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The Phytochemistry of Cherokee Aromatic Medicinal Plants

Background: Native Americans have had a rich ethnobotanical heritage for treating diseases, ailments, and injuries. Cherokee traditional medicine has provided numerous aromatic and medicinal plants that not only were used by the Cherokee people, but were also adopted for use by European settlers in North America. Methods: The aim of this review was to examine the Cherokee ethnobotanical literature and the published phytochemical investigations on Cherokee medicinal plants and to correlate phytochemical constituents with traditional uses and biological activities. Results: Several Cherokee medicinal plants are still in use today as herbal medicines, including, for example, yarrow (Achillea millefolium), black cohosh (Cimicifuga racemosa), American ginseng (Panax quinquefolius), and blue skullcap (Scutellaria lateriflora). This review presents a summary of the traditional uses, phytochemical constituents, and biological activities of Cherokee aromatic and medicinal plants. Conclusions: The list is not complete, however, as there is still much work needed in phytochemical investigation and pharmacological evaluation of many traditional herbal medicines.


Introduction
Natural products have been an important source of medicinal agents throughout history and modern medicine continues to rely on traditional knowledge for treatment of human maladies [1]. Traditional medicines such as Traditional Chinese Medicine [2], Ayurvedic [3], and medicinal plants from Latin America [4] have proven to be rich resources of biologically active compounds and potential new drugs. Several plant-derived drugs are in use today, including, for example, vinblastine (from Catharanthus roseus (L.) G. Don, used to treat childhood leukemia); paclitaxel (from Taxus brevifolia Nutt., used to treat ovarian cancer); morphine (from Papaver somniferum L., used to treat pain); and quinine (from Cinchona spp., used to treat malaria) [5]. Not only are phytochemicals useful medicines in their own right, but compounds derived from them or inspired by them have become useful medicines [6,7]. For example, Artemisia annua L., a plant originally used in Traditional Chinese Medicine to treat fever, is the source of artemisinin, a clinically-useful antimalarial sesquiterpenoid [8]; the antihypertensive drug reserpine, isolated from the roots of Rauvolfia serpentina (L.) Benth. ex Kurz., has been used in Ayurveda to treat insanity, epilepsy, insomnia, hysteria, eclampsia, as well as hypertension [9]; Dysphania ambrosioides (L.) Mosyakin and Clemants (syn. Chenopodium ambrosioides L.) is used in several Latin American cultures as an internal anthelmintic and external antiparasitic [4] and has shown promise for treatment of cutaneous leishmaniasis [10]. The biological activity of D. ambrosioides has been attributed to the monoterpenoid endoperoxide ascaridole.
Unfortunately, much of the traditional medicine knowledge of Native North American peoples has been lost due to population decimation and displacement from their native lands by European conquerors (see, for example: [11][12][13][14]). Nevertheless, there are still some remaining sources of Unfortunately, much of the traditional medicine knowledge of Native North American peoples has been lost due to population decimation and displacement from their native lands by European conquerors (see, for example: [11][12][13][14]). Nevertheless, there are still some remaining sources of information about Native American ethnobotany [15,16]. In addition, there are several sources of Cherokee ethnobotany [17][18][19][20][21][22].
The Cherokee Native Americans are a tribe of Iroquoian-language people who lived in the southern part of the Appalachian Mountain region in present-day northern Georgia, eastern Tennessee, and western North Carolina and South Carolina at the time of European contact [13] ( Figure 1A). During and after the American Revolution, Cherokee wars with European settlers resulted in the surrender of vast amounts of territory. Gold was discovered on Cherokee land in north Georgia and the Treaty of New Echota (1835) ceded all Cherokee land east of the Mississippi River to the United States. Congress passed the Indian Removal Act in 1830, and the forced eviction of as many as 16,000 Cherokee took place during the fall and winter of 1838-1839 to a new territory in north-eastern Oklahoma (Fibure 1B). During this "Trail of Tears", an estimated one-fourth of the Cherokee died. However, at the time of the removal, a few hundred Cherokee successfully escaped to the mountains of western North Carolina, forming what is now the Eastern Band of Cherokee Indians.
In this review, I have consulted the ethnobotanical sources for plants used in Cherokee traditional medicine [15][16][17][18][19][20][21][22][23][24] and I have carried out a literature search using Google Scholar, PubMed, ResearchGate, and Science Direct for phytochemical analyses on the plant species. Note that in many instances, the phytochemistry was determined by plants not collected in the south-eastern United States; many of the species have been introduced to other parts of the world and some species are native to other continents besides North America. The phytochemistry, therefore, may be affected by the different geographical and climatic conditions [25]. Sources reporting the phytochemical constituents, regardless of geographical origin, have been included.

Cherokee Aromatic Medicinal Plants and Their Phytochemical Constituents
The plants used by the Cherokee people for traditional medicines for which the phytochemistry has been investigated are summarized in Table 1.

Cherokee Aromatic Medicinal Plants Currently in Use as Herbal Medicines
3.1. Achillea millefolium L.
Achillea millefolium (yarrow) is native to temperate regions of the Northern Hemisphere but has been introduced worldwide [510]. The traditional medical uses of A. millefolium have been reviewed and the plant has been used since ancient times as a wound-healing agent and to treat gastrointestinal complaints [510][511][512]. Consistent with this, the Cherokee have also used A. millefolium as an antihemorrhagic; for healing wounds, treating bloody hemorrhoids and bloody urine, and for bowel complaints [15,17,510]. In addition, infusions of A. millefolium have been used as a treatment for fever [15,17,510]. Yarrow extract has shown spasmogenic effects on murine and human gastric antrum, consistent with its traditional use to treat dyspepsia [513]. In a double-blind clinical trial, A. millefolium ointment was shown to reduce pain, inflammation, and ecchymosis in episiotomy wound healing [514].
The essential oils of A. millefolium have shown wide variation depending on geographical location and growing season. Volatile oil samples from Turkey [48] and Macedonia [51] were dominated by 1,8-cineole and camphor, whereas the essential oil from Lavras, Brazil, was rich in chamazulene [49]. The essential oil from Lithuania showed wide variation in composition depending on morphological type (flower color) as well as plant phenology [50]; γ-terpinene and cadinene (isomer not identified) were the major components during the flowering phase, but β-pinene was abundant during the vegetative phase. Conversely, A. millefolium leaf essential oil from Portugal was rich in 1,8-cineole during the flowering phase, but germacrene D dominated the oil during the vegetative phase [53].

Caulophyllum thalictroides (L.) Michx.
A decoction of the roots of C. thalictroides (blue cohosh) has been used by the Cherokee as an anticonvulsive (to treat "fits and hysterics") and antirheumatic [15]. The plant is also used as a gynecological aid, to promote childbirth and to treat womb inflammation [15]. These traditional uses are in apparent contrast to the observed toxic effects (convulsions, respiratory paralysis) of the plant observed in range animals such as sheep [108]. The rhizome of C. thalictroides contains several quinolizidine alkaloids, including N-methylcytisine (also known as caulophylline), baptifoline, anagyrine, and lupanine [108,110,112]. N-Methylcytisine is known to stimulate the central nervous system, and in high doses causes convulsions followed by paralysis [532]. Acute lupanine toxicity is characterized by neurotoxic effects including decreased cardiac contractility, blocking of ganglionic transmission and contraction of uterine smooth muscle [533]. This latter effect explains the traditional Cherokee use to promote childbirth. Apparently, lupanine, in lower doses, does not exhibit sub-chronic, chronic, reproductive, or mutagenic toxic effects [533]. Both N-methylcytisine [110] and anagyrine [534] have been shown to be teratogenic, however. The aporphine alkaloid magnoflorine, on the other hand, has shown sedative and anxiolytic effects [535] and may be responsible for the anti-convulsive and sedative uses of C. thalictroides in Cherokee traditional medicine.
Lee and co-workers [115] have shown that the oleanolic acid glycosides caulosides A-D exert anti-inflammatory effects by way of inhibiting expression of inducible nitric oxide synthase (iNOS) and the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). The anti-inflammatory effects of C. thalictroides triterpene saponins are consistent with the Cherokee traditional uses to treat rheumatism and inflammation. Black cohosh (C. racemosa) has been a popular herbal supplement for many years [536]. The plant is reputed to possess anti-inflammatory, diuretic, sedative, and antitussive activities [511], and the root has been reported to have estrogenic activity [537][538][539]. Fukinolic acid [137] and formononetin [511] have been reported to be estrogenic constituents of C. racemosa rhizome. The traditional Cherokee use of C. racemosa rhizome to stimulate menstruation [15] is consistent with the reported estrogenic activity. There have been conflicting reports regarding the estrogenic activity of C. racemosa rhizome, however [540][541][542], and a survey of 13 populations of C. racemosa in the eastern United States failed to detect the presence of formononetin [543]. Molecular docking studies have suggested that C. racemosa triterpenoids are unlikely estrogen receptor binding agents, but any estrogenic activity of C. racemosa extract is probably due to phenolic components such as cimicifugic acid A, cimicifugic acid B, cimicifugic acid G, cimiciphenol, cimiciphenone, cimiracemate A, cimiracemate B, cimiracemate C, cimiracemate D, and fukinolic acid [544]. Although recent evidence suggests the estrogen receptor not to be a target of C. racemosa phytochemical constituents, other biomolecular targets may be involved. Rhizome extracts of C. racemosa have been shown to interact with the serotonin receptor [545], the µ-opioid receptor [546,547] as well as the γ-aminobutryic acid type A (GABA A ) receptors [548]. Modulation of these receptors may contribute to some of the biological effects of C. racemosa extracts.
Reviews of several randomized clinical trials have failed to demonstrate efficacy of C. racemosa on menopausal symptoms [549,550]. However, one randomized, placebo-controlled double-blind clinical trial with menopausal women, concluded that C. racemosa extract showed superiority over a placebo in ameliorating menopausal disorders [551]. Clinical studies have generally suggested C. cimicifuga use to be safe, but there have been some case reports indicating safety concerns [552].
The Cherokee have also used infusions of C. racemosa rhizome to treat rheumatism, coughs, and colds [15]. Aqueous extracts of C. racemosa have demonstrated reduction of the release of pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interferon-gamma (IFN-γ) in whole blood, and the prominent active component responsible was isoferulic acid [553]. The ethyl acetate fraction of the aqueous extract of C. racemosa was also shown to suppress the release of TNF-α, due to cimiracemate A [554]. Aqueous extracts reduced inducible nitric oxide synthase (iNOS) protein expression as well as iNOS mRNA levels, but did not inhibit iNOS enzymatic activity; the triterpenoid glycoside 23-epi-26-deoxyactein was found to be the active principle in the extract [555]. These effects likely explain the anti-inflammatory activities of C. racemosa and their traditional uses to treat rheumatism and other inflammatory diseases.

Hamamelis virginiana L.
Hamamelis virginiana, American witch hazel, is a shrub or small tree, native to eastern North America. Several Native American tribes have used the plant for numerous medicinal purposes. Decoctions of the bark or the stems of witch hazel have been used as a topical lotion for cuts, bruises, insect bites, external inflammations, and other skin problems [15]. In addition, the Cherokee people took infusions of witch hazel for periodic pains, to treat colds, sore throats, and fevers. Modern uses of witch hazel include treatment of hemorrhoids, inflammation of the mouth and pharynx (leaf only), inflammation of the skin, varicose veins, wounds and burns [537]. Hamamelis virginiana leaves contain up to 10% tannins, including gallic acid, polygalloylglucose, hamamelitannin and analogs, flavonoids, and proanthocyanidins [511], which are responsible for the observed astringent, anti-inflammatory, and hemostatic effects [537]. The bark also contains hamamelitannin and analogs, and proanthocyanidins [511].
The aqueous ethanol extract of H. virginiana showed anti-inflammatory activity in the croton oil mouse ear edema test [556] as well as the induced rat paw edema assay, confirming its use as an anti-inflammatory agent [557]. The extract also showed notable antiviral activity against Herpes simplex virus type 1 (HSV-1) [556]. Hamamelitannin and galloylated proanthocyanidins from H. virginiana were found to be potent inhibitors of 5-lipoxygenase (5-LOX) [558]. Hamamelis proanthocyanidins were found to stimulate cell growth of keratinocytes, enhancing cell growth, and are likely responsible for the dermatological use of tannin-containing witch hazel preparations [559]. Hamamelis tannins have also shown cytotoxic activity against HT-29 human colorectal adenocarcinoma cells [223] and antiviral activity against influenza A virus and human papillomavirus [560].
The anti-inflammatory activity of witch hazel was demonstrated in a clinical study using a lotion prepared from H. virginiana distillate, which showed suppression of erythema after ultraviolet (UVB) light exposure [561]. Similarly, in a clinical trial with patients suffering from atopic eczema, a cream containing H. virginiana distillate significantly reduced skin desquamation, itching and redness [562]. Of course, H. virginiana distillate will not contain tannins.

Hydrastis canadensis L.
Goldenseal (Hydrastis canadensis), a perennial herb in the Ranunculaceae, is native to eastern North America from Ontario, Canada, south to Alabama and Georgia [563]. The Cherokee used the root decoction of goldenseal as a tonic and wash for local inflammations; took the root decoction orally to treat cancer, dyspepsia, and general debility [15]. Goldenseal is still used in herbal medicine to control muscle spasms, treat cancer, increase blood pressure, treat gastrointestinal disorders, manage painful and heavy menstruation, treat infections topically, and reduce swelling [537,564].

Panax quinquefolius L.
American ginseng (Panax quinquefolius) is a member of the Araliaceae and is native to eastern North America [585]. Ginseng root from P. ginseng or P. notoginseng, has been used for thousands of years in the Asian traditional medicine. Panax quinquefolius is currently cultivated in the United States, Canada, and China, and is used as a medical tonic worldwide. Native Americans have used P. quinquefolius for numerous medical problems as well as a general tonic [15], and European settlers had also utilized this plant for similar purposes [586]. The Cherokee used the root as an expectorant, to treat colic, oral thrush, and as a general tonic [15].
The phytochemistry and pharmacology of P. quinquefolius has been reviewed several times [333,339,341,342]. The major components in P. quinquefolius roots are triterpenoid glycosides, the ginsenosides, as well as several polyacetylenes. The ginsenosides have shown anti-inflammatory, antiproliferative, hepatoprotective, cardioprotective, neuroprotective, cholesterol-lowering, and cognitive improvement [340].
Several clinical trials have been carried out using P. quinquefolius extracts. In terms of cognitive function, a randomized, double-blind, placebo-controled crossover trial, P. quinquefolius extract showed significant improvement in working memory, choice reaction time and "calmness" [587]. A clinical trial to study the effects of P. quinquefolius extract on cancer-related fatigue showed a promising significant trend in relieving fatigue [588]. Panax quinquefolius extracts were found to be clinically effective in preventing upper respiratory infections in healthy adult senior citizens [589,590].

Sanguinaria canadensis L.
Bloodroot (Sanguinaria canadensis, Papaveraceae) is native to eastern North America [591]. The plant has been used by Native Americans as a traditional medicine for a variety of ailments [455]. The Cherokee used a decoction of the root, in small doses, for coughs, lung inflammations, and croup, and a root infusion was used as a wash for ulcers and sores [15]. The roots are rich in isoquinoline alkaloids, including sanguinarine, chelerythrine, sanguilutine, chelilutine, sanguirubine, chelirubine, protopine, and allocryptopine [455]. The traditional Cherokee uses of bloodroot as a cough medicine/respiratory aid as well as for treating ulcers and sores can be attributed to the antimicrobial activities of the isoquinoline alkaloids [592]. Thus, for example, sanguinarine has shown antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) [593], biofilm-forming Candida spp. [594], Mycobacterium spp. [452], and Helicobacter pylori [453].

Scutellaria lateriflora L.
Infusions of the roots of blue skullcap (Scutellaria lateriflora, Lamiaceae) were used by the Cherokee for monthly periods and to treat diarrhea; root decoctions were used as an emetic to expel afterbirth and to remedy breast pains [15]. Interestingly, the aerial parts, rather than the roots, are currently used as an herbal medicine as an anxiolytic, sedative and antispasmodic [511,537,595,596].
The flavonoids scutellarin and baicalin and the phenylpropanoid ferulic acid have shown in vitro estrogenic effects [597,598], and may be responsible for the traditional Cherokee uses of S. lateriflora.
Consistent with the current herbal medicinal use of S. lateriflora, the plant has shown anti-convulsant activity in rodent models of acute seizures, attributable to the flavonoid constituents [474]. Baicalin has shown anti-convulsant activity in pilocarpine-induced epileptic model in rats [599], and wogonin has shown anti-convulsant effects on chemically-induced and electroshock-induced seizures in rodents [600]. In addition, scutellarin has shown relaxant activity using rodent aorta models [601,602], while wogonin showed smooth muscle relaxant activity in rat aorta [603] and rat uterine smooth muscle [604]. On the other hand, both baicalin and baicalein inhibited NO-mediated relaxation of rat aortic rings [605]. Baicalein and baicalin have shown anxiolytic activity [606]. Apparently, baicalin and wogonin exert their anxiolytic effects through allosteric modulation of the GABA A receptor by way of interaction at the benzodiazepine site [607,608]. Conversely, baicalein promotes anxiolytic effects via interaction with non-benzodiazepine sites of the GABA A receptor [609]. There have apparently been no clinical trials on the root extracts of S. lateriflora. However, in randomized, double-blind, placebo-controlled crossover clinical trials, the anxiolytic effects of S. lateriflora herbal treatments significantly enhanced overall mood without reducing cognition or energy [610,611].

Conclusions
This is not a complete list of the phytochemistry of Cherokee aromatic medicinal plants. Numerous plants described in the Cherokee ethnobotanical literature [15][16][17][18][19][20][21][22][23][24] have not been investigated for phytochemical constituents or pharmacological activity. In addition, in many instances the phytochemistry is not sufficiently characterized, particularly in terms of the plant tissues used in Cherokee traditional medicine. In this review, there are numerous instances where the phytochemical constituents and the biological activities associated with them correlate with the traditional Cherokee uses of the plant, but there are several instances where there is no apparent correlation. Therefore, much work is needed to add to our knowledge of the pharmacological properties of the chemical components, not to mention potential synergistic or antagonistic interactions.
Funding: This research received no external funding.