以高通量方法筛选红桑叶的抗菌活性并进行活性组分的分离研究

1、以高通量方法筛选红桑叶的抗菌活性并进行活性组分的分离研究    ).  Controls were also plated along with wells that did not show activity around the active ones (e.g. If fraction 31 showed activity we would plate fraction 30, 31, and 32.)  The plates were incubated at 37 in an in anaerobic condition f

2、or 48 hours.  The colonies (CFU) were counted on each agar plate after the incubation.2  RESULTSA simple HTS screening method could be successfully used to screen antibacterial activity of a large plant extract library. Among the 163 plant extract, the Morus rubra extract showed the signif

3、icant anti-bacterial activity.  The Morus rubra extract was further purified by HPLC into 71 fractions and tested again for antibacterial activity. The HPLC profile was shown in Figure 1.2.1  HPLC purification The result of the HPLC was showed in Figure 1, where 20 g Morus rubra extract so

4、lution was injected. 71 fractions were collected roughly 1 fraction/minute. The HPLC ran for 80 minutes at 1 ml per ml.  A methanol/water gradient was used.Figure 1  The HPLC Chromatograph of Morus rubra leaves extract fractions（略）2.2  HPLC fraction HTS resultsThe HPLC fractions were

5、tested again for antibacterial activity (Figure 2a).  There were three fractions (fraction 4, 20, and 31) which showed significant anti-bacterial activity, with fraction 31 showing the most bioactivity.  The results of each fractions anti-bacterial activity in vitro could be sees in Figure

6、 2a and Figure 2b.Figure 2a  The HTS results of the Morus rubra leaves HPLC fractions(1-71) against S. mutans. One l of each fraction was added into the well of the 96-well plate. （略）Figure 2b  The HTS results of the Morus rubra leaves MUL HPLC fractions(1-71) against S. mutans. （略）The wel

7、ls in this plate have been treated with S. mutans and the Morus rubra HPLC fractions (1-71 reading left to right).  The wells in this plate have been treated with S. mutans and the HPLC fractions (1-71 reading left to right).  The resazurin indicates that fraction 31 exhibits the most inhi

8、bition, followed by fraction 20 and fraction 4. The blue color of resazurin indicated the fraction 31 significantly inhibited the growth S. mutans.Ten l of a control well without Morus rubra fraction (Right) or blue well with Morus rubra HPLC fraction #31 (Left) was diluted to 10-4 and 50 l was dist

9、ributed onto a blood agar plate. The left plate contained significant less S. mutans CFUs than the right plate.2.3  Blood agar plating resultsThe 3 wells (fraction 4, 20, and 31) from the 96 well-plate that showed antibacterial activity in presence of resazurin were plated on blood agar plates

10、in 10-3 to 10-4 dilution.  After incubation, the cfu (colony forming units) on each plate were counted and plotted.  Fraction 31 (the darkest color well) allowed no bacterial colonies, 100% inhibition against oral bacteria A. actinomycetemcomitans (Figure 3a).  This cfu data confirmed

11、 the data of HTS data.Again, fraction 31 showed a significant inhibitory activity against the S. mutans.  The plate of fraction 31 had an average of 41 cfus compared to over 3800 cfus of the control plate.  This indicated 99% of the S. mutans was inhibited (Figure 3b).  Fraction 31 of

12、 Morus rubra had a broad bioactivity and showed inhibitory activity against both oral pathogens, S. mutans and A. actinomycetemcomitans.Figure 3a  The Blood Agar Plating results of the fractions with the anti-bacterial activity in HTS (against A. aetinomycetemcomi-tans). （略）C= controls plated a

13、fter incubation.Figure 3b  The Blood Agar Plating results of the fractions with the anti-bacterial activity in HTS(against S.mutans).（略）C=controls plated after incubation.3  DISCUSSIONOur study demonstrated that our simple and fast HTS method could be a useful tool for screening for plant-

14、based antibiotics as the method enabled us to screen a plant extract library quickly.  The HTS identified Morus rubra plant leaf as a potential source for future plant-based antibiotics as it exhibited a significant antibacterial activity.  The screening method was sensitive and cost-effec

15、tive for the initial screening. Resazurin detection method has been used for detecting bacterial contamination in food industry. Resazurin dye is much simple and less time-consuming.  The live bacteria could metabolize the blue resazurin into pink color, while the dead bacteria could not. 

16、 After incubation in the presence of resazurin, bacteria could be judged by dead or alive simply by looking at the color of the resaurin dye.  Our study demonstrated that our HTS method was a useful tool for plant-based antibiotics as our HTS method enabled us to screen a plant extract library

17、and identify Morus rubra plant leaf as a potential source for future plant-based antibiotics.The experimental data showed that the Morus rubra leaves had strong antibacterial effect against oral pathogens S. mutans and A. actinomycetemcomitans.  The data indicated that the fraction 31 could com

18、plete or near complete inhibition against both of the bacterial species, S. mutans and A. actinomycetemcomitans. The fraction appears to be bactericidal and had a broad killing capability.Morus rubra is commonly known as red mulberry tree. Morus rubra is Morus rubra L. species, Morus L. genus, Morac

19、eae family. Morus is broadly distributed on the earth. Morus rubra is one of the dozen species of Morus L genus. The main chemical components in Morus leaves are flavanones, flavonoid glycosides, alkaloids, Polysaccharide, sterol, volatile oil and vitamins. The pharmacological studies showed that th

20、e Morus rubra leaves had multi functions, such as hypoglycemic effect, lipids-decreasing effects, antihyperlipidemia effect, anti- inflammatory, anti-cancer and anti-virus 1120.  Little research has been on its antibacterial activity against oral pathogens.Periodontal disease has been called a

21、silent epidemic.  Nearly 25% of adults from the ages 33 to 45 suffer from destructive periodontal disease21.  Many more suffer from gingivitis, and other bacteria related inflammatory oral diseases.  Thus, identifying and developing more effective therapeutic regimens targeting aspect

22、s of the microbial infection and inflammatory responses in oral cavity would provide a significant benefit to these individuals. Gingivitis and periodontitis affect as large a proportion of the global population as any disease known to mankind.  Thus, approaches to identify and characterize nov

23、el natural products that can be provided to the population through oral medicaments would constitute a significant contribution to the health care enterprise.  Natural pharmaceuticals with the ability to improve oral health without the adverse effect of antibiotics, such as the Morus rubra, cou

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