In vitro antimicrobial activity of Alpinia zerumbet and A. purpurata nonpolar fraction of leaf extract

This study aimed to evaluate the in vitro antimicrobial activity of fractions of leaf hydroalcoholic crude extracts: hexane, dichloromethane, ethyl acetate and butanolic of Alpinia zerumbet and A. purpurata by the agar drop diffusion method in order to screen the main compounds involved in antimicrobial activity. Leaves of A. zerumbet (Pers.) B.L. Burtt et R.M. Sm. and A. purpurata (Vieill) K. Schum adult plants were collected and then dried and macerated in 70% ethanol. Leaf extracts were further partitioned using solvents in increasing polarity. Dichloromethane fractions were analyzed by gas chromatography/mass spectrometry (GC/MS). The major compound in the dichloromethane fraction of both species was the hexadecanoic acid (palmitic acid). By drop diffusion assay, the antimicrobial activity of crude extract, as well as fractions of hexane, dichloromethane, ethyl acetate and butanolic, was evaluated against pathogenic bacteria and fungi. No bacteria were inhibited. However, the dichloromethane fraction exhibited promising antifungal activity against the following fungi tested: Cryptococcus neoformans, Fonsecaea pedrosoi, Trichophytoon rubrum, Microsporium canis and M. gypseum.


Introduction
The traditional use of species of Zingiberaceae in the treatment of disease, in food and as aromatic is an which have been tested against microorganisms, such as bacteria, fungi, yeast, and parasitic protists [1][2][3][4][5] .
Alpinia zerumbet and A. purpurata (Zingiberaceae) are perennial plants widely distributed in subtropical and tropical regions [6] . These species are commonly used for ornamental, medicinal, and aromatic purposes owing to their essential oils. A. zerumbet is widely used in folk medicine [7,8] to treat ulcer [9] , cardiovascular hypertension [10] and muscular aches and pains [11] . It has been shown that different substances from A. zerumbet and A. purpurata have pronounced antimicrobial and antioxidant activity, particularly those composed of nonpolar compounds [12][13][14] .
Some fungal and bacterial species cause a broad range of diseases in human and plants, but studies have shown the potential of plant metabolites in combating microorganisms. Plants present a high diversity of bioactive chemicals that are good alternatives for the control of microorganisms in contrast to the use of common fungicides and antibiotics that can result in resistance [15,16] . Resistant strains are common and appear periodically, representing a huge global challenge [17] .
Therefore, this study aimed to evaluate the in vitro antimicrobial activity of fractions of leaf hydroalcoholic crude extracts: hexane, dichloromethane, ethyl acetate and butanolic of A. zerumbet and A. purpurata by the agar drop diffusion method in order to screen the main compounds involved in antimicrobial activity.

Plant material
Leaves of Alpinia zerumbet and A. purpurata were collected from adult plants in November 2014 on the campus of the Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil). Voucher specimens were identified and deposited at the Herbarium of Rio de Janeiro Botanical Garden under accession numbers RB 433485 and RB 433484, respectively.

Plant material extraction and fractionation
Leaves of adult plants were collected in the morning, and then plant material was dried for 3 days at 50 o C and extracted by maceration in 70% ethanol for a week at room temperature (25 o C±2 o C). The total hydroalcoholic extracts were concentrated in a rotational evaporator under reduced pressure at 60ºC, and the residues were resuspended in methanol: water (9:1, v/v). Extracts were submitted to successive partitioning in different solvents of increasing polarity between n-hexane, dichloromethane, ethyl acetate and n-butanol. The solutions were completely evaporated to give the respective fractions. Each solvent extraction was carried out five times. gas, helium at 1 mL/min; injector temperature, 270°C; ion-source temperature 280°C; column temperature, 60ºC to 290ºC at 5°C/min. Mass spectra were obtained by electron impact ionization (EI) at a scan rate of 0.5 scans/s and fragments from 40 to 500 Da. The identification of the major constituents was confirmed by comparison of their retention time (RT) and mass spectra with those from the NIST database, Wiley Libraries, Adams [18] , and the literature.

Agar drop diffusion method
The antimicrobial assay was carried out by the agar drop diffusion method described by Hili et al. [19] . The microorganisms tested included bacteria, as follows: Gram-negative Escherichia coli, Gram-positive,

methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Streptococcus mutans, and
Lactobacillus casei, as well as the following fungi: Cryptococcus neoformans T444, Candida albicans, Fonsecaea pedrosoi, Trichophyton rubrum, Microsporum canis and M. gypseum. Microorganisms (2 x 10 5 cells) were spread over an agar plate surface containing Brain Heart Infusion (BHI) agar medium for bacteria, Sabouraud Dextrose Agar medium for yeast, and Potato Dextrose Agar for filamentous fungi, respectively. For antimicrobial activity, crude extracts were tested, along with hexane, dichloromethane, ethyl acetate and nbutanol fractions. As standard, the flavonoid rutin (Merck ® ) was used by its previously identification in extracts of A. zerumbet and A. purpurata [20,21] . The nonpolar samples were initially solubilized in DMSO (50 mg/ml) and The diameter of inhibition zone (cm) was measured after each incubation period.

Results and Discussion
Different groups of compounds were identified, such as hydrocarbons, fatty acids, ester and alcohol (TABLE 1). A. zerumbet and A. purpurata leaf extracts contain palmitic acid (n-Hexadecanoic acid), as the main component, and other fatty acids, such as stearic acid, nonadecanoic acid and oleic acid. Fatty acids are distributed in natural fats and dietary oils, and studies show their antibacterial and antifungal properties [22] .
Fatty acids can act as anionic surfactants and have antibacterial and antifungal properties at low pH, rendering the cell membrane more permeable to these compounds [23,24] . Besides palmitic and stearic acids, oleic acid found in dichloromethane extracts of A. purpurata has been used to control cucumber powdery mildew and is also known to have potential antibacterial and antifungal activity [22] .  antibiofilm and antivirulence [25] . Crude extracts and fractions of A. zerumbet and A. purpurata leaves did act against a wide variety of human pathogenic fungi. In support of antifungal activity, studies have demonstrated the potential of extracts and fractions of different organs of plants of Zingiberaceae: Alpinia, Zingiber, Elettaria, and Curcuma species [4] . Recently, Mathew and Victório [5] verified that rhizome methanolic extracts of Alpinia calcarata exhibit significant antifungal effects against Aspergillus flavus, A. niger, A.
fumigatus, Rhizopus stolonifer and Candida albicans. Biological activity against the bacteria tested was not found. However, in studies with different species of Zingiberaceae, crude extracts of rhizome were reported to be active against bacteria, but almost completely inactive against fungi [26] .
canis and M. gypseum. Different from crude extracts, nonpolar fractions have a major concentration of some compounds similar to those found for essential oils, which have shown high antimicrobial activity, indicating that polarity seems to be correlated with biological effects against fungi [13,14] . In the same way, Habsah et al. [26] found the dichloromethane extracts to be much stronger than methanol extracts of rhizomes of species albicans [27] . In addition, some studies have shown palmitic acid (n-Hexadecanoic acid) and others fatty acids with an antifungal action against the yeast of C. albicans [32] , in despite of founds results. Besides that, antifungal activities of palmitic acid against some phytophatogenic and others fungal strains have also been described [33,34] . This substance has been devoted to act specially in the spore germination inhibition of a large amount of fungi [35] . According to these observations, it is possible to speculate the contribution of n-Hexadecanoic acid in the antifungal microbial activity of leaves from A. zerumbet and A. purpurata.  Negative control for all microorganisms tested indicated its respective growth in the absence of test sample applied.

Conclusion
In the current study, nonpolar fractions from leaf extracts evidenced a positive antifungical activity against C. neoformans T444, F. pedrosoi, T. rubrum, M. canis and M. gypseum. These results provide additional data in support of using products from dichloromethane fractions of A. zerumbet and A. purpurata as an alternative against yeast and filamentous fungi. Such findings further support the use of plant metabolites as alternatives. However, more studies must be carried out to isolate metabolites from dichloromethane fractions (nonpolar) and, thus, to determine if crude plant extracts have greater in vitro antifungical activity than isolated constituents at an equivalent dose.