflux method to extract *Ulva lactuca* extract for antibacterial experiments, and examines its antibacterial stability under various factors. The results show that it retains good antibacterial activity and stability even after treatment with different temperatures, UV irradiation times, pH values, and metal ions. This study enriches the basic research on *Ulva lactuca*, provides a scientific basis for further research and development of this medicinal material, and offers a reference for researchers to find healthy, safe, and synthetic drug alternatives from natural products as natural preservatives.

Claims

1. The application of *Ulva prostrata* extract in the preparation of antibacterial drugs, characterized in that: The application is that the extract of *Ulva zebrina* has antibacterial effects against Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Micrococcus luteus. The extract of the Wind Chaser Umbrella was prepared according to the following steps: (1) Take 0.5g of dried Chasing Wind Umbrella into a 250mL round bottom flask, add 53% ethanol, weigh it, the ratio of Chasing Wind Umbrella to 53% ethanol is 1g:31mL, place it in a 67℃ constant temperature water bath for 237min, then make up the weight loss with 53% ethanol, shake well, filter, take the filtrate, and obtain the initial extract of Chasing Wind Umbrella. (2) Take the initial extract of the wind-chasing umbrella, concentrate it under reduced pressure, evaporate it to dryness in a water bath, and then fully dissolve it in sterile water to obtain a solution with a concentration of 100 mg / mL. Filter it through a 0.22 μm filter membrane to remove bacteria, and the wind-chasing umbrella extract is obtained.

2. An antibacterial drug, characterized in that: The drug includes the extract of *Ulva prostrata* as described in claim 1.

3. An antibacterial drug, characterized in that: The main active ingredient in the drug is the *Ulva prostrata* extract as described in claim 1.

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