Extract of eucheuma and preparation method and application thereof
Extracts of *Symplocos edulis* were prepared by extracting the whole plant with water or ethanol, which solved the problem of pathogen resistance and provided the potential of a broad-spectrum antibacterial agent, especially with significant antibacterial effects against a variety of pathogens.
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
- 2024-06-28
- Publication Date
- 2026-07-24
AI Technical Summary
The increasing drug resistance of pathogens in existing technologies makes infectious diseases difficult to cure, necessitating the development of novel antibacterial products.
The whole plant of *Symplocos lucida* was extracted with water or ethanol as solvent, and *Symplocos lucida* extract was prepared by hot reflux extraction to obtain water extract and alcohol extract, which were used to prepare antibacterial products.
The extract of *Staphyllum oxypetalum* showed significant antibacterial effects against various pathogens such as Staphylococcus aureus and Escherichia coli, providing a new research direction for antibacterial products.
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Figure CN118845861B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of phytochemistry, and more specifically to extracts of *Phyllostachys edulis*, their preparation methods, and their application in the preparation of antibacterial products. Background Technology
[0002] Pathogenic infections pose a direct threat to human and animal health, and in severe cases can be life-threatening. Therefore, prevention and control of pathogenic infections are crucial. Treatment for pathogens primarily relies on antibiotics, but inappropriate use of antibiotics can lead to increased drug resistance in pathogens, making otherwise treatable infectious diseases difficult to cure.
[0003] Staphylococcus aureus 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. Methicillin-resistant Staphylococcus epidermidis is a pathogen closely associated with drug-resistant infections, especially common in hospital-acquired infections, and often exhibits resistance to multiple antibiotics. Klebsiella pneumoniae is easily transmitted in hospital settings and frequently causes serious infections such as pneumonia, urinary tract infections, and sepsis. Its multidrug resistance is becoming a serious public health problem globally, limiting treatment options. Streptococcus pneumoniae is one of the leading pathogens causing community-acquired pneumonia, meningitis, otitis media, and sepsis. Its increasing drug resistance, including resistance to multiple antibiotics such as penicillin, has increased the challenge of treatment.
[0004] The increasing drug resistance of pathogens has become a challenging problem in clinical treatment, necessitating the development of novel antibacterial products. Plant-derived antibacterial agents are natural, safe, and environmentally friendly, showing broad application prospects in various fields such as medicine, food, and agriculture. Therefore, identifying plants with antibacterial activity or their effective components is particularly important.
[0005] *Hippuris vulgaris* L., a plant belonging to the genus *Hippuris* in the family Hippuriaceae, is a Tibetan medicine with effects such as clearing heat, soothing the liver, and benefiting the lungs. It is mainly used to treat pulmonary tuberculosis cough and tuberculosis fever. To date, no antibacterial effects of *Hippuris vulgaris* have been found in this field. Summary of the Invention
[0006] To address the aforementioned technical problems, the present invention aims to provide a *Taxus chinensis* extract, its preparation method, and its applications. The extract is obtained by extraction with water or ethanol as a solvent. The antibacterial activity of the extract is studied using 12 common animal pathogens as activity test targets, and it is found that the *Taxus chinensis* extract has the potential to be used as a broad-spectrum antibacterial agent.
[0007] The technical solution adopted by this invention to achieve its technical objectives is as follows:
[0008] This invention provides a method for preparing extracts of *Symplocos buergeriana*, comprising: cutting the whole herb of *Symplocos buergeriana* into small pieces, extracting twice by hot reflux with distilled water and 75% ethanol as extraction solvents respectively, combining the filtrates, evaporating and concentrating under reduced pressure, evaporating the solvent to prepare a dry extract, thereby obtaining water extract and alcohol extract of *Symplocos buergeriana*.
[0009] Preferably, the amount of distilled water and 75% ethanol is 10 times.
[0010] Preferably, the first hot reflux extraction takes 2 hours, and the second takes 1 hour.
[0011] Preferably, the product concentrated by vacuum evaporation is placed in an evaporating dish and the solvent is evaporated at a low temperature of 50°C to 60°C in a water bath.
[0012] The present invention also provides an extract of *Taxus chinensis*, which is prepared by the above-described method.
[0013] The present invention also provides the application of the above-mentioned *Tamarix chinensis* extract as an antibacterial product.
[0014] Preferably, the aqueous extract and alcoholic extract of *Symplocos lucida* have significant antibacterial effects against both Staphylococcus aureus and Escherichia coli.
[0015] Preferably, the aqueous extract and alcoholic extract of *Staphyllum truncatum* also exhibit antibacterial activity against the following pathogens: *Staphylococcus epidermidis*, methicillin-resistant *Staphylococcus epidermidis*, *Dystrophococcus occulta*, *Bacillus*, *Pseudomonas aeruginosa*, *Enterococcus faecalis*, *Micrococcus luteus*, and *Acinetobacter baumannii*.
[0016] Preferably, the ethanol extract of *Cephalotaxus fortunei* also has antibacterial activity against *Streptococcus pneumoniae* and *Klebsiella pneumoniae*.
[0017] Preferably, the antibacterial product includes antibacterial drugs or antibacterial cleaning products.
[0018] The beneficial effects of this invention are as follows:
[0019] This invention obtains *Symplocos buergeriana* extract using water or ethanol as solvents. It has been found that the extract inhibits *Staphylococcus aureus*, *Escherichia coli*, *Staphylococcus epidermidis*, methicillin-resistant *Staphylococcus epidermidis*, *Dystrophococcus*, *Bacillus*, *Pseudomonas aeruginosa*, *Enterococcus faecalis*, *Micrococcus luteus*, *Acinetobacter baumannii*, *Streptococcus pneumoniae*, and *Klebsiella pneumoniae*. This invention shows great potential for future research into antibacterial drugs, antibacterial cleaning products, and other antibacterial products, providing a new approach for the full utilization of *Symplocos buergeriana* resources. Attached Figure Description
[0020] Figure 1 Schematic diagram of the 96-well plate micro-broth dilution method.
[0021] Figure 2 Antibacterial effect of water extract of *Cephalotaxus fortunei* against 12 pathogens.
[0022] Figure 3 Antibacterial effect of 75% ethanol extract of *Stachys chinensis* against 12 pathogens.
[0023] Figure 4 Comparison of antibacterial effects between the aqueous extract and the 75% ethanol extract of *Sinocyclocheilus spp.* (The horizontal axis only shows the bacterial species whose MIC values for the plant extract have been determined). Detailed Implementation
[0024] To more clearly illustrate the present invention, the invention will be further described in detail below with reference to embodiments and accompanying drawings. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.
[0025] Example 1 Preparation of *Cephalotaxus fortunei* extract
[0026] Plant samples: The whole plant of *Cephalotaxus fortunei* used in this example was produced in Nanjing, Jiangsu Province, and was provided by the Aquatic Plant Experiment Group of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province.
[0027] Cut the dried whole plant of *Echinochloa crus-galli* into small pieces, and place approximately 200-250 g of each into round-bottom flasks. Use 10 times the volume of distilled water and 10 times the volume of 75% ethanol as extraction solvents, respectively, and allow to soak thoroughly overnight in a cool place. Perform hot reflux extraction twice (for distilled water extraction, use an electric heating mantle with a temperature set at 100-105℃; for 75% ethanol extraction, use a water bath with a temperature set at 80-90℃), the first time for 2 hours and the second time for 1 hour. After cooling, filter to remove residue, combine the filtrates, and concentrate under reduced pressure using a rotary evaporator to obtain at least a small amount of liquid. Place the concentrate in an evaporating dish and evaporate the solvent in a water bath at 50-60℃ to obtain a dry extract, yielding both water and ethanol extracts, which are stored at -20℃.
[0028] Example 2 Antibacterial effect of *Phyllostachys edulis* extract
[0029] 1. Experimental Materials and Instruments
[0030] 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.
[0031] Test sample: The extract of *Taxobacterium truncatum* prepared in Example 1.
[0032] Experimental reagents: sodium chloride, thiazolyl blue (MTT), peptone, beef extract, agar powder, PBS buffer.
[0033] Experimental equipment: electronic balance (1 / 100,000), clean workbench, pipette, 96-well plate, autoclave, ultraviolet spectrophotometer, constant temperature incubator, shaking incubator.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 2. Determination of Minimum Inhibitory Concentration (MIC) using the micro-broth dilution method
[0039] 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°C 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°C.
[0040] 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 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.
[0041] Preparation of test solutions: 192 mg of each of the extracts of *Taxobacterium tumefaciens* obtained in Example 1 were 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 sterile conditions.
[0042] Determination of minimum inhibitory concentration (MIC): The MIC was determined in a 96-well microtiter plate using the two-fold dilution method. Figure 1 As shown, 100 μL of liquid culture medium was added to wells 2-11 of a 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, and after mixing with the culture medium, 100 μL was pipetted from well 2 to well 3, and the mixture was repeatedly pipetted and mixed several times in each well. This process was repeated serially until well 10, and the remaining 100 μL was discarded. Freshly prepared 10 μL of the drug solution was then inoculated into 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. The plant 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.
[0043] 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 all positive wells showed bacterial growth and obvious turbidity, and the negative blank wells were clear and sterile, 10 μL of MTT solution was added to each well for staining. After the addition was complete, the plate was incubated at 37 ℃ for 1–2 h. It was then removed and placed against a white background. The wells were visually observed to see if they were stained blue. Blue staining indicated bacterial growth, while no staining indicated sterility. The experimental results were recorded. The lowest concentration of wells showing no blue-purple formazan crystals was recorded as the MIC of the *Cephalotaxus fortunei* extract.
[0044] 3. Experimental Results
[0045] This application uses water and 75% ethanol as solvents to extract *Tamarix chinensis* plants, and conducts antibacterial tests on the obtained dry extracts. The extracts showed good antibacterial effects against various pathogenic bacteria in the tested animals, especially against two common pathogens, *Staphylococcus aureus* and *Escherichia coli*. The test results are as follows: Figure 2 and 3 As shown, the MIC values are statistically summarized in Table 1.
[0046] A comparative study was conducted on the antibacterial effects of the aqueous extract and the 75% ethanol extract of *Cephalotaxus fortunei*. The results are as follows: Figure 4 As shown.
[0047] The aqueous extract of *Staphyllum oxypetalum* showed good antibacterial effects against *Staphylococcus aureus* and *Escherichia coli*, with a minimum inhibitory concentration (MIC) of 2 mg / mL for both. At a concentration of 16 mg / mL, it exhibited antibacterial activity against *Staphylococcus epidermidis*, methicillin-resistant *Staphylococcus epidermidis*, *Dystrophococcus occulta*, *Bacillus*, and *Pseudomonas aeruginosa*. At a concentration of 32 mg / mL, it showed antibacterial activity against *Enterococcus faecalis*, *Micrococcus luteus*, and *Acinetobacter baumannii*.
[0048] The ethanol extract of *Stachys chinensis* showed good antibacterial activity against 12 tested pathogens. The MIC values against *Streptococcus pneumoniae* and *Klebsiella pneumoniae* were both 32 mg / mL. It also showed good antibacterial activity against *Staphylococcus epidermidis*, methicillin-resistant *Staphylococcus epidermidis*, *Dystrophococcus occulta*, and *Bacillus*, with MICs of 8 mg / mL. The antibacterial effect against *Escherichia coli* and *Staphylococcus aureus* was the most significant, with MICs as low as 0.25 mg / mL and 0.5 mg / mL, respectively.
[0049] Table 1. MIC determination results of aqueous extract and 75% ethanol extract of *Cephalotaxus fortunei*.
[0050]
[0051] Overall, the extract of *Staphyllum oxypetalum* showed significant antibacterial effects against pathogens such as Staphylococcus aureus and Escherichia coli.
[0052] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.
Claims
1. The application of *Stachys chinensis* extract in the preparation of antibacterial products, wherein the bacteria are: *Staphylococcus epidermidis*, methicillin-resistant *Staphylococcus epidermidis*, *Stachys occulta*, *Bacillus*, *Pseudomonas aeruginosa*, *Enterococcus faecalis*, *Micrococcus luteus*, and *Acinetobacter baumannii*, and the preparation method of the *Stachys chinensis* extract includes: The whole plant of *Symplocos lucida* was chopped and extracted twice by hot reflux with distilled water and 75% ethanol as extraction solvents. The filtrates were combined, concentrated by vacuum evaporation, and the solvent was evaporated to make a dry extract, thus obtaining the water extract and alcohol extract of *Symplocos lucida*.
2. The application according to claim 1, characterized in that, The bacteria in question is also Klebsiella pneumoniae.
3. The application according to claim 1, characterized in that, The antibacterial products include antibacterial drugs or antibacterial cleaning products.