Essential Oil Compositions and Antifungal Activity of Sunflower (Helianthus) Species Growing in North Alabama

Abstract

Helianthus species are North American members of the Asteraceae, several of which have been used as traditional medicines by Native Americans. The aerial parts of two cultivars of Helianthus annuus, “Chianti” and “Mammoth”, and wild-growing H. strumosus, were collected from locations in north Alabama. The essential oils were obtained by hydrodistillation and analyzed by gas chromatography—mass spectrometry. The Helianthus essential oils were dominated by monoterpene hydrocarbons, in particular α-pinene (50.65%, 48.91%, and 58.65%, respectively), sabinene (6.81%, 17.01%, and 1.91%, respectively), β-pinene (5.79%, 3.27%, and 4.52%, respectively), and limonene (7.2%, 7.1%, and 3.8%, respectively). The essential oils were screened against three opportunistic pathogenic fungal species, Aspergillus niger, Candida albicans, and Cryptococcus neoformans. The most sensitive fungus was C. neoformans with minimum inhibitory concentration (MIC) values of 78, 156, and 78 μg/mL, respectively.

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 [9,10,11]. 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°38′29″N, 86°24′39″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°42′42″N, 86°32′35″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. Chemical compositions of Helianthus annuus “Chianti”, H. annuus “Mammoth”, and H. strumosus aerial parts essential oils.

RI a	RI b	Compound	Percent Composition c
			H. annuus “Chianti”	H. annuus “Mammoth”	H. strumosus
800	797	(3Z)-Hexenal	0.06 ± 0.01	Tr d	tr
801	801	Hexanal	0.35 ± 0.02	0.24 ± 0.04	0.41 ± 0.03
810	796	2-Hexanol	---	---	0.07 ± 0.00
849	846	(2E)-Hexenal	1.13 ± 0.05	0.83 ± 0.05	1.96 ± 0.03
861	854	(2E)-Hexenol	---	---	tr
864	863	1-Hexanol	---	---	0.09 ± 0.00
921	921	Tricyclene	0.37 ± 0.00	0.21 ± 0.01	0.18 ± 0.00
924	924	α-Thujene	0.17 ± 0.00	0.23 ± 0.01	0.1
932	932	α-Pinene	50.65 ± 0.32	48.91 ± 0.64	58.65 ± 0.14
946	945	α-Fenchene	---	---	tr
948	946	Camphene	7.26 ± 0.03	3.72 ± 0.03	3.38 ± 0.02
952	953	Thuja-2,4(10)-diene	0.05 ± 0.01	---	tr
971	969	Sabinene	6.81 ± 0.04	17.01 ± 0.18	1.91 ± 0.00
977	974	β-Pinene	5.79 ± 0.04	3.27 ± 0.01	4.52 ± 0.02
988	988	Myrcene	0.42 ± 0.01	0.30 ± 0.03	9.79 ± 0.03
1004	1003	p-Mentha-1(7),8-diene	---	---	tr
1006	1002	α-Phellandrene	---	0.08 ± 0.01	0.05 ± 0.01
1008	1008	δ-3-Carene	---	---	tr
1016	1014	α-Terpinene	---	tr	---
1024	1020	p-Cymene	0.06 ± 0.03	0.09 ± 0.01	0.07 ± 0.00
1028	1024	Limonene	7.20 ± 0.03	7.11 ± 0.11	3.79 ± 0.01
1030	1025	β-Phellandrene	0.24 ± 0.00	0.21 ± 0.14	0.29 ± 0.01
1031	1026	1,8-Cineole	0.06 ± 0.00	0.07 ± 0.02	tr
1034	1032	(Z)-β-Ocimene	---	---	tr
1044	1044	(E)-β-Ocimene	---	tr	0.41 ± 0.01
1057	1054	γ-Terpinene	0.10 ± 0.00	0.25 ± 0.01	tr
1069	1065	cis-Sabinene hydrate	---	---	tr
1084	1086	Terpinolene	0.10 ± 0.01	0.16 ± 0.01	tr
1099	1099	α-Pinene oxide	0.10 ± 0.01	---	tr
1105	1100	Nonanal	---	---	tr
1109	1108	p-Mentha-2,8-dien-1-ol	0.36 ± 0.00	0.10 ± 0.02	tr
1112	1114	(3E)-4,8-Dimethyl-1,3,7-nonatriene	0.09 ± 0.01	0.13 ± 0.02	0.18 ± 0.00
1121	1124	Chrysanthenone	0.05 ± 0.00	---	---
1127	1122	α-Campholenal	0.33 ± 0.01	0.05 ± 0.01	0.06 ± 0.01
1140	1135	trans-Pinocarveol	0.37 ± 0.05	tr	0.06 ± 0.02
1141	1137	cis-Verbenol	0.10 ± 0.01	---	tr
1145	1140	trans-Verbenol	1.50 ± 0.02	0.24 ± 0.02	0.26 ± 0.00
1163	1160	Pinocarvone	0.11 ± 0.01	tr	tr
1171	1165	Borneol	0.73 ± 0.01	0.07 ± 0.02	0.12 ± 0.00
1180	1174	Terpinen-4-ol	0.09 ± 0.01	0.19 ± 0.01	tr
1187	1179	p-Cymen-8-ol	0.05 ± 0.01	---	---
1195	1186	α-Terpineol	---	tr	---
1195	1195	Myrtenal	0.29 ± 0.02	tr	0.10 ± 0.01
1206	1201	Decanal	tr	---	---
1207	1204	Verbenone	0.28 ± 0.04	0.11 ± 0.01	0.07 ± 0.00
1219	1215	trans-Carveol	0.16 ± 0.00	---	tr
1283	1287	Bornyl acetate	7.13 ± 0.04	3.02 ± 0.04	4.97 ± 0.01
1294	1298	trans-Pinocarvyl acetate	0.08 ± 0.01	tr	tr
1382	1387	β-Bourbonene	0.21 ± 0.02	0.18 ± 0.01	tr
1386	1387	β-Cubebene	tr	tr	tr
1387	1389	β-Elemene	0.05 ± 0.01	0.17 ± 0.01	tr
1392	1392	(Z)-Jasmone	---	---	tr
1416	1419	β-Ylangene	0.07 ± 0.01	0.15 ± 0.01	tr
1417	1417	β-Caryophyllene	0.33 ± 0.03	0.54 ± 0.09	0.84 ± 0.00
1427	1434	γ-Elemene	---	---	tr
1428	1431	β-Gurjunene (=Calarene)	0.62 ± 0.01	0.86 ± 0.01	tr
1430	1432	trans-α-Bergamotene	0.06 ± 0.00	0.14 ± 0.03	tr
1442	1442	6,9-Guaiadiene	tr	tr	---
1446	1453	Geranyl acetone	tr	tr	---
1454	1452	α-Humulene	0.19 ± 0.02	0.29 ± 0.01	0.20 ± 0.00
1479	1484	Germacrene D	3.32 ± 0.03	6.84 ± 0.09	3.68 ± 0.02
1487	1489	β-Selinene	0.12 ± 0.01	tr	tr
1493	1493	epi-Cubebol	0.12 ± 0.04	---	---
1494	1500	Bicyclogermacrene	---	0.16 ± 0.01	0.07 ± 0.01
1513	1514	Cubebol	0.14 ± 0.02	tr	tr
1516	1522	δ-Cadinene	tr	0.07 ± 0.00	tr
1547	1548	Elemol	---	tr	0.07 ± 0.01
1559	1561	(E)-Nerolidol	0.10 ± 0.02	0.09 ± 0.03	0.64 ± 0.03
1575	1574	Germacrene D-4β-ol	0.37 ± 0.02	0.46 ± 0.00	0.46 ± 0.00
1581	1582	Caryophyllene oxide	0.16 ± 0.09	tr	0.37 ± 0.01
1608	1608	Humulene epoxide II	---	---	0.05 ± 0.01
1636	1639	Caryophylla-4(12),8(13)-dien-5β-ol	---	---	tr
1638	1643	Hedycaryol	---	---	0.10 ± 0.01
1641	1638	τ-Cadinol	0.18±0.01	tr	0.14 ± 0.01
1654	1649	β-Eudesmol	0.10 ± 0.02	---	0.16 ± 0.03
1655	1652	α-Cadinol	---	tr	tr
1663	1665	Intermediol	tr	0.56 ± 0.01	---
1683	1685	Germacra-4(15),5,10(14)-trien-1α-ol	0.13 ± 0.04	---	0.51 ± 0.01
1686	1687	Eudesma-4(15),7-dien-1β-ol	---	---	0.14 ± 0.00
1689	1690	(Z)-trans-α-Bergamotol	---	---	0.12 ± 0.01
1699	1695	6-epi-Shyobunol	---	---	0.05 ± 0.01
		Monoterpene hydrocarbons	79.21	81.56	83.13
		Oxygenated monoterpenoids	11.80	3.85	5.64
		Sesquiterpene hydrocarbons e	4.97	9.40	4.79
		Oxygenated sesquiterpenoids e	1.39	1.11	2.79
		Green leaf volatiles	1.54	1.07	2.53
		Others	0.091	0.13	0.18
		Total Identified	99.01	97.12	99.05

^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.

(average of three measurements ± standard deviations) are based on peak integration without standardization.

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

Material	Fungal Species
	Aspergillus niger	Candida albicans	Cryptococcus neoformans
H. annuus “Chianti”	625	625	78
H. annuus “Mammoth”	625	625	156
H. strumosus	625	1250	78
(+)-α-Pinene	625	313	20
(−)-α-Pinene	156	625	39
(−)-β-Pinene	156	625	39
(+)-Limonene	1250	625	78
(−)-Limonene	2500	1250	313
Amphotericin B	0.78	0.78	1.56

^a Each MIC determination was carried out in triplicate.

. 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 suggest 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.

4. Conclusions

Helianthus essential oils have been shown to be rich in α- and β-pinenes, sabinene, and limonene, and have demonstrated poor antifungal activities against A. niger and C. albicans, but promising activity against C. neoformans (although much lower activity than the reference antifungal drug amphotericin B). These and other monoterpene-rich essential oils deserve further exploration as alternative and complementary agents to combat fungal infections; further studies against more susceptible fungi are recommended.