Phenolics from Mikania micrantha and Their Antioxidant Activity

A phytochemical study on the aerial parts of Mikania micrantha led to the isolation of two new phenolic compounds, benzyl 5-O-β-d-glucopyranosyl-2,5-dihydroxybenzoate (1) and (7S,8R)-threo-dihydroxydehydrodiconiferyl alcohol 9-acetate (2), together with twelve known compounds, benzyl 2-O-β-d-glucopyranosyl-2,6-dihydroxybenzoate (3), 4-allyl-2,6-dimethoxyphenol glucoside (4), (+)-isolariciresinol (5), icariol A2 (6), 9,10-dihydroxythymol (7), 8,9,10-trihydroxythymol (8), caffeic acid (9), p-coumaric acid (10), ethyl protocatechuate (11), procatechuic aldehyde (12), 4-hydroxybenzoic acid (13), and hydroquinone (14). Their structures were elucidated on the basis of extensive spectroscopic analysis. Except 8 and 9, all the other compounds were isolated from this plant species for the first time. The antioxidant activity of those isolated compounds were evaluated using three different assays. Compounds 1, 2, 3, 9, 10, 13, and 14 demonstrated significant 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) free radical cation scavenging activity ranging from SC50 0.31 to 4.86 µM, which were more potent than l-ascorbic acid (SC50 = 10.48 µM). Compounds 5, 9, 11, and 12 exhibited more potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (SC50 = 16.24–21.67 µM) than l-ascorbic acid (39.48 µM). Moreover, the ferric reducing antioxidant power (FRAP) of compounds 2, 5, 9, and 11 were discovered to be also comparable to or even more potent than l-ascorbic acid.


Introduction
Mikania micrantha H. B. K., belonging to the Asteraceae family, is a fast-growing perennial creeping vine indigenous to Central and South America. This plant is known as an invasive species in Southeast Asia and the Pacific region, including southern China [1]. The rapid spread of M. micrantha in South China has caused great damage to agriculture and forestry, as well as the ecological balance in invaded areas [1]. M. micrantha has long been used as a traditional herbal medicine in Jamaica to treat skin medicine in Jamaica to treat skin itches and athlete's foot [2]. Previous phytochemical studies have reported some structurally diverse chemicals from this plant, including sesquiterpene lactones, flavonoids, steroids, diterpene glucosides, and phenolic compounds, some of which have shown significant bioactivities [3][4][5][6][7][8][9][10][11][12]. Our recent study on this plant also revealed a group of rare C-9 hydroxylated ent-kaurene diterpene glucosides [13]. During our ongoing phytochemical research on invasive plants in China [14][15][16][17][18][19], two new (1 and 2) and twelve known (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) phenolic compounds (Figure 1), were further isolated from the aerial parts of M. micrantha. Herein, we report the isolation and structure elucidation of these compounds, as well as their antioxidant activity.
All of the isolated compounds were measured for their antioxidant activity by using three different in vitro assays, i.e., ABTS radical cation (ABTS •+ ) scavenging assay, DPPH radical (DPPH•) scavenging assay, and FRAP assay, with L-ascorbic acid as a reference compound. As shown in Table  2, new compounds 1 and 2, and known compounds 3, 9, 10, 13, and 14 demonstrated ABTS radical cation scavenging activity with SC50 values ranging from 0.31 to 4.86 µ M, which were more potent than L-ascorbic acid (SC50 = 10.48 µ M). Compounds 5, 9, 11, and 12 exhibited more potent DPPH radical scavenging activity (SC50 = 16.24-21.67 µ M) than L-ascorbic acid (SC50 = 39.48 µ M). Moreover, the revealed ferric reducing antioxidant power (FRAP) of compounds 2, 5, 9, and 11 were also comparable to or even more potent than the reference compound.  Table 1) showed signals of an acetoxy methyl at δ H 2.02 (3H, s), two methoxy groups at δ H 3.83 (3H, s) and 3.89 (3H, s), and five aromatic protons. The 13 C-NMR spectrum (Table 1), coupled with HSQC analysis, exhibited the signals of twenty-two carbons in total, comprising three methyls, two oxymethylenes [δ C 64.3 (C-9 ) and 66.7 (C-9)], nine methines (including five aromatic methanes), and eight quaternary carbons [including a carboxyl group at δ C 172.7 and seven aromatic quaternary carbons]. Detailed analysis of the NMR data indicated that compound 2 closely resembled threo-dihydroxydehydrodiconiferyl alcohol [20][21][22], with the only difference of the hydroxyl group at C-9 being replaced by an acetoxy group in 2. This deduction was consistent with the molecular formula and in accord with the significant HMBC correlations (Figure 2) from δ H 4.33 and 4.44 (C-9) to δ C 172.7. The relative configuration of H-7 and H-8 was determined to be trans, as supported by the NOE correlations of H-7/H-9 and H-8/H-6, H-2 ( Figure 2). The absolute configuration of C-7 and C-8 in 2 was assigned to be S and R based on the positive CD Cotton effects at 292 nm and 240 nm and the negative Cotton effect at 224 nm [23,24]. Furthermore, according to literature reports about that the chemical shifts of the C-7 and C-8 and the value of ∆δ C8 -C7 are characteristically different for the threo and erythro isomers, the relative configuration of C-7 and C-8 in 2 could then be determined to be threo based on its 13 C-NMR data at δ C 75.3 (C-7 ) and 77.6 (C-8 ) with the value of ∆δ C8 -C7 > 2.0 ppm [25][26][27]. Hence, compound 2 was elucidated as (7S,8R)-threo-dihydroxydehydrodiconiferyl alcohol 9-acetate.
All of the isolated compounds were measured for their antioxidant activity by using three different in vitro assays, i.e., ABTS radical cation (ABTS •+ ) scavenging assay, DPPH radical (DPPH • ) scavenging assay, and FRAP assay, with L-ascorbic acid as a reference compound. As shown in Table 2, new compounds 1 and 2, and known compounds 3, 9, 10, 13, and 14 demonstrated ABTS radical cation scavenging activity with SC 50 values ranging from 0.31 to 4.86 µM, which were more potent than L-ascorbic acid (SC 50 = 10.48 µM). Compounds 5, 9, 11, and 12 exhibited more potent DPPH radical scavenging activity (SC 50 = 16.24-21.67 µM) than L-ascorbic acid (SC 50 = 39.48 µM). Moreover, the revealed ferric reducing antioxidant power (FRAP) of compounds 2, 5, 9, and 11 were also comparable to or even more potent than the reference compound. Generally, free radicals and reactive oxygen species (ROS) are formed unceasingly in human body and the normal presence of free radicals can produce beneficial oxidation during physiological events. However, excessive generation of free radicals in human body will bring harmful oxidation to organisms, which is recognized as a leading cause of a variety of chronic diseases such as atherosclerosis, angiocardiopathy and cancer [38,39]. It is well known that natural antioxidants can help prevent oxidation and help regulate immune function. This study, to some extent, indicate that the invasive plant M. micrantha is rich in structurally diverse natural antioxidants, at least in antioxidant phenolic compounds, which are potential functional chemicals beneficial for human health worthy of further investigation.

General Experimental Procedures
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DRX-500 NMR spectrometer

Plant Material
The aerial parts of M. micrantha were collected from Guangzhou, China, in July 2014, and identified by Prof. Hong-Feng Chen, South China Botanical Garden, Chinese Academy of Sciences (CAS). A voucher specimen (No. 20140705) was deposited at the Laboratory of Bioorganic Chemistry of the South China Botanical Garden, CAS.

ABTS Radical Cation Scavenging Assay
The ABTS radical cation (ABTS •+ ) scavenging activity of the isolated compounds was evaluated following the procedures as previously described [40]. Briefly, potassium persulfate was added to 7 mM of ABTS •+ , and the mixture was allowed to stand in the dark at room temperature for 12-16 h before use. The ABTS •+ solution was diluted with phosphate-buffered saline (PBS, pH 7.4) to provide an absorbance of 0.70 ± 0.02 at 734 nm. The diluted ABTS •+ solution (190 µL) was added to sample fractions (10 µL) in DMSO at different concentrations. Each treatment was conducted in triplicate. After a mixing time of 10 s and an incubation period of 6 min at 37 • C in the dark, the absorbance in each well was read at 415 nm on a Genios microplate reader (Tecan). L-Ascorbic acid was used as a positive control. The inhibitory rates of ABTS •+ were calculated according to the following formula: ABTS scavenging rate (%) = [1 − (absorbance of compound − absorbance of blank)/absorbance of control] × 100. SC 50 values were calculated and expressed as means ± SD in micromolar.

DPPH Radical Scavenging Assay
Scavenging activity of the compounds towards DPPH radicals was carried out by the method as previously described [40]. DPPH radical solution was freshly prepared with MeOH to 0.1 mM. Test compounds were dissolved in DMSO and diluted two-fold to six serial concentrations. The DPPH solution (180 µL) and sample solution (20 µL) were mixed in 96-well plates. L-Ascorbic acid was dissolved in methanol and used as a positive reference. The control group contained DMSO instead of the compound solution, and the blank group contained methanol in place of the DPPH solution. Each treatment was performed in quadruplicate. The plates were incubated at 37 • C for 30 min in the dark. The absorbance in each well was read at 515 nm on a Genios microplate reader (Tecan). The inhibitory rates of DPPH radicals were calculated according to the formula: DPPH scavenging rate (%) = [1 − (absorbance of compound − absorbance of blank)/absorbance of control] × 100. SC 50 values (the concentrations required to scavenge 50% DPPH radicals present in the test solution) were calculated and expressed as means ± SD in micromolar.

FRAP Assay
Ferric reducing ability of the compounds was conducted according to the procedures as previously described [40]. FRAP reagent was made freshly by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM hydrochloric acid, and 20 mM aqueous ferric chloride (FeCl 3 ) solution in a 10:1:1 (v/v) ratio. The TPTZ solution was prepared on the same day. Test compounds were dissolved in methanol and diluted 2-fold to six concentrations. Twenty microliters of the compound solution and 180 µL of FRAP reagent were mixed in 96-well plates. L-Ascorbic acid was dissolved in methanol and used as a positive reference. Each treatment was conducted in quadruplicate. The plates were incubated at 37 • C for 30 min in the dark. The absorbance of the product (ferrous TPTZ complex) in each well was read at 595 nm using a Genios microplate reader (Tecan Group, Mannedorf, Switzerland). One milliliter of ferrous sulfate (FeSO 4 ) at six different concentrations and 1 mL of 10 mM TPTZ and 10 mL of 300 mM acetate buffer (pH 3.6) were used for a calibration curve. FRAP values were calculated and expressed as means ± the standard deviation (SD) in millimoles of Fe (II) per gram.

Conclusions
Fourteen phenolic compounds, including two new ones-1 and 2, were isolated from the aerial parts of M. micrantha. Their structures were identified by analysis of their spectroscopic data. Except 8 and 9, all the other compounds were isolated from this plant species for the first time. Bioassays revealed that seven compounds demonstrated good ABTS radical cation scavenging activity more potent than L-ascorbic acid, and four compounds exhibited more potent DPPH radical scavenging activity than L-ascorbic acid. Moreover, the ferric-reducing antioxidant power (FRAP) of four compounds were comparable to or even more potent than L-ascorbic acid. This study indicates that the invasive plant M. micrantha is rich in structurally diverse phenolic compounds with functional potential beneficial for human health and is worthy of further investigation.
Supplementary Materials: The following are available online: HR-ESI-MS and NMR spectra data of compounds 1 and 2 as supporting information.