羅漢果葉莖根的抑菌活性組分的研究

1、羅漢果葉莖根的抑菌活性組分的研究         09-09-07 15:02:00     作者：周英，黃赤夫     編輯：studa20【摘要】  目的通過以活性為導(dǎo)向的提取分離的方法，首次研究羅漢果全植株中的抑菌活性組分。 方法 通過提取得到羅漢果全植株各個(gè)部分果實(shí)、葉、莖和根的提取物，然后運(yùn)用一種快速顏色指示法和血瓊脂法研究檢測它們的抑菌活性。結(jié)果羅漢果的非果實(shí)部分及果實(shí)對于口腔細(xì)菌轉(zhuǎn)糖鏈球菌S.mutans具有很強(qiáng)

2、的抑菌活性。為了進(jìn)一步研究并分離活性組分，將其粗提物通過Amberlite XAD樹脂進(jìn)行分離純化，再通過同樣的檢測方法進(jìn)行抗菌活性測定，對有活性的組分通過血瓊脂法進(jìn)行驗(yàn)證。結(jié)論羅漢果葉、莖和根（非果實(shí)部分）以及果實(shí)都具有較強(qiáng)的抑菌活性；羅漢果的非果實(shí)部分具有進(jìn)一步開發(fā)為抗菌藥物和保健品的潛力。 【關(guān)鍵詞】  羅漢果全植株; 抗菌染色檢測方法; Amberlite XAD樹脂; 抑菌活性Abstract:ObjectiveThis research intended to study and identify the antibacterial activity of Lo Han

3、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 detecti

4、on 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 antib

5、acterial 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 v

6、arious 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 activity1  Introduction 

7、   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 sm

8、all-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

9、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 th

10、e 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 frui

11、ts have been studied 25.  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 frui

12、ts 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

13、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 ac

14、tivities, 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 activit

15、ies 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 antim

16、icrobial 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 Methods2.1  InstrumentsAmberlite XAD purification column, Phenomenex Gemini 5 l C18, System: Waters 510 pumps 2, Waters 484 Tunable Absorbance Detecto

17、r; 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

18、, 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 pur

19、chased 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 m

20、ortar 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, an

21、d root were obtained through the lyophilization process and were re-suspended in 100l 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

22、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 co

23、lumn 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

24、 using the HTS (high-throughput screening) procedure described below 6.  Lyophilized extracts were dissolved in 50 l of 50% EtOH and tested for bioactivity as following procedure by adding 1 l of each extract to selected wells of a 96-well plate containing 100l of TSBYE medium and 10% of bacter

25、ia from the overnight culture. The plate was then incubated in an anaerobic chamber between 16 and 18 hours. After incubation, 6 l 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 meta