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       【摘要】 
       目的通过以活性为导向的提取分离的方法，首次研究罗汉果全植株中的抑菌活性组分。 方法 通过提取得到罗汉果全植株各个部分—果实、叶、茎和根的提取物，然后运用一种快速颜色指示法和血琼脂法研究检测它们的抑菌活性。结果罗汉果的非果实部分及果实对于口腔细菌转糖链球菌S.mutans具有很强的抑菌活性。为了进一步研究并分离活性组分，将其粗提物通过Amberlite XAD树脂进行分离纯化，再通过同样的检测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。结论罗汉果叶、茎和根（非果实部分）以及果实都具有较强的抑菌活性；罗汉果的非果实部分具有进一步开发为抗菌药物和保健品的潜力。
       【关键词】  罗汉果全植株; 抗菌染色检测方法; Amberlite XAD树脂; 抑菌活性
       Identification of the Antibacterial Activity from Leaf, Vine and Root of Lo Han Kuo (Momordica grosvenori)
       ZHOU Ying, HUANG Chifu
       (1.Department of Pharmaceutical Engineering, College of Life Science, Guizhou University, Guiyang, 550025, China; 2.College of Dentistry, University of Kentucky, Lexington, Kentucky 40503, U.S.A.)
       Brief introduction of author：ZHOU Ying, 1971-, Female, Ph. D. Associate Professor of Guizhou University, Supervisor for postgraduates. Brief introduction of author：ZHOU Ying, 1971-, Female, Ph. D. Associate Professor of Guizhou University, Supervisor for postgraduates.
       Abstract:ObjectiveThis research intended to study and identify the antibacterial activity of Lo Han Kuo plant (Momordica grosvenori) by extracting and purifying bioactive substances from various parts of Lo Han Kuo plant.  MethodsVarious extracts were prepared from Lo Han Kuo fruit, leaf, vine, and root, and the antibacterial activity of these extracts were tested using a colorimetric detection method and agar plating.ResultsExtracts of Lo Han Kuo all parts exhibited strong antibacterial activity against the oral bacteria, S. mutans.  To further isolate the bioactive fractions, the extracts were purified with Amberlite chromatography and the purified fractions were tested for antibacterial activity. The positive fractions showing the antibacterial activity were further tested by blood agar plating method. ConclusionThe experimental data demonstrated that extracts of Lo Han Kuo leaves, vines, fruit and roots exhibited strong antibacterial activities. This study suggests that various parts of Lo Han Kuo have the potential to be used for pharmaceutical preparations and dietary supplements to treat various human ailments, such as infection.
       Key words:Momordica grosvenori, Colorimetric detection method，Amberlite chromatography, Antibacterial activity
       1  Introduction
        Throughout the history of China people have used a wide variety of plants and herbs to help prevent/treat chronic and acute diseases, improve immunity to infection.  One plant that has been commonly used is the Lo Han Kuo fruit (Momordica grosvenori).  Lo Han Kuo fruits are small-sized fruits with very sweet taste.  Lo Han Kuo fruits are native to Guangxi Guilin region, also grown in Hunan, Guizhou, Jiangxi and many other provinces.  The Lo Han Kuo fruit has been utilized for centuries as traditional medicine to treat sore throat and coughing.  Lo Han Kuo fruits are typically harvested in late fall and dried immediately.  The dried Lo Han Guo fruits are used as whole or grinded as powder, typically used in beverage, such as tea and soup.
       
       Many attempts to identify the active compounds from Lo Han Kuo fruits were made over in the early fifties. Those efforts resulted in the identification of intensive sweetening components, known as mogrosides (mogroside I to VI).  These compounds are triterpenoids, with glucose moiety attached to a chemical structure called mogrol group ［1］. The biological activities of Lo Han Guo fruits have been studied ［2～5］.  Previous research demonstrated Lo Han Kuo fruit extract had antibacterial activity.  Although some publications report some bioactivities of Lo Han Kuo fruits, little research has been done on the non-fruit parts of Lo Han Kuo.  Traditionally, Lo Han Guo fruits are harvested in late fall and the dried vine/leaf are cut off.  The roots are covered with soil and protected from severe winter cold condition.  As little studies have been done on the non-fruit part, the Lo Han Kuo leaf and vine are not utilized at all.  Very little bioactivity are known about the non-fruit parts of Lo Han Kuo.
       
       This study aims to prepare the extracts and study the antimicrobial activity of Lo Han Guo leaf, vine, and root extracts. Our experimental data indicated that extracts of Lo Han Kuo leaf, vine, and root have potent antimicrobial activities, especially against oral organisms, such as Streptococcus mutans and A. actinomycetemcomitans. The potent anti-microbial activities are found in the fruit, leaf, vine, and root of Lo Han Kuo plant.  This is the first experimental study that demonstrated the potent antibacterial activities existing in the non-fruit parts of Lo Han Kuo.  The potent anti-microbial chemicals are not the sweetening agents, such as the mogrosides of the fruit as the bioactivity widely exists in the leaf, vine, and root of Lo Han Kuo.  The usage of non-fruit part of Lo Han Kuo to produce antimicrobial chemicals will have major economic impact as the leaf/vine is very cheap or it can be obtained at a very low cost.
       2  Materials and Methods
       2.1  InstrumentsAmberlite XAD purification column, Phenomenex Gemini 5 μl 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; Amberlite XAD resin, Methanol, Resazurin and Ethanol were purchased from Sigma, USA. Blood agar plates were obtained from Remel Company, USA.
       2.2  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.
       2.3  Lo Han Kuo extractionFruit, leaf, vine, and root of Lo Han Kuo were collected from field and dried.  Plant extracts were prepared from Lo Han Kuo parts by crushing 10 grams of dry Lo Han Kuo materials using a mortar and pestle, and followed by adding 20 ml of 50% EtOH.  Extracts of various Lo Han Kuo parts were allowed to shake for 24 hours in a flask.  After filtered through filter paper, 1 ml of filtered extract was then lyophilized 8 hours into dry powder. The dried extracts of leaf, vine, and root were obtained through the lyophilization process and were re-suspended in 100μl of 50% EtOH.
       2.4  Chromatography purification procedure100 grams of Lo Han Kuo leaf, vine, and root were separately grinded and boiled in 500 ml of water for 30 min. The water extraction was repeated for three times.  The extract solution was filtered and the supernatant was saved. 1000 ml of the supernatant were passed through a 100 ml nonpolar Amberlite XAD column which was pre-equilibrated with water.  Then, the column was washed with 1000 ml water to remove nonbinding materials.  The column was eluted with 10% to 100% ethanol solution.  The 200 ml of 10 fractions were collected and analyzed for antibacterial activity.
       2.5  Bioassay screening of Lo Han Kuo fractions1 ml of each fraction purified from Amberlite XAD chromatography was lyophilized and tested against S. mutans using the HTS (high-throughput screening) procedure described below ［6］.  Lyophilized extracts were dissolved in 50 μl of 50% EtOH and tested for bioactivity as following 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 plate was then incubated in an anaerobic chamber between 16 and 18 hours. After incubation, 6 μl 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.  A reading using a Dynex 96 well plate reader was 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.
       2.6  Blood agar platingFractions with blue colors 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.  The plates were incubated at 37℃ in an anaerobic condition for 48 hours.  The colonies (CFU) were counted on each agar plate after the incubation.
       3  Results
        The effect of various LHK extracts on the growth of S. mutans was examined. The extracts of Lo Han Kuo leaf, vine, stem and root demonstrated significant inhibition of S. mutans growth.  Fig. 1 shows antibacterial activity of Lo Han Kuo leaf extract. As compared to negative control, the strongest bioactivity fractions are from 60% to 100% ethanol.  Fig. 2 shows that Lo Han Kuo fruit extract has similar antibacterial activity profile.  As compared to negative control, the strongest bioactivity fractions are from 70% to 100% ethanol.  Antibacterial activity of Lo Han Kuo root extract also are from 70% to 100% ethanol (Fig. 3).  Fig. 4 shows antibacterial activity of Lo Han Kuo vine extract, the strongest bioactivity fractions are in the range from 60% to 100% ethanol. 
       
       To further investigate the chemical property of the bioactive components of Lo Han Kuo, the bioactive fraction 90% and 100% of the leaf extract were collected.  After removing the ethanol, fractions were fractionated by ether three times, an ether and water fractions were obtained.  The ether fraction had the significant antibacterial activity, while the water fraction did not have any antibacterial activity (Fig. 5).  The antibacterial components appeared to be low polar compounds.
       4  Discussion
        Extracts of Lo Han Kuo leaf, vine, root and fruit exhibited antibacterial activity against S. mutans.  The various parts of Lo Han Kuo share the similar antibacterial profiles ranging from 60% ethanol to 100% ethanol elution.  The similar profiles suggest that those extracts possibly contain similar antibacterial compounds in leaf, vine, root and fruit. This study demonstrated that all parts of Lo Han Kuo share similar antibacterial activity.  Ongoing research work is focused in isolating and identifying those bioactive compounds from Lo Han Kuo plant. 
       
       Dental caries is the predominant cause of tooth loss in children and young adults ［7］.  Caries is a bacterial infection caused by specific bacteria, albeit, this is not limited to a monospecific pathogen［8］. Plant-based "antibiotics" products from Lo Han Kuo plant 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.  Lo Han Kuo plant haa been used as medicine in China to treat sore throat and Strep bacterial infection for centuries.  The antibacterial components isolated from Lo Han Kuo plants can serve as an important source for future therapeutic treatment of oral diseases.  Although extracts of Lo Han Kuo leaf, vine, root and fruit exhibited antibacterial activity against S. mutans, they could be tested against many other infectious and oral bacteria. 
       
       Acknowledgements:This research was supported by the grant R41DE17265-01 from the NIH/NIDCR, U. S. A.
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