Antimicrobial Constituents of the Leaves of Mikania micrantha H. B. K

Background To isolate plant-derived compounds with antimicrobial activity from the leaves of Mikania micrantha, to determine the compounds configuration, and to evaluate their antimicrobial activity against eight plant pathogenic fungi (Exserohilum turcicum, Colletotrichum lagenarium, Pseudoperonispora cubensis, Botrytis cirerea, Rhizoctonia solani, Phytophthora parasitica, Fusarium solani, and Pythium aphanidermatum,) and four plant pathogenic bacteria (gram negative bacteria: Ralstonia dolaanacearum, Xanthomonas oryzae pv. Oryzae, Xanthomonas Campestris pv. Vesicatoria, and Xanthomonas campestris pv. Citri), and four bacteria (gram positive bacteria: Staphyloccocus aureus, Bacillus subtilis, Micrococcus luteus, and Bacillus cereus). Methods and Results Antimicrobial constituents of the leaves of M. micrantha were isolated using bioactivity- guided fractionation. The antifungal activity of the isolated compounds was evaluated by the inhibit hypha growth method and inhibit spore germination method. Characterization of antibacterial activity was carried out using the minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs). MIC and MBC were determined by the broth microdilution method. Six compounds – deoxymikanolide, scandenolide, dihydroscandenolide, mikanolide, dihydromikanolide, and m - methoxy benzoic acid – have been isolated from leaves of Mikania micrantha H. B. K. Deoxymikanolide, scandenolide, and dihydroscandenolide were new compounds. The result of bioassay showed that all of isolated compounds were effective against tested strains and deoxymikanolide showed the strongest activity. Conclusions and Significance The leaves of M. micrantha may be a promising source in the search for new antimicrobial drugs due to its efficacy and the broadest range. Meanwhile, adverse impact of M. micrantha will be eliminated.


Introduction
Mikania micrantha H. B. K. (Asteraceae), commonly known as mile-a-minute weed, is an extremely fast-growing, perennial creeping weed. Its native distribution is in Central and South America. It has been present in south China since the 1980s and has caused adverse impact on agricultural production in the established area [1]. So, the weed has been listed as one of the world's 100 worst invasive alien species by Invasive Species Specialist Group of IUCN [2]. The leaves of M. micrantha, commonly known as 'guaco', are used to make a poultice for snake bites and scorpion sting, decoction of the leaves is used to bathe rashes, and skin itches [3]. In Jamaica, its most popular uses are for wound dressings and promote the healing of sores as folk medicine [4]. In addition, it has been attracted the attention of natural products chemists because of its antibacterial, antitumor, cytotoxic, analgesic, inflammatory, antiproliferative, and phytotoxic activities [5][6][7][8][9][10][11][12][13]. By consulting literatures, sterols [14], [15], diterpenes [15], polyphenols [16], flavonoids [17][18][19], and sesquiterpene lactones [4], [14], [20] have been isolated from M. micrantha. However, the antimicrobial constituents were ambiguous, though mikanolide and dihydromikanolide as antimicrobial ingredient of M. micrantha were reported. With the aim of identifying those potentially additional and bioactive natural products of this plant, a phytochemical study has been carried out bioactivity-guided fractionation and resulted in the isolation of three new sesquiterpene lactones, named deoxymikanolide, scandenolide, and dihydroscandenolide (1)(2)(3), together with three known compounds (4)(5)(6). Compound 6 was named as mmethoxy benzoic acid, which was the first reported occurrence from this plant. Herein, the isolation and structural elucidation of these compounds, as well as their antimicrobial activities against eight bacteria and eight fungi, were described. This study provides first comprehensive description about the antimicrobial constituent and antimicrobial activities in an attempt to improve the understanding of the practicality of M. micrantha.

Extraction and isolation
The air-dried leaves powder of M. micrantha (2.0 kg) was ultrasonic extracted with chloroform (6L63) at ambient temperature. Actived carbon was added in the extract for decolorizing, then filtered in order to get rid of actived carbon and undissolved substance. The extract was concentrated in vacuo to give 122.60 g of the crude residue (yield 6.13%). The crude residue (120 g) was subjected to chromatography on silica gel (100-200 mesh) eluting successively with gradient carbon tetrachloride -acetonemethanol (35:2:1, 35:5:1, 35:5:2 35:6:2, 35:6:3, 35:6:4, 35:6:5, v/v) to give fractions A-1 to A-30. A-11 (1.8434 g) was rechromatographed on a silica gel column eluted with chloroform: ethyl acetate (4:1 v/v) followed by crystallization from chloroform to give compound 1 (0.8310 g 0.04155% yield) and compound 2 (0.3512 g 0.01756% yield). A-14 (1.2306 g) was rechromatographed on a silica gel column eluted with petroleum ether: ethyl acetate (5:1 v/v) followed by crystallization from chloroform to give compound 3 (0.4432 g 0.02216% yield). A-16 (5.7534 g) was rechromatographed on a silica gel column eluted with petroleum ether: ethyl acetate (1:1 v/v) followed by crystallization from acetone to give compound 4 (2.7364 g 0.13682% yield). A-17 (2.5618 g) was rechromatographed on a silica gel column eluted with chloroform: ethyl acetate (1:1 v/v) followed by crystallization from acetone to give compound 5 (0.8041 g 0.040205% yield). A- Single crystal X-ray diffraction analysis of 1, 2, and 3 Upon crystallization from CHCl 3 -EtOAc (1:1) using the vapor diffusion method, colorless needles of compound 1 were obtained. A suitable crystal was selected and performed on a SuperNova, Dual, and Cu at zero, Atlas diffractometer. The crystal was kept at 100.01(10) K during data collection. Using Olex2 [21], the structure was solved with the XS [22], structure solution program using Direct Methods and refined with the XL [22] refinement package using Least Squares minimisation. Crystal data: The final R 1 was 0.0764 (. 2sigma (I)) and wR 2 was 0.3147. In the structure refinements, nonhydrogen atoms were placed on the geometrically ideal positions by the ''ride on'' method. Hydrogen atoms bonded to oxygen were located by the structure factors with isotropic temperature factors. Crystallographic data for 1 have been deposited at the Cambridge Crystallographic Data Centre (deposition No. CCDC-939627).
Upon crystallization from CHCl 3 -EtOAc (2:1) using the vapor diffusion method, colorless needles of compound 3 were obtained. A suitable crystal was selected and performed on a SuperNova, Dual, and Cu at zero, Atlas diffractometer. The crystal was kept at 180.00(10) K during data collection. Using Olex 2 [21], the structure was solved with the Superflip [23] structure solution program using Charge Flipping and refined with the XL [22] refinement package using Least Squares minimisation. Crystal data: C 17 H 20 O 7 , M = 336.33, space group monoclinic, P2 1 ; unit cell dimensions were determined to be a = 6.2346 (3)  Antibacterial activity assay MIC and MBC of the compounds were assessed using the broth microdilution method recommended by the National Committee for Clinical Laboratory Standards [24], [25]. Inoculums of the microorganism were prepared from 24 h Mueller-Hinton Broth (MHB) cultures and suspensions were adjusted with turbidity equivalent to that of a 0.5 McFarland standard. The compounds dissolved in 5% dimethylsulfoxide (DMSO), were first diluted to the highest concentration (2000 mg/L) to be tested, and then serial twofold dilutions were made in a concentration range from 15.625 to 1000.0 mg/L in 5 ml sterile test tubes containing the medium. The 96-well plates were prepared by dispensing into each well 95 mL of nutrient broth and 5 mL of the inoculum. A 100 mL aliquot from the stock solutions of each isolates was added into the first wells. Then, 100 mL from the serial dilutions were transferred into 6 consecutive wells. The last well containing 195 mL of the medium without the compounds and 5 mL of the inoculum on each strip was used as negative control. The final volume in each well was 200 mL. Broth with 5 mL of DMSO was used as blank test. Plates were covered and incubated for 12 h at 37uC. After incubation, the lowest concentration of tested samples, which did not show any visual growth after macroscopic evaluation, was determined as MIC. Using the results of the MIC assay, the concentrations showing complete absence of visual growth of bacteria were identified and 10 mL of each culture broth was transferred on to the agar plates and incubated for the specified time and temperature as mentioned above. The complete absence of growth on the agar surface in the lowest concentration of sample was defined as the MBC. All tests were performed in triplicate.

Antifungal activity assay
Antifungal activities of the compounds was tested using the inhibit hypha growth method. The compounds were prepared with different concentrations using dimethyl sulfoxide (DMSO) and diluted by Potato Dextrose Agar (PDA) medium as 500, 250, 125, 62.5, and 31.25 mg/L. The PDA mediums mixed with the compounds were dumped respectively into petri dishes (9 cm diameter) as plating. An agar plug of fungal inoculums (6 mm in diameter) was removed from a previous culture of the fungal strains tested and placed upside down in the center of the petri dishes. The same amount of DMSO and distilled water which   were used to replace the compounds were added respectively into the PDA mediums as the blank test and negative control. Each treatment was done with three replicates. All of materials were subjected to autoclaving at 121uC for 30 min. The means diameter of fungal colony was measured by criss-cross method with calipers by incubated at 28uC.

Inhibit spore germination activity assay
The spore suspensions of Botrytis cinerea, Glomerella cingulata, Exserohilum turcicum, Fusarium solani were obtained from their respective 10 days old cultures. The spore suspension was adjusted with sterile distilled water to a concentration of approximately 1.0610 5 spore/mL. The compounds dissolved in 5% dimethyl sulfoxide (DMSO), and then serial twofold dilutions with the spore suspensions were made in a concentration range from 125.0 to 2000.0 mg/L. 100 mL of the spore suspensions were taken onto the glass slide and incubated at 2462uC for 24 h. About 200 spores were counted and percentage of spore germination was calculated. The same amount of DMSO and distilled water were used as negative control and blank test. All experiments were conducted in triplicate. All of materials were subjected to autoclaving at 121uC for 30 min.

Statistical analysis
Data were analyzed using SPSS 10.0 (Chicago, IL, USA). The data were considered statistically significant for P values #0.05.
Compounds 4-6 ( Figure 1) were, respectively, identified as mikanolide, dihydromikanolide [19], and m -methoxy benzoic acid by comparison of their spectral data with those reported in the literature. 1 H and 13 C NMR spectral data of these compounds were shown in Table 1. Particularly, this is the first reported occurrence of m -methoxy benzoic acid from this plant.
All 6 compounds were subjected to quantitative bioassays for antimicrobial activity. DMSO used at 2.5% (v/v) showed no antimicrobial activity against all tested microorganisms (data not shown). As shown in Table 2, 3, and 4, all isolates were found to possess antimicrobial activity. Among these compounds, 1 was shown to possess significantly more antibacterial activity compared with the others against tested bacteria grown in suspension; the MIC values for tested strains (n = 8) ranged from 62.5 to 125 and the MBC values ranged from 125 to 250 ( Table 2). The others were showed moderate activity against all tested bacteria. By the inhibit hypha growth method, it was seen that EC 50 values of 6 isolated for tested fungi (n = 4) ranged from 82.40 to 330.10 mg/L in different times (Table 3). It is observed that antifungal susceptibility of 6 isolated was not significantly different. The results of inhibit spore germination assay showed that 1 was observed the highest inhibition activity; IC 50 values ranged from 21.44 to 53.18 mg/L (Table 4). Compound 6 was weakest in activity.

Discussion
In recent decade, problems of multidrug resistant microbes have reached an alarming level in around the world. These pose a serious challenge to the scientific community hence emphasis has been laid on the development of new antimicrobial agents [28], [29]. One possible strategy is the rational localization of bioactive products from folk medicines, with the hope that systematic screening of these will result in the discovery of novel effective Table 2. Inhibitory effects of compound 1-6 isolated from leaves of M. micrantha against tested bacteria strains.   compounds with potent and useful activities against microbes. Now, there is an ever-increasing demand for plant-based therapeutics in both developing and developed countries due to a growing recognition that they are natural products, non-narcotic and, in most cases, easily available at affordable prices; they also have no side effects. In China, since its introduction in the late 1980s and the early 1990s, M. micrantha has drastically infested Guangdong Province and caused significant damage to forests, farmlands, and orchards; and led to great loss of native species diversity, significant decline of microbial communities and food web stability; and even altered mineral cycling [30]. Though, M. micrantha was used in the traditional pharmacopoeia in some countries. So, the aims of this study not only develop to new antimicrobial agents, but also providing theoretic basis for the rational exploiting and controlling of M. micrantha.
Early reports on M. micrantha revealed its antimicrobial action; mikanolide and dihydromikanolide were major antimicrobial activity constituents [4]. In the present study, antimicrobial activity compounds were further isolated and the 6 compounds were obtained using bioactivity-guided fractionation, including 3 new sesquiterpene dilactones -deoxymikanolide, scandenolide, and dihydroscandenolide, and 3 known isolates -mikanolide, dihydromikanolide, and m -methoxy benzoic acid. Structure elucidation of 3 new sesquiterpene dilactones were determinated according to 1D, 2D NMR, IR, and single-crystal-X-ray diffraction study. In order to reveal effective substances for bioactivities in vitro, 6 isolates were tested for antibacterial activities against four gram-negative and four gram-positive bacteria by the broth microdilution methods. The obtained MIC and MBC values ranged from 62.5 to 1000 mg/L, especially, deoxymikanolide possessed the highest antimicrobial activity; the others only demonstrated moderate antimicrobial activities. Antifungal activities of 6 isolates were described against eight plant pathogens fungi. The results showed that EC 50 values ranged from 82.40 to 330.10 mg/L in different times by the inhibit hypha growth method and IC 50 values ranged from 21.44 to 255.37 mg/L by inhibit spore germination method. This study showed that antimicrobial constituents of M. micrantha were separated. In addition, structures of five compounds possess special guaianolide skeleton, familiar structure of the compounds were reported in Mikania species [8], [31][32][33]. So, the skeleton as parent structure should synthesis and structure-function relationship should further study. Occurrences of dilactone in this special guaianolide skeleton suggest that the problem of water-solubility of these active compounds will been solved by hydrolysis reaction, but bioactivity of the compounds will been tested in future experiment.
The results further demonstrated the broadest range of antimicrobial properties of M. micrantha. Considering the broadest range of antimicrobial properties, this would suggest that M. micrantha might be useful as a broad-spectrum antimicrobial. The mechanism of action will be done in further study on the basis of in vitro experimentation for development of new products. The isolation and bioactivity results will provide scientific foundation for rational development and utilization of this plant. If possible from collected specifically for controlling pathogen agent use, in order to manage indirectly and reduce adverse impact about M. micrantha.