TWI722282B - Rheum officinale extract and use thereof for manufacturing compoitions for inhibiting biofilm formation of malassezia furfur - Google Patents

Abstract

The present invention discloses a Rheum officinale extract and its use for manufacturing compositions for inhibiting biofilm formation of Malassezia furfur. The Rheum officinale extract is prepared by the steps of extracting a Rheum officinale material by ethanol to obtain an ethanol extract; and extracting the ethanol extract by an ethyl acetate/ water solution to obtain an ethyl acetate extract and a water extract, wherein the ethyl acetate extract contains the Rheum officinale extract having polyphenols. An effective dose of the Rheum officinale extract is applied to a subject in need for inhibiting growth and biofilm formation of Malassezia furfur. Accordingly, the present invention has efficacy of improving skin conditions caused by Malassezia furfur.

Description

Rhubarb extract and its use in preparing biofilm composition for inhibiting dander spore bacteria

The present invention relates to a rhubarb extract and its use in preparing a composition for inhibiting Malassezia furfur (Malassezia furfur) biofilm, especially a rhubarb extract extracted by a specific preparation method, which can effectively improve the individual Related skin diseases caused by dander spores.

Biofilm (biofilm) refers to the secretion of extracellular polymeric substance (EPSs) containing polysaccharides and proteins after the growth of certain microbial groups, which improves the tolerance of microorganisms to adverse factors such as the external environment or antibiotics. degree. Biofilms can be formed on biological or non-biological surfaces, such as the surface of human skin or the surface of water pipes, and are likely to cause harm to the human body.

Malassezia furfur is an oleophilic fungus that uses lipase to decompose the oil secreted by the human body. If the skin's oil secretion is excessive, it will cause a large number of dander spores to grow, which will cause the skin oil to be decomposed and produced. Fatty acids that cause skin inflammation, which in turn leads to damage to human skin tissues. At present, it is known that skin diseases related to the massive growth of dander spores include folliculitis, seborrheic dermatitis, dandruff, sweat spots, psoriasis and so on. And because the dander spores will form a biofilm on the surface of the skin, it will increase the difficulty of treatment.

The current drugs used to treat dander spore infection or dander spore overgrowth include econazole or itraconazole, which mainly inhibit fungal cell membrane synthesis to achieve the effect of inhibiting fungal growth. However, these drugs have side effects such as nausea, vomiting, abdominal pain, or itching. Because the drugs are metabolized by the liver, they are also likely to cause adverse reactions to patients with abnormal liver function.

US Patent Publication No. US 9648876 B2 is a method for inhibiting the formation of biofilms, which will use compounds such as xanthochymol and garcinol purified from Garcinia xanthochymus to inhibit white rosary beads. Bacteria and its biofilm formation; in addition, the application of US Patent Publication No. US 20150190447(A) uses myrtle plant extracts to inhibit the formation of Propionibacterium acnes biofilm; apparently, The use of plant-derived compounds or extracts to control the formation of microbial biofilms has become a new research direction.

Today, the inventors have made improvements in view of the insufficiency of the existing medicinal compositions for controlling the formation of dander spore biofilms, so he has a tireless spirit and is assisted by his wealth of professional knowledge and years of practical experience. , And based on this research and creation of the present invention.

The present invention relates to a rhubarb (Rheum officinale ) extract and its use in preparing a composition for inhibiting Malassezia furfur (Malassezia furfur) biofilm. It mainly refers to the extract obtained by extracting rhubarb through specific steps The compound has the effect of inhibiting the formation of Malassezia furfur biofilm, and can be used to improve skin diseases related to Malassezia furfur.

The present invention provides a rhubarb ( Rheum officinale ) extract, and its preparation method comprises: first extracting a rhubarb material with ethanol to obtain an ethanol extract; and using the ethanol extract with a volume ratio of 2:1 ethyl acetate/ The aqueous solution is partitioned and extracted to separate an ethyl acetate phase and an aqueous phase. The ethyl acetate phase contains an ethyl acetate partition extract. The rhubarb extract of the present invention is an ethyl acetate partition extract.

The present invention also provides a use of rhubarb ( Rheum officinale ) extract in the preparation of a composition that inhibits Malassezia furfur (Malassezia furfur) biofilm. An effective dose of rhubarb extract is administered to a desired individual to To achieve the purpose of inhibiting the formation of dander spore biofilm.

One embodiment of the present invention, in the embodiment, the first extract containing 355.30 ± 0.74 μ g / mg of total polyphenols.

In an embodiment of the present invention, the minimal inhibitory concentration of the first extract of Bacillus dander (minimal inhibitory concentrations) is 0.1 μg/mL.

In one embodiment of the present invention, the first extract further inhibits the growth of Bacillus cereus , Pseudomonas aeruginosa and Staphylococcus aureus .

In one embodiment of the present invention, the first extract further scavenges free radicals and inhibits the release of nitric oxide from cells, so as to achieve anti-oxidation and anti-inflammatory effects.

In this way, the rhubarb extract in this case can be used as a pharmaceutical composition or a cosmetic composition for inhibiting the biofilm of dander spores, so as to achieve the effect of improving skin diseases related to danders spores.

Figure 1: Schematic diagram of the biofilm staining of Bacillus dander of the present invention.

The second figure; the pH value of the present invention affects the biofilm formation of Bacillus dander.

Figure 3: Analysis diagram of the influence of temperature on the biofilm formation of dander spores in the present invention.

The fourth figure: the analysis diagram of the biofilm formation rate of the dander spore bacteria of the present invention.

Figure 5: Analysis of the inhibition of biofilm formation by the Chinese herbal medicine extract of the present invention.

Figure 6: Analysis of the inhibition of biofilm formation by the rhubarb extract of the present invention.

Figure 7: An analysis diagram of the Rhubarb extract of the present invention inhibiting the growth of environmental microorganisms.

The purpose of the present invention and its structural and functional advantages will be described based on the structure shown in the following drawings and specific embodiments, so that the review committee can have a deeper and specific understanding of the present invention.

The present invention is a rhubarb ( Rheum officinale ) extract. The preparation method includes: extracting the rhubarb material with ethanol to obtain an ethanol extract; and distributing the ethanol extract with a volume ratio of 2:1 ethyl acetate/water solution Extraction is divided into an ethyl acetate phase and an aqueous phase, wherein the ethyl acetate phase contains an ethyl acetate partition extract, and the rhubarb extract of the present invention is the ethyl acetate partition extract. When the rhubarb extract of the present invention is administered to a desired individual at an effective dose, the formation of the biofilm of dander spore bacteria can be inhibited to achieve the antibacterial effect. Among them, the total polyphenol content of rhubarb extract is 355.30±0.74μg/mg.

The present invention also provides the use of the above-mentioned rhubarb extract in the preparation of a composition for inhibiting the growth of Malassezia furfur or the formation of biofilms, wherein the Rhubarb extract has a minimum inhibitory concentration of 0.1μg/ mL, has an excellent inhibition rate of dander spore biofilm formation, and can further inhibit the growth of Bacillus cereus , Pseudomonas aeruginosa and Staphylococcus aureus ; in addition, rhubarb extract series It further scavenges free radicals and inhibits the release of nitric oxide from cells to achieve anti-oxidation and anti-inflammatory effects.

In addition, the following specific examples can further prove the scope of practical application of the present invention, but it is not intended to limit the scope of the present invention in any form.

Experiment 1. Establishing the biofilm growth model of Bacillus dander

The purpose of this experiment is to establish a biofilm culture model of Bacillus dander, and screen out Bacillus dander strains with high biofilm formation rate and high infectivity for subsequent testing of the efficacy of various plant extracts.

Malassezia furfur BCRC 22243 was purchased from the Bioresource Conservation Research Center of Food Industry Development Institute (Hsinchu, Taiwan). The strain was first inoculated into MEB (malt extract broth) medium and cultured at 30°C Cultivate in a box for 2~3 days to activate the strains, and then smear the bacteria on MEA medium (malt extract agar) with an inoculation loop to obtain purified strains; in the experiment, select a single colony from the MEA medium to the MEB medium for culture . MEB culture medium and MEA culture medium are conventional technologies, so I won't repeat them here.

The following experiments are repeated more than twice, and the difference between the groups is analyzed by One-Way ANOVA, and marked with English lowercase letters. If the group marked with the same letter indicates that there is no significant difference, mark a different letter The group represents a significant difference between the two; Student unpaired t test is also used for analysis between groups, expressed as mean ± standard error of the mean (mean ± SEM), when the p value is less than 0.05 ( p <0.05) or the p value is less than When 0.01 ( p <0.01), it means that the experimental group has a statistical difference compared with the control group.

(1) Staining condition screening

Take the dander spore bacterial solution that reaches the log phase in the growth phase, take the bacterial solution containing 1 X 10 ⁶ CFU of the bacteria to a 96-well culture plate, incubate at 30°C for 24 hours, and absorb the suspended bacterial solution. To remove non-adherent bacteria, stain with 0.01%, 0.05%, and 0.1% crystal violet solution respectively. After staining for 10 minutes, wash three times with potassium dihydrogen phosphate buffer solution; suck up the diluent, dissolve the cell membrane with 95% ethanol, and measure _{The absorbance value (OD 595} ) of the dissolution solution at a wavelength of 595 nm. Please refer to the first figure. For dyeing with 0.01% crystal violet solution, the color is too light and the OD ₅₉₅ value is too low; for dyeing with 0.1% crystal violet solution, the color is too dark and the OD ₅₉₅ value is too high. It is high and may reach the critical value of the instrument. Therefore, the results obtained by dyeing with 0.01% crystal violet solution or 0.1% crystal violet solution are not easy to distinguish the difference with the naked eye or the instrument; in contrast, dyeing with 0.05% crystal violet solution In addition, both the color observed by the naked eye and _{the value of OD 595} are relatively moderate, so it is more suitable for detecting the formation of dander spore biofilm.

(2) The influence of pH value on the formation of dander spore biofilm

Take the dander spore bacterial solution that reaches the log phase in the growth phase, and take the bacterial solution containing 1 X 10 ⁶ CFU bacteria to a 96-well culture plate, and set the pH to pH 4.7, pH 7 and pH 9 Incubate the MEB culture medium at 30°C for 24 hours, blot the suspended bacteria liquid to remove non-adherent bacteria; stain with 0.05% crystal violet solution for 10 minutes, then wash three times with the diluent, and blot the diluent dry; % Ethanol solution dissolves the cell membrane and dissolves the crystal violet; measuring the absorbance of the dissolving solution at a wavelength of 595nm as a criterion for evaluating the formation of biofilms. Please refer to the second figure. Under the pH 4.7, pH 7 and pH 9 environment, the OD ₅₉₅ values obtained after dyeing are 3.015, 0.530 and 0.475 in order, and they are also statistically significant. Therefore, It can be seen that the environment of pH 4.7 is more suitable for the growth of dander spores.

(3) The influence of temperature on the growth of dander spores

Take the dander spore bacterial solution that reaches the log phase in the growth phase, take the bacterial solution containing 1 X 10 ⁶ CFU bacteria to a 96-well culture plate, and incubate at 30°C or 37°C for 24 hours ; Blot the suspended bacteria liquid to remove non-adherent bacteria; stain with 0.05% crystal violet solution for ten minutes; remove the crystal violet solution, wash three times with the diluent, suck up the diluent; dissolve the cell membrane with 95% ethanol Crystal violet dissolves to detect absorbance at 595nm wavelength. Please refer to the third figure. The OD ₅₉₅ value of the group cultured at 30°C is 1.568, which is significantly higher than the OD ₅₉₅ value of the group cultured at 37°C of 1.245 (p <0.01), indicating that the suitable growth temperature for dander 30°C. According to the literature, the average temperature of the human body surface is also around 30°C, so it can be inferred that the dander spore bacteria are more likely to grow on the human body surface and cause diseases.

(4) Screening of dander spores with high biofilm formation rate

First spread the dander spores on the MEA medium. After it grows into a single colony, pick out 10 single colonies, inoculate them in a 96-well culture plate containing MEB culture solution, and incubate at 30°C for 24 hours; The liquid is sucked dry to remove non-adherent bacteria, and then stained with 0.05% crystal violet solution for 10 minutes; remove the crystal violet solution, wash three times with the diluent, and blot the diluent; dissolve the cell membrane with 95% ethanol solution and remove the crystal violet Dissolve, measure the absorbance at a wavelength of 595nm. Please refer to the fourth figure. Arrange the results of 10 strains according to the OD ₅₉₅ value. After subtracting the highest absorbance value from the lowest absorbance value, the calculated difference value is divided into quarters (quartiles) ); _Those whose OD 595 value falls in the lowest interval (called the first quartile) are called low biofilm forming strains, and those that fall in the second and third quartiles are called Medium biofilm-forming strains, and the OD ₅₉₅ value falls in the fourth quartile are called high biofilm-forming strains. After several experiments, the Bacillus spores with high biofilm formation obtained by screening were used as the strains for subsequent experiments.

Experiment 2: Antibacterial test of Chinese herbal medicine extract

(1) Anti-dander spore biofilm formation test

The dry materials of Saposhnikovia , Rheum officinale , Schizonepeta tenuifolia , Coptis chinensis , and Paeonia suffruticosa Andr. are ground, and after preliminary extraction with 95% ethanol, The obtained ethanol extract was tested against dander spore biofilm formation.

Inoculate 1 X 10 ⁶ CFU of high biofilm-forming dander spores into a 96-well plate, and then add ethanol extracts of various Chinese herbal medicines at a concentration of 1 mg/mL, and use the optimal culture conditions obtained in Experiment 1 (pH 4.6). , 30℃) for 24 hours; blot the suspension culture to remove non-adherent bacteria, and stain with 0.05% crystal violet solution for 10 minutes; remove the excess crystal violet solution, and wash three times with the diluent, and suck the diluent dry ; Dissolve the cell membrane with 95% ethanol solution and dissolve the crystal violet, and measure the absorbance of the dissolving solution at a wavelength of 595nm. The control group is the group without added plant extracts, and the results obtained in the control group and the experimental group are calculated with the following formula to obtain the biofilm formation rate: biofilm formation rate (%) = (experimental group OD ₅₉₅ value/control group OD ₅₉₅ value) X 100%

Please refer to the fifth figure. Compared with the control group, the biofilm formation rate of the ethanol extract of rhubarb is only 60.57% of that of the control group, the ethanol extract of Paeonia Suffruticosa is 89.09%, and the ethanol extract of Parsnip is 82.34%; and Coptidis Ethanol extracts and ethanol extracts of Nepeta sinensis could hardly inhibit the formation of dander spore biofilms. The biofilm formation rate of the ethanol extract group of Coptis Rhizoma was 100%, and that of ethanol extracts of Nepeta sinensis was 98.44%.

The ethanol extract of rhubarb was further partitioned and extracted with ethyl acetate/water solution with a volume ratio of 2:1 to obtain an ethyl acetate phase and an aqueous phase. After the ethyl acetate phase was concentrated under reduced pressure, rhubarb acetic acid was obtained. The ethyl ester partitions the extract. The ethanol extract of rhubarb and the ethyl acetate extract of rhubarb were partitioned, and the biofilm formation rate of Bacillus dander was tested by the above-mentioned experimental method. Please refer to the sixth figure. Under the condition of using a concentration of 1mg/mL, the biofilm formation rate of the ethyl acetate partitioned extract of rhubarb is 0%, which is much lower than the 60.57% of the ethanol extract of rhubarb, which means that rhubarb acetic acid The ethyl ester distribution extract has excellent anti-dander spore biofilm formation effect.

(2) The minimum inhibitory concentration of dander spores of rhubarb extract

The minimum inhibitory concentration refers to the minimum concentration required to inhibit the growth of microorganisms; first take 1 X 10 ⁶ CFU of dander spores and add it to a 96-well plate, and then add different concentrations of rhubarb ethanol extract or rhubarb ethyl acetate Ester distributes the extract and cultures it for 24 hours. After the culture, the bacterial solution is serially diluted and smeared on the agar gum culture plate, cultivated overnight and counted the number of colonies, and obtained the lowest concentration of rhubarb extract that has the effect of inhibiting colony growth . Please refer to Table 1. The minimum inhibitory concentration of dander spores in ethanol extract of rhubarb is 5μg/mL, and the ethyl acetate partitioned extract of rhubarb is 0.1μg/mL, showing that the ethyl acetate partitioned extract of rhubarb has excellent properties. Inhibit the growth of dander spores.

(3) The environmental microbial inhibition rate of the ethyl acetate distribution extract of rhubarb

Microorganisms used in this test were purchased from Biological Resources Preservation Research Center, Institute of Food Industry development, containing Bacillus cereus (Bacillus cereus BCRC 10603), Escherichia coli (Escherichia coli BCRC 10675), Pseudomonas aeruginosa (Pseudomonas aeruginosa BCRC 10944), salmonella ( Salmonella choleraesuis BCRC 10744) and Staphylococcus aureus BCRC 12657. The test method is briefly described as follows: Take 5 X 10 ⁵ CFU of different bacterial liquids and add them to a 96-well plate, and then add 1 mg/mL rhubarb ethyl acetate Ester partition extracts, co-cultivation for 18-24 hours; take out the bacterial liquid, perform serial dilution and smear the bacterial liquid on a plate medium, observe the number of colonies formed after 18-24 hours of cultivation to obtain the total bacterial count; among them, Escherichia coli The culture temperature of Pseudomonas aeruginosa, Salmonella and Staphylococcus aureus is 37℃, and the culture temperature of Cactus bacteria is 30℃. In this experiment, the group without addition of rhubarb ethyl acetate distribution extract was used as the control group. For the calculation method of the antibacterial rate, please refer to the following formula: Inhibition rate (%) = (number of bacteria in the control group-number of bacteria in the experimental group )/Control group number of bacteria X 100%

Please refer to the seventh figure. The ethyl acetate distribution extract of rhubarb has the worst effect on the inhibition of Salmonella. The inhibition rate is only about 20-30%, and the inhibition rate for E. coli is about 63%. However, for Cactus bacillus and Pseudomonas aeruginosa The antibacterial rate of Bacillus and Staphylococcus aureus can be as high as about 99%. It is known that the ethyl acetate partition extract of rhubarb also has an inhibitory effect on environmental microorganisms.

Experiment 3: The effect of rhubarb extract in scavenging free radicals

DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable free radical with a maximum absorbance at a wavelength of 517nm; DPPH will form an electron pair with antioxidants, and the color will change from blue-violet to pale yellow, absorbing wavelength The ability of 517nm also changes, so the ability of the test substance to scavenge free radicals can be calculated by the change of the absorbance at 517nm. Partition the extracts of different concentrations of rhubarb ethanol extract and rhubarb ethyl acetate, add a freshly prepared 100μM DPPH ethanol solution, mix well, in a dark environment, 37 ℃ for 30 minutes, and then detect with a spectrophotometer Measure the absorbance value at 517nm wavelength, and make a calibration curve to calculate the concentration of Nepeta extract (IC ₅₀ ) with a free radical inhibition rate of 50%. The group without any substances is used as a negative control group to calculate the inhibition rate, the formula is As follows: DPPH inhibition rate (%)=[(negative control group absorbance value-experimental group absorbance value)/negative control group absorbance value] X 100%

Please refer to Table 2. The IC _{50 of the ethanol extract of rhubarb is 28.42±3.07μg/mL, the IC 50} of the ethyl acetate distribution extract of rhubarb is 7.89±0.12μg/mL, and the vitamin E is used as the positive control group. The results show Rhubarb ethyl acetate partition extract does have good antioxidant capacity.

Experiment 4. Anti-inflammatory effect of rhubarb extract

The mouse macrophage cell line RAW 264.7 (BCRC 60001) purchased from the BCRC Biological Resources Conservation and Research Center was cultured in DMEM medium containing 10% Fetal bovine serum and kept at 37°C, 5% CO _2. Incubate in a constant temperature cell incubator for 2-3 days. When the cells grow to 8-9 percent full, they can be subcultured.

(1) Cell survival test

The MTT assay is used to test the viability of the cells. The principle of this test is that the mitochondria of living cells will reduce the MTT compound (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) The purple crystal is then dissolved with dimethyl sulfide (DMSO), and its absorbance at a wavelength of 550 is measured to obtain the content of the purple crystal, and the level of cell metabolic activity is known, and then the level of cell metabolic activity is taken as the cell The basis for survival; therefore, the more purple crystals are generated, the higher the absorbance at wavelength 550, which means the higher the survival rate of cells; the MTT method is now widely used It is used to test cell survival rate and test the toxicity of a compound or substance to cells.

Culture RAW 264.7 cells in a 96-well plate with 5 x 10 ⁴ cells/well and 100μL culture medium/well. _{After culturing for 24 hours at 37℃ and 5% CO 2} , replace the new culture medium and add different concentrations Partition extract of rhubarb ethyl acetate; add 10ng/mL lipopolysaccharide (LPS) after incubating for 30 minutes; after incubating for 24 hours, remove the culture medium and add 0.5mg/mL MTT solution, React at 37°C for 3 hours; centrifuge for 5 minutes, remove the supernatant, and add 100 μL of DMSO to each culture well to dissolve the crystals in the cells, and shake for 5 minutes; measure the absorbance at a wavelength of 550 nm. Take the absorbance of the cells without any treatment as the control group with cell survival rate of 100%, and use the following formula to calculate the cell survival rate of the experimental group, as follows: Cell survival rate (%) = (absorbance value of the experimental group / absorbance value of the control group )X 100%

The experimental results showed that the cell survival rate of the 10ng/mL LPS treatment group was 95.90±16.53%. The cells that added 25μg/mL and 50μg/mL rhubarb ethyl acetate to distribute the extract had a survival rate of more than 80%. The cell survival rate of 50μg/mL Rhubarb ethyl acetate partitioned extract was 94.64±8.24%, indicating that the rhubarb ethyl acetate partitioned extract was not cytotoxic to RAW 264.7.

(2) Anti-inflammatory test

After the conditions in RAW 264.7 Cells at 5 x 10 ⁴ cells / hole, 100 μ L culture medium / well in 96-well plates of cultured at 37 ℃, the culture environment of 5% CO ₂ for 24 hours and replaced with new culture solution was added Different concentrations of rhubarb ethyl acetate partition extract; after 30 minutes of incubation, add 10ng/mL lipopolysaccharide (LPS); after 20 hours of incubation, add Griess reagent and shake for 5 minutes, then measure the wavelength of 550nm The absorbance value, and _{the absorbance value of the standard solution of sodium nitrite (NaNO 2} ) as the calibration line, to test the nitrite release amount of the cells of different treatments, to represent the cell's nitric oxide (NO ) Release amount.

Please refer to Table 3. The experimental results show that the NO release of the LPS treatment group has a significant increase compared with the control group, but after the Rhubarb ethyl acetate distribution extract is processed, the NO release of the cells will decrease. , And there is a phenomenon of dose dependence; among them, in the group of 50μg/mL rhubarb ethyl acetate partitioning extract, the NO release of cells decreases more obviously, indicating that the rhubarb ethyl acetate partitioning extract has anti-inflammatory properties effect.

Experiment 5. Determination of total polyphenols in rhubarb extract

Folin-Ciocalteu colorimetric method was used to measure the total polyphenol content in rhubarb extract. Because Folin-Ciocalteu reagent contains tungstomolybdic acid, a blue compound will be formed when tungstomolybdic acid is reduced. The color intensity is similar to that of the test substance. The content of polyphenols is proportional; in this test, gallic acid is used as the standard product, and the relative content of total polyphenols in the test substance can be calculated after the calibration line is drawn. The experimental method is briefly described as follows: add Folin-Ciocalteu reagent to a 96-well plate, add 0.5mg/mL rhubarb ethanol extract or ethyl acetate partition extract, or different concentrations of gallic acid (gallic acid) standards After mixing uniformly, react for 10 minutes, then add 2% sodium carbonate (Na ₂ CO ₃ ) solution, mix uniformly and react for 10 minutes; measure the absorbance at 630nm wavelength; draw a standard curve based on the measurement result of gallic acid , And then compare the absorbance value of the rhubarb extract with the standard curve to obtain its total polyphenol content, which is expressed as "total polyphenol equivalent", which means "1 milligram (mg) rhubarb extract and how many micrograms (μg) of gallic acid has the same effect of reducing tungstomolybdic acid." The results are shown in Table 4; the total polyphenol content of the ethanol extract of rhubarb is 126.29±1.52μg/mg), and the ethyl acetate partition extract of rhubarb contains a high amount of total polyphenols, the content is 355.30±0.74μg/mg.

As can be seen from the above implementation description, compared with the prior art, the present invention has the following advantages:

1. The Rhubarb ethyl acetate distribution extract of the present invention has an extremely low minimum inhibitory concentration on dander spores, only 0.1 μg/mL, and has an excellent rate of inhibiting the formation of dander spores biofilm.

2. The Rhubarb ethyl acetate distribution extract of the present invention can further inhibit the growth of Cactus bacillus, Pseudomonas aeruginosa and Staphylococcus aureus, and is a multifunctional antibacterial substance.

3. The Rhubarb ethyl acetate distribution extract of the present invention further has the effects of scavenging free radicals and inhibiting the release of NO from cells, so it can be used for anti-oxidation and anti-inflammatory in addition to antibacterial applications.

In summary, the rhubarb extract of the present invention and its use in the preparation of the composition for inhibiting dander spores can indeed achieve the expected use effects through the above-disclosed embodiments, and the present invention has not been disclosed. Before the application, Cheng has fully complied with the provisions and requirements of the Patent Law. If you file an application for a patent for invention in accordance with the law, you are kindly requested to review and grant a quasi-patent.

However, the above-mentioned explanations are only the preferred embodiments of the present invention, and are not intended to limit the scope of protection of the present invention; it; those who are familiar with the art, based on the characteristic scope of the present invention, etc. Effective changes or modifications should be regarded as not departing from the design scope of the present invention

Claims (3)

A kind of rhubarb ( Rheum officinale ) extract is used to prepare a biofilm composition that inhibits Malassezia furfur (Malassezia furfur), wherein the Rheum officinale has a minimum inhibitory concentration of 0.1 μg/mL and is It is prepared by the following steps: Step 1: Extract the rhubarb material with ethanol to obtain the rhubarb ethanol extract; and Step 2: Extract the rhubarb ethanol with a volume ratio of 2:1 ethyl acetate/water solution Extract to obtain an ethyl acetate phase and an aqueous phase, wherein the ethyl acetate phase contains an ethyl acetate partition extract, and the rhubarb extract is the ethyl acetate partition extract; wherein the large The yellow extract contains 355.30±0.74μg/mg total polyphenols. The use according to claim 1, wherein the rhubarb extract further inhibits the growth of Bacillus cereus , Pseudomonas aeruginosa and Staphylococcus aureus . The use according to claim 1, wherein the rhubarb extract further has the function of scavenging free radicals and inhibiting the release of nitric oxide from cells, so as to achieve anti-oxidation and anti-inflammatory effects.

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