The Volatile Phytochemistry of Seven Native American Aromatic Medicinal Plants

As part of our evaluation of essential oils derived from Native American medicinal plants, we have obtained the essential oils of Agastache foeniculum (Pursch) Kuntze (Lamiaceae), Gaultheria procumbens L. (Ericaceae), Heliopsis helianthoides (L.) Sweet (Asteraceae), Liatris spicata (L.) Willd. (Asteraceae), Pycnanthemum incanum (L.) Michx. (Lamiaceae), Smallanthus uvedalia (L.) Mack. ex Mack. (Asteraceae), and Verbena hastata L. (Verbenaceae) by hydrodistillation. The essential oils were analyzed by gas chromatographic techniques. The essential oil of A. foeniculum was dominated by estragole (88–93%), while methyl salicylate (91%) dominated the G. procumbens essential oil. Germacrene D was the major component in H. helianthoides (42%) and L. spicata (24%). 1,8-Cineole (31%) and α-terpineol (17%) were the main compounds in P. incanum essential oil. The essential oil of S. uvedalia showed α-pinene (24%), perillene (15%), and β-caryophyllene (17%) as major components. Verbena hastata essential oil was rich in 1-octen-3-ol (up to 29%) and palmitic acid (up to 22%). Four of these essential oils, H. helianthoides, L. spicata, P. incanum, and V. hastata, are reported for the first time. Additionally, the enantiomeric distributions of several terpenoid components have been determined.


Introduction
Plants have been used in traditional medicine since prehistoric times. The therapeutic properties of medicinal plants are generally attributed to secondary metabolites produced by the plants as protection against pathogens and herbivory. As with many other aboriginal peoples, Native North Americans have used plants as medicines throughout their history.
Although not as extensively documented as traditional Chinese medicine or Ayurvedic medicine, there are several sources of information regarding Native American ethnopharmacology [1][2][3]. As part of our ongoing investigations of the essential oil compositions of Native American medicinal plants, we have collected and examined seven aromatic medicinal plants growing in the southeastern United States.
Agastache foeniculum (Pursch) Kuntze (Lamiaceae) is native to north central United States and southern Canada, but has been recorded in southern Alabama [4]. The plant has been introduced to Europe, particularly the U.K., the Netherlands, and Germany, as an ornamental [5]. Cheyenne and Chippewa Native Americans used an infusion of the leaves of A. foeniculum as a cold medicine [2]. Essential oil compositions of A. foeniculum have been extensively studied, and the oils are typically dominated by methyl chavicol (estragole) with smaller amounts of (E)-anethole [6]. Nonvolatile phytochemicals from A. foeniculum include flavonoids (apigenin, quercetin), polyphenolics (rosmarinic acid, caffeic acid), pentacyclic triterpenoids (α-amyrin, β-amyrin), and sterols (campesterol, campestanol, sitosterol, stigmasterol, stigmastanol) [6]. oils of these seven plant species, to determine their chemical compositions as well as the enantiomeric distributions of terpenoid constituents.

Results and Discussion
The essential oils of each species were obtained by hydrodistillation of dried plant material (Table 1). The volatile materials reported in this research were obtained by hydrodistillation with continuous solvent extraction using a Likens-Nickerson apparatus. Many researchers consider these products not to be true "essential oils", but rather "volatile fractions".

Agastache foeniculum (Pursch) Kuntze (Lamiaceae)
The aerial parts of three different plant samples of A. foeniculum were collected from cultivated plants in Newville, Alabama. Hydrodistillation gave pale yellow essential oils in yields ranging from 1.48% to 2.30% yield. The essential oil compositions are compiled in Table 2.
The major component of G. procumbens essential oil, methyl salicylate, is well-known as an anti-inflammatory, antipyretic, analgesic agent [53], and accounts for the traditional use of the herb by Native Americans. Methyl salicylate is a common flavoring and fragrance ingredient in cosmetics, shampoos, toilet soaps, and other toiletries, however, it is also a potentially hazardous intoxicant [54][55][56].

Liatris spicata (L.) Willd. (Asteraceae)
The essential oil composition of L. spicata essential oil is presented in Table 5. The major components were germacrene D (23.7%), myrcene (13.7%), α-pinene (8.1%), and caryophyllene oxide (5.9%). Apparently, there have been no previous reports on the essential oil composition of L. spicata.    Table 6 shows the chemical composition of the essential oil from the aerial parts of P. incanum growing wild in South Carolina. The essential oil was rich in oxygenated monoterpenoids, including 1,8-cineole (30.7%), α-terpineol (16.9%), borneol (8.2%), and cissabinene hydrate (5.6%). The sesquiterpene hydrocarbons (E)-β-caryophyllene (11.0%), and germacrene D (5.0%) were also relatively abundant. To our knowledge, this is the first reported analysis of P. incanum essential oil. Volatiles obtained from a diethyl ether extract have been analyzed by gas chromatography-olfactometry to determine the key odorants [20]. Although the percentages of the volatiles were not reported, the enantiomeric distributions of several components were determined (Table 6). 1-Octen-3-ol, isomenthone, αterpineol, and pulegone showed comparable enantiomeric distributions between P. incanum essential oil and the volatiles from the previously published diethyl ether extract [20]. Concentrations of α-pinene, linalool, and menthol were too low in this current study to obtain reliable enantiomeric distributions for comparison.

Plant Material
The aerial parts of A. foeniculum, G. procumbens, and H. helianthoides were obtained from plants cultivated in at Kirkland Gardens, in Newville, Alabama ( Table 1). The cultivated plants were started from commercially available seeds (A. foeniculum, Homegrown Seed Company, and H. helianthioides, Prairie Moon Nursery), tubers (L. spicata, Wal-Mart), or young plants (G. procumbens, The Home Depot). All the plants were grown in full sun, except the G. procumbens, which was located in a partially shaded location (4 h/day average sunlight), and all were watered at least once a week. The plants were cultivated directly in the ground, which was clayey-loamy sand, which was amended with composted chicken manure, worm castings, kelp meal, and bone meal at time of planting. Pycnanthemum incanum and S. uvedalia were collected in the wild from a fully shaded forest understory roadside location near a small waterfall in northern South Carolina. The plants were located beside highway 276 near the North Carolina-South Carolina border (see Table 1). The soil was a thick loam with a lot of leaf litter. Verbena hastata was collected in the wild near a disturbed fence-line area with full sun between a bovine field and a paved county road in rural Newville, Alabama ( Table 1). Specimens of each plant were collected during the flowering phase (Table 1). Voucher specimens of P. incanum (SKL83120), S. uvedalia (SKL31820), and V. hastata (SKL51321) were deposited in the University of Alabama in Huntsville Herbarium (HALA); cultivated plants were not vouchered. For each species, the plant material was air-dried in the laboratory (around 23 • C) for 10 days. The dried plant materials of each species were chopped and hydrodistilled using a Likens-Nickerson apparatus with continuous extraction with dichloromethane for 4 h. The dichloromethane was evaporated using a stream of dry nitrogen to give the essential oils (Table 1).

Gas Chromatographic Analysis
The essential oils were analyzed by gas chromatography-mass spectrometry (GC-MS), gas chromatography-flame ionization detection (GC-FID), and chiral GC-MS as previously described [77].
GC-FID: Shimadzu GC 2010 equipped with flame ionization detector, a split/splitless injector, and Shimadzu autosampler AOC-20i, with a ZB-5 capillary column (30 m length × 0.25 mm inner diameter) with a (5% phenyl)-polymethylsiloxane stationary phase and a film thickness of 0.25 µm; oven temperature was programmed the same as above for GC-MS; injector temperature = 250 • C, detector temperature = 280 • C, N 2 carrier gas, and flow rate = 1.0 mL/min. The composition percentages were calculated from raw peak areas without standardization.

Conclusions
This report presented the essential oil compositions of seven aromatic medicinal plants used by Native Americans. Four of these essential oils, Heliopsis helianthoides, Liatris spicata, Pycnanthemum incanum, and Verbena hastata, were reported for the first time. Additionally, the enantiomeric distributions of several terpenoid components have been determined. The chemical compositions presented add to our knowledge of the phytochemistry of the medicinal plants.
Funding: This research received no external funding. Data Availability Statement: All data are presented in this report.