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

amy54321 · 发表于 2014-8-18 18:28:35

【摘要】  目的通过一种简单快速的高通量方法筛选具有抗菌活性的植物提取物，并通过此种方法从有活性的粗提物中纯化活性组分。方法首先建立植物提取物库和抗菌的高通量筛选方法。然后针对转糖链球菌Streptococcus mutans和伴放线放线杆菌Actinobacillus actinomycetemcomitans进行高通量筛选。结果通过筛选发现红桑叶具有很强的抑菌活性。为了进一步研究其活性，将其粗提物通过HPLC进行分离得到71个组分，再通过同样的高通量筛选方法进行抗菌活性测定，对有活性的组分通过血琼脂法进行验证。实验数据表明红桑叶的一个组分#31对两种细菌有几乎完全的抑制（抑制率>99%），而另外两个组分对伴放线放线杆菌Actinobacillus actinomycetemcomitans有部分抑制。结论所建立的高通量筛选方法简便快捷，筛选结果稳定可靠，可用于大规模的植物提取物的抗菌活性筛选。红桑叶具有很强的抗菌活性，且可分离得到活性组分，红桑叶具有进一步开发为抗菌药物和保健品的潜力。

【关键词】  红桑叶；高通量抗菌筛选；抗菌活性；抑制率

Identification of the Antibacterial Activity of Morus rubra Leaves by a High-Throughput Screening Method

　　Abstract:ObjectiveThis research intended to test the antibacterial activity of a plant extract library using a simple and fast colorimetric detection method and identify those plant extracts with bioactivity, and to further purify those bioactive fractions of the selected plant extracts. MethodsWe first created a plant extract library and developed an antimicrobial High Throughput Screening (HTS) method. Then, the plant extracts were tested for antibacterial activity against two species of bacteria, S. mutans and A. actinomycestemcomitans. ResultsThe Morus rubra leaf extract was selected because it had the strongest biological activity against the oral bacteria tested. To further study the Morus rubra leaves, the extract was separated into small fractions via high-performance liquid chromatography (HPLC). The fractions were tested against bacteria S. mutans and A. actinomycestemcomitans using HTS and the antibacterial activity of those selected bioactive fractions was further confirmed by blood agar plating method.  One fraction of Morus rubra leaves, #31, exhibited near complete inhibiton (99% or better) for both bacteria, while two other fractions exhibited some, but not complete, inhibition of A. actinomycestemcomitans. ConclusionThe HTS can be used to screen for a large plant extract library due to its fast and simple procedure. Morus rubra leaves exhibited the strongest antibacterial activity and the active fraction was obtained. The Morus rubra leaves have potential usage to treat bacterial infection in the future.

　　Key wordsMorus rubra leaves；  High-Throughput antibacterial Screening, Antibacterial activity, Inhibition rate

　　Plant-based "antibiotics" products could be important alternatives to the traditional antibiotics to combat the recent development of antibiotics-resistant bacterial strains due to the overuse of over-the-counter antibiotics ［1，2］.  Plant materials and plant parts have been used as medicine in China to treat diseases for centuries.  Plants are the rich source for drug discovery due to the secondary metabolites produced.  Plant-based "antibiotics" products can serve as an important source for future therapeutic treatment of infectious diseases ［3，4］.

　　Screening for antibiotics from a vast plant source can be a daunting task because there are hundreds of plant species and hundreds of plant secondary metabolites even within a single plant ［5，6］. Using the traditional screening methods to identify antibacterial phytochemicals from a plant extract library can be time-consuming and very expensive.  Thus, a simple, cost-effective, and fast high-throughput antimicrobial screening method is needed for using the plant extract library for drug discovery source and purification process. In this study, we report a fast and simple colorimetric HTS for screening plant extract library for plant-based antibiotics using resazurin dye as an indicator. Resazurin detection method was used for detecting bacterial contamination in food industry and infectious disease detection ［7，8］.  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.  After incubation in the presence of resazurin, bacteria could be judged by dead or alive simply by looking at the color of the resazurin dye.

　　To sum up, we developed an antimicrobial High Throughput Screening (HTS) method for plant drug discovery.  We tested a plant extract library consisting of 163 plant species for antimicrobial activity against two species of oral bacteria:  Streptococcus mutans (a bacteria involved in dental caries formation) and Actinobacillus actinomycestemcomitans (strongly associated with periodontitis).  Morus rubra leaves is the extract that indicated strong biological activity against the three species of bacteria tested.  However, there were few studies on the Morus antimicrobial properties and the existing research is either very general or doesn't focus on the qualities［9，10］. In this study, we expanded our research on the Morus rubra leaves extract by separating out its properties via HPLC.  This allowed us to isolate the active fraction in the crude extract and give us the opportunity to further characterize its properties.

　　1  MATERIALS AND METHODS

　　1.1  Plant materialPlant materials were collected in the park and surroundings of Lexington, Kentucky and identified by the Prof. Huang in University of Kentucky, U.S.A.

　　1.2  Bacterial strain and growth conditionTwo oral bacterial species were used in this study. A. actinomycetemcomitans (ATCC 714) and Streptococcus mutans(ATCC 25175), were purchased from ATCC. TSBYE media and Anaerobe Broth were purchased from Oxoid Ltd. Growth conditions were at 37℃ in anaerobic condition.

　　1.3  InstrumentsHPLC Column: Phenomenex Gemini 5μl C18, System: Waters 510 pumps (2), Waters 484 Tunable Absorbance Detector; Microplate reader: MRX plate reader (Dynatech Laboratories). Evaporation equipment: Savant SC-110 Speed Vac, Savant RT-100 Refrigerated Condensation with a Savant VP 100 oil pump; water purification equipment: Milli-Q.

　　1.4  ChemicalsMethanol, Resazurin and Ethanol were purchased from Sigma, USA.  Blood agar plates were obtained from Remel Company, USA.

　　1.5  Plant extract libraryPlant extracts were prepared from plant materials by crushing 2 grams of plant material using a mortar and pestle and followed by adding 8 ml of 50% EtOH.  Each individual extract is allowed to shake for 24 hours in a 6-well tissue culture plate before being filtered through filter paper. Filtered extracts are then lyophilized 16 to 24 hours into dry powder. The dried extracts obtained through the lyophilization process are re-suspended in 100μl of 50% EtOH. A total of 163 plant extracts were prepared this way.

　　1.6  HTS screening of extract libraryLyophilized extracts were tested for activity through the HTS procedure by adding 1 μl of each extract to selected wells of a 96 well plate containing 100μl of TSBYE medium and 10% of bacteria from the overnight culture. The plates were then incubated in an anaerobic chamber between 16 and 18 hours. After incubation, 3μl of the colorimetric indicator resazurin was added to each well. The live bacteria could metabolize the blue resazurin into pink color, while the dead bacteria could not. The plate was then allowed to incubate for the resazurin to be metabolized by the bacteria providing the change in color. Wells containing the S. mutans required 1 hr of incubation and A. actinomycestemcomitans required 2 hours.  A reading using a Dynex 96 well plate reader is taken for each bacteria at 600nm.  Typically wells with viable cell will range in pink color giving an OD reading of 0.200 to 0.600.  Wells with nonviable bacteria will be blue giving an OD reading above 2.000. The resazurin screening method is simple and fast.

　　1.7  HPLC procedureEach fraction from the HPLC was tested against S. mutans and A. actinomycetemcomitans using the HTS procedure listed above. The plant extract was to separate with the HPLC as following.

　　Column: Phenomenex Gemini 5μl C18；System: Waters 510 pumps (2), Waters 484 Tunable Absorbance Detector；  Detector: UV 280nm； Injection Volume：20 μl； Flow rate：1.0 ml/min.

　　Table 1  HPLC Gradient（略）

　　Twenty 20μg of a selected plant extract were injected into HPLC and 71 fractions (1 ml per fraction) were collected roughly 1 fraction/minute where the fraction number correlates with the minute in which it was separated.  This allows us to identify the peak and each fraction was collected as 1 ml solution.  Each fraction was dried using a Savant drying machine.  Each fraction was re-suspended in 25μl of 50% ethanol solution.

　　1.8  Blood agar platingFractions in the 96-well plate that showed biological activity via the HTS were diluted 10000x and plated in duplicate on blood agar plates (Remel).  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 for 48 hours.  The colonies (CFU) were counted on each agar plate after the incubation.

　　2  RESULTS

　　A 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 significant 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 μg Morus rubra extract solution 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 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 fraction's anti-bacterial activity in vitro could be sees in Figure 2a and Figure 2b.

　　Figure 2a  The HTS results of the Morus rubra leaves HPLC fractions(1-71) against S. mutans. One μl 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 wells 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 inhibition, followed by fraction 20 and fraction 4. The blue color of resazurin indicated the fraction 31 significantly inhibited the growth S. mutans.

　　Ten μl 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 μl was distributed 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 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 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 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 after 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  DISCUSSION

　　Our study demonstrated that our simple and fast HTS method could be a useful tool for screening for plant-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-effective 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.  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 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 complete 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, Moraceae 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 the Morus rubra leaves had multi functions, such as hypoglycemic effect, lipids-decreasing effects, antihyperlipidemia effect, anti- inflammatory, anti-cancer and anti-virus ［11～20］.  Little research has been on its antibacterial activity against oral pathogens.

　　Periodontal disease has been called a silent epidemic.  Nearly 25% of adults from the ages 33 to 45 suffer from destructive periodontal disease［21］.  Many more suffer from gingivitis, and other bacteria related inflammatory oral diseases.  Thus, identifying and developing more effective therapeutic regimens targeting aspects 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 novel 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, could be exploited for future new antimicrobial medicine and new usage of the Morus leaves.

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[药学] 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