Application of blumea balsamifera extract in in-vitro antibiosis and antioxidation

Abstract

The invention provides an application of a blumea balsamifera extract in in-vitro antibiosis and antioxidation. The blumea balsamifera extract comprises blumea balsamifera oil, blumea balsamifera powder and / or blumea balsamifera tablets; the invention relates to a method for inhibiting bacteria, which comprises the following steps of: inhibiting bacillus subtilis, staphylococcus epidermidis, candida tropicalis and / or malassezia furfur, and further comprises the following steps: inhibiting bacillus subtilis, staphylococcus epidermidis, Candida tropicalis and / or malassezia furfur, and further comprises the following steps of: inhibiting bacteria, namely inhibiting bacillus subtilis, staphylococcus epidermidis, Candida tropicalis and / or malassezia furfur. The invention further discloses the effectiveness and antioxidant characteristics of the blumea balsamifera oil, the blumea balsamifera powder and the blumea balsamifera tablets in the aspect of inhibiting specific pathogenic bacteria, a new thought is provided for application of different extracts of the blumea balsamifera in the fields of inhibiting specific pathogenic bacteria infection and resisting oxidation, and a new way is opened up for comprehensive development and utilization of blumea balsamifera resources.

Description

Technical Field

[0001] The invention relates to the field of medical technology, in particular to the field of antibacterial and antioxidant technologies of traditional Chinese medicine, and in particular to the application of Blumea balsamifera extract in in vitro antibacterial and antioxidant activities. Background Art

[0002] Blumea balsamifera (L.) DC. is the fresh or dried aerial part of Blumea balsamifera (L.) DC., a plant of the Asteraceae family. It has a unique aromatic smell and has the effects of dispelling wind and dampness, promoting blood circulation and detoxifying, reducing swelling and relieving pain, resisting bacteria and inflammation, and clearing the throat. In Hainan, Guizhou, Yunnan, Guangdong and other provinces in southern China, due to its special geographical location and climatic conditions, Blumea balsamifera has gradually developed into an important economic crop. The whole plant contains rich volatile components, and three different volatile substances can be extracted by steam distillation under the same process conditions. Among them, after heating and sublimation treatment, its fresh branches and leaves are converted into powdery crystals, called "wormwood powder"; and after the wormwood powder is pressed and deoiled, the block crystals formed are called "wormwood tablets". In addition, the oil obtained during the pressing process is called "wormwood oil". It is worth mentioning that wormwood tablets have been included in the Chinese Pharmacopoeia and are an important source of natural borneol. Blumea Balsamifera is widely used in the fields of medicine and chemical industry. As the raw materials of mugwort powder, mugwort tablets and mugwort oil, more than 30 Chinese patent medicines have been developed, such as Yan Lishuang oral drops, Yan Kang lozenges, Jinhoujian spray, etc. At the same time, Blumea Balsamifera Oil has also been included in the "Catalogue of Names of Used Cosmetic Raw Materials (2021 Edition)" and has been widely used in the chemical and fragrance industries. Studies in recent years have shown that Blumea Balsamifera is rich in volatile oils, flavonoids and sesquiterpenoid compounds, which exhibit multiple pharmacological activities such as antibacterial, anti-inflammatory, analgesic and antiviral. However, there is still a lack of literature on the research of three different volatile extracts of Blumea Balsamifera (mugwort oil, mugwort powder, and mugwort tablets) against specific pathogens (for example, Malassezia furfur) and their antioxidant activities. Therefore, in-depth research on the application of Blumea Balsamifera extracts in antibacterial and antioxidant activities has important scientific value and practical significance. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention aims to provide an application of Blumea balsamifera extract in in vitro antibacterial and antioxidant activities.

[0004] In order to achieve the above purpose, the scheme of the present invention is as follows:

[0005] The invention discloses an application of an extract of Artemisia annua Balsamifera in preparing an in vitro antibacterial and / or antioxidant product. The extract of Artemisia annua Balsamifera includes Artemisia annua oil, Artemisia annua powder and / or Artemisia annua tablets; the antibacterial agent includes antibacterial agents against Bacillus subtilis, Staphylococcus epidermidis, Candida tropicalis and / or Malassezia furfur.

[0006] Preferably, the mugwort powder refers to: powdery crystals obtained after the fresh branches and leaves of Artemisia balsamifera are subjected to heating and sublimation treatment; mugwort flakes are block crystals formed after the mugwort powder is pressed and deoiled; mugwort oil is the oil obtained during the pressing process of mugwort powder.

[0007] Preferably, the anti-oxidation comprises scavenging DPPH free radicals and / or scavenging ABTS free radicals.

[0008] Preferably, when the concentration of wormwood oil is ≥25 mg / mL, the ability to scavenge DPPH free radicals is the highest; when the concentration of wormwood oil is ≥0.16 mg·mL -1 When the concentration of wormwood oil is ≥16mg / ml, the total antioxidant capacity is the highest.

[0009] Preferably, for resistance to Bacillus subtilis, the concentration of wormwood oil, wormwood powder or wormwood tablets is ≥1 / 2MIC, and the MIC is 0.3125 mg / mL; for resistance to Staphylococcus epidermidis, the concentration of wormwood oil or wormwood tablets is ≥0.625 mg / mL, and the concentration of wormwood powder is ≥1.25 mg / mL; for resistance to Candida tropicalis, the concentration of wormwood oil is ≥0.625 mg / mL, the concentration of wormwood powder is ≥1 / 2MIC, the MIC is 0.3125 mg / mL, and the concentration of wormwood tablets is 0.3125 mg / mL; for resistance to Malassezia furfur, the concentration of wormwood oil is ≥1.25 mg / mL, and the concentrations of wormwood powder and wormwood tablets are ≥0.625 mg / mL.

[0010] Preferably, for resistance to Bacillus subtilis, the concentration of wormwood powder is 2.5 mg / mL, and the concentration of wormwood tablets is 0.625 mg / mL; for resistance to Staphylococcus epidermidis, the concentration of wormwood oil is ≥0.625 mg / mL, the concentration of wormwood powder is ≥1.25 mg / mL, and the concentration of wormwood tablets is ≥0.625 mg / mL; for resistance to Candida tropicalis, the concentration of wormwood oil is ≥0.625 mg / mL, the concentration of wormwood powder is ≥1 / 2MIC, the MIC is 0.3125 mg / mL, and the concentration of wormwood tablets is 0.3125 mg / mL; for resistance to Malassezia furfur, the concentration of wormwood oil is ≥1.25 mg / mL, and the concentrations of wormwood powder and wormwood tablets are ≥0.625 mg / mL.

[0011] Preferably, the antibacterial agent is against Candida tropicalis or Malassezia furfur.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention further reveals the effectiveness of mugwort oil, mugwort powder and mugwort tablets in resisting specific pathogenic bacteria and their antioxidant properties. The results show that mugwort oil, mugwort powder and mugwort tablets exhibit certain inhibitory activity against Bacillus subtilis, Staphylococcus epidermidis, Candida tropicalis and Malassezia furfur, and there are significant differences in the inhibitory effects of different strains. In addition, mugwort oil is more prominent in antioxidant activity.

[0014] The invention provides a new idea for the application of different Blumea balsamifera extracts in the fields of inhibiting infection by specific pathogenic bacteria and anti-oxidation, and also opens up a new path for the comprehensive development and utilization of Blumea balsamifera resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Antibacterial effect of different extracts of Blumea balsamifera on skin pathogens.

[0016] Figure 2 :Effects of different mass concentrations of Blumea balsamifera extracts on the growth curves of skin pathogens.

[0017] Figure 3 : DPPH free radical scavenging ability of different Blumea balsamifera extracts.

[0018] Figure 4 : ABTS free radical scavenging ability of different Blumea balsamifera extracts.

[0019] Figure 5 : Total antioxidant capacity of different extracts of Blumea balsamifera. DETAILED DESCRIPTION

[0020] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0021] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0022] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0023] Experimental example:

[0024] 1 Materials and Instruments

[0025] 1.1 Materials and reagents

[0026] 1 Materials and reagents

[0027] Mugwort oil (AY), mugwort powder (AF) and mugwort tablets (AP) were purchased from Guizhou Aiyuan Ecological Pharmaceutical Development Co., Ltd. and identified by Yu Fulai, a researcher at the Institute of Tropical Crops Variety Resources, Chinese Academy of Tropical Agricultural Sciences, as different extracts of Blumea balsamifera (L.) DC., a plant of the genus Blumea in the Asteraceae family. The samples were stored in the Southern Medicine and Health Research Center of the Institute.

[0028] Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC15442), Bacillus subtilis (ATCC6633), Staphylococcus epidermidis (ATCC12228), Candida albicans (ATCC10231) were purchased from Guangdong Huankai Biotechnology Co., Ltd.; Candida tropicalis (ATCC750), Malassezia furfur furfur (ATCC44344) was purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd.; MH agar, MH broth, YM agar, YM broth, nutrient medium (NA), nutrient broth (NB), modified Dixon agar (MD), 2693 modified Dixon (mDixon), soybean casein agar (TSA), tryptone soy broth (TSB), Tween 40 were purchased from Qingdao Haibo Biotechnology Co., Ltd.; 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,6-di-tert-butyl-4-methylphenol (BHT) were purchased from Tokyo Chemical Industry Co., Ltd.; 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt] acid, ABTS] were purchased from Beijing Solebow Technology Co., Ltd.; total antioxidant capacity (FRAP method) test kit was purchased from Suzhou Keming Biotechnology Co., Ltd.; ascorbic acid VC, Tween 80, dimethyl sulfoxide (DMSO), and anhydrous ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd.; levofloxacin and fluconazole were purchased from Beijing Solebow Technology Co., Ltd.; and ketoconazole was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0029] 1.2 Instruments and Equipment

[0030] Sartarius CPA225D electronic analytical balance (Sartorius (Beijing) Scientific Instrument Co., Ltd.), SW-CJ-1F clean bench (Suzhou Guangyuan Purification Technology Co., Ltd.), SpectraMax iD3 multifunctional microplate reader (MeiGu Molecular Instrument (Shanghai) Co., Ltd.), BM2100 optical microscope (Zhejiang Xinfeng Medical Equipment Co., Ltd.), DNP-9052BS-Ⅲ electric constant temperature incubator, HZQ-X160 constant temperature shaker (Shanghai Shenmiao Medical Equipment Manufacturing Co., Ltd.), LX-B50L portable pressure steam sterilizer (Hefei Huatai Medical Equipment Co., Ltd.).

[0031] 2. Methods

[0032] 2.1 Antimicrobial activity assay

[0033] 2.1.1 Activation of bacterial strains and preparation of bacterial suspension

[0034] Take out the standard strain frozen in -80℃ glycerol and inoculate it into liquid culture medium, place it in a constant temperature incubator with 90r / min shaking culture until the corresponding time for recovery, the bacterial culture temperature is 37℃, the fungal culture temperature is 28℃, and the culture temperature of Malassezia furfur is 30℃, then streak it onto solid culture medium to culture monoclonal bacteria, pick monoclonal bacteria with good growth and inoculate it into corresponding liquid culture medium, and culture it until the logarithmic growth phase of each strain. The concentration of bacterial suspension is calculated by bacterial turbidity meter for bacteria and hemocytometer for fungi, and then diluted with corresponding liquid culture medium to a colony count of 1×10 6 cfu / mL bacterial suspension for later use.

[0035] 2.1.2 Drug sensitivity test

[0036] The implementation standard of antimicrobial susceptibility test issued by the Clinical and Laboratory Standards Institute (CLSI) of the United States (CLSI) (CLSI M100 S24) was used to detect the sensitivity of different extracts of Blumea balsamifera and antibiotics to skin pathogens. First, Blumea balsamifera oil, wormwood powder and wormwood tablets were prepared with DMSO to prepare drug solutions with a mass concentration of 1.5 g / mL. Under sterile conditions, 60 μL of the prepared bacterial suspension was evenly applied to the surface of the culture medium (culture dish = 6 cm) by paper agar diffusion method, and placed for 2-3 minutes. After the bacterial solution on the surface of the plate was absorbed, 35 μL of the drug solution was taken with a pipette and dropped on a sterile filter paper (diameter 8 mm). After the drug solution was completely absorbed, the drug loading of the paper was 52.5 mg / piece. The paper was attached to the culture medium plate coated with the bacterial solution and the name of the drug solution was marked. The solvent DMSO was used as a negative control, and the corresponding antibiotics were used as positive controls. After the culture was completed at the corresponding temperature, the diameter of the inhibition zone in the central area was measured using a vernier caliper, and three parallels were set for each treatment group.

[0037] 2.1.3 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

[0038] Refer to the method of Yahia et al. and make some modifications, and use the micro-broth dilution method. First, use DMSO + Tween 80 to dissolve wormwood oil, wormwood powder, and wormwood tablets respectively, add water to make up to 20mg / mL and 50mg / mL for standby use, and ensure that the final concentrations of Tween 80 and DMSO are ≤2% and ≤1%, respectively. Under sterile conditions, add 100μL of liquid culture medium to each well of the first three rows A / B / C in a 96-well plate, take 100μL of the drug solution to the first well of the first three rows, dilute each well by multiples until the last well (the 12th well) draws 100μL and discards it, so that the drug solution concentration is gradiently diluted, and then add 100μL / well of the diluted bacterial suspension, set up three parallels for each sample; then set up a negative control (culture medium + drug solution) and a positive control (culture medium + bacterial solution). Then place the 96-well plate in an incubator and culture it for the corresponding time. After the culture is completed, use an enzyme reader to measure the absorbance value OD of the 96-well plate 600 , the inhibition rate was calculated according to formula (1), and the sample concentration with an average inhibition rate of 80% was the MIC value of the tested bacteria. Finally, the agar plate method was used, and 100 μL of each well with a concentration greater than MIC was applied to the corresponding solid culture medium. After a certain period of culture according to the specific bacterial culture conditions, the bacterial growth was observed. The lowest drug concentration when the colonies on the plate were ≤5 was the MBC value. Three parallels were set for each treatment group.

[0039] Inhibition rate (%) = (positive control OD 600 -Medicine solution OD 600 ) / (positive control OD 600 - Negative control OD 600 )×100(1)

[0040] 2.1.4 Determination of inhibition curve

[0041] In order to further study the effects of different extracts of Blumea balsamifera on the growth of skin pathogens, the OD of bacterial solution under different drug concentrations was measured. 600The law of the change of the value over time can intuitively reflect the growth changes of skin pathogens under the action of different extracts of Blumea Balsamifera. The antibacterial growth curve experiment was carried out by selecting strains with better antibacterial effects of different extracts of Blumea Balsamifera. Prepare a sample mother solution with a concentration of 20 mg / mL according to the method of 2.1.3 for use; set up drug groups with different extracts of Blumea Balsamifera, a positive control group (culture medium + bacterial solution) and a negative control group (culture medium + sample solution). Since the sample solution is milky white, it will affect the determination of the sample absorbance value, so a negative control is required. First, add liquid culture medium and bacterial suspension in a ratio of 1:1, and then add a certain amount of sample mother solution to make the final concentrations of wormwood oil, wormwood powder and wormwood tablet drug groups 1 / 2MIC, MIC and 2MIC, respectively. Oscillate and culture according to the culture conditions in 2.11. Measure the absorbance value of bacteria within 36 hours, and measure the absorbance value of fungi within 48 hours. Take samples every 2 hours in the first 12 hours, and then take samples every 12 hours. Use the microplate reader to measure the absorbance value OD 600 , solution OD 600 = Sample OD 600 -Negative OD 600 , with OD 600 The growth curve was drawn with the logarithm as the ordinate and the sampling time as the abscissa, and three parallel groups were set up for each treatment group.

[0042] 2.2 Determination of the antioxidant activity of different extracts of Blumea balsamifera in vitro

[0043] 2.2.1 Determination of DPPH free radical scavenging ability

[0044] Referring to the method of Ajay et al., the DPPH free radical scavenging method was used to determine the DPPH free radical scavenging ability of wormwood oil, wormwood powder and wormwood tablets, and VC and BHT were used as control samples and positive control solutions. First, a certain amount of wormwood oil, wormwood powder, wormwood tablets, VC and BHT were weighed and prepared into sample solutions of different gradient concentrations with corresponding solvents, and then a 0.1mM DPPH ethanol solution was prepared and sealed in a 4°C refrigerator away from light for standby use. Then a 96-well plate was plated, and the 96-well plate was divided into a control group A0 (100μL DPPH ethanol solution + 100μL sample solvent), a sample group A1 (100μL DPPH ethanol solution + 100μL sample solution) and a blank group A2 (100μL sample solution + 100μL sample solvent). Each sample was paralleled 3 times and operated in the dark. After the plate was loaded, it was kept at room temperature and away from light for 30min, and the absorbance was measured at a wavelength of 517nm using an enzyme reader, and the average value was taken to calculate the DPPH scavenging rate of each concentration. Calculate the clearance rate (I%) according to formula (2), draw a line graph of drug mass concentration and clearance rate, draw a regression equation and calculate IC 50 .

[0045] I%=[1-(A1-A2) / A0]×100% (2)

[0046] 2.2.2 Determination of ABTS free radical scavenging ability

[0047] First, weigh a certain amount of mugwort oil, mugwort powder, mugwort tablets, VC and BHT and use the corresponding solvents to prepare sample solutions and positive control solutions of different concentrations. Then, use distilled water to prepare 7mmol / L ABTS solution and 2.45mmol / L potassium persulfate solution, respectively, mix them in a 1:1 ratio, and let them stand for 12-16h at room temperature in the dark to obtain ABTS + · solution. Dilute the ABTS+· solution with anhydrous ethanol so that its absorbance at a wavelength of 734nm is 0.7±0.02 working solution, and store the working solution in a light-proof 4℃ refrigerator for later use. Then, spot the 96-well plate. The 96-well plate is divided into a control group A0 (ABTS working solution 100μL + sample solvent 100μL), a sample group A1 (ABTS working solution 100μL + sample solution 100μL) and a blank group A2 (sample solution 100μL + sample solvent 100μL). The operation is done in a dark environment. After the plate is placed at room temperature in a dark environment for 10 minutes, the absorbance is measured at a wavelength of 734nm using an enzyme reader. Each sample solution is repeated 3 times, and the average value is taken to calculate the clearance rate of ABTS at each concentration. The clearance rate (I%) is calculated according to formula (3), a line graph is drawn between the drug mass concentration and the clearance rate, and a graph is drawn between the logarithm of the mass concentration and the clearance rate. A regression equation is drawn and the IC is calculated. 50 .

[0048] I%=[1-(A1-A2) / A0]×100% (3)

[0049] 2.2.3 Total antioxidant capacity

[0050] The total antioxidant capacity (iron reducing capacity assay (FRAP method) kit instructions were used for determination. First, a certain amount of wormwood oil, wormwood powder, VC and BHT were weighed and prepared into sample solutions and positive control solutions of different concentrations with corresponding solvents. Then, reagents 1, 2 and 3 in the kit were prepared into a mixed solution (FRAP working solution) in a ratio of 10:1:1. The FRAP working solution must be prepared and used immediately, and preheated at 37°C before use. Then, a 96-well plate was plated. First, 10 μL of each sample solution of different concentrations was aspirated into a 96-well plate, and then 190 μL of FRAP working solution was added. The mixture was mixed thoroughly and allowed to stand at room temperature for 20 minutes. The absorbance was measured at a wavelength of 593 nm using an ELISA reader. Anhydrous ethanol was used as a blank control. Each sample solution was repeated 3 times and the average value was taken. According to the FeSO4-7H2O standard curve y=1.2416x+0.0134 provided by the kit, the correlation coefficient R 2= 0.9996, where y is the absorbance difference ΔA, x is the Trolox concentration (μmol / ml), and the standard curve absorbance difference range is between 0.14 and 1.25. The calculated absorbance difference is inserted into the standard curve, and the result is expressed as the equivalent antioxidant capacity of Trolox per milliliter of sample.

[0051] 3. Results and Analysis

[0052] 3.1 Drug sensitivity test

[0053] According to the sensitivity judgment standard of inhibition zone by Li Meng et al.: the diameter of inhibition zone is ≥20mm, extremely sensitive; the diameter of inhibition zone is 15~<20mm, highly sensitive; the diameter of inhibition zone is 10~<15mm, moderately sensitive; the diameter of inhibition zone is <10mm, lowly sensitive, and 0 is resistant. The results are shown in Table 1 and Figure 1 .from Figure 1 From the above, except for Pseudomonas aeruginosa, the drug-sensitive papers of other drug groups all showed inhibition zones of varying sizes with clear boundaries. Gram-positive bacteria are more sensitive to different extracts of Blumea Balsamifera than Gram-negative bacteria. The sensitivity of Gram-positive bacteria to different extracts of Blumea Balsamifera is wormwood oil>wormwood powder>wormwood tablets. In particular, wormwood oil is extremely sensitive to Bacillus subtilis, with an inhibition zone diameter of 21.00±3.35mm. There is no significant difference in the sensitivity of Gram-negative bacteria to different extracts of Blumea Balsamifera. Escherichia coli shows low sensitivity, and Pseudomonas aeruginosa shows drug resistance. In terms of fungi, different extracts of Blumea Balsamifera show certain drug sensitivity, and the sensitivity of different drugs varies greatly. Among them, the sensitivity of fungi to wormwood oil and wormwood tablets is tropical Candida>Malassezia furfur>Candida albicans; 3 strains of fungi show moderate sensitivity to wormwood powder, and there is no significant difference in sensitivity. Overall, the sensitivity of fungi to drugs is wormwood oil>wormwood tablets>wormwood powder.

[0054] Table 1 Average inhibition zone diameters of different extracts of Blumea balsamifera against different skin pathogens

[0055]

[0056] Note: - indicates undetermined. The positive control concentration is levofloxacin (20 μg / ml); ketoconazole (20 mg / ml) is a positive control for Candida albicans and Candida tropicalis, and fluconazole (3 mg / ml) is a positive control for Malassezia furfur. Different lowercase letters in the same row indicate differences between different samples of the same species.

[0057] 3.2 Determination of MIC and MBC

[0058] MIC refers to the lowest concentration of antimicrobial drugs that can inhibit the growth of bacteria. The lower the MIC value, the stronger the activity of the antimicrobial drug against specific strains. As shown in Table 2, different extracts of Blumea balsamifera showed different degrees of inhibition on 5 pathogenic bacteria and 3 fungi, with MIC ranging from 0.3125 to 6.25 mg / ml. On Gram-positive bacteria, wormwood oil had the best inhibitory effect on Bacillus subtilis, with a MIC value of 0.3125 mg / ml. The inhibitory effect on Staphylococcus epidermidis was relatively poor, with a MIC value of 2.5 mg / ml, which was consistent with the results of drug sensitivity. The inhibitory effect of wormwood powder and wormwood tablets on Gram-positive bacteria was also good, with a MIC value of 0.625 to 1.25 mg / ml, but it was not consistent with the experimental results of drug sensitivity. Similarly, in Gram-negative bacteria, from the drug sensitivity test, Pseudomonas aeruginosa also showed resistance to the three extracts. The MIC values ​​showed that the three extracts had a certain inhibitory effect on Gram-negative bacteria. Ai tablets had the best inhibitory ability against Escherichia coli, with a MIC value of 0.625 mg / ml. In fungi, overall, the antibacterial activity of the three extracts against fungi was the best with Ai powder, with a MIC value of 0.3125-125 mg / ml and an MBC value of 0.625-2.5 mg / ml. Ai tablets were better than Ai oil, which was inconsistent with the results of drug sensitivity tests. The reason may be that the diameter of the inhibition zone of different antibacterial drugs may be different due to the influence of the diffusion rate of the drug in agar. It can be seen that the diffusion rate in agar is Ai oil> Ai powder> Ai tablets, so there will be inconsistency with the results of drug sensitivity tests.

[0059] MBC is the lowest drug concentration at which an antibacterial drug can kill more than 99.9% of the tested bacteria. The lower the value, the better the effect on bacteria and fungi. As shown in Table 2, the three extracts of Blumea balsamifera showed different bactericidal abilities against five strains of skin pathogenic bacteria and three strains of fungi, with MBC values ​​ranging from 0.625 to 12.5 mg / ml. Among them, the bactericidal ability against Pseudomonas aeruginosa was relatively weak, with MBC values ​​greater than 5 mg / ml.

[0060] Table 2 MIC and MBC test results of different extracts of Blumea balsamifera against skin pathogens

[0061]

[0062] 3.3 Effects of different extracts of Blumea balsamifera on the growth curve of pathogenic bacteria

[0063] Depend on Figure 2It can be seen that the bacterial positive control group entered the rapid growth logarithmic phase after 2 hours, the bacterial liquid concentration gradually increased, Bacillus subtilis reached a peak at 24 hours, then began to decline or stabilize, and the bacteria began to enter the decline period; while Staphylococcus epidermidis was in the rapid growth logarithmic phase between 2 hours and 36 hours, and the growth rate was relatively reduced after 24 hours. The bacterial liquid concentration of the fungal positive control group hardly increased in the first 12 hours, entered the rapid growth logarithmic phase from 12 to 24 hours, and most fungi entered the decline phase from 12 to 36 hours and then reproduced again after 36 to 48 hours. After adding different extracts of Blumea balsamifera with mass concentrations of 1 / 2MIC, MIC and 2MIC, the growth of bacteria and fungi showed different inhibitory effects, and the number of viable bacteria was significantly less than that of the positive control group, and with the increase of drug concentration, the inhibitory effect of the growth curve increased. At 1 / 2MIC mass concentration, the growth of Bacillus subtilis was inhibited to a certain extent before 2 hours, the number of live bacteria decreased, and then entered the logarithmic growth phase. The inhibitory effect was 1 / 2MIC Ai tablets (0.3125mg / mL) > 1 / 2MIC Ai oil (0.15625mg / mL) > 1 / 2MIC Ai powder (0.3125mg / mL). However, the number of live bacteria of Staphylococcus epidermidis decreased significantly at a mass concentration of 1 / 2MIC. The positive drug began to enter the logarithmic growth phase after 2 hours, Ai powder and Ai tablets entered the logarithmic growth phase after 12 hours, and Ai oil entered the logarithmic growth phase after 24 hours. As the drug mass concentration increased to MIC and 2MIC, the bacterial growth rate slowed down, and the number of live bacteria decreased significantly. Under the action of MIC, the number of most bacteria stabilized at a relatively low level in the later stage. Under the action of 2MIC, each bacteria directly entered the decay phase and was killed, showing the high efficiency of different extracts of Ai balsam pear against bacterial pathogens.

[0064] For fungi, their growth began to be inhibited after adding different mass concentrations of Blumea balsamifera extracts. The number of live bacteria hardly increased, and showed a significant decline in the first 2 hours, and then stopped growing in the first 36 hours. At a mass concentration of 1 / 2MIC, after 36 hours, moxa tablets and moxa powder showed a slight growth trend, but the growth rate was significantly lower than that of the positive control; under the action of MIC and 2MIC, the fungi directly entered the decay stage and were all killed, indicating that different extracts of Blumea balsamifera have a significant inhibitory effect on fungi such as Candida albicans.

[0065] 2.2 Determination of the antioxidant activity of different extracts of Blumea balsamifera in vitro

[0066] 2.2.1 Determination of DPPH free radical scavenging ability

[0067] The antioxidant activities of different extracts of Blumea balsamifera determined by DPPH method are shown in Table 3 and Figure 3As shown, among the three kinds of Artemisia annua extracts, only Artemisia argyi oil has the ability to scavenge DPPH free radicals, but it is lower than the positive controls VC and BHT. It can be seen from the regression equation that there is a significant linear relationship between the mass concentration of Artemisia argyi oil samples and the DPPH free radical scavenging rate, while the free radical scavenging ability of Artemisia argyi powder and Artemisia argyi tablets does not show a dose-effect relationship with their mass concentration, and the DPPH free radical scavenging rate does not show a significant change with the increase of sample mass concentration. When the concentration of Artemisia argyi oil is 25mg / ml, its ability to scavenge DPPH free radicals is equivalent to that of VC with a mass concentration of 8μg / ml and BHT with a mass concentration of 64μg / ml, and the scavenging rate is greater than 80%. In order to further compare the ability of the three Artemisia annua extracts to scavenge DPPH free radicals, the half inhibitory concentration (half inhibitory concentration, IC 50 ) is the determination index, IC 50 The smaller the value, the stronger its ability to scavenge DPPH free radicals. As shown in Table 3, the IC 50 were 2.657, 0.0018, and 0.0115 mg / mL, respectively, while the IC values ​​of moxa powder and moxa tablets were 50 The value is too large, indicating that these two Blumea balsamifera extracts have no ability to scavenge DPPH free radicals.

[0068] Table 3 The ability of different extracts of Blumea balsamifera to scavenge DPPH free radicals

[0069]

[0070] 2.2 Determination of ABTS free radical scavenging ability

[0071] Regression equation and IC of different extracts of Blumea balsamifera for scavenging ABTS free radicals 50 The values ​​are shown in Table 4. From the regression equation, it can be seen that the mass concentration of different extracts of Artemisia balsamifera oil, Artemisia balsamifera powder, and Artemisia balsamifera tablets has a good linear relationship with the ABTS free radical scavenging rate, showing an obvious dose-effect relationship. IC values ​​of Artemisia balsamifera oil, Artemisia balsamifera powder, Artemisia balsamifera tablets, 50 0.049, 0.817, 14.158, 0.00055, 0.002 mg·mL respectively -1 .Depend on Figure 4 It can be seen that the maximum concentration of the abscissa range of wormwood oil is 10 mg·mL -1 , when the mass concentration is 0.16 mg·mL -1 When the clearance rate is greater than 95.12%, the clearance rate tends to be stable. The maximum concentration of the abscissa range of moxa powder and moxa tablets is 100 mg·mL -1 When the concentration of moxa powder is 3.125 mg·mL -1 When the clearance rate is greater than 82.69%, the mass concentration is greater than 6.25 mg·mL -1, the clearance rate tends to be stable; when the mass concentration of Ai tablets is greater than 40 mg·mL -1 After that, the clearance rate is greater than 80% and the mass concentration is greater than 50 mg·mL -1 After that, the clearance rate tended to be stable. From the above analysis, it can be seen that different extracts of Artemisia balsamifera have a certain ability to scavenge ABTS free radicals, and the scavenging ability is Artemisia balsamifera oil>Artemisia balsamifera powder>Artemisia balsamifera tablets, but there is a certain gap between the ability to scavenge ABTS free radicals and the positive control.

[0072] Table 4 Ability of galangal essential oil to scavenge ABTS free radicals

[0073]

[0074] 2.3 Total antioxidant capacity assay (FRAP)

[0075] Depend on Figure 5 It can be seen that wormwood oil has a strong total antioxidant capacity, and as the mass concentration increases, the total antioxidant capacity increases, while wormwood powder and wormwood tablets have no obvious changes with the increase of mass concentration. -1 When the Trolox value was 0.61 μmol / ml, the total antioxidant capacity was 0.1 mg·mL -1 VC, 0.5mg·mL -1 The total antioxidant capacity of BHT is equivalent.

[0076] 3. Conclusion and Analysis

[0077] In this study, two Gram-positive bacteria, two Gram-negative bacteria and three fungi were selected as skin pathogen test bacteria. The results showed that different extracts of Blumea Balsamifera (Baimei oil, wormwood powder, and wormwood tablets) showed different degrees of inhibition on these seven skin pathogens, and there were differences in antibacterial ability. Among them, the diameter of the inhibition zone ranged from 9.4±0.17 to 30.85±4.89 mm, the minimum inhibitory concentration (MIC) was 0.3125 to 6.25 mg / ml, and the minimum bactericidal concentration (MBC) value was 0.0625 to 12.5 mg / ml, indicating that these three Blumea Balsamifera extracts had good antibacterial activity and inhibition against bacterial and fungal pathogens. From the analysis of the growth curve, the inhibitory effect of different Blumea Balsamifera extracts on pathogens showed concentration dependence. In the antibacterial experiment, the results of the agar well diffusion method showed that the three Blumea Balsamifera extracts had no inhibition zone on Pseudomonas aeruginosa, but showed inhibitory activity in the broth dilution method. This may be due to the effect of drug molecular size, as larger molecules have lower diffusion in agar. In the MIC and MBC determinations, samples that were in direct contact with bacterial cells showed more effective results.

[0078] This study used the DPPH free radical scavenging method, ABTS free radical scavenging method and total antioxidant capacity (FRAP) to evaluate the antioxidant capacity of three different extracts of Artemisia annua. The results showed that Artemisia annua oil showed strong activity in scavenging DPPH free radicals and ABTS free radicals, and its total antioxidant capacity was also relatively good. In contrast, Artemisia annua powder and Artemisia annua tablets did not show significant effects in scavenging DPPH free radicals and total antioxidant capacity, and only showed a certain scavenging ability for ABTS free radicals.

[0079] Mugwort oil, mugwort powder and mugwort tablets are volatile natural products extracted under the same process conditions by industrial steam distillation. However, there are significant differences in the antibacterial and antioxidant activities of the three extracts, and the main reason for this phenomenon is that their chemical compositions are different. Previous studies have shown that mugwort oil, mugwort powder and mugwort tablets only contain borneol as a common component, but the content of borneol is significantly different. The content of borneol in mugwort powder is 81.40%, that in mugwort tablets is 76.07%, and that in mugwort oil is only 13.36%. In terms of chemical composition, mugwort oil contains at least 56 compounds, which is significantly higher than the 19 compounds in mugwort powder and the 8 compounds in mugwort tablets. These results indicate that the antibacterial properties and antioxidant activities of the three extracts of Blumea balsamifera are determined by the synergistic effects between different components, so their biological activities may be better than those extracts with a single component or fewer types.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of Blumea balsamifera extract in in vitro antibacterial and / or antioxidant activities, characterized in that: The Blumea balsamifera extract includes wormwood oil, wormwood powder and / or wormwood tablets; the antibacterial agent includes resistance to Bacillus subtilis, Staphylococcus epidermidis, Candida tropicalis and / or Malassezia furfur.

2. The use according to claim 1, characterized in that: The mugwort powder refers to: the powdery crystals obtained after the fresh branches and leaves of Artemisia balsamifera are heated and sublimated; the mugwort flakes are block crystals formed after the mugwort powder is pressed and deoiled; the mugwort oil is the oil obtained in the process of pressing the mugwort powder.

3. The use according to claim 1, characterized in that: The anti-oxidation includes scavenging DPPH free radicals and / or scavenging ABTS free radicals.

4. The use according to claim 3, characterized in that: When the concentration of wormwood oil was ≥25 mg / mL, the DPPH free radical scavenging ability was the highest; when the concentration of wormwood oil was ≥0.16 mg·mL -1 When the concentration of wormwood oil is ≥16mg / ml, the total antioxidant capacity is the highest.

5. The use according to claim 1, characterized in that: For resistance to Bacillus subtilis, the concentration of wormwood oil, wormwood powder or wormwood tablets is ≥1 / 2MIC, and the MIC is 0.3125 mg / mL; for resistance to Staphylococcus epidermidis, the concentration of wormwood oil or wormwood tablets is ≥0.625 mg / mL, and the concentration of wormwood powder is ≥1.25 mg / mL; for resistance to Candida tropicalis, the concentration of wormwood oil is ≥0.625 mg / mL, the concentration of wormwood powder is ≥1 / 2MIC, the MIC is 0.3125 mg / mL, and the concentration of wormwood tablets is 0.3125 mg / mL; for resistance to Malassezia furfur, the concentration of wormwood oil is ≥1.25 mg / mL, and the concentrations of wormwood powder and wormwood tablets are ≥0.625 mg / mL.

6. The use according to claim 1, characterized in that: For resistance to Bacillus subtilis, the concentration of wormwood powder is 2.5 mg / mL, and the concentration of wormwood tablets is 0.625 mg / mL; for resistance to Staphylococcus epidermidis, the concentration of wormwood oil is ≥0.625 mg / mL, the concentration of wormwood powder is ≥1.25 mg / mL, and the concentration of wormwood tablets is ≥0.625 mg / mL; for resistance to Candida tropicalis, the concentration of wormwood oil is ≥0.625 mg / mL, the concentration of wormwood powder is ≥1 / 2MIC, the MIC is 0.3125 mg / mL, and the concentration of wormwood tablets is 0.3125 mg / mL; for resistance to Malassezia furfur, the concentration of wormwood oil is ≥1.25 mg / mL, and the concentrations of wormwood powder and wormwood tablets are ≥0.625 mg / mL.

7. The use according to claim 1, characterized in that: The antibacterial agent is anti-tropical Candida or Malassezia furfur.

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