Flavanone Glycosides, Triterpenes, Volatile Compounds and Antimicrobial Activity of Miconia minutiflora (Bonpl.) DC. (Melastomataceae)

Chemical composition of the essential oils and extracts and the antimicrobial activity of Miconia minutiflora were investigated. The flavanone glycosides, pinocembroside and pinocembrin-7-O-[4″,6″-HHDP]-β-D-glucose, were identified, along with other compounds that belong mainly to the triterpene class, besides the phenolics, gallic acid and methyl gallate. Sesquiterpenes and monoterpenes were the major compounds identified from the essential oils. Screening for antimicrobial activity from the methanolic extract of the leaves showed that the MIC and MMC values against the tested microorganisms ranged from 0.625 to 5 mg·mL−1 and that the extract was active against microorganisms, Staphyloccocus aureus, Escherichia coli, and Bacillus cereus.


Introduction
Melastomataceae comprises 166 genera and around 4500 species [1]. About 1470 species were cataloged in Latin America distributed in countries such as Brazil, Uruguay, Mexico, Argentina, Colombia, Ecuador, and Venezuela [2]. More than 1400 species within 69 genera occur in Brazil spread throughout the Amazon to the Uruguay frontier [3]. Miconia is the most representative genus of Melastomataceae, with a wide distribution in the American continent including 1057 species and representing the largest genus of woody flowering plants with a distribution restricted to tropical America [4].
Some Miconia species were widely used in folk medicine to treat diarrhea and stomachache [5]. Isolated compounds and Miconia extracts have demonstrated diverse pharmacological activities. The aqueous extract of M. latecrenata (DC.) Naudin leaves showed a high antioxidant effect and high antibacterial activity [4]. The cytotoxic and mutagenic potential of extracts from M. cabucu Hoehne, M. rubiginosa (Bonpl.), M. stenostachya DC., and M. albicans (Sw.) Steud were investigated and the results confirmed the safe use of Miconia extracts and reinforced the therapeutic properties and their protective effects on doxorubicin-induced mutagenicity [6]. The ethanolic leaf extract of M. albicans shows an anti-arthritic profile [7]. The ethanolic extract of M. willdenowii Klotzsch ex Naudin showed schistosomicidal [8], antimicrobial activities, and anti-L. amazonensis effects, as well as evidence that the most abundant constituent, the benzoquinone derivative primin, is the major bioactive metabolite [9].
This work reports the chemical composition of extracts and essential oils and the antimicrobial activity of the methanolic extract from the leaves of Miconia minutiflora (Bonpl.) DC. A survey of the literature shows one study on the volatiles of M. minutiflora inflorescences, which were characterized by the presence of α-copaene and β-caryophyllene as major constituents [13]. Another study on M. minutiflora showed the anti-inflammatory and antinociceptive effects of the leaf methanol extract; in the same work, the authors tentatively identified the compounds casuarinin (4 isomers), ellagic acid, HHDP-galloylglucose (one isomer), myricetin-galloyl-deoxihexoside (one isomer), myruianthic acid (two isomers), and arjunolic acid (seven isomers) using UPLC-DAD-QTOF-MS/MS [14]. Effects of the extracts of seven Miconia species, including M. minutiflora, were evaluated on Lactuca sativa seeds and seedlings growth, the extract of M. minutiflora showed no allelopathic effects on the rootlets of the tested plant [15].

Antimicrobial Activity
The minimal inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) of the methanol extract from M. minutiflora against the tested microorganisms ranged from 0.625 to 5 mg·mL −1 , these data are shown in Table 2  Rodrigues and coworkers (2008) tested the dichloromethane extract of M. cabucu against C. albicans and obtained an MIC value of 1.5 mg·mL −1 and the methanol extract of M. stenostachya against B. cereus showed an MIC of 3.0 mg·mL −1 [29]. The ethanol extract of M. albicans and M. rubiginosa showed antimicrobial activity using the well diffusion method [30].
The antimicrobial activity observed for the methanol extract of M. minutiflora can be explained by the presence of substances in the studied extracts indicating that this species is a valuable source for the discovery of new antimicrobial products.

Plant Material
Leaves and stems of M. minutiflora were collected in the Municipality of Belém (Estrada do Paiol, Km 5), State of Pará, Brazil in June 2010 for the study of its non-volatile compounds. A voucher specimen was identified and deposited at the Herbarium of the Museu Paraense Emílio Goeldi (Belém-Pará-Brazil) under the reference number MG-204.906. Another collection (leaves, primary and secondary branches) was taken in the same municipality at the Museu Paraense Emílio Goeldi-research campus in November 2014 for the identification of the volatile compounds; this specimen was identified at the same herbarium by comparison with the same voucher.

Extraction, Isolation, and Identification of the Non-Volatile Compounds
Leaves (2.00 Kg) and stems (2.00 Kg) of M. minutiflora were extracted by maceration with hexane (7 days × 2) and MeOH (14 days × 2) at room temperature. The filtrates were concentrated under reduced pressure to yield the hexane extracts (30.00 g of leaves extract and 7.00 g of stems extract) and the methanolic extracts (267.00 g of leaves extract and 118.00 g of stems extract). Part of the methanolic extracts (40.00 g each) was suspended in MeOH-H 2 O 3:1 and extracted with CH 2 Cl 2 , EtOAc, and n-BuOH yielding the leaves phases (CH 2 Cl 2 phase: 7.00 g, EtOAc phase: 13.87 g, n-BuOH: 8.00 g) and the stems phases (CH 2 Cl 2 phase: 3.00 g, EtOAc phase: 2.54 g, n-BuOH: 1.19 g) of the methanolic extracts.
The hexane extracts of the leaves (20.00 g) and of the stems (6.00 g), the CH 2 Cl 2 phase of the stems (3.00 g), and the EtOAc phase of the leaves (14.03 g) were purified using column chromatography (CC) over silica gel using mixtures of hexane-EtOAc and EtOAc-MeOH with increasing polarity. When necessary, the resulting fractions were rechromatographed using similar techniques. The hexane extract of the leaves afforded M1 (1401 mg), S1 (

Extraction of the Essential Oils
Samples of leaves, primary and secondary branches (120 g) were hydrodistilled for 3 h, using a Clevenger-type apparatus with maintenance of the refrigeration water at 15 • C in accordance with the works described in the literature [38,39].

Analysis of the Essential Oils
The chemical composition of the volatile compounds of the Miconia minutiflora (Bonpl.) DC. (Melastomataceae) was analyzed using gas chromatography coupled to mass spectrometry, using a Thermo DSQ-II system equipped with a DB-5MS silica capillary column (30 m × 0.25 mm; 0.25 mm). For this analysis, the same protocols described previously by our research group were followed [40,41]. The volatile compounds present in the essential oil were identified by comparison with the literature [42,43].

Well Diffusion Test
The antimicrobial activity of the methanol extract was first evaluated using the well diffusion test as follows. A swab of the microorganism was transferred to 6 mL of a 0.45% saline solution and the resulting suspension was adjusted to 0.1 mL of a 1.5 × 108 cels·mL −1 (bacteria) and 1.5 × 105 cels·mL −1 (yeast). The cells suspension was added to 120 mL of MHA. The resulting mixture was transferred to Petri dishes (100 mm). After cooling the mixture, six equidistant wells (6 mm in diameter) received 65 µL of the methanol extract, at 200 mg·mL −1 in DMSO-water 1:1. Positive controls were chloramphenicol at 30 µg·mL −1 for bacteria and nystatin at 10 µg·mL −1 for yeast. The negative control was DMSO. Petri dishes were incubated at 37 • C for 24 h (bacteria) and at 28 • C for 48 h (yeast). The results were reported as the diameter of the zone of inhibition (in mm) (CLSI, 2003, with adaptations) [44].

Minimum Inhibitory Concentration (MIC) and Minimal Microbicidal Concentration (MMC)
The minimum inhibitory concentration (MIC) and minimal microbicidal concentration (MMC) of the methanol extract of the leaves of M. minutiflora were performed following the same protocols described in previous works [45,46].

Conclusions
This study showed that the extracts of M. minutiflora are an important source of glycosylated flavanones, which are very often identified from Miconia. This is the first time that the flavanones, pinocembroside and pinocembrin-7-O-[4",6"-HHDP]-β-glucose, were isolated from a Miconia species. Compounds (3Z)-hexenol, 1-octen-3-ol, (3Z)-hexenylbutanoate, cis-3-hexenyl isovalerate, (3Z)-hexenyl hexanoate, and phytol were the major constituents of the essential oils from the leaves, while n-hexadecanoic acid and (9Z,12Z)-octadecadienoic acid were the major constituents from the primary branches and (9Z,12Z)-octadecadienoic acid, dodecanoic acid, tetradecanoic acid and n-hexadecanoic acid were the major constituents from the secondary branches. The antimicrobial screening showed that the leaves' methanol extract is active against E. coli, S. aureus, and B. cereus and these activities can be in part explained by the presence of known bioactive compounds in the extracts.