Application of mulberry leaf extract in preparation of antibacterial product
Through the thermal reflux extraction method of mulberry leaf extract, the problem of difficult inhibition of drug-resistant pathogens in the prior art was solved, and mulberry leaf extract with inhibitory effect was prepared for preparing antibacterial products and providing alternative antibacterial agents The new approach significantly improves the inhibitory effect of target pathogens.
Patent Information
- Application Number
- CN202411329014.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-24
- Publication Date
- 2024-12-24
AI Technical Summary
The prior art is difficult to effectively inhibit drug-resistant pathogens, such as methicillin-resistant Staphylococcus epidermis, Enterococcus faecium, Garcinia micrococci, Acinetobacter baumannii, Klebsiella pneumoniae and Streptococcus pneumoniae, leading to infectious diseases treatment difficulty.
Mulberry leaf extract with antibacterial activity was prepared by using the method of extracting mulberry leaf extract through heat reflux, using distilled water or 75% ethanol as the extraction solvent, and used to prepare antibacterial products.
Mulberry leaf extract shows significant inhibitory effects on target pathogens, providing a new way to replace antibacterial agents, and has great development potential, helping to develop and utilize mulberry leaf plant resources.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of traditional Chinese medicine extracts, and in particular relates to application of mulberry leaf extracts in preparing antibacterial products. Background Art
[0002] Pathogenic infections pose a direct threat to human health, and in severe cases may be life-threatening. Prevention and control of pathogenic infections is of vital importance. The use of antibiotics for prevention and treatment is currently a commonly used and effective measure, but irrational use of antibiotics may lead to increased resistance of pathogens to drugs, making infectious diseases that should be treatable difficult to cure. For example: Klebsiella pneumoniae often causes serious infections such as pneumonia, urinary tract infections and sepsis, and its multidrug resistance is becoming a serious public health problem worldwide. Streptococcus pneumoniae can cause pneumonia, meningitis, otitis media and sepsis. Its current resistance to multiple antibiotics such as penicillin has increased the challenge of treatment. Methicillin-resistant Staphylococcus epidermidis is a pathogen closely related to drug-resistant infections and shows resistance to multiple antibiotics.
[0003] Some plants have certain antibacterial activity and low toxicity, and can be an alternative to antibiotic treatment. Morusfolium is the dried leaves of Morus alba L., a plant of the Moraceae family. It has antioxidant, anti-inflammatory, hypoglycemic and lipid-lowering functions, but there is a lack of research on its inhibitory activity against some bacteria, such as methicillin-resistant Staphylococcus epidermidis, Enterococcus faecium, Micrococcus luteus, Klebsiella pneumoniae, Sarcina, Acinetobacter baumannii, and Streptococcus pneumoniae. Summary of the invention
[0004] In view of the shortcomings of the prior art, the present invention provides the use of mulberry leaf extract in the preparation of antibacterial products. The present invention uses 7 common animal pathogens as activity test objects to study the activity of mulberry leaf extract in inhibiting these bacteria, providing a new way to find alternative antibacterial agents.
[0005] The technical solution provided by the present invention is as follows:
[0006] The present invention provides an application of a mulberry leaf extract in preparing an antibacterial product. The preparation method of the mulberry leaf extract comprises the following steps:
[0007] The dried mulberry leaves are crushed into flakes, and an extraction solvent equivalent to 7 to 10 times the weight of the mulberry leaf fragments is added. After soaking in an extraction container for 12 to 18 hours, hot reflux extraction is performed twice, each time for 2 hours. After cooling, the residue is filtered out, the filtrate is combined, and concentrated into a small amount of liquid by reduced pressure evaporation. After that, the solvent is evaporated at 50°C to 60°C to make a dry paste to obtain mulberry leaf extract.
[0008] Furthermore, the extraction solvent includes distilled water or 75% ethanol.
[0009] Furthermore, when the extraction solvent is water, the heat reflux temperature is 100-105°C, and when the extraction solvent is 75% ethanol, the heat reflux temperature is 80-90°C.
[0010] Furthermore, the bacteria are selected from one or more of Gram-negative bacteria and Gram-positive bacteria.
[0011] Furthermore, the Gram-negative bacteria are selected from Klebsiella pneumoniae and Acinetobacter baumannii, and the Gram-positive bacteria are selected from Micrococcus luteus, Enterococcus faecium, methicillin-resistant Staphylococcus epidermidis, Sarcina, and Streptococcus pneumoniae.
[0012] Furthermore, the pathogens targeted by the antibacterial product include bacteria.
[0013] Furthermore, the antibacterial product includes an antibacterial drug, an antibacterial cleaning product or an antibacterial feed additive.
[0014] Furthermore, the antibacterial drug comprises an effective dose of mulberry leaf extract and pharmaceutically acceptable excipients.
[0015] Beneficial Effects
[0016] The present invention obtains mulberry leaf extract by extracting with water or ethanol as solvent, and it is found that the mulberry leaf extract has inhibitory effects on methicillin-resistant Staphylococcus epidermidis, Sarcina, Enterococcus faecium, Micrococcus luteus, Acinetobacter baumannii, Streptococcus pneumoniae and Klebsiella pneumoniae. The mulberry leaf extract has great development potential in the research of future antibacterial drugs, antibacterial feed additives and other antibacterial products, is helpful to better develop and utilize mulberry leaf plant resources, and provides a new way to find alternative antibacterial agents. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is a graph showing the antibacterial effect of mulberry leaf water extract on 7 pathogens.
[0018] Figure 2 This is a graph showing the antibacterial effect of mulberry leaf alcohol extract on 7 pathogenic bacteria. DETAILED DESCRIPTION
[0019] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.
[0020] Example 1 Preparation of mulberry leaf extract
[0021] Plant samples: The mulberry leaves in this embodiment were purchased from Beijing Tong Ren Tang Nanjing Pharmacy, manufacturer: Hebei Ruida Pharmaceutical Co., Ltd., batch number: 151221201.
[0022] The above mulberry leaf medicinal materials were crushed, and about 200g of each was taken into a round-bottom flask. 8 times the amount of distilled water or 8 times the amount of 75% ethanol was used as the extraction solvent. After soaking in a cool place overnight for about 15 hours, it was fully infiltrated and extracted twice with hot reflux, each time for 2 hours. After cooling, the residue was removed by suction, and the filtrate was combined and concentrated to a small amount of liquid by a rotary evaporator under reduced pressure. The solvent was evaporated in an evaporating dish at 50℃~60℃ in a water bath to prepare a dry paste, and the mulberry leaf water extract and alcohol extract were obtained respectively.
[0023] In this embodiment, when the extraction solvent is water, the heat reflux temperature is 100-105°C, and when the extraction solvent is 75% ethanol, the heat reflux temperature is 80-90°C.
[0024] Example 2 Antibacterial effect of mulberry leaf extract
[0025] 1. Experimental materials and instruments
[0026] Experimental strains: Seven pathogens, including methicillin-resistant Staphylococcus epidermidis, Sarcina, Enterococcus faecium, Micrococcus luteus, Acinetobacter baumannii, Streptococcus pneumoniae and Klebsiella pneumoniae, were provided by the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province.
[0027] Test samples: mulberry leaf water extract and mulberry leaf alcohol extract prepared in Example 1.
[0028] Experimental reagents: sodium chloride, peptone, beef extract, agar powder.
[0029] Experimental equipment: electronic balance (one hundred thousandth), clean workbench, pipette, 96-well plate, high-pressure steam sterilizer, UV spectrophotometer, constant temperature incubator, shaking incubator.
[0030] Nutrient broth (NB): weigh 5 g sodium chloride, 3 g beef extract and 10 g peptone in an appropriate amount of distilled water, sonicate until completely dissolved, add distilled water to 1000 mL, dispense into 250 mL conical flasks, sterilize at 121°C and set aside.
[0031] Nutrient agar medium (NA): weigh 5 g sodium chloride, 3 g beef extract, 10 g peptone and 17 g agar powder in an appropriate amount of distilled water, sonicate and heat to dissolve, add distilled water to make it 1000 mL, divide it into 250 mL conical bottles, sterilize at 121°C and set aside.
[0032] Solid plate culture medium: Take the above sterilized nutrient agar medium, heat it until it is completely dissolved, pour about 10 mL into a 9 cm round plastic culture dish in an ultra-clean workbench, spread it flat, and wait for it to cool and solidify before inoculation.
[0033] 2. Agar dilution method to determine the minimum inhibitory concentration MIC
[0034] Rejuvenation of bacterial strains: Take the bacterial liquid stored in the laboratory and inoculate it on NA plate culture medium by the plate streak method. Incubate it in a constant temperature incubator at 37℃ for 16-24h. Pick a single colony and streak it on the plate. Repeat the above steps several times until a colony with good growth condition is cultured. Seal the plate with sealing film and store it in a 4℃ refrigerator.
[0035] Preparation of bacterial suspension: Pick several colonies with similar morphological characteristics from the NA medium cultured for 16 to 24 hours, inoculate them into 50 mL of NB medium, and culture them overnight in a shake flask at 37°C and 200 rpm to rapidly amplify the bacteria. Using the blank medium as a control, adjust the concentration of the bacterial suspension at 600 nm so that the absorbance of the bacterial suspension is between 0.08 and 0.10, and the bacterial density is about 1×10 8 CFU / mL. Then the bacterial solution was diluted step by step to obtain a density of 1×10 4 CFU / mL bacterial solution, ready for use.
[0036] Determination of minimum inhibitory concentration: The minimum inhibitory concentration (MIC) was determined in a square culture dish using the agar dilution method. A certain amount of the mulberry leaf extract (water extract and alcohol extract) obtained in Example 1 was accurately weighed and evenly dispersed into the nutrient agar medium. The extract was poured into a 10 cm square plastic culture dish in a clean bench and flattened. After cooling and solidification, it was used for inoculation. k (k=1,2,3……7) are 32.0, 16.0, 8.0, 4.0, 2.0, 1.0, 0.5 mg / mL respectively, and a growth control without drug and only inoculated bacteria is set. Use a pipette to draw 2 μL of bacterial suspension and inoculate it on the drug-containing culture dish. Each bacteria is inoculated 3 times in parallel. After the inoculation point is dry, all culture dishes are moved to a constant temperature incubator. After culturing at 37℃ for 16-24 hours, the bacterial growth is observed. The concentration with no bacterial growth is recorded as MIC.
[0037] 3. Experimental results
[0038] The present invention uses water and 75% ethanol as solvents to extract mulberry leaves, and uses the obtained mulberry leaf water extract and alcohol extract to conduct antibacterial tests, which show good antibacterial effects on 7 tested pathogens. The test results are as follows: Figures 1-2 As shown, the MIC values are statistically shown in Table 1.
[0039] Table 1 MIC determination results of mulberry leaf water extract and mulberry leaf alcohol extract
[0040]
[0041] The results showed that mulberry leaf water extract and alcohol extract had different degrees of inhibitory effect on the tested pathogens. The MIC values of Klebsiella pneumoniae, Micrococcus luteus and Enterococcus faecium were all 32.0 mg / mL, and the MIC values of methicillin-resistant Staphylococcus epidermidis and Sarcina were all 16.0 mg / mL. Among them, the inhibitory effect of alcohol extract on Streptococcus pneumoniae and Acinetobacter baumannii was better than that of water extract. The MIC value of alcohol extract on Streptococcus pneumoniae and Acinetobacter baumannii was 16.0 mg / mL, and the MIC value of water extract on Streptococcus pneumoniae and Acinetobacter baumannii was 32.0 mg / mL.
[0042] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may 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 mulberry leaf extract in the preparation of antibacterial products, characterized in that: The preparation method of the mulberry leaf extract comprises: The dried mulberry leaves are crushed into flakes, and an extraction solvent equivalent to 7 to 10 times the weight of the mulberry leaf fragments is added. After soaking in an extraction container for 12 to 18 hours, hot reflux extraction is performed twice, each time for 2 hours. After cooling, the residue is filtered out, the filtrate is combined, and concentrated into a small amount of liquid by reduced pressure evaporation. After that, the solvent is evaporated at 50 ℃ ~ 60 ℃ to make a dry paste to obtain mulberry leaf extract.
2. The use of the mulberry leaf extract in the preparation of antibacterial products according to claim 1, characterized in that: The extraction solvent includes distilled water or 75% ethanol.
3. The use of the mulberry leaf extract according to claim 2 in the preparation of an antibacterial product, characterized in that: When the extraction solvent is water, the reflux temperature is 100~105 ℃, and when the extraction solvent is 75% ethanol, the reflux temperature is 80~90 ℃.
4. The use of the mulberry leaf extract according to claim 1 in preparing an antibacterial product, characterized in that: The bacteria are selected from one or more of Gram-negative bacteria and Gram-positive bacteria.
5. The use of the mulberry leaf extract according to claim 4 in the preparation of an antibacterial product, characterized in that: The Gram-negative bacteria are selected from Klebsiella pneumoniae and Acinetobacter baumannii, and the Gram-positive bacteria are selected from Micrococcus luteus, Enterococcus faecium, methicillin-resistant Staphylococcus epidermidis, Sarcina, and Streptococcus pneumoniae.
6. The use of the mulberry leaf extract according to claim 1 in preparing an antibacterial product, characterized in that: The pathogens targeted by the antibacterial product include bacteria.
7. The use of the mulberry leaf extract according to claim 1 in preparing an antibacterial product, characterized in that: The antibacterial products include antibacterial drugs, antibacterial cleaning products or antibacterial feed additives.
8. The use of the mulberry leaf extract according to claim 7 in preparing an antibacterial product, characterized in that: The antibacterial drug comprises an effective dose of mulberry leaf extract and pharmaceutically acceptable auxiliary materials.