Echinacea plants as antioxidant and antibacterial agents: From traditional medicine to biotechnological applications

Wiley Online Library Phytotherapy Research REVIEW Full Access Echinacea plants as antioxidant and antibacterial agents: From traditional medicine to biotechnological applications Mehdi Sharifi‐Rad Dima Mnayer Maria Flaviana Bezerra Morais‐Braga Joara Nályda Pereira Carneiro Camila Fonseca Bezerra Henrique Douglas Melo Coutinho Bahare Salehi Miquel Martorell María del Mar Contreras Azam Soltani‐Nejad Yoshie Adriana Hata Uribe Zubaida Yousaf Marcello Iriti Javad Sharifi‐Rad ... See fewer authors First published: 10 May 2018 https://doi.org/10.1002/ptr.6101 Cited by: 9 UBC eLink Sections ePDFPDF Tools Share Abstract The genus Echinacea consists of 11 taxa of herbaceous and perennial flowering plants. In particular, Echinacea purpurea (L.) Moench is widely cultivated all over the United States, Canada, and in Europe, exclusively in Germany, for its beauty and reported medicinal properties. Echinacea extracts have been used traditionally as wound healing to improve the immune system and to treat respiratory symptoms caused by bacterial infections. Echinacea extracts have demonstrated antioxidant and antimicrobial activities, and to be safe. This survey aims at reviewing the medicinal properties of Echinacea species, their cultivation, chemical composition, and the potential uses of these plants as antioxidant and antibacterial agents in foods and in a clinical context. Moreover, the factors affecting the chemical composition of Echinacea spp. are also covered. 1 INTRODUCTION The genus Echinacea belongs to the family Asteraceae. It consists of 11 taxa (McKeown, 1999), and all are herbaceous and perennial flowering plants (Pellati, Benvenuti, Melegari, & Lasseigne, 2005). In particular, Echinacea purpurea (L.) Moench is described by straight stem up to 2 m in height, alternate leaves on long stalks, coarse hairs and solitary spiny, as well as reddish‐orange flowers surrounded by purple bracts. This purple coneflower is cultivated widely all over the United States, Canada, and Europe, exclusively in Germany, for its beauty and its reported medicinal properties (Barrett, 2003). In this sense, this medicinal plant is famous for its effects on the immune system (Barrett, 2003). The first historical reports of Echinacea were by Clayton in the Flora Virginica (1762) and by Schöpf in the Materia Medica Americana (1787; Flannery, 1999). History of using Echinacea as an immunomodulator dates back to 1913, when von Unruh published his report on increasing phagocytosis of tuberculosis bacteria (von Unruh, 1913). Echinacea was mentioned in the National Formulary of the United States from 1916 to 1950 (Barrett, 2003). During the 20th century, German scientists were leaders in the research about Echinacea. US markets and US researchers were able to catch up in the last decade or so (Barrett, 2003). Echinacea products sale annually about $300 million in the US alone (Brevoort, 1998). In addition to support for a healthy immune system, Echinacea extracts have been traditionally used in wound healing. This effect is attributed to its polysaccharide fraction (echinacin B) by producing a hyaluronic acid–polysaccharide complex that stimulates wound healing, leads to the inhibition of hyaluronidase, and promotes fibroblasts growth (Newall, Anderson, & Phillipson, 1996). Furthermore, Echinacea has been used for its ability in healing sore throats, coughs, and various other respiratory symptoms caused by bacterial infections (M. Sharma, Vohra, Arnason, & Hudson, 2008). Besides the presence of high molecular weight polysaccharides, other chemical components of Echinacea species are volatile terpenes, such as germacrene D, polyacetylenes, highly unsaturated alkamides, phenolic compounds, and glycoproteins (Mistrikova & Vaverkova, 2006; Pellati et al., 2005). The interest in Echinacea plants has led to publish scientific studies to demonstrate their pharmacological properties. Echinacea may stimulate monocytes and natural killer cells, which are the first line of immune defense in the body against infections (Mistrikova & Vaverkova, 2006). This immunomodulatory activity of E. purpurea extracts was examined treating leukemic mice for 50 days, and the results showed an enhanced immune status and a prolonged life span (Currier & Miller, 2001). The antimicrobial activity of different Echinacea species and their chemical constituents against several microorganisms has been studied. As an example, echinacoside exhibited in vitro antimicrobial activity against Staphylococcus aureus (6 mg were as effective as one unit of penicillin). E. purpurea can inhibit Legionella pneumophila, the causal organism for pneumonia, by inhibiting the induction of proinflammatory cytokines (Hudson, 2011). The inhibition of Pseudomonas aeruginosa and Escherichia coli with polyacetylenes derived from the roots of Echinacea angustifolia and E. purpurea was reported. Furthermore, n‐hexane extracts of Echinacea spp. had inhibitory activity against the fungi Saccharomyces cerevisiae, Candida shehata, Candida kefir, Candida albicans, Candida steatulica, and Candida tropicalis under near ultraviolet irradiation (phototoxicity) and to a lower extent without irradiation (conventional antifungal activity). This phototoxic activity of Echinacea spp. was ascribed to ketoalkenes and ketoalkynes present in the roots (Mistrikova & Vaverkova, 2006). In addition, E. purpurea increased chemotoxicity in neutrophils and also exhibited cytotoxicity toward cancer cells (WEHI 164 cells) and cells infected either with the parasite Leishmania enriettii or with yeast C. albicans. S. aureus, C. albicans, and S. cerevisiae showed higher sensitivity for Echinacea compared with two tested antibiotics, erytromycin and tylosin tartrate (Stanisavljević, Stojičević, Veličković, Veljković, & Lazić, 2009). E. purpurea has also the ability to interfere with viruses during their initial contact with host cells and during the spread of virus from infected cells whereas being much less effective against intracellular virus (Hudson, 2011). For example, E. purpurea stopped the binding of influenza A virus with receptors of the host cell, thus interfering the virus entry into the cells (Pleschka, Stein, Schoop, & Hudson, 2009). This plant is also effective against Hemophilus influenzae, a pathogen associated with otitis media, chronic bronchitis, and pneumonia, as well as against herpes simplex virus (Vimalanathan et al., 2005). Moreover, cichoric acid, another phenolic component of Echinacea, was able to inhibit HIV‐1 integrase and replication (Stanisavljević et al., 2009). 2 CULTIVATION OF PLANTS BELONGING TO GENUS ECHINACEA As noted before, the genus Echinacea is native to North America, and it is represented by nine species: E. purpurea, Echinacea laevigata (C.L. Boynton & Beadle) S.F. Blake, Echinacea paradoxa Britton, Echinacea atrorubens (Nutt.) Nutt., E. angustifolia, Echinacea tennesseensis (Beadle) Small, Echinacea sanguinea Nutt., Echinacea pallida (Nutt.) Nutt., and Echinacea simulata McGregor (Binns, Baum, & Arnason, 2002; Foster, 1991; McGregor, 1968). Most of its species are perennials and are found to be drought tolerant. Their seeds germinate within 10–20 days and plants flower 90 to 120 days after seed sowing. These species usually require 30–60 mm/year of precipitations and prefer sunlight with normal to alkaline pH of substrate. E. angustifolia and E. pallida grow easily in gravelly clay type soil with good drainage, whereas E. purpurea prefers sandy and loamy soils. The latter aforementioned species, that is, E. purpurea, E. angustifolia, and E. pallida, are commercially important in terms of their herbal products (Schulthess, Giger, & Baumann, 1991). A product named echinacin, obtained from plant's fresh juice, has been prescribed in Germany for many years (Blumenthal, 1998; Hobbs, 1989; Leslie, 1995). Many species have been described as the most important plants used by the Native Americans for the treatment of many diseases, including colds, tonsillitis, toothaches, bowel pain, snakebites, rabies, seizures, wound infections, septic conditions, and cancer (Foster, 1991; Hobbs, 1989). Echinacea species are also cultivated for ornamental purposes (Sari, Morales, & Simon, 1999), as commented before. Therefore, the cultivation of these species needs to be increased due to its increasing market demand (T. S. Li, 1998). However, a number of problems have been reported due to the low seed germination rate and plant susceptibility to biotic stresses (Smith‐Jochum & Albrecht, 1986, 1988). Indeed, only 0–6% and 0–2% of germination were observed when E. angustifolia seeds were placed under light or dark conditions, respectively (Baskin, Baskin, & Hoffman, 1992). In addition, plants are highly susceptible to fungal infections, aster yellows, beetles, aphids, and hoppers, in particular E. purpurea (Sari et al., 1999). To date, no resistant cultivar is available. The cultivation of E. purpurea with 20, 40, or 60 cm distance and in rows 50 cm apart produces more elongated plants with higher biomass in dry weight. However, the highest total biomass per unit of production area is obtained at 20 cm spacing. Although fertilizers are generally not required for the growth of these species, the supplementation of nitrogen fertilizers seems to enhance the plant growth and yield. Also, applying low levels of potassium provides better results than the addition of nitrogen alone. Thus, a balanced N:K ratio can be relevant (Shalaby, El‐Gengaihi, Agina, El‐Khayat, & Hendawy, 1997). 3 CHEMICAL COMPOSITION OF ESSENTIAL OILS OBTAINED FROM ECHINACEA SPP. PLANTS The Echinacea genus is valuable for its healthy properties (Erenler et al., 2015; Yamada, Hung, Park, Park, & Lim, 2011) which are mostly related to their chemical components (Wiesner & Knöss, 2017). These valuable compounds were obtained by various extraction techniques (solid phase extraction, solvent extraction, supercritical extraction, distillation, etc.) and were evaluated for their chemical structure and biological properties (Erenler et al., 2015). Many components were isolated from roots or aerial parts of the plants. These mainly included volatile compounds, alkylamides, polyphenols, caffeic acid derivatives, polysaccharides, alkaloids, and many other different structures (Głowniak, Zgórka, & Kozyra, 1996; Yu et al., 2013). Regarding the volatile compounds, essential oils (EOs) are considered as potential medicinal agents (Salehi et al., 2017; J. Sharifi‐Rad, Sureda, et al., 2017; M. Sharifi‐Rad, Varoni, et al., 2017; M. Sharifi‐Rad et al., 2018; Yu et al., 2013). The EOs from Echinacea plants can be obtained from different parts of the plants, such as roots, leaves, flowers, and aerial parts, and with different yields (Mazza & Cottrell, 1999). In general, the EOs yields are 0.05–0.48% in fresh materials and 0.1%–1.25% in dried materials (Mistrikova & Vaverkova, 2006). Nevertheless, the yield varied considerably among the species; for example, a yield of 1.85% for E. purpurea was reported in dry flower heads (Holla, Vaverkova, Farkas, & Tekel, 2005) and even less than 0.1% for E. angustifolia roots (Aiello et al., 2015). In addition, the chemical composition of the EOs can vary considerably among the Echinacea species, and this is due to many intrinsic and extrinsic factors such as genetic traits, geographical location, climatic changes, growing conditions, seasonal variation, and time of harvest (Sasidharan, Venugopal, & Menon, 2012; Stoyanova, Konakchiev, Damyanova, Stoilova, & Suu, 2006; Sukatta, Rugthaworn, Punjee, Chidchenchey, & Keeratinijakal, 2009; Wang et al., 2006). The major volatile compounds of these EOs are presented in Figure 1. image Figure 1 Open in figure viewerPowerPoint Major volatile compounds of the Echinacea genus essential oils As commented before, among the Echinacea species, E. purpurea is the most known and used as a medicinal plant to treat common diseases. Its volatile composition and biological properties have been recently investigated (Chiellini et al., 2017). For example, different parts of E. purpurea plants were hydrodistillated and tested for their anti‐inflammatory and analgesic activities. The main components found in the leaf and root EOs included germacrene D (18.1% and 20.3%, respectively), naphthalene (7.8% and 6.4%, respectively), caryophyllene oxide (11.3% and 12.2%, respectively), α‐phellandrene (6.9% and 6.6%, respectively), α‐cadinol (9.1% and 5.9%, respectively), and caryophyllene (4.5% and 4%, respectively, Nyalambisa et al., 2017). Another study reported by Mazza and Cottrell (1999) showed that the major compound found in E. purpurea root EO was α‐phellandrene, whereas, in flower, leaf, and stem tissues, it was β‐myrcene. In this study, germacrene D was measured in very low levels (Mazza & Cottrell, 1999). On the contrary, another study showed that the hydrodistillated EO from the flower heads contained germacrene D (4.8%) and α‐phellandrene (4.3%), together with palmitic acid (8.3%), nerolidol (6.6%), α‐pinene (5.1%), and β‐pinene (4.1%; Holla et al., 2005). According to Lepojević et al. (2017), the EO extracted from the aerial parts of E. purpurea using supercritical carbon dioxide included terpenoids, long‐chain hydrocarbons, and phytosterols. Moreover, the most abundant terpenoids were sesquiterpene hydrocarbons and oxygenated sesquiterpenes (Lepojević et al., 2017). Germacrene D was also present in a high concentration (57%) in the flower heads of this species cultivated in Iran (Mirjalili, Salehi, Badi, & Sonboli, 2006) and used to treat skin disorders (Schmidt, Noletto, Vogler, & Setzer, 2007). E. purpurea growıng in Turkey was hydrodistillated and analyzed for its chemical composition. Again, germancrene D (11.3%) was the major component, followed by caryophyllene oxide (8.7%), β‐caryophyllene (7.2%), α‐cadinol (6.3%), naphthalene (3.3%), and α‐phellandrene (2.9%) (Diraz, Karaman, & Koca, 2012). From these studies, it can be highlighted that the EOs obtained from E. purpurea showed a high variability in their chemical composition, though, in general, the sesquiterpene germacrene D was the most abundant compound. Therefore, the EOs obtained from E. purpurea have to be standardized before using, and more scientific evidences about their bioactivity should be collected. The composition of the EOs also varies between species. As an example, the composition of the EOs obtained by hydrodistillation from the flower heads of E. purpurea, E. pallida, and E. angustifolia cultivated in Iran was compared. Sesquiterpene hydrocarbons were the main group of compounds found in E. purpurea (70.9%), E. angustifolia (70%), and E. pallida (62.6%). The abundance of germacrene D was high in the EO of E. purpurea (57%), as expected, and it was found as a principal component in E. pallida (51.4%) and E. angustifolia (49.6%) too. The monoterpene hydrocarbons were observed mainly in E. purpurea (6.4%) and E. angustifolia (1.2%) EOs, whereas these compounds were absent in E. pallida (Mirjalili et al., 2006). A study reported by Mistrikova and Vaverkova (2006) showed that E. purpurea EO contained borneol, bornyl acetate, pentadeca‐8‐(Z)‐en‐2‐one, germacrene D, caryophyllene, and caryophyllene epoxide, whereas E. angustifolia and E. pallida EOs contained, among other compounds, pentadeca‐(1.8Z)‐diene (44%), 1‐pentadecene, ketoalkynes, and ketoalkenes. In another study, the EO obtained by hydrodistillation from the roots of E. pallida showed the presence of 8‐pentadecene‐2‐one (26.10%), cyclopentadecanone (18.32%), and β‐caryophyllene (2.67%), whereas, in the EO of E. atrorubens roots, 8‐pentadecene‐2‐on (24.30%) and cyclopentadecanone (21.25%) were the major compounds (Vaverkova et al., 2007). Noteworthy, pathogen infections represent other exogenous factors that contribute to the phytochemical variation in plant tissues. Hudaib, Fiori, Bellardi, Rubies‐Autonell, and Cavrini (2002) compared the composition of the EOs obtained from the hydrodistilled aerial parts of E. purpurea grown in Italy, healthy and infected by the cucumber mosaic cucumovirus. The infected plants yielded a lower amount of EO that showed significant quantitative differences in the composition. In particular, germacrene D, the most abundant component in healthy plants (57.8%), was present in a lower amount in the infected material (52.6%). Other minor compounds were found in both healthy and infected plant samples, including α‐pinene (2.3% and 7.5%, respectively), myrcene (1.7 and 8.8%, respectively), β‐caryophyllene (3.1 and 2.1%, respectively), and δ‐cadinene (2.7 and 1.5%, respectively). Other factors to be considered are the climatic conditions. A study conducted by Thappa et al. (2004) on the overmature flower heads of E. purpurea under subtropical climate was carried out to compare the effect of climatic changes on the EO composition. Their results showed that the most abundant terpene found in the EO was germacrene D, which showed a steady rise from 7.2% in June to 33.5% in December. These results confirm that climatic factors, including temperature and humidity, greatly affect both the EO amount and composition. 4 OTHER COMPONENTS Other constituents of Echinacea spp. plants contribute to their bioactivity, including high molecular weight polysaccharides, lipophilic components (polyacetylenes and highly unsaturated alkamides), caffeic acid derivatives, and glycoproteins (Pellati et al., 2005). Polysaccharides include inulin, arabinorhamnogalactans, and heteroxylans, which exhibit immunostimulatory and anti‐inflammatory activities. Several factors have an effect on these plant chemicals: the genetic traits of Echinacea species, the part of the plant (leaves, flowers, stems, or roots), the growing, drying and storage conditions, and the method of extraction (Gray, Pallardy, Garrett, & Rottinghaus, 2003). In this sense, although aerial parts contain mainly alkylamides and polyacetylenes, caffeic and ferulic acid derivatives, polysaccharides, and glycoproteins, the roots contain more volatile components and pyrrolizidine alkaloids, such as tussilagine and isotussilagine (Stanisavljević et al., 2009). Another example is alkylamides; these compounds are present in different structural forms in the roots of Echinacea spp. plants, and they have a similar pattern in the aerial parts of the different species (Bauer & Remiger, 1989). Furthermore, these chemical constituents fluctuate during the E. purpurea growth cycle, both quantitatively and qualitatively (Letchamo, Livesey, Arnason, Bergeron, & Krutilina, 1999); indeed, these compounds decline slowly in the aerial parts and increase in the roots as the plant matures (Mistrikova & Vaverkova, 2006). Freshly harvested plants are more effective than the stored ones. This is probably due to degradation of bioactive constituents during long storage. Interestingly, polyacetylenes were only found in the dried roots of E. pallida and not in fresh ones; therefore, it is believed that this type of compounds can be artifacts formed during storage (Mistrikova & Vaverkova, 2006). However, polyacetylenes decreased significantly in long‐term storage. Besides polyacetylenes, alkylamides are also highly sensitive to auto‐oxidation, making them highly reliant on processing and storage conditions (Mistrikova & Vaverkova, 2006). The alkylamides are constituted of highly unsaturated carboxylic acids and an amine compound, either isobutylamine or 2‐methylbutylamine. The presence of these unsaturated bonds makes them susceptible to oxidation, but they are safe in the form of natural plant matrix (Bone, 1997). Other components present in purple coneflower are fructose and fructan polymers. These compounds increase in E. purpurea during the winter, and this increase occurs later in the season in the case of E. angustifolia. In addition, fructans are present in the aerial parts of E. purpurea 10 times less than in the roots, whereas these compounds were almost absent in stems of E. angustifolia. Besides all of the above mentioned compounds, other constituents of Echinacea plants include phytosterols, n‐alkanes, and inorganic elements, for example, potassium, calcium, magnesium, iron (III), aluminum sulfate, carbonate, chloride, and silicate, which are also believed to play a role in the biological properties of these plants (Bauer, 1999). Concerning the effect of the extraction method used, most studies focused on either an aqueous “pressed juice” or ethanol extract from the aerial parts or roots of the dried plants. The main chemical components, such as caffeic acid derivatives, alkylamides, and polysaccharides, can differ noticeably between these types of preparations (Hudson, 2011). 5 THE GENUS ECHINACEA IN TRADITIONAL MEDICINE Three of the most used medicinal plant species belonging to the Echinacea genus are E. purpurea, E. angustifolia, and E. pallida, known all of them as purple coneflower, black sampson, pale purple coneflower, among others (Barnes, Anderson, Gibbons, & Phillipson, 2005; Barrett, 2003; Flannery, 1999; Gurib‐Fakim, 2006). The indigenous traditional healers of the native North American tribes such as Cheyenne, Choctaw, Dakota, Delaware, Fox Kiowa, Montana, Omaha Pawnee, Ponca, Sioux, and Winnebago prescribed different preparations of E. angustifolia, E. purpurea, and E. pallida for pain relief, skin inflammatory conditions, wound treatment, as an antidote against various poisons, snakebites, for symptoms associated with the common cold, infectious diseases, and among others (Barrett, 2003; Borchers, Keen, Stern, & Gershwin, 2000; Flannery, 1999; Gurib‐Fakim, 2006; Guz, Puk, Walczak, Oniszczuk, & Oniszczuk, 2014; Guz, Sopinska, & Oniszczuk, 2011). Echinacea spp. are ranked in third position on the 25 best‐selling herbal dietary supplements in the US mainstream multioutlet channel in 2015 being retail sales of 60.1 million US dollars in rounded figures (Andrew & Izzo, 2017). The parts generally used of this plant are roots and aerial parts, and the condition most frequently used is cold as immunostimulant. Even though the origin of the Echinacea genus is located in North America, these plants have been widely used in other countries and cultures. Traditional knowledge was introduced to the European settlers that documented it in different ways. As commented before, some uses of E. purpurea were mentioned in Flora Virginica, edited by Laurens Thodore Grenovius in 1762. This report was later cited in 1787, in the Materia Medica Americana by Johann Schöpf. Since then, physicians and medical botanists prescribed Echinacea due to its therapeutic properties. In 1880, its large‐scale production as a tincture started by John Lloyd, and hence, Echinacea gained popularity, and its therapeutic efficacy was known. Consequently, the aforementioned species were included in the fourth edition of The National Formulary in 1916. They remained in this compendium until 1947 in its eighth edition (Flannery, 1999; Lans, 2016). Traditionally, Echinacea species have been described as “anti‐infective” agents, though the modern interest in the genus Echinacea is focused on its immunomodulatory activity (Barnes et al., 2005; Barrett, 2003; Šutovská et al., 2015; Torkan, Khamesipour, & Katsande, 2015). Noteworthy, the antimicrobial and immunomodulatory activities may be related in plants, possibly due to synergistic effects on host immune system and against microbes (Patwardhan & Gautam, 2005; Tan & Vanitha, 2004). Indeed, it is important to notice the holistic approach followed by traditional healing systems. For instance, in the Indian Ayurveda system of medicine, E. angustifolia is known as an immunomodulator to treat respiratory conditions. Similarly, in the traditional Chinese medicine, E. purpurea is named Song Guo Ju and is prescribed to prevent and treat upper respiratory tract infections (Kumar et al., 2012; J. Li, Li, & Zhang, 2015; Lü et al., 2015; Patwardhan & Gautam, 2005). 6 THE GENUS ECHINACEA PLANTS AS ANTIOXIDANTS AND FOOD PRESERVATIVES Preservatives can be defined as “substances which prolong the shelf life of foodstuffs by protecting them against deterioration caused by microorganisms” (Ohlsson & Bengtsson, 2002). The interest toward safer, more effective, and economical sources of natural antioxidants and preservatives is increasing (Salehi et al., 2018; J. Sharifi‐Rad, Ayatollahi, et al., 2017; J. Sharifi‐Rad, Salehi, Schnitzler, et al., 2017). The natural preservatives, generally, possess little or no harmful effects (J. Sharifi‐Rad, Salehi, Varoni, et al., 2017; J. Sharifi‐Rad, Salehi, Stojanović‐Radić, et al., 2017). These chemical agents can be obtained from plants, animals, microbes, and their metabolites. Natural preservatives are divided into three groups based on mode of action: (a) antimicrobials, which inhibit the bacterial and fungal growth; (b) antioxidants, which hinder product oxidation; and (c) inhibitors of ripening and enzymatic processes which occur in harvested foods (Anupama, Sharmaa, & Garima, 2010). In general, extracts of E. purpurea, as potential natural preservatives, have shown antioxidant and antimicrobial activities (Sabouri, Barzegar, Sahari, & Naghdi Badi, 2012). This section is focused on the antioxidant potential of Equinacea plants; the Table 1 summarize the experiment related to the antioxidant and other biological activities. Table 1. Biological activities of Echinacea spp Biological activity Compound or extract Experimental model Key results Reference Antioxidant activity Ethanol extract of E. purpurea DPPH assay Antioxidant power higher than ascorbic acid, EC50 value of 3.02 and 15.36 ml/L, respectively. Georgieva et al. (2014) Ethanol extract of E. purpurea aerial parts DPPH assay EC50 value of 76.7 μg/ml Jukić et al. (2015) Methanol extracts of E. angustifolia, E. pallida, and E. purpurea roots DPPH assay The average EC50 values for E. purpurea, E. pallida, and E. angustifolia were 134, 167, and 231 μg/ml, respectively. Pellati et al. (2004) Food preservatives Methanol extract of E. purpurea Antioxidant agent in cakes and compared with BHA Slow increase in peroxide values compared with the control sample and BHT. Sabouri et al. (2012) Antibacterial activity E. angustifolia DC. In vitro (respiratory tract and skin pathogens) and clinical trials In vitro results of antimicrobial activity are not confirmed in clinical trials. European Medicines Agency, CoHMP, (2012) Six Echinacea commercial ethanol extracts (E. angustifolia and E. purpurea roots and E. purpurea aerial parts) 15 bacterial strains L. pneumophila, C. difficile, P. acne, and H. influenza reduced approximately three log10 reductions in colony forming unit by the products. M. Sharma et al. (2008) Ethanol extract of E. purpurea aerial parts and roots L. pneumophila, S. pyogenes, M. smegmatis, and H. influenza H. influenzae and L. pneumophila were also readily inactivated, and their proinflammatory responses reversed. S. Sharma, Anderson, Schoop, and Hudson (2010) Ethanol extract of E. purpurea aerial parts and roots Standard strains and seven clinical isolates of P. acnes Bacterial growth decreased at 40 mg/ml and was completely inhibited at 160 mg/ml. M. Sharma, Schoop, Suter, and Hudson (2011) Ethanol extract E. angustifolia roots S. aureus No antimicrobial activity. Snowden et al. (2014) Hydroethanol extracts of E. purpurea aerial parts E. coli, P. aeruginosa, B. subtilis, and S. aureus E. coli and B. subtilis were significantly inhibited; the other strains were sensitive. Stanisavljević et al. (2009) E. angustifolia extracts (petroleum ether, methanol, and water) B. subtilis, S. aureus, E. coli, and S. epidermidis The petroleum ether and methanol extract showed a MIC ranged from 400 to 500 μg/ml and 300 to 500 μg/ml, respectively. The aqueous extract exhibited a lower inhibition, MIC ranged from 200 to 300 μg/ml. Rehman, Sudhaker, Roshan, and Khan (2012) Fermented extract of E. purpurea (5% w/v fermented with Lactobacillus plantarum) E. coli, E. aerogenes, E. durans, Y. enterocolitica, W. confusa, L. lactis, P. jensenii, L. sakei, and B. megaterium Growth inhibition of most of the strains tested. No activity was observed for L. sakei. Rizzello et al. (2013) Note. DPPH = 2,2‐diphenyl‐1‐picrylhydrazyl; BHA = butylated hydroxyanisole; BHT = butylated hydroxytoluene; MIC = minimum inhibitory concentration. Nowadays, there is an increasing demand for natural antioxidants. This is explained by the fact that the synthetic ones, such as butylated hydroxyanisole, butylated hydroxytoluene, and tert‐butyl hydroquinone, can cause harmful effects (Sabouri et al., 2012). As a note, E. purpurea proved to be nontoxic or very slightly toxic when examined in mice, rat, and also humans, even administered intravenously at high doses (Stanisavljević et al., 2009). Oxidative stress is an imbalance status where the formation of reactive oxygen species overpasses the cellular antioxidant capacity. This phenomenon has turned to a concern and represented the topic of plenty of researches in the field of food and nutrition sciences (Lee, Lin, Yu, & Lee, 2017). The antioxidant activity of E. purpurea extracts has been already reported in several studies. In this way, Georgieva, Christova‐Bagdassarian, and Atanassova (2014) evaluated the antiradical capacity of an E. purpurea ethanol extract using the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay. The antioxidant power was higher than that showed by ascorbic acid (vitamin C), and it was related to the total phenol content (353.6 mg gallic acid equivalents/100 ml). These components can act in several ways: as reducing agents, hydrogen donors, singlet oxygen quenchers, and metal chelating agents (Georgieva et al., 2014). Pellati, Benvenuti, Magro, Melegari, and Soragni (2004) reported that methanol extracts of E. angustifolia, E. pallida, and E. purpurea roots as well as Echinacea herbal medicines scavenged the synthetic free radical DPPH (EC50 from 104 to 2,165 μg/ml). Moreover, these authors quantified the caffeoyl derivatives, that is, caftaric acid, chlorogenic acid, caffeic acid, cynarin, echinacoside, and cichoric acid (Figure 2), present in the extracts (ranging from 1.08 to 23.23 mg/g), which contributed to the observed antioxidant activity. Among them, the most active compound was echinacoside (EC50 6.6 μM; Pellati et al., 2004). Therefore, in view of the radical scavenger activity of caffeic acid derivatives, Echinacea root extracts have interest as a source of them (Pellati et al., 2004). In another study, an ethanol extract of E. purpurea aerial parts showed an EC50 value of 76.7 μg/ml, in the DPPH assay, and efficiently scavenged superoxide anion radicals. These effects were attributed to antioxidant compounds, including phenolic compounds (Jukić, Habeš, Aldžić, Durgo, & Kosalec, 2015). Extracts of the roots of E. purpurea, E. angustifolia, and E. pallida exhibited antioxidant properties in a free radical scavenging assay and in a lipid peroxidation assay. Cichoric acid and verbascoside predominated in E. purpurea roots, cynarine, and dodeca‐2E,4E,8Z,10Z/E‐tetraenoic acid isobutylamide were the major components of E. angustifolia roots, whereas echinacoside and 6‐O‐caffeoylechinacoside were the most abundant in E. pallida roots (Sloley et al., 2001). image Figure 2 Open in figure viewerPowerPoint Chemical structures of caffeoyl derivatives found in Echinacea roots Lipid peroxidation and fungal growth limit the shelf‐life in cake production, and so they are critical issues to be controlled. In a study carried out by Sabouri et al. (2012), a methanol extract of E. purpurea was evaluated as an antioxidant agent in cakes and compared with butylated hydroxyanisole. The antioxidant activity was determined by measuring peroxide and thiobarbituric acid values during 60 days storage at 25 °C. The E. purpurea‐treated samples showed a slow increase in peroxide values compared with the control sample (without any antioxidant and preservative) and butylated hydroxytoluene (100–200 ppm) during the storage time. A similar trend was found for the acidity values. Moreover, the extract used at 1,000 and 1,500 ppm improved color, texture, taste, and overall acceptability of cakes compared with the synthetic antioxidant. This extract was also effective against the growth of C. albicans and S. cerevisiae. Therefore, E. purpurea extracts are suitable as replacer for synthetic antioxidants and antimicrobial preservatives, though its concentration should be controlled to avoid pro‐oxidant effects (Sabouri et al., 2012). In conclusion, the antioxidant activity of ethanolic and methanolic extracts of E. purpurea has been already reported in several studies using methods such DPPH assay. 7 ANTIBACTERIAL ACTIVITIES OF THE GENUS ECHINACEA PLANTS There exist several reports on the traditional uses of plants belonging to the genus Echinacea for the treatment of diseases related to bacterial infections. Three species, E. purpurea, E. angustifolia, and E. pallida, were traditionally used as antibacterial herbal remedies. Tribess et al. (2015) discussed the use of E. purpurea in Brazil for the treatment of infections, where leaf infusion is applied externally in the infected area. However, authors did not specify the type of infections, and therefore, the topical use is probably indicated for skin lesions (Tribess et al., 2015). In Germany, Jeschke et al. (2009) mentioned the use Echinacea spp. plants as herbal remedies both alone and in association with other species. In particular, the syrup Contramutan N is made with the species E. angustifolia and is indicated for the treatment of fever and acute upper respiratory infections. However, its composition (the parts of the plant used) and the form of preparation are not mentioned. Furthermore, E. pallida (ex herba 20%, HAB, Vs. 12c, 1.0 g/10 g), Calendula officinalis (20%, HAB, Vs. 12c, 1.0 g per 10 g), and other plants are formulated in the Echinacea Mund und Rachen spray used for the treatment of acute pharyngitis. A number of studies have been carried out to investigate and to prove the traditionally reported antibacterial activity of the plants belonging to the genus Echinaceae and their preparations. The antibacterial activity of the fermented extract of E. purpurea (5% w/v fermented with Lactobacillus plantarum) was investigated by Rizzello et al. (2013), though they did not mention the plant part used. The disc diffusion and broth microdilution assays were carried out on E. coli, Enterobacter aerogenes, Enterococcus durans, Yersinia enterocolitica, Weissella confusa, Leuconostoc lactis, Propionibacterium jensenii, Lactobacillus sakei, and Bacillus megaterium bacterial strains. The results showed that the fermented extract inhibited the growth of most of the strains tested, with the most significant result obtained for the B. megaterium and L. lactis strains with inhibition halos >3.5 and 2.5–3.5 mm, respectively. No activity was observed for L. sakei (Rizzello et al., 2013). S. Sharma et al. (2010) investigated the bactericidal activity of the ethanol extract (65%) from freshly harvested aerial parts and roots of E. purpurea, at concentrations of 40 and 120 mg of dry mass/ml by microdilution in tubes using light and dark assays. The extract was assayed against strains responsible for respiratory infections: L. pneumophila, Streptococcus pyogenes, Mycobacterium smegmatis, and H. influenzae. S. pyogenes, H. influenzae, and L. pneumophila strains were sensitive to the highest concentration of the extract. At the lowest concentration, the extract was not effective, as it did not significantly inhibit bacterial growth. There was no significant difference in activity in relation to the presence or absence of light (S. Sharma et al., 2010). These authors (M. Sharma et al., 2011) also evaluated the antibacterial activity of the ethanol extract from the roots and aerial parts of E. purpurea, commercially produced as a natural remedy (Echinaforce®), by serial dilution method. They performed experiments on standard strains and seven clinical isolates of Propionibacterium acnes, a major causal agent of acne. The tested concentrations of the extract were 40–160 mg/ml of dry mass per volume, as indicated by the manufacturer. Bacterial growth decreased at 40 mg/ml and was completely inhibited at 160 mg/ml. In another study using the same methodology, M. Sharma et al. (2008) analyzed six different Echinacea commercial ethanol extracts (detailed specifications and product batches were not reported), including extracts from E. angustifolia and E. purpurea roots and E. purpurea aerial parts. Five out of 15 bacterial strains were susceptible to the products, namely, L. pneumophila, Clostridium difficile, P. acne, and H. influenzae, with approximately three log10 reductions in the colony forming units (M. Sharma et al., 2008). Stanisavljević et al. (2009) using an agar diffusion experiment evaluated the hydroethanol extracts (obtained by conventional and ultrasonic extractions) of the aerial parts of E. purpurea (20 mg/ml). The standard strains used were E. coli ATCC 25922, P. aeruginosa ATCC 9027, Bacillus subtilis ATCC 6633, and S. aureus ATCC 6538. The diameters of the inhibition zone of the microorganisms were higher for conventional extracts. All the strains were sensitive, though only E. coli (11.2 ± 0.2 mm) and B. subtilis (10.9 ± 0.1 mm) were significantly inhibited. Rehman et al. (2012) verified the antibacterial potential of E. angustifolia extracts obtained by solvents in increasing order of polarity, that is, petroleum ether, methanol, and water, evaluating them in different concentrations (0.5, 1.5, and 1.5 mg/ml), against B. subtilis, S. aureus, E. coli, and Staphylococcus epidermidis. The part of the plant from which the extracts were prepared was not mentioned. The aqueous extraction was less effective than that of other solvents. The petroleum ether extract showed inhibition halos of 8.0, 12.0, and 18.0 mm (in increasing concentration) against S. aureus, and, against B. subtilis, halos were 7.0, 13.0, and 18.0 mm, which were the most sensitive strains. The minimum inhibitory concentration (MIC) ranged from 400 to 500 μg/ml. For the methanol extract, the most significant inhibition halos were for S. aureus (6.0, 11.0, and 16.0 mm) and E. coli (8.0, 11.0, and 16.0 mm), and the MIC varied from 300 to 500 μg/ml. The aqueous extract exhibited a lower inhibition when compared with the other extracts (S. aureus, 3.0, 6.0, and 10.0 mm, and S. epidermidis, 3.0, 7.0, and 10.0 mm), although the MIC ranged from 200 to 300 μg/ml (Rehman et al., 2012). Conversely, Snowden et al. (2014) tested the ethanol extract (95% ethanol/distilled water/glycerol) from E. angustifolia roots, and no antimicrobial activity against S. aureus was documented using the broth dilution methodology (Snowden et al., 2014). Respiratory infections may be caused by a number of pathogenic bacteria, such as Streptococcus pneumonie, H. influenzae, Moraxella catarralis, S. pyogenes, Bordetella pertusis, Mycoplasma pneumonie, S. aureus, P. aeruginosa, Burkholderia cepacia (Cappelletty, 1998; Tümmler & Kiewitz, 1999), and among others. In this regard, Echinacea spp. plants exhibited a promising antibacterial potential against microorganisms involved in these infections, as demonstrated by M. Sharma et al. (2008), Stanisavljević et al. (2009), and Rehman et al. (2012). In addition, L. pneumophila, the responsible microorganism for pneumonia, can be also inhibited by E. purpurea (Hudson, 2011). Nevertheless, for S. aureus, controversial results have been reported, probably due to differences among the Echinacea species, plant parts, extraction procedures, and consequently, chemical compositions. Unfortunately, no phytochemical information was provided in most of these studies. Anyway, M. Sharma et al. (2008) suggested that neither alkylamides nor polysaccharides were individually responsible for the direct bactericidal activities reported. Besides, no correlation between bactericidal activity and caffeoyl derivatives was found (M. Sharma et al., 2008). On the contrary, Stanisavljević et al. (2009) correlated the antimicrobial activity to total phenol and flavonoid contents. A number of microorganisms, including P. acnes, S. aureus, S. pyogenes, P. aeruginosa, Pasteurella multocida, Capnocytophaga canimorsus, Bartonella spp., Klebsiella rhinoscleromatis, and Vibrio vulnificus, form an important group of skin pathogenic bacteria (Chiller, Selkin, & Murakawa, 2001; Stulberg, Penrod, & Blatny, 2002). M. Sharma et al. (2008), S. Sharma et al. (2010), M. Sharma et al. (2011), Stanisavljević et al. (2009), and Rehman et al. (2012) showed that active constituents with antimicrobial properties against skin pathogens are present in Echinacea spp. plants, thus corroborating the reported ethnomedicinal use of this genus for the treatment of skin infections. Although it seems that species of the genus Echinacea can exert a promising in vitro antimicrobial activity against respiratory tract and skin pathogens, E. angustifolia and E. purpurea did not confirmed these results in clinical trial (European Medicines Agency, CoHMP, 2012). Therefore, more studies in humans are required to evaluate the effectiveness of Echinacea species against the aforementioned infectious diseases. 8 CONCLUSIONS AND FUTURE PERSPECTIVES The main chemical components of Echinacea species are volatile terpenes, such as germacrene D, high molecular weight polysaccharides, polyacetylenes, highly unsaturated alkamides, phenolic compounds, and glycoproteins. However, these constituents showed high chemical variability, due to several endogenous and exogenous factors, including genetic traits, plant organs (leaves, flowers, stems, or roots), climatic factors, growing, drying and storage conditions, type of extraction, and diseases affecting the plant. Therefore, another crucial aspect is the standardization of the extracts and the EOs obtained from Echinacea plants before using. The genus Echinacea were traditionally used in the United States. Then, due to its popularity as effective herbal remedy, its use was disseminated to other countries. Today, it is the basic ingredient of several supplements prescribed to boost the immune system or treat respiratory disorders. Indeed, several preclinical studies have shown the antimicrobial activity of Echinacea preparations against pathogens responsible for respiratory diseases. In addition, skin pathogens represent potential targets. However, further clinical studies are needed to corroborate these effects in humans. Similarly, further studies are required to ascertain the mainly bioactive phytochemicals responsible for the observed antimicrobial activity. Among other bioactivities reported for these plants, their antioxidant properties are interesting to be exploited in the food industry. The in vitro antiradical capacity of E. purpurea extracts has been documented in several studies, scavenging DPPH and superoxide anion radicals and decreasing peroxides levels. Phenolic compounds, mainly, caffeoyl derivatives, were indicated as the potential active components. These compounds can act in several ways: as reducing agents, hydrogen donors, singlet oxygen quenchers, and metal chelating agents. Therefore, the use of Echinacea products in foods seems to be promising as natural antioxidant agents and antimicrobial preservatives, particularly as novel components of active packaging systems. Finally, the use extraction methods alternative to the conventional ones, using food grade solvents and green extraction methods, for example, supercritical fluid extraction using CO2 or pressurized water extraction, should also be promoted. CONFLICT OF INTEREST The authors declare that they have no competing interests. REFERENCES Notes : Note. DPPH = 2,2‐diphenyl‐1‐picrylhydrazyl; BHA = butylated hydroxyanisole; BHT = butylated hydroxytoluene; MIC = minimum inhibitory concentration. Citing Literature Publication cover image Volume32, Issue9 September 2018 Pages 1653-1663 Figures References Related Information Metrics Citations: 9 Details Copyright © 2018 John Wiley & Sons, Ltd. Keywords Asteraceae Echinacea purpurea ethnobotany ethnopharmacology herbal remedies immunomodulatory activity Publication History Issue Online: 06 September 2018 Version of Record online: 10 May 2018 Manuscript accepted: 05 April 2018 Manuscript revised: 01 April 2018 Manuscript received: 26 November 2017 About Wiley Online Library Privacy Policy Terms of Use Cookies Accessibility Help & Support Contact Us Opportunities Subscription Agents Advertisers & Corporate Partners Connect with Wiley The Wiley Network Wiley Press Room Copyright © 1999-2018 John Wiley & Sons, Inc. All rights reserved back