Application of the extract of the plant of the genus lindera in preparing bacteriostatic products
By preparing clove and knotweed extract, the problems of pathogen resistance and the insufficiency of existing antibacterial agents have been solved, achieving effective inhibition of a variety of pathogens and providing an alternative to natural antibacterial agents.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
- Filing Date
- 2024-07-05
- Publication Date
- 2026-07-21
AI Technical Summary
In the current technology, the increasing resistance of pathogens to antibiotics makes many infectious diseases difficult to treat, and existing research on antibacterial agents is insufficient to show the antibacterial activity of Polygonum syringum.
Using clove and knotweed extract as an antibacterial agent, the clove and knotweed extract was prepared by hot reflux extraction with water or 75% ethanol as solvent. The prepared clove and knotweed extract showed significant antibacterial effects against a variety of pathogens, including Gram-negative and Gram-positive bacteria.
Clove extract has significant antibacterial effects against various pathogens such as Staphylococcus aureus and Escherichia coli, providing a new alternative to traditional antibiotics and reducing the risk of drug resistance.
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Figure CN118766977B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of traditional Chinese medicine extract technology, and in particular relates to the application of clove and knotweed extract in the preparation of antibacterial products. Background Technology
[0002] Pathogenic infections pose a direct threat to human health, and in severe cases can be life-threatening. Therefore, prevention and control of pathogenic infections are crucial. Using antibiotics is one effective measure, but inappropriate use of antibiotics can lead to increased drug resistance in pathogens, making otherwise treatable infectious diseases difficult to cure.
[0003] For example: Staphylococcus aureus ( Staphylococcus aureus Staphylococcus epidermidis is a highly pathogenic bacterium that can cause a wide range of clinical infections, including skin and soft tissue infections, osteomyelitis, endocarditis, and sepsis. Several drug-resistant strains have emerged clinically, including methicillin-resistant and vancomycin-resistant strains. Staphylococcus epidermidis Methicillin-resistant Staphylococcus epidermidis is a type of skin flora commonly found in medical device-related infections. Its multidrug-resistant strains are widely distributed in healthcare settings, increasing the complexity of treatment. Staphylococcus epidermidis Escherichia coli (E. coli) is a pathogen closely associated with drug-resistant infections, especially in hospital-acquired infections, and often exhibits resistance to multiple antibiotics. Escherichia coli Klebsiella pneumoniae is one of the main pathogens causing urinary tract infections and often leads to gastroenteritis and sepsis. Drug resistance in Escherichia coli is constantly increasing, with strains producing extended-spectrum β-lactamases and galactosidases being particularly common. Klebsiella pneumoniae Pseudomonas aeruginosa is easily transmitted in hospital settings, often causing serious infections such as pneumonia, urinary tract infections, and sepsis. Its multidrug resistance is becoming a serious public health problem globally, limiting treatment options. Pseudomonas aeruginosa Streptococcus pneumoniae (S. pneumoniae) is one of the leading pathogens causing hospital-acquired pneumonia, urinary tract infections, and wound infections due to its high drug resistance. Streptococcus pneumoniae It is one of the main pathogens causing community-acquired pneumonia, meningitis, otitis media and sepsis. Its drug resistance is becoming increasingly common, including resistance to multiple antibiotics such as penicillin, which increases the challenge of treatment.
[0004] Plant extracts are products obtained by extracting active ingredients from natural plants. They are one of the main forms of transforming medicinal plant resources. Natural products are rich in various active ingredients such as flavonoids, terpenes, and polyphenols, and possess functions such as antibacterial, antiviral, antioxidant, and immunomodulatory effects. Some medicinal plants, as naturally derived antibacterial agents, have certain antibacterial activity with low side effects and toxicity, and can serve as alternative treatment options, reducing dependence on traditional antibiotics.
[0005] clove knotweed ( Ludwigia ovalis *Polygonum styracifolium* (Miq.) is a plant belonging to the genus *Polygonum* in the family Onagraceae. It has stems that root at the nodes, and its leaves are ovate. It mainly grows in moist, shallow water areas. *Polygonum styracifolium* is primarily used as an adjunct treatment for lung-heat cough, sore throat, red and swollen eyes, damp-heat diarrhea, jaundice, painful urination, edema, leukorrhea, hematemesis, hematuria, intestinal bleeding, boils, scabies, traumatic injuries, external bleeding, and snake / insect bites, as well as rabies bites. *Polygonum styracifolium* possesses certain pharmacological activities, but research on its antibacterial activity is relatively lacking. Summary of the Invention
[0006] To address the shortcomings of existing technologies, this invention provides the application of clove and knotweed extract in the preparation of antibacterial products. This invention uses 12 common animal pathogens as activity test targets to study the antibacterial activity of clove and knotweed extract, which helps to better develop and utilize plant resources and provides a new approach to finding alternative antibacterial agents.
[0007] The technical solution provided by this invention is as follows: This invention provides the application of clove and knotweed extract in the preparation of antibacterial products, wherein the preparation method of the clove and knotweed extract includes the following steps: S1. Take dried *Syringa ovata*, a plant of the genus *Syringa* in the family Onagraceae, crush it, and pass it through a 40-mesh sieve to obtain fragments of *Syringa ovata*. S2: Weigh an appropriate amount of *Syringa ovata* fragments obtained in S1, add an extraction solvent equivalent to 9-11 times the weight of the *Syringa ovata* fragments, soak in the extraction container for 12 hours, and then perform hot reflux extraction twice. The first hot reflux extraction is for 2 hours, and the second hot reflux extraction is for 1 hour. After cooling, filter to remove residue, combine the filtrates, concentrate under reduced pressure, and evaporate the solvent at 50℃~60℃ to obtain a dry extract, which is then stored at -20℃.
[0008] Furthermore, the extraction solvent includes distilled water or 75% ethanol.
[0009] Furthermore, the reflux temperature is 100~105℃ when the extraction solvent is water, and 80~90℃ when the extraction solvent is 75% ethanol.
[0010] Furthermore, the bacteria are selected from one or more Gram-negative and Gram-positive bacteria.
[0011] Furthermore, the Gram-negative bacteria are selected from Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii, and the Gram-positive bacteria are selected from Staphylococcus aureus, Micrococcus luteus, Bacillus, Enterococcus faecalis, Staphylococcus epidermidis, methicillin-resistant Staphylococcus epidermidis, Micrococcus occulta, and Streptococcus pneumoniae.
[0012] Furthermore, the antibacterial product targets pathogens including bacteria.
[0013] Furthermore, the antibacterial products include antibacterial drugs, antibacterial cleaning products, or antibacterial feed additives.
[0014] Furthermore, the antibacterial agent comprises an effective dose of clove extract and pharmaceutically acceptable excipients.
[0015] Beneficial effects
[0016] This invention obtains clove and knotweed extract by extraction with water or ethanol as solvent. It was found that the clove and knotweed extract has inhibitory effects on Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis, methicillin-resistant Staphylococcus epidermidis, Micrococcus aureus, Bacillus, Pseudomonas aeruginosa, Enterococcus faecalis, Micrococcus luteus, Acinetobacter baumannii, Streptococcus pneumoniae, and Klebsiella pneumoniae. It has significant development potential in future research on antibacterial drugs, antibacterial feed additives, and other antibacterial products, contributing to better development and utilization of clove and knotweed plant resources and providing a new avenue for finding alternative antibacterial agents. Attached Figure Description
[0017] Figure 1 This is a schematic diagram illustrating the antibacterial effect of water extract of Syringa ovata on 12 pathogenic bacteria.
[0018] Figure 2 This is a graph showing the antibacterial effect of water extract of Syringa ovata on 12 pathogens.
[0019] Figure 3 This is a schematic diagram illustrating the antibacterial effect of a 75% ethanol extract of *Polygonum ovatum* against 12 pathogenic bacteria.
[0020] Figure 4 This is a graph showing the antibacterial effect of a 75% ethanol extract of Syringa ovalis against 12 pathogens.
[0021] Figure 5 This is a schematic diagram comparing the antibacterial effects of the water extract and the 75% ethanol extract of *Syringa ovoidea*. (The left image shows the water extract of *Syringa ovoidea*, and the right image shows the 75% ethanol extract of *Syringa ovoidea*. Note: The horizontal axis only shows the bacterial species whose MIC values for the plant extracts have been determined.) Detailed Implementation
[0022] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.
[0023] Example 1: Preparation of clove and knotweed extract
[0024] Plant samples: The whole plant of Polygonum hydropiper used in this example was produced in Nanjing, Jiangsu Province, and was provided by the Aquatic Plant Resources and Development and Utilization Project Team of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province.
[0025] Take dried whole clove knotweed, pulverize it, and pass it through a 40-mesh sieve to obtain clove knotweed fragments. Take about 200 g to 250 g of each and place them in a round-bottom flask. Use 10 times the amount of distilled water or 10 times the amount of 75% ethanol as the extraction solvent, and soak them thoroughly overnight in a cool place. Perform hot reflux extraction twice, the first time for 2 hours and the second time for 1 hour. After cooling, filter to remove the residue, combine the filtrates, and concentrate the liquid under reduced pressure using a rotary evaporator to obtain at least a small amount of liquid. Place the liquid in an evaporating dish and evaporate the solvent in a water bath at 50 ℃ to 60 ℃ to obtain a dry extract, thus obtaining the water extract and alcohol extract of clove knotweed. Store them in a refrigerator at -20 ℃.
[0026] In this embodiment, the reflux temperature is 100~105℃ when the extraction solvent is water, and 80~90℃ when the extraction solvent is 75% ethanol.
[0027] Example 2: Antibacterial effect of clove and knotweed extract
[0028] 1. Experimental Materials and Instruments
[0029] The experimental strains, including Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis, methicillin-resistant Staphylococcus epidermidis, Micrococcus occulta, Bacillus, Pseudomonas aeruginosa, Enterococcus faecalis, Micrococcus luteus, Acinetobacter baumannii, Streptococcus pneumoniae, and Klebsiella pneumoniae, were provided by the Jiangsu Institute of Botany, Chinese Academy of Sciences.
[0030] Test samples: water extract and alcohol extract of Polygonum clove prepared in Example 1.
[0031] Experimental reagents: sodium chloride, thiazolyl blue (MTT), peptone, beef extract, agar powder, PBS buffer.
[0032] Experimental equipment: electronic balance (1 / 100,000), clean workbench, pipette, 96-well plate, autoclave, ultraviolet spectrophotometer, constant temperature incubator, shaking incubator.
[0033] Nutrient Broth (NB): Weigh 5 g sodium chloride, 3 g beef extract and 10 g peptone into an appropriate amount of distilled water, sonicate until completely dissolved, add distilled water to 1000 mL, dispense into 250 mL Erlenmeyer flasks, sterilize at 121 ℃ and use for later use.
[0034] Nutrient agar (NA): Weigh 5 g sodium chloride, 3 g beef extract, 10 g peptone and 17 g agar powder into an appropriate amount of distilled water, sonicate and heat to dissolve, add distilled water to make 1000 mL, dispense into 250 mL Erlenmeyer flasks, sterilize at 121 ℃ and use for later use.
[0035] Solid plate culture medium: Take the sterilized nutrient agar culture medium mentioned above, heat it until completely dissolved, pour about 10 mL into a 9 cm plastic petri dish in a clean bench, spread it evenly, and wait for it to cool and solidify before inoculation.
[0036] MTT solution: Accurately weigh an appropriate amount of MTT according to the usage amount, add it to PBS buffer to prepare a 5 mg / mL solution, and use immediately after preparation.
[0037] 2. Determination of Minimum Inhibitory Concentration (MIC) using the micro-broth dilution method
[0038] Strain rejuvenation: Take the bacterial culture stored in the laboratory and inoculate it onto NA agar plates using the streak plate method. Incubate at 37 ℃ for 16-24 h in a constant temperature incubator. Pick a single colony and streak it onto a plate. Repeat the above steps several times until colonies with good growth are cultured. Seal the petri dish with sealing film and store it in a refrigerator at 4 ℃.
[0039] Preparation of bacterial suspension: Several morphologically similar colonies were picked from NA medium cultured for 16–24 h and inoculated into 50 mL of NB medium. The culture was then incubated overnight at 37 ℃ and 200 rpm to allow for rapid bacterial amplification. Using a blank medium as a control, the bacterial concentration was adjusted at 600 nm to achieve an absorbance between 0.08 and 0.10, at which point the bacterial density was approximately 1 × 10⁻⁶. 8 CFU / mL. The bacterial culture was then diluted 1000-fold to obtain a density of 1×10⁻⁶. 5 Prepare and use fresh bacterial suspensions at CFU / mL.
[0040] Preparation of test solutions: 192 mg of the clove and Polygonum extract obtained in Example 1 was accurately weighed and dissolved in 3 mL of sterile NB medium to make a final concentration of 64 mg / mL. The solution was prepared and used immediately under aseptic conditions.
[0041] Determination of Minimum Inhibitory Concentration (MIC): The MIC was determined in a 96-well microtiter plate using a two-fold dilution method. 100 μL of liquid culture medium was added to wells 2-11 of the 96-well plate, and 200 μL of culture medium was added to well 12 as a negative control. 100 μL of the drug solution was added to wells 1-2, mixed with the culture medium, and then 100 μL was pipetted from well 2 to well 3, mixing several times in each well. This serial dilution was continued up to well 10, and the remaining 100 μL was discarded. Freshly prepared 10 μL of culture medium was then added to wells 1-11. 5 100 μL of CFU / mL bacterial suspension was used, with well 11 serving as a positive control without the added drug. To avoid difficulty in observing the results due to the dark color of the extract, a control row without bacterial suspension was set up for comparison. The bacterial suspension was replaced with an equal volume of liquid culture medium, resulting in a liquid volume of 200 μL per well. Extract concentration... C k (k=1,2,3……10) were 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 mg / mL, respectively. The containers were covered and incubated at 37 ℃ for 16–24 h.
[0042] MTT is a dye that acts on the mitochondria of living cells. It reduces to water-insoluble blue-purple formazan crystals, which then deposit in the cells, facilitating the observation of bacterial growth and the antibacterial effect of the solution. After removing the 96-well plate, when positive wells show bacterial growth and obvious turbidity, and negative blank wells are clear and sterile, 10 μL of MTT solution is added to each well for staining. After addition, the plate is incubated at 37 ℃ for 1–2 h. It is then removed and placed against a white background. The wells are visually observed to see if they are stained blue. Blue staining indicates bacterial growth, while no staining indicates sterility. The results are recorded. The lowest concentration of wells showing no blue-purple formazan crystals is recorded as the MIC of the clove extract.
[0043] 3. Experimental Results
[0044] This application uses water and 75% ethanol as solvents to extract the whole herb of *Syringa ovata*, and conducts antibacterial tests on the obtained extracts. The extracts showed good antibacterial effects against a variety of tested pathogens, especially against two common pathogens, *Staphylococcus aureus* and *Escherichia coli*. The test results are as follows: Figures 1-5 As shown, the MIC values are statistically summarized in Table 1.
[0045] Table 1. MIC determination results of aqueous extract and 75% ethanol extract of Polygonum ovalis 'Ovum'
[0046]
[0047] A comparative study was conducted on the antibacterial effects of the aqueous extract and the 75% ethanol extract of Polygonum syringum. The results are as follows: Figure 5 As shown.
[0048] The results showed that the aqueous extract of *Polygonum ovatum* exhibited varying degrees of inhibitory effects on pathogens other than *Streptococcus pneumoniae* and *Klebsiella pneumoniae*. The MIC values for methicillin-resistant *Staphylococcus epidermidis*, *Micrococcus*, *Bacillus*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii* were 8 mg / mL, while the MIC values for *Staphylococcus epidermidis*, *Enterococcus faecalis*, and *Micrococcus luteus* were 16 mg / mL. A concentration of 1 mg / mL of the aqueous extract showed significant inhibitory effects on *Escherichia coli* and *Staphylococcus aureus*. The ethanolic extract of *Polygonum ovatum* showed certain inhibitory effects on all tested pathogens, especially *Escherichia coli* and *Staphylococcus aureus*, with extremely significant inhibitory effects, and MIC values of 0.125 mg / mL and 0.5 mg / mL, respectively.
[0049] In summary, the water extract of *Polygonum ovatum* showed good antibacterial effects against 10 pathogens, with MICs all below 16 mg / mL; its alcohol extract had a broader antibacterial spectrum, effective against all tested pathogens, with the best antibacterial effects against *Escherichia coli* and *Staphylococcus aureus*, with MICs of 0.125 mg / mL and 0.5 mg / mL, respectively.
[0050] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.
Claims
1. The application of clove and knotweed extract in the preparation of antibacterial products, characterized in that, The preparation method of the clove and knotweed extract includes the following steps: S1. Take dried *Syringa ovata*, a plant of the genus *Syringa* in the family Onagraceae, crush it, and pass it through a 40-mesh sieve to obtain fragments of *Syringa ovata*. S2: Weigh an appropriate amount of *Syringa ovata* fragments obtained in S1, add an extraction solvent equivalent to 9-11 times the weight of the *Syringa ovata* fragments, soak in the extraction container for 12 hours, and then perform hot reflux extraction twice. The first hot reflux extraction is for 2 hours, and the second hot reflux extraction is for 1 hour. After cooling, filter to remove residue, combine the filtrates, concentrate under reduced pressure, and evaporate the solvent at 50℃~60℃ to obtain a dry extract, which is then stored at -20℃. The extraction solvent is distilled water or 75% ethanol.
2. The application of the clove and knotweed extract according to claim 1 in the preparation of antibacterial products, characterized in that, When the extraction solvent is water, the reflux temperature is 100~105℃; when the extraction solvent is 75% ethanol, the reflux temperature is 80~90℃.
3. The application of the clove and knotweed extract according to claim 1 in the preparation of antibacterial products, characterized in that, The bacteria are selected from one or more Gram-negative and Gram-positive bacteria.
4. The application of the clove and knotweed extract according to claim 3 in the preparation of antibacterial products, characterized in that, The Gram-negative bacteria are selected from Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii; the Gram-positive bacteria are selected from Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Enterococcus faecalis, Staphylococcus epidermidis, methicillin-resistant Staphylococcus epidermidis, Micrococcus occulta, and Streptococcus pneumoniae.
5. The application of the clove and knotweed extract according to claim 1 in the preparation of antibacterial products, characterized in that, The antibacterial products target pathogens including bacteria.
6. The application of the clove and knotweed extract according to claim 1 in the preparation of antibacterial products, characterized in that, The antibacterial products include antibacterial drugs, antibacterial cleaning products, or antibacterial feed additives.
7. The application of the clove and knotweed extract according to claim 6 in the preparation of antibacterial products, characterized in that, The antibacterial drug comprises an effective dose of clove extract and pharmaceutically acceptable excipients.