Diversity of antioxidant ingredients among Echinacea species

https://doi.org/10.1016/j.indcrop.2021.113699Get rights and content

Highlights

  • Non-target metabolomic was adopted to explore the difference among Echinacea.

  • Chicoric acid, caftaric acid and echinacoside were the main difference factors.

  • These components were also the active ingredients for antioxidant activity.

  • Different Echinacea can be used for different purposes according to composition.

  • Chicoric acid diversity was determined by precursor content and genes expression.

Abstract

Echinacea species are important medicinal plants with significant therapeutic potential and are widely used in the pharmaceutical industry. Different Echinacea species exhibit various chemical compositions and bioactivities. In the present study, the chemical differences, antioxidant ingredients, and diversity mechanisms of Echinacea species were studied. The aerial parts and roots of six different Echinacea species (E. purpurea (L.) Moench; E. pallida (Nutt.) Nutt.; E. angustifolia DC.; E. atrorubens (Nutt.) Nutt.; E. paradoxa (Norton) Britton var. paradoxa; E. sanguinea Nutt.) were collected for investigation. Through non-target metabolomics following targeted quantitative analysis, chicoric acid, caftaric acid, and echinacoside were found to be the main different components of Echinacea species. Coincidentally, these three chemicals were also the dominant antioxidant ingredients of Echinacea extracts, as determined by correlation analysis between chemical contents and in vitro antioxidant activities. Based on the cloning, sequencing, and measurement of the identified chicoric acid biosynthetic genes, the diversity of chicoric acid and caftaric acid among Echinacea species was determined by the precursor content, as well as by the expression levels of key biosynthetic genes. Overall, these results clarified the chemical differences and the possible mechanisms, as well as the bioactive ingredients, in Echinacea species and can guide the selection of Echinacea species for different industrial applications.

Introduction

The genus Echinacea, belonging to the Asteraceae family, is a group of nine species native to mid-latitude North America (Tang et al., 2017). The distribution of Echinacea covers a wide range of moisture and temperature regimes in North America, from the relative warmth of central Texas, Georgia, and Alabama to the cooler weather of Montana, North Dakota, Minnesota, and Canada (Kindscher, 2016). In the past, Echinacea was used by Native Americans for various ailments, including mouth sores, colds, and cough (Borchers et al., 2000). Echinacea-derived products are widely used as daily supplements worldwide and are marketed and used as immunostimulants to treat and prevent the common cold, influenza, and upper respiratory tract infections (Cao and Kindscher, 2016). Owing to the health benefits, products from Echinacea have drawn increasing attention globally and have become a massive industry. In 2019, in the US market alone, the sales of products from three commonly used species, including E. angustifolia (Ean), E. pallida (Epa), and E. purpurea (Epu) reached 120 million US dollars, an increase of 4.9% compared with the previous year. In addition, in the first half of 2020, Echinacea sales grew sharply by 90.9%, which may be due to the COVID-19 pandemic (Smith et al., 2020).

Modern pharmacology studies on Echinacea have identified many bioactivities, such as antioxidant, immunomodulatory, anti-inflammatory, antifungal, and antiviral activities (Barrett, 2003; Binns et al., 2002; Melchart et al., 1995). Caffeic acid derivatives, alkamides, polysaccharides, polyacetylenes, polyenes, flavonoids, and terpenoids have been linked to these bioactivities (Cao and Kindscher, 2016). In addition, synergistic effects of alkamides, caffeic acid derivatives, and polysaccharides have been reported in E. purpurea (Dalby-Brown et al., 2005). Two important caffeic acid derivatives from Echinacea, chicoric acid and echinacoside, have been widely studied for the remarkable bioactivity and are assumed to be the active ingredients of Echinacea (Aiello et al., 2015; Naveed et al., 2018; Silva et al., 2014). The production, stabilization, and changes during storage have been extensively studied (Bergeron et al., 2002; Dalby-Brown et al., 2005; Lin et al., 2011). The chemical compositions of the three commonly used Echinacea species (Ean, Epa, and Epu) were different, resulting in various bioactivities (Barnes et al., 2010; Erenler et al., 2015; Perry et al., 2001; Sloley et al., 2001; Thomsen et al., 2012). On a broader level, the genome size and chloroplast genome of the Echinacea genus have been compared (Jedrzejczyk, 2020; Zhang et al., 2017).

Recently, the complete biosynthesis pathway of chicoric acid in Echinacea has been successfully elucidated (Fu et al., 2021). Chicoric acid and its two substrates, caftaric acid and chlorogenic acid, originate from phenylpropanoid metabolism. Hydroxycinnamoyl-CoA: quinate/shikimate hydroxycinnamoyl transferase (HCT) catalyzes the synthesis of caffeoyl CoA, the most important precursor; hydroxycinnamoyl-CoA: tartaric acid hydroxycinnamoyl transferase (HTT) catalyzes caffeoyl CoA and tartaric acid to synthesize caftaric acid; hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase (HQT) combines caffeoyl CoA and quinic acid into chlorogenic acid; and caftaric acid and chlorogenic acid are used as acyl acceptor and acyl donor, respectively, by chicoric acid synthase (CAS) to generate chicoric acid. These four biosynthetic enzymes complete the specific chicoric acid biosynthesis and allow the study of the intrinsic determinants of chicoric acid diversity in Echinacea (Fu et al., 2021).

In the present study, the chemical differences among six Echinacea species, namely, Epu, Epa, Ean, E. atrorubens (Eat), E. paradoxa var. paradoxa (Epp), and E. sanguinea (Esa), were compared using non-target metabolomics. The active antioxidant ingredients were determined based on the positive relationship between the observed antioxidant activity and the sum of each active ingredient’s antioxidant contribution. Finally, the potential mechanisms of chicoric acid and caftaric acid diversity among Echinacea species were investigated. All these results will significantly promote the research into Echinacea species, such as the selection of materials for special applications.

Section snippets

Reagents

MS-grade methanol, acetonitrile, and formic acid were purchased from Thermo Fisher Scientific Inc. (MA). Chlorogenic acid (98%, CAS: 327-97-9) was obtained from Chengdu Herbpurify Co., Ltd. (Chengdu, China). Caftaric acid (98%, CAS: 67879-58-7), chicoric acid (98%, CAS: 6537-80-0), echinacoside (98%, CAS: 82854-37-3), fluorescein sodium salt, 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium

The chemical diversity of Echinacea species

Six of the nine known Echinacea species, namely, Epu, Epa, Ean, Eat, Epp, and Esa were germinated and cultivated in a greenhouse under the same conditions for 2 months. The phenotypes of the Echinacea species are shown in Fig. 1a. Among them, Epu showed a distinct phenotype with more fibrous roots and wide blades. The aerial parts and roots were separately extracted and analyzed.

First, a liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based non-target metabolomic analysis was

Conclusion

Echinacea species have received increasing attention owing to the health benefits and great potential for industrial applications. Diversity in chemical composition exists within the genus. In the present study, Echinacea species were cultivated under the same conditions to exclude environmental effects on the chemical contents. The chemical diversity was comprehensively studied and analyzed. Echinacea sanguinea has considerable potential for the production of chicoric acid, in addition to the

CRediT authorship contribution statement

Rao Fu: Conceptualization, Methodology, Investigation, Resources, Data curation, Writing - original draft, Writing - review & editing, Visualization, Project administration, Funding acquisition. Pingyu Zhang: Methodology, Investigation, Resources. Zongbi Deng: Methodology, Investigation, Resources. Ge Jin: Methodology, Investigation, Resources. Yiran Guo: Conceptualization, Writing - review & editing, Project administration, Funding acquisition. Yang Zhang: Conceptualization, Writing - review &

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgments

This work was financially supported by the Natural Science Foundations of China (No. 31800258 and 31670352) and the China Postdoctoral Science Foundation Grant (No. 2018M631080).

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