阅读说明：本技术 一种抑制铜绿假单胞菌生长及其生物膜形成的苦楝果提取物的制备方法和应用 (Preparation method and application of chinaberry fruit extract for inhibiting growth of pseudomonas aeruginosa and biofilm formation thereof ) 是由 彭红 周刚 王颖思 施庆珊 谢小保 于 2020-06-12 设计创作，主要内容包括：本发明公开了一种抑制铜绿假单胞菌生长及其生物膜形成的苦楝果提取物的制备方法和应用。经试验证明,本发明的苦楝果提取物在含量为0.5-1.0g/mL能有效抑制铜绿假单胞菌浮游菌的生长及其生物膜的形成,在含量为0.25-0.5g/mL对铜绿假单胞菌浮游菌的抑制效果较差,但能较好抑制铜绿假单胞菌生物膜的形成。本发明的原材料天然环保,抑菌抑膜效果显著,有益于人体健康。苦楝果提取物可应用于制备清洗压疮、糖尿病足溃疡、下肢动静脉溃疡等慢性伤口、疥疮、头癣、湿疮、湿疹痛痒等的药物、食品保鲜防腐剂或日化产品抗菌剂。(The invention discloses a preparation method and application of a melia azedarach fruit extract for inhibiting growth of pseudomonas aeruginosa and biofilm formation thereof. Tests prove that the melia azedarach fruit extract can effectively inhibit the growth of pseudomonas aeruginosa planktonic bacteria and the formation of a biofilm thereof when the content is 0.5-1.0g/mL, has poor inhibition effect on the pseudomonas aeruginosa planktonic bacteria when the content is 0.25-0.5g/mL, and can better inhibit the formation of the pseudomonas aeruginosa biofilm. The raw materials of the invention are natural and environment-friendly, have obvious bacteriostatic and membrane inhibiting effects and are beneficial to human health. The melia azedarach fruit extract can be used for preparing medicaments, food fresh-keeping preservatives or daily chemical product antibacterial agents for cleaning chronic wounds such as pressure sores, diabetic foot ulcers, lower limb arteriovenous ulcers and the like, scabies, tinea capitis, eczema, pain and itch and the like.)

1. A preparation method of chinaberry fruit extract for inhibiting growth of pseudomonas aeruginosa and biofilm formation thereof is characterized by comprising the following steps: cleaning and drying the chinaberry fruits to constant weight, and crushing to obtain chinaberry fruit powder; adding water into Melia azedarach fruit powder, extracting with ultrasound-assisted water extraction method to obtain extractive solution, centrifuging the extractive solution, collecting supernatant, and concentrating the supernatant to obtain Melia azedarach fruit extract.

2. The method of claim 1, comprising the steps of: cleaning and drying the chinaberry fruits to constant weight, and crushing the chinaberry fruits to particles with the diameter less than or equal to 1mm by using a high-speed crusher to obtain chinaberry fruit powder; mixing fructus Toosendan powder with distilled water at a ratio of 100g/L, ultrasonic extracting at room temperature for 30min, boiling for 30min, ultrasonic extracting for 30min, and filtering to obtain extractive solution; adding distilled water into the residue, boiling for 30min, filtering to obtain extractive solution, mixing the two extractive solutions, centrifuging at 5000r/min for 10min, collecting supernatant, vacuum filtering the supernatant under reduced pressure, vacuum concentrating, and sterilizing to obtain Melia azedarach fruit extract.

3. The method of claim 2, wherein the ultrasonic extraction is performed at a frequency of 40kHz and a power of 100W.

4. A neem fruit extract prepared by the process of any one of claims 1 to 3.

5. The Melia azedarach fruit extract according to claim 4, wherein the content of the Melia azedarach fruit extract is 0.25-1.0 g/mL.

6. Use of the extract of melia azedarach fruit obtained by the process according to any one of claims 1 to 3 for inhibiting the growth of pseudomonas aeruginosa and the formation of biofilm thereon.

7. The use of the extract of melia azedarach fruit obtained by the process according to any one of claims 1 to 3 for the manufacture of a medicament, food preservative or daily chemical antibacterial agent for cleansing pressure sores, diabetic foot ulcers, lower extremity arteriovenous ulcers, scabies, tinea capitis, eczema and itching.

Technical Field

The invention belongs to the fields of medicine, daily chemicals, plants and microorganisms, and particularly relates to a preparation method and application of a chinaberry fruit extract for inhibiting growth of pseudomonas aeruginosa and biofilm formation thereof.

Background

The chemical bactericide is subjected to various degrees of drug resistance caused by a plurality of microorganisms due to long-term large-scale and improper use, and meanwhile, the residue of the chemical bactericide can also threaten human health, so that the chemical bactericide is restricted and banned in various fields, and researchers are prompted to excavate more active ingredients from the existing natural plants and Chinese herbal medicines with excellent bactericidal functions. Particularly, the nopell professor extracting artemisinin which can effectively inhibit malaria from artemisia annua obtains a nobel physiology or medical prize, and Chinese herbal medicine preparations and therapies appear in diagnosis and treatment schemes for a plurality of times in the process of preventing and treating novel coronavirus, which can bring natural products of China to new climax for research, development and utilization of bacteriostasis and virus inhibition.

However, up to now, researchers have found that more than about 1600 plants worldwide contain bactericidal, insecticidal and antiviral substances, and among them, most of them are meliaceae plants, mainly including melia azedarach, toosendan and neem. Chinaberry is common in the south provinces of the yellow river in China. A plurality of documents report that the melia azedarach has multiple activities of bacteriostasis, sterilization, desinsectization, disinsection, antivirus, antitumor and the like on various microorganisms. The study of scholars shows that the ethanol extract of the chinaberry tree has good bacteriostatic activity on aspergillus niger and trichoderma viride, and the chinaberry infusion has certain bacteriostatic effect on trichophyton xanthum, trichophyton concentricum, sphagnum schoenleinii, trichophyton austochiensis, trichophyton ferruginese, trichophyton lanuginese, dermatophyton rubrum, nocardia stellata and the like.

Bacterial Biofilm (BF) is a form of survival of bacteria, and there is data indicating that up to 95% of bacteria in nature exist in the form of Bacterial biofilm, a population of bacteria that are attached to the surface of biological or non-biological materials, are encapsulated by extracellular macromolecules secreted by themselves, and have certain three-dimensional structural characteristics. The formation of the biofilm results in the generation of bacterial drug resistance, and researches show that the drug resistance of the bacterial biofilm is 10-1000 times higher than that of the bacterial biofilm in a planktonic state. The formation of bacterial biofilm can also lead to a number of hazards, such as corrosion of attached material surfaces, contamination of medical devices and the initiation of human disease. The chronic wound bacterial biofilm is a membranous tissue formed by bacteria attached to a wound bed and fused with extracellular matrix components secreted by the bacteria, and the chronic wound is easy to infect and even form a bacterial biofilm which is difficult to treat due to long duration and multiple influencing factors, so that the treatment difficulty is increased, and the healing effect is influenced. Clinically, it is often seen in chronic wounds such as pressure sores, diabetic foot ulcers, lower limb arteriovenous ulcers and the like.

Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA) is commonly called Pseudomonas aeruginosa and is an important pathogenic bacterium for secondary infection of a series of serious suppurative infections, such as bronchiectasis, chronic bronchitis, cystic pulmonary fibrosis and other basic diseases, and the Pseudomonas aeruginosa is easy to form a biological membrane in vivo and in vitro. Many infections caused by this bacterium occur in debilitating or immunocompromised hospitalized patients and are the second most common causative bacteria of intensive care unit infections. Patients suffering from metabolic diseases, hematopathy and malignant tumors, and patients after operation or after certain treatments are very easy to be infected with the bacterium, the infection of wounds after operation is often caused, burn wounds are also easy to be infected with copper green pseudomonas, bedsores, abscess, suppurative otitis media and the like can also be caused, and the caused infection focus can cause bacteremia and septicemia and can cause death. There is therefore a need to screen for drugs or methods that inhibit the growth of pseudomonas aeruginosa, thereby reducing the risk of this bacterium. Many researches have proved that the PA generates drug resistance to various antibiotics and other bactericidal drugs, and if active ingredients with killing effect on the PA can be found from natural plant extracts and developed and utilized, the drug spectrum for preventing and treating the bacteria can be greatly expanded. Antibiotic treatment of bacterial biofilms has been controversial, and it has been reported in literature that although antibiotics can kill streptococcus, they promote other bacteria such as pseudomonas aeruginosa and serratia serrata to form biofilms, and these bacteria are planted in the deep layer of wounds, so that antibiotics are difficult to act, and bacteria persist, thereby delaying the healing of wounds. Diseases can also affect the survival or absence of bacterial biofilms, e.g. streptococci in wounds 63 times as large as in non-diabetic patients.

Disclosure of Invention

The first purpose of the invention is to provide a preparation method of chinaberry fruit extract for inhibiting the growth of pseudomonas aeruginosa and the formation of biofilm thereof, which comprises the following steps: cleaning and drying the chinaberry fruits to constant weight, and crushing to obtain chinaberry fruit powder; adding water into Melia azedarach fruit powder, extracting with ultrasound-assisted water extraction method to obtain extractive solution, centrifuging the extractive solution, collecting supernatant, and concentrating the supernatant to obtain Melia azedarach fruit extract.

Preferably, the preparation method comprises the following steps: cleaning and drying the chinaberry fruits to constant weight, and crushing the chinaberry fruits to particles with the diameter less than or equal to 1mm by using a high-speed crusher to obtain chinaberry fruit powder; mixing fructus Toosendan powder with distilled water at a ratio of 100g/L, ultrasonic extracting at room temperature for 30min, boiling for 30min, ultrasonic extracting for 30min, and filtering to obtain extractive solution; adding distilled water into the residue, boiling for 30min, filtering to obtain extractive solution, mixing the two extractive solutions, centrifuging at 5000r/min for 10min, collecting supernatant, vacuum filtering the supernatant under reduced pressure, vacuum concentrating, and sterilizing to obtain Melia azedarach fruit extract.

The ultrasonic extraction frequency in the preparation method is 40kHz, and the power is 100W.

The second object of the present invention is to provide a neem fruit extract prepared by the above preparation method.

The content of the melia azedarach fruit extract is 0.25-1.0 g/mL.

The third purpose of the invention is to provide the application of the chinaberry fruit extract prepared by the preparation method in inhibiting the growth of pseudomonas aeruginosa and the formation of a biofilm thereof.

The melia azedarach fruit extract can be used for preparing medicaments, food fresh-keeping preservatives or daily chemical product antibacterial agents for cleaning pressure sores, diabetic foot ulcers, lower limb arteriovenous ulcers, scabies, tinea capitis, eczema and eczema pain and itch.

According to the characteristics of active ingredients in the melia azedarach fruits, the invention is provided with a specific extraction method, so that the extraction rate of the active ingredients is improved, and the inhibition effect of the melia azedarach fruit extract on the growth of pseudomonas aeruginosa and the biofilm formation is improved.

The raw materials of the invention are derived from plants, are natural and environment-friendly, have obvious bacteriostatic effect, are beneficial to human health, can be applied to preparing medicaments for cleaning wounds, food fresh-keeping preservatives or daily chemical product antibacterial agents, and have wide application range and prospect.

Drawings

FIG. 1 shows the bacteriostatic effect of Melia azedarach fruit extract on Pseudomonas aeruginosa. (A) The inhibition effect of pseudomonas aeruginosa on bacterial plankton in 24h (8 parallels in each experimental concentration, n is 8) is obtained by culturing the melia azedarach extract in different concentrations, and the inhibition effect of pseudomonas aeruginosa in 24h (8 parallels in each experimental concentration, n is 8) is obtained by culturing the melia azedarach extract in different concentrations.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

When the test strain in the following examples is pseudomonas aeruginosa (p.aeruginosa ATCC 9027), the test method is: the test strains were cultured at 37 ℃ for 1 day in a slant medium of nutrient agar medium (NA) in this example, then washed with PBS and diluted to OD₆₀₀Working solution of bacterial suspension was prepared as 1.0, while sterile distilled water was used as blank. Adding certain concentration of Melia azedarach fruit extract into culture system according to gradient dilution method, sucking 150 μ L into 96-well cell culture plate (each experiment concentration is 8 in parallel, n is 8), placing the culture plate into 37 deg.C constant temperature incubator, standing and culturing for 24 hr, taking out 96-well plate, and measuring absorbance (OD) at 600nm of bacterial liquid with full wavelength microplate reader₆₀₀) Comparing the absorbance value with that of a blank control to evaluate the inhibition effect of the chinaberry fruit extract on bacterial plankton; then, removing planktonic bacteria, gently cleaning the plate holes, air drying, adding 200 μ L of 1% (v/w) crystal violet staining solution into each plate hole, standing and staining for 30 minutes, removing the crystal violet staining solution, washing the plate holes with sterile water, air drying at room temperature, decolorizing with 200 μ L of 95% ethanol for 30 minutes, and measuring the absorbance value (OD) at 595nm by using a full-wavelength microplate reader₅₉₅) The inhibition of bacterial biofilm formation by the extract of neem fruit was evaluated in comparison to the absorbance values of the blank control.

The following abbreviations are used in the specification: ATCC is shorthand for American type culture Collection, PA is Pseudomonas aeruginosa, NA is nutrient agar medium, PBS is phosphate buffer saline (phosphate buffer saline), temperature is given in degrees Celsius (C.), min is minutes, d is day, mL is mL, μ L is μ L, mg is mg, g is g, OD is OD₆₀₀Refers to the absorbance, OD, of the solution at a wavelength of 600nm₅₉₅Refers to the absorbance of the solution at 595 nm.