Volatile Compositions and Antifungal Activities of Native American Medicinal Plants: Focus on the Asteraceae

In the past, Native Americans of North America had an abundant traditional herbal legacy for treating illnesses, disorders, and wounds. Unfortunately, much of the ethnopharmacological knowledge of North American Indians has been lost due to population destruction and displacement from their native lands by European-based settlers. However, there are some sources of Native American ethnobotany remaining. In this work, we have consulted the ethnobotanical literature for members of the Asteraceae used in Cherokee and other Native American traditional medicines that are native to the southeastern United States. The aerial parts of Eupatorium serotinum, Eurybia macrophylla, Eutrochium purpureum, Polymnia canadensis, Rudbeckia laciniata, Silphium integrifolium, Smallanthus uvedalia, Solidago altissima, and Xanthium strumarium were collected from wild-growing plants in north Alabama. The plants were hydrodistilled to obtain the essential oils and the chemical compositions of the essential oils were determined by gas chromatography–mass spectrometry. The essential oils were tested for in-vitro antifungal activity against Aspergillus niger, Candida albicans, and Cryptococcus neoformans. The essential oil of E. serotinum showed noteworthy activity against C. neoformans with a minimum inhibitory concentration (MIC) value of 78 μg/mL, which can be attributed to the high concentration of cyclocolorenone in the essential oil.


Introduction
Many aspects of modern medicine have relied on the traditional knowledge of native cultures, including, for example, traditional Indian medicine (Ayurveda) [1], traditional Chinese medicine (TCM) [2], and traditional Islamic medicine [3]. Unfortunately, many of the traditional uses of medicinal plants are being lost due to several reasons. Recent generations are less interested in traditional knowledge, and habitat destruction and forced migration have reduced access to medicinal plants. The Native Americans of North America also had rich traditions of medicinal plant use. However, much of this knowledge has been lost due to population declines and displacement from native lands. Nevertheless, there are still some existing references to the ethnobotanical uses of medicinal plants by Native Americans [4].
Eupatorium serotinum Michx., "late boneset", is native to eastern North America and ranges from Texas, Oklahoma, and Kansas, to the Atlantic coast and from the Gulf of Mexico north to Wisconsin and Michigan [5]. The Houma people of Louisiana used a decoction of the flowers to treat typhoid fever [6]. Extracts of the aerial parts of E. serotinum have yielded germacranolide sesquiterpenoids [7,8].

Results and Discussion
The essential oils from E. serotinum, E. macrophylla, E. purpureum, P. canadensis, R. laciniata, S. integrifolium, S. uvedalia, S. altissima, and X. strumarium were obtained from the fresh aerial parts of the plants by hydrodistillation, generally in low yield. The essential oils were analyzed by GC and GC-MS (Tables 1, 3-9, and 11).
Monoterpene hydrocarbons, limonene (28.66%), β-pinene (8.57%), and terpinolene (5.35%), and germacrane sesquiterpenes, germacrene D (19.81%), and germacrene B (7.07%), were the major components in the essential oil of E. macrophylla (Table 3). To our knowledge, there are no reports on essential oil compositions of any Eurybia species. The major components in the essential oil of E. purpureum were the green leaf volatiles (2E)-hexenal (60.59%) and hexanal (6.78%), along with the aromatic compounds eugenol (11.68%) and methyl salicylate (10.31%; Table 4). There have apparently been no previous reports on the essential oil composition of E. purpureum or any other Eutrochium species. There are numerous reports on Eupatorium essential oils, however (see above). α-Phellandrene (28.30%), α-pinene (19.71%), and germacrene D (11.42%) were the major components in the essential oil from the aerial parts of P. canadensis (Table 5). The volatile chemical profile of P. canadensis in this current work is in marked contrast to our previous report on this species [45]. Previous samples were rich in the sesquiterpene hydrocarbons germacrene D (63.6% and 44.5%) and β-caryophyllene (15.9% and 14.8%). The differences in compositions are likely due to seasonal variation (the current sample was collected in July, 2018, while the previous samples were collected in September, 2015, and December, 2016, respectively). We cannot rule out, however, chemical profile differences attributable to environmental differences or biotic differences (e.g., genetics, herbivory, or pathogen stress).

Plant Material
Aerial parts of each plant were collected from various sites in north Alabama (Table 14). Plants were identified by S.K. Lawson and voucher specimens were deposited in the University of Alabama in Huntsville herbarium (HALA). The fresh plant material (aerial parts) were chopped and hydrodistilled using a Likens-Nickerson apparatus with continuous extraction with CH 2 Cl 2 for three hours. The solvent was evaporated to give pale yellow essential oils (Table 14).

Gas Chromatography-Mass Spectrometry
The Asteraceae essential oils were analyzed by GC-MS using a Shimadzu GC-MS-QP2010 Ultra fitted with a Phenomenex ZB-5ms column as previously described [104]. Identification of the essential oil components was determined by comparison of their retention indices, determined with respect to a homologous series of n-alkanes and their mass spectral fragmentation patters with those from available databases (Adams [105], NIST17 [106], and FFNSC 3 [107]) or in our in-house library [108].

Gas Chromatography-Flame Ionization Detection
Quantification of the essential oils was determined by GC-FID using a Shimadzu GC 2010 instrument fitted with a ZB-5 column [104], using the same parameters that were used for the GC-MS. The concentrations (average of three measurements ± standard deviations) are based on peak integration without standardization.

Antifungal Screening
The essential oils were screened for antifungal activity against Aspergillus niger (ATCC 16888), Candida albicans (ATCC 18804), and Cryptococcus neoformans (ATCC 24607) using the microbroth dilution method as previously described [109]. Amphotericin B was used as the positive control and RPMI medium was used as the negative control. The antifungal assays were carried out in triplicate.

Conclusions
There is much intraspecific variation in essential oil compositions of these members of the Asteraceae. Much of the variation can be attributed to geographical location or seasonal variation.
Eupatorium serotinum essential oil showed notable antifungal activity against Cryptococcus neoformans. However, the yield of this essential oil (0.013%) is too low to be considered as pharmacologically useful. If suitable sources of the major component cyclocolorenone can be identified, then this compound may serve as important antifungal template for further elaboration.