Germacrane Sesquiterpene Dilactones from Mikania micrantha and Their Antibacterial and Cytotoxic Activity

Four new germacrane sesquiterpene dilactones, 2β-hydroxyl-11β,13-dihydrodeoxymikanolide (1), 3β-hydroxyl-11β,13-dihydrodeoxymikanolide (2), 1α,3β-dihydroxy-4,9-germacradiene-12,8:15,6-diolide (3), and (11β,13-dihydrodeoxymikanolide-13-yl)-adenine (4), together with five known ones (5–9) were isolated from the aerial parts of Mikania micrantha. Their structures were elucidated on the basis of extensive spectroscopic analysis. Compound 4 is featured with an adenine moiety in the molecule, which is the first nitrogen-containing sesquiterpenoid so far isolated from this plant species. These compounds were evaluated for their in vitro antibacterial activity against four Gram-(+) bacteria of Staphyloccocus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA), Bacillus cereus (BC) and Curtobacterium. flaccumfaciens (CF), and three Gram-(–) bacteria of Escherichia coli (EC), Salmonella. typhimurium (SA), and Pseudomonas Solanacearum (PS). Compounds 4 and 7–9 were found to show strong in vitro antibacterial activity toward all the tested bacteria with the MIC values ranging from 1.56 to 12.5 µg/mL. Notably, compounds 4 and 9 showed significant antibacterial activity against the drug-resistant bacterium of MRSA with MIC value 6.25 µg/mL, which was close to reference compound vancomycin (MIC 3.125 µg/mL). Compounds 4 and 7–9 were further revealed to show in vitro cytotoxic activity toward human tumor A549, HepG2, MCF-7, and HeLa cell lines, with IC50 values ranging from 8.97 to 27.39 μM. No antibacterial and cytotoxic activity were displayed for the other compounds. The present research provided new data to support that M. micrantha is rich in structurally diverse bioactive compounds worthy of further development for pharmaceutical applications and for crop protection in agricultural fields.


Introduction
Mikania micrantha H. B. K. (Asteraceae) is a perennial creeping vine indigenous to Central and South America. However, for the people in some other tropical and subtropical areas of the world, especially in Southeast and South Asia, Australia, and some Pacific islands, this plant is more often recognized by the local people as a harmful exotic invasive plant [1]. In those new Asia-Pacific habitats far away from its native America homeland, M. micrantha grows very fast and has been significantly hampering the normal growth of other local plants and thereby seriously threatening the local biodiversity, due to which M. micrantha has now been recorded as one of the 100 most invasive species in the world [2]. It was reported that the first appearance of M. micrantha in China was in the 1980s and now it has spread widely and rapidly in a large area of southern China; its long-lasting [2]. It was reported that the first appearance of M. micrantha in China was in the 1980s and now it has spread widely and rapidly in a large area of southern China; its long-lasting invasion has caused huge economic losses to the local agricultural and forestry production, and seriously damaged the original ecological balance [1,3].
Apart from the recognition of this plant as a very successful exotic invasive plant species, it is very interesting to know that, in Mexico, M. micrantha has long been used as a traditional folk remedy for the treatments of skin diseases, snake bites, and scorpion stings [4]. In addition, this plant has long been used in Jamaica as a traditional herbal medicine to treat skin itches and athlete's foot [5]. In the past twenty years or so, some bioactive screening studies on exploring the potential values of this plant species have indicated that M. micrantha possesses a wide range of biological activities, including antibacterial, antitumor, analgesic, cytotoxic, and phytotoxic activities [5][6][7][8][9][10][11][12][13][14]. These literature reports indicated that potentially rich biologically active chemical constituents would certainly exist in this plant. To date, some phytochemical studies conducted by several research groups in different countries have discovered a series of structurally diverse chemicals from this plant species, including terpenoids, steroids, flavonoids, and phenolic compounds, but only a small part of those identified compounds were addressed for their biological activities [11,[15][16][17][18][19][20][21][22][23][24].
Recently, in our pre-test, we noticed that the extracts of the aerial part material of this plant collected in Guangzhou, China, showed obviously detectable antibacterial and cytotoxic activity, and therefore we initiated a phytochemical study on this plant, by which a group of rare C-9 hydroxylated ent-kaurene diterpene glucosides and some phenolic compounds with antioxidant activities were revealed [25,26]. During our ongoing effort to clarify those potential antibacterial and cytotoxic natural products in this plant, a series of germacrane sesquiterpene dilactones, including four new (1)(2)(3)(4) and five known ones (5)(6)(7)(8)(9) (Figure 1), were further isolated and identified from the aerial parts of M. micrantha. The new structures were established based on detailed analysis of their MS and NMR spectra (see Supplementary Materials). Among those isolates, compound 4 is novelly featured with an adenine moiety in the molecule which is the first nitrogen-containing sesquiterpenoid so far isolated from this plant species. The in vitro bioassays further revealed that part of these germacrane sesquiterpene dilactones showed significant antibacterial (including anti-MRSA) and cytotoxic activities. In this paper, we describe the isolation and structure elucidation of these compounds, as well as the evaluation for their in vitro antibacterial and cytotoxic activity.

Results and Discussion
The air-dried and powdered leaf material of M. micrantha was extracted with 95% ethanol (in water). The resultant crude ethanol extract was then suspended with water and sequentially partitioned with petroleum ether (PE), ethyl acetate (EtOAc), and n-butanol (n-But), respectively, to generate the PE, EtOAc, and n-But extracts. The obtained EtOAc extract was then subjected to a series of column chromatographic steps over silica gel, ODS, and Sephadex LH-20 to afford the four new (1-4) and five known (5-9) germacrane sesquiterpene dilactones.
All nine germacrane sesquiterpenoids were evaluated for their in vitro antibacterial activity against four Gram-(+) bacteria of Staphyloccocus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA), Bacillus cereus (BC), and Curtobacterium flaccumfaciens (CF), and three Gram-(-) bacteria of Escherichia coli (EC), Salmonella typhimurium (ST), and Pseudomonas solanacearum (PS), by using a microdilution titer method as described in the experimental section [29]. As shown in Table 3, compounds 4 and 7-9 were found to display significant in vitro antibacterial activity toward all the tested bacteria with MIC values ranging from 6.25 to 12.5 µg/mL, which were close to that of the reference compounds kanamycin sulfate and vancomycin, while no detectable antibacterial activity was displayed for the other compounds. Among the seven test microorganisms, SA, BC, EC, and ST were human pathogenic bacteria, and CF and PS were two Agro-pathogenic bacteria capable of widely causing crop infection diseases. The exhibition of strong and broad-spectrum antibacterial activity of compounds 4 and 7-9 against all the test human pathogenic and agro-pathogenic bacteria in this bioassay indicated that these four compounds were not only valuable to be developed in medical usage as antibacterial agents for the treatment of human infectious diseases, but also valuable to be applied in agriculture for crop protection. It is worth noting that compounds 4 and 7-9 showed significant antibacterial activity against MRSA. MRSA infection is responsible for a rapidly increasing number of serious infectious diseases severely threatening global public health [30,31], and its control and clinic therapy are urgently lacking effective and safe anti-MRSA agents. The strong anti-MRSA activity of these four compounds suggested that they have the potential to be developed as an effective anti-MRSA agent. Compounds 1-9 were further screened for their in vitro cytotoxic activity against human tumor A549, HepG2, MCF-7, and HeLa cell lines, using a microdilution titer technique as recently we described [32]. The resulting IC 50 values are displayed in Table 4, compared to Adriamycin as positive control. Compounds 4 and 7-9 were found to obviously show cytotoxic activity against all the four tested cancer cell lines, with IC 50 values ranging from 8.97 to 27.39 µM. No obvious cytotoxic activity was displayed for the other compounds. The bioassay data indicated that compounds 4 and 7-9 were bioactive compounds with certain anticancer potentials highly valuable to be further developed as therapeutic drugs or health care agents for cancer treatment or prevention. Based on comparison of the chemical structures and their antibacterial and cytotoxic activities of compounds 1-6 (except 4) versus 7-9, we can find out that the existence of the α-methylene-γ-lactone moiety, i.e., the carboxyl group of O=C(12) conjugated with the exocyclic double bond -C(11)=C(13)H 2 , would be essential for this type of germacrane sesquiterpenoid to display strong antibacterial potential. It was reported that the mechanism for those sesquiterpenoids, characterized with a α-methylene-γ-lactone moiety in the molecule, to display strong antibacterial and cytotoxic activity, it would be due to the potential Michael-type addition reactions that easily happen in vivo by nucleophiles, such as thiol groups of cysteine residues in proteins, to react with the α-methylene-γ-lactone moiety [33]. However, at this point, it was exceptional for compound 4 which also showed strong antibacterial and cytotoxic activity toward all the seven bacterial strains, but evidently, this compound did not contain the aforementioned α-methylene-γ-lactone moiety in the structure. In fact, compound 4 was novelly characterized with a nitrogen-containing adenine motif attaching at C-13 in the molecule, which suggested that compound 4 might employ a different mechanism to show its antibacterial and cytotoxic activity, and the present data supported a presumption that the introduction of nitrogen atom or nitrogencontaining moiety into the structure might be another effective way to remarkably enhance the antibacterial and cytotoxic potentials of sesquiterpenoids, at least reasonable for this type of sesquiterpene dilactones [34].
As a very successful invasive plant, M. micrantha can annually produce a huge plant biomass at its invasion areas, but this biomass resource has yet not been well-developed and utilized so far. In recent years, some phytochemical studies on M. micrantha have revealed a variety of chemical constituents, with some of them addressed with biological activities, and those findings also indicated the existence of variability in the chemical constituent compositions among various populations of this plant species distributed in different parts of the world. In the present study, the obtained data provided new evidences to supported that the populations of M. micrantha growth in Guangzhou area in China is rich in bioactive natural products, at least rich in antibacterial and cytotoxic chemicals, potentially highly valuable to be developed in medical usage or in agriculture for crop protection.

Plant Material
The aerial part materials of M. micrantha was collected at the South China Botanical Garden, Guangzhou, Guangdong Province, P.R. China, and identified by Dr. Xinsheng Qin at the College of Forestry and Landscape Architecture, South China Agricultural University. A voucher specimen (No.20190623) was deposited in the phytochemical laboratory at the College of Forestry and Landscape Architecture, South China Agricultural University.

Extraction and Isolation
The air-dried aerial part materials of M. micrantha (18.5 kg) were powdered and extracted three times with 95% EtOH at room temperature for 3 days each time. After evaporation to remove the solvent ethanol under reduced pressure, the produced viscous concentrate was suspended in water (3 L) and sequentially partitioned with petroleum ether (3 L × 3) and EtOAc (3 L × 3) to afford the corresponding petroleum ether-soluble extract (520 g) and the EtOAc-soluble fraction extract (260 g). The EtOAc-soluble extract was subjected to silica gel column chromatography (CC), eluted with an increasing polarity gradient solvent system of CHCl 3 /MeOH (from 98:2 to 70:30, v/v, each 15 L) to afford fractions Fr. 1 -Fr. 8 Tables 1 and 2. 3β-Hydroxyl-11β, 13

Antibacterial Assay
The antibacterial activity of the nine germacrane sesquiterpenoids 1-9 was evaluated by using a method as we described recently [29], with slight modifications. In brief, 100 µL indicator solution (resazurin in sterile water, 100 µg/mL) was first placed into each of the sterility control wells (11th column) on 96 well plates, and the indicator solution (about 7.5 mL, 100 µg/mL) was mixed with test bacteria (5 mL, 10 6 cfu/mL, OD 600 = 0.07) followed by transferring (100 µL, each) to growth control wells (12th column) and all test wells (1-10th column). Then, each of 100 µL of test compounds (0.8 mg/mL) in beef extract peptone medium, along with the positive control solutions (prepared by kanamycin sulfate and vancomycin instead of the test compounds) and the negative control solution (3% DMSO in beef extract peptone medium), were applied to the wells in the 1st column of the plates. Once all samples (test compounds) and controls were properly applied to the 1st column of wells on the plate, half of the homogenized content (100 µL solution) was then parallel transferred from the 1st column wells to the 2nd column wells, and each subsequent well was treated similarly (doubling dilution) up to the 10th column, followed by discarding the last 100 µL aliquot. Finally, the plates were incubated at 37 • C for about 5-6 h until the color of growth control change to pink. The lowest concentration for each test compound at which color change occurred was recorded as the MIC value of the test compound. In the bioassay, a total of seven microorganisms including four Gram-(+) bacteria of Staphyloccocus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA), Bacillus cereus (BC), Curtobacterium flaccumfaciens (CF), and three Gram-(-) bacteria of Escherichia coli (EC), Salmonella typhimurium (SA), and Pseudomonas solanacearum (PS) were used in the bioassay.

Cytotoxic Assay
The cytotoxic activity of sesquiterpenoid compounds 1-9 against human tumor A549, HepG2, MCF-7, and HeLa cell lines were assayed by using 96 well plates according to a literature MTT method with slight modification [32]. Briefly, the cells for the test were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere with 5% CO 2 at 37 • C. Each well of 96-well cell culture plates was seeded with 100 µL adherent cells (5 × 10 4 cell/mL) and placed in an atmosphere with 5% CO 2 at 37 • C for 24 h to form a monolayer on the flat bottoms. Subsequently, the supernatant in each well was removed and 100 µL fresh medium and 100 µL medium containing one of the test compounds was added. Then, the test plate was incubated in a humidified atmosphere with 5% CO 2 at 37 • C for three days. Afterwards, 20 µL MTT at concentration 5 mg/mL in DMSO was added into each well and incubated for 4 h. Carefully, the supernatant in each well was removed and 150 µL DMSO was added. Then, the plate was vortex shaken for 15 min to dissolve blue formazan crystals. The OD (optical density) value of each well was tested on a Genois microplate reader (Tecan GENios, Männedorf, Switzerland) at 570 nm. All the tests were carried out by three individual experiments and adriamycin was applied as a positive control. In a test, for each of the tumor cell lines, each of the test compounds was set at concentrations 50, 25, 12.5, 6.25, 3.125, and 1.5625 µg/mL. The inhibitory rate of tumor cell growth was calculated by the formula: Inhibition rate (%) = (OD control -OD treated )/OD control × 100%, and the IC 50 values were calculated by SPSS 16.0 statistic software. The four tumor cell lines were obtained from the Kunming Institute of Zoology, CAS. The resulting IC 50 values listed in Table 4 were based on three individual experiments and represented as means ± standard deviation (SD).

Conclusions
Four new germacrane sesquiterpene dilactones, trivially named dihydrodeoxymikanolide M (1), dihydrodeoxymikanolide N (2), dihydrogermacradienolide (3) and dihydrodeoxymikadenine (4), along with five known ones (5-9) were isolated from the aerial parts of Mikania micrantha. Their structures were determined by extensive spectroscopic analysis. Compound 4 features an adenine moiety in the molecule, which is the first nitrogen-containing sesquiterpenoid so far isolated from this plant species. Bioassays indicated that compounds 4 and 7-9 were active to show strong in vitro antibacterial activity toward all the seven tested bacteria with the MIC values ranging from 1.56 to 12.5 µg/mL. Notably, compounds 4 and 9 showed significant antibacterial activity against the drug-resistant bacterium of MRSA with MIC value close to reference compound vancomycin. Compounds 4 and 7-9 were further revealed to show in vitro cytotoxic activity toward human tumor A549, HepG2, MCF-7 and HeLa cell lines, with IC 50 values ranging from 8.97 to 27.39 µM. No antibacterial and cytotoxic activity were displayed for the other compounds. The current research provided new data to support that M. micrantha is rich in structurally diverse bioactive compounds worthy of further development in medicinal or healthcare application and for crop-protection in agricultural fields.