**1. Introduction**

*Helianthus* L., the sunflowers, is a genus in the family Asteraceae, tribe Heliantheae, made up of 51 North American species [1]. *Helianthus annuus* L. (common sunflower) is native to North America and the current range of wild forms of *H. annuus* are central and western United States, southern Canada, and northern Mexico [2]. The common sunflower is one of the earliest domesticated plants in the Americas. There is evidence that the plant was domesticated in Tabasco, Mexico, around 2600 B.C. [3], and independently in the southeastern United States around 2800 B.C. [2,4]. Several Native American tribes used *H. annuus* in traditional medicine [5]. For example, the White Mountain Apache used a poultice of the crushed plants to treat snakebites; the Hopi used the plant as a spider bite medicine; the Jemez applied the juice of the plant to cuts; the Pima used a decoction of the leaves to treat fevers [5]; and the Zuni natives of New Mexico used the roots to treat rattlesnake bites [6]. In addition, *H. annuus* is used as a traditional herbal medicine in many locations where it has been introduced. Ethiopians use *H. annuus* in teas to treat food poisoning [7]. In Bangladesh the seeds and/or the flowers are crushed and used for snake bites, scorpion bites, and a variety of other ailments, such as burning sensation in the vagina and worms in the ears [8].

*Helianthus strumosus* L. (woodland sunflower) is a rhizomatous perennial plant, growing up to two meters tall and is native to eastern North America [9–11]. These plants are strongly aromatic. Leaves are up to 10 cm long and cuneate to subcordate in shape. The composite flower heads can be

up to 9 cm at the peduncle. The ray flowers are a dark yellow color with orange-brown disc flowers in the center. These flowers are common along roadsides and in open fields and are sometimes found in forests. The Iroquois used a decoction of the roots as an anthelmintic [5].

Invasive fungal infections are becoming increasingly common in immunocompromised patients, such as those receiving cancer chemotherapy, transplant patients receiving immunosuppressant drugs, and HIV patients [12]. The predominant fungal pathogens are *Aspergillus* spp. [13,14] and *Candida* spp. [15,16] among others [12]. *Aspergillus niger* is a haploid filamentous parasitic fungus that is commonly known for the disease "black mold" on fruits, vegetables, and nuts [17]. *Aspergillus* conidia (fungal "spores") are environmentally widespread and inhalation can lead to opportunistic pulmonary aspergillosis, chiefly attributed to *A. fumigatus*, *A. flavus*, and *A. tubingensis*, as well as *A. niger* [18]. In immunocompromised individuals, however, the infection can progress to invasive systemic aspergillosis [19]. *Candida albicans* is another opportunistic pathogenic fungus that commonly colonizes the human body [20]. The organism can cause superficial infections of the mucosa, but can lead to invasive candidiasis in immunocompromised patients [21]. Cryptococcosis is a fungal infection caused by *Cryptococcus neoformans* [22]. The fungus is widespread in the environment and typically enters the body through inhalation where it can cause pulmonary infection [23]. However, the organism has the ability to cross the blood brain barrier and in immunocompromised patients, cryptococcosis can lead to cryptococcal meningoencephalitis with increased intracranial pressure [24,25]. As part of our continuing investigation of antifungal activity of essential oils [26] as well as essential oils from the Asteraceae growing in north Alabama [27], we have collected and analyzed the essential oils from the aerial parts of *H. annuus* and *H. strumosus*, and we have carried out in vitro antifungal screening of the essential oils against *A. niger*, *C. albicans*, and *C. neoformans*.

#### **2. Materials and Methods**

#### *2.1. Plant Materials*

The two cultivars of *H. annuus* ("Chianti" and "Mammoth") were cultivated, grown without fertilizer or pesticides, in a rural area near Gurley in north Alabama (34◦3829"N, 86◦2439"W, elevation 199 m) and the aerial parts were collected on 4 and 6 August 2018. Aerial parts of *H. strumosus* were collected on 10 August 2018 from wild-growing plants near Huntsville, Alabama (34◦4242"N, 86◦3235"W, elevation 354 m). The plants were identified by S.K. Lawson. Voucher specimens have been deposited in the herbarium of the University of Alabama in Huntsville (20180729-183243 and 20190402-111732). The fresh plant materials (78.14, 80.32, and 65.47 g, respectively) were hydrodistilled using a Likens–Nickerson apparatus with continuous extraction with dichloromethane for 3 h. The dichloromethane was carefully evaporated, and the residual essential oils weighed using an analytical balance to give the essential oils (82.3, 20.3, and 24.0 mg, respectively).

#### *2.2. Gas Chromatographic—Mass Spectral Analysis*

The *Helianthus* essential oils were analyzed by GC-MS with a Shimadzu GCMS-QP2010 Ultra with a ZB-5 capillary column as previously described [28]. Identification of the chemical components was carried out by comparison of the retention indices, calculated with respect to a homologous series of normal alkanes using the arithmetic index [29], and by comparison of their mass spectra with those reported in the Adams [30], NIST17 [31], FFNSC 3 [32], and our own in-house library [33]. Concentrations shown in Table 1 (average of three measurements ± standard deviations) are based on peak integration without standardization.


**Table 1.** Chemical compositions of *Helianthus annuus* "Chianti", *H. annuus* "Mammoth", and *H. strumosus* aerial parts essential oils.


**Table 1.** *Cont.*

a RI = Retention index determined with reference to a homologous series of *n*-alkanes on a ZB-5 column. b RI values from the databases (NIST17 [31], FFNSC 3 [32], Adams [30], or Satyal [33]). c Average of three measurements ± standard deviations. d tr = "trace" (<0.05%). e Sesquiterpenoids are considered tentatively identified based on MS and RI.

#### *2.3. Antifungal Screening Assays*

The *Helianthus* essential oils were screened for antifungal activity against *Aspergillus niger* (ATCC 16888), *Candida albicans* (ATCC 18804), and *Cryptococcus neoformans* (ATCC 24607) using the broth dilution technique as previously described [26,34]. Antifungal screening was carried out in triplicate.

#### **3. Results and Discussion**

#### *3.1. Essential Oil Compositions*

Hydrodistillation of *Helianthus* aerial parts gave pale yellow essential oils in 0.105%, 0.025%, and 0.037% yield ( *w*/*w*) for *H. annuus* "Chianti", *H. annuus* "Mammoth", and *H. strumosus*, respectively. The essential oil compositions for the three essential oils are compiled in Table 1. A perusal of the table reveals that the three *Helianthus* essential oils are qualitatively similar. The major components for *H. annuus* "Chianti" were α-pinene (50.65%), camphene (7.26%), limonene (7.20%), bornyl acetate (7.13%), sabinene (6.81%), and β-pinene (5.79%). The essential oil of *H. annuus* "Mammoth" was also dominated by α-pinene (48.91%), followed by sabinene (17.01%), limonene (7.11%), and germacrene D (6.84%). *H. strumosus* essential oil was also rich in α-pinene (58.65%), as well as myrcene (9.79%) and bornyl acetate (4.97%).

The compositions of *H. annuus* essential oils cultivated in north Alabama are very similar to those reported by Adams and co-workers for populations growing in the southern plains of the United States [35]. The essential oils of *H. annuus* from Pisa, Tuscany, Italy [36]; Lagos, Nigeria [37]; or from western United States [35] had much lower concentrations of α-pinene and correspondingly higher concentrations of germacrene D. In marked contrast to the essential oils of *Helianthus*, essential oils of *Rudbeckia fulgida* Aiton and *Rudbeckia hirta* L. (Asteraceae, Heliantheae) from north Alabama were devoid of α-pinene, but rich in sesquiterpene hydrocarbons [27].

## *3.2. Antifungal Activity*

The *Helianthus* essential oils were screened for antifungal activity against three potentially pathogenic fungi, *Aspergillus niger*, *Candida albicans*, and *Cryptococcus neoformans*, as shown in Table 2. The most susceptible fungus was *C. neoformans*. Both *H. annuus* "Chianti" and *H. strumosus* essential oils showed minimum inhibitory concentration (MIC) values of 78 μg/mL. It is tempting to sugges<sup>t</sup> that the major component, α-pinene, is responsible for the observed anti-*Cryptococcus* activity; all three *Helianthus* essential oils have around 50% α-pinene. Furthermore, α-pinene has shown antifungal activity against *C. neoformans* with an MIC around 70 μg/mL [38,39]. In addition, α-pinene-rich (46.1% α-pinene) commercial *Myrtis communis* essential oil showed a similar antifungal activity against *C. neoformans* (MIC = 78 μg/mL) [26]. Conversely, commercial *Cupressus sempervirens* essential oil, with 49.7% α-pinene was less active against *C. neoformans* (MIC = 313 μg/mL) [26]. There may be synergistic or antagonistic effects of α-pinene with minor components. Limonene [39,40] and β-pinene [39], have also shown antifungal activity against *C. neoformans*; camphene, however, was inactive [41]. Although we do not know which of the enantiomers is present in the *Helianthus* essential oils, we have screened both (+)- and (−)-α-pinene, (+)- and (−)-limonene, and (−)-β-pinene against the three fungal strains, as shown in Table 2. Consistent with previous investigations, (−)-β-pinene and (+)-limonene both showed activity against *C. neoformans* with MIC values of 39 and 78 μg/mL. Furthermore, both enantiomers of α-pinene were active against *C. neoformans*; MIC = 20 and 39 μg/mL for (+)- and (−)-α-pinene, respectively.


**Table 2.** Antifungal activities (minimum inhibitory concentration (MIC), μg/mL) of *Helianthus* essential oils and major components a.

> a Each MIC determination was carried out in triplicate.
