A Comparison of the Antimicrobial Activities of Cultivated Echinacea angustifolia (Purple Coneflower) versus Wild E. angustifolia

Ech2Marlee Finley, Levi Binstock, and Mandy Guinn
Environmental Science Department, United Tribes Technical College, Bismarck, ND 58504

Abstract

Medicinal plants have been have been used for centuries to treat various diseases across the world. While plant extracts have been used to synthesize modern commercial drugs, so far only a small percent of traditionally prescribed plant medicines have been studied for their therapeutic value. In recent years, the American public has become enamored with herbal remedies, yet there continues to be a relative scarcity of scientific research. Echinacea (purple coneflower) has received global attention because of its potential for medicinal value. Extensive laboratory and clinical research on Echinacea angustifolia in the last few years in Germany has confirmed its immunostimulatory, antiviral, and antibacterial benefit to humans. The purpose of this study is to use the agar-well diffusion method to compare antimicrobial activity of cultivated and wild E. angustifolia. We hypothesize that cultivated E. angustifolia will show more antimicrobial activity against five different strains of bacteria (two Gram-negative, three Gram-positive) due to being cultivated under ideal conditions.

Introduction

Despite tremendous progress in human medicine, infectious diseases caused by bacteria, fungi, viruses and parasites are a growing concern for public health as human population increases. The complete eradication of infectious diseases is almost impossible. Bacteria continue to evolve new modes of resistance in response to overuse of antibiotics. Antimicrobial resistance in bacteria is a rising global concern (Siddiqi et al. 2011). The impacts of these threats are particularly damaging for developing countries due to the lack of sanitations, availability of medicines, and the emergence of widespread drug resistance (Okeke et al. 2005). Because of the rapid evolution of microbes, it is critical that research focuses on finding new antimicrobial substances (Clardy & Walsh 2004).

Plants are a future source of antimicrobial agents in different countries (Alviano & Alviano 2009). About 60 to 90% of populations in developing countries use plant-derived medicine. Traditional cultures use plant extracts as herbal medicine for the treatment of infectious diseases (Alviano & Alviano 2009; Malini et al. 2013; Zhang et al. 2006). Plants are rich in a variety of phytochemicals including tannins, alkaloids, and flavonoids which have been found in vitro to have antimicrobial properties (Dorman & Deans 2000; Talib and Mahasneh 2010). Although the mechanism of action and efficacy of these herbal extracts in most cases are unknown, these preparations can mediate important host responses (Cruz et al. 2007; Ruberto et al. 2000).

In recent years, the American public has become enamored with herbal remedies, yet there remains a scarcity of scientific research (Lindenmuth & Lindenmuth 2000). Consumer interest in herbs and botanicals is growing; sales of herbal products have grown by about 10-15% per year since the passage of Dietary Supplements Health and Education Act (DSHEA) in 1994. Barrett et al. (1999) reports several estimates, ranging from 10%, 15%, and 32%, of Americans who use medicinal botanicals in a given year.

Yet, most medical professionals caution against such self-medication and many herbal products contain warnings, especially for pregnant or nursing women.

Preparations of three Echinacea species, commonly known as purple coneflower, have become the bestselling herbal immunostimulants in Europe and North America. Most products are manufactured from the parts of the plant which are above ground and/or root of E. purpurea or from the roots of E. angustifolia and E. pallida (Lindenmuth & Lindenmuth 2000). In 1998, Echinacea was the tenth most important medicinal plant sold in Europe with annual sales of about $120 million. In North America, Echinacea is listed as the first among 11 top-selling herbal extracts (Yu & Kaarlas 2004). Retail sales of Echinacea products are more than $158 million annually in the USA and have been estimated at $1.3 billion annually worldwide (Blumenthal 2003).

Echinacea is a North American native plant used for medicinal purposes by indigenous Americans (Hill et al. 1996). Prior to European colonization a number of Native American nations, including the Blackfoot, Cheyenne, Choctaw, Comanche, Dakota, Delaware, and Lakota, used various Echinacea preparations for a variety of purposes (Flannery 1999; Foster 1991). Echinacea preparations in various preparations were used to treat such problems as wounds, snakebites, tonsillitis, headache, and cold symptoms (Hobbs 1989).

The Cheyenne people of Oklahoma and Montana prepared an aqueous infusion of the leaf and root of E. angustifolia for treatment of sore throat (Moerman 1998). The Dakota used the freshly scraped root as a remedy for rabies, snakebite, and situations where a wound had putrefied (Smith 1928). The Dakota applied the root (probably ground up) to areas of inflammation to relieve the sensation of burning by its “feeling of coolness” (Gilmore 1913). The Lakota used a compress of chewed root to soothe bites and stings and made a tincture to boost immunity and relieve toothaches, sore throats, and stomach-aches (Different Cloud-Jones and Flying By 1998). The Kiowa have used the purple coneflower root as a cough medicine since ancient times (Vestal and Schultes 1939). The Crow chewed the root for colds and drank a tea prepared from the root for colic (Hart 1976). Hidatsa warriors were known to chew small pieces as a stimulant when traveling all night (Nickel 1974). The Comanche used the root for treating sore throat and toothache (Carlson and Jones 1939). Many of these traditional uses are still practiced.

Medicinal plants have been used for centuries to treat various diseases all over the world (Vaghasiya & Chanda 2007). Only a small percentage of traditionally prescribed plant medicines have been studied in a lab setting to determine their therapeutic value. Plant phytochemicals could provide alternative classes of antibiotics having different target sites than current antibiotics, which may be effective against resistant pathogens (Oskay & Sari 2007). Recent laboratory and clinical research on E. angustifolia in the last few years in Germany has confirmed its immunostimulatory, antiviral, and antibacterial benefit to humans (Bauer & Wagner 1991; Bodinet & Beuscher 1991; Bodinet et al. 1993; Parnharm 1996).

Cultivation of Echinacea in small beds may allow for much easier harvesting and use of this medicinal plant by a wider range of people. The aim of this study is to determine if cultivated E. angustifolia has similar antimicrobial properties to its wild-grown counterpart. The activity of the extracts from cultivated versus wild E. angustifolia against several Gram-positive and Gram-negative bacterial strains will be tested using a well-diffusion assay test. We expect that the cultivated plants will have increased antimicrobial activity due to being grown under ideal conditions.

Methods

Collection of Plant Material—

Whole plant samples of wild E. angustifolia will be collected from known established sites near Mandan, North Dakota. Cultivated varieties of E. angustifolia will be purchased from a commercial supplier (morningskygreenery.com). Fresh root samples will be washed under running tap water, air dried and then stored in airtight bottles in a cool, dark place.

Extraction of Plant Material—

Extraction procedures will replicate Parekh and Chanda’s (2006) study on plant antimicrobial activity. The root sample will be homogenized to a fine powder and stored in airtight bottles until future testing can be completed. When all the samples have been processed, 10 g of the air-dried powder will be placed in 100 ml of methanol in a conical flask. The flask will be plugged with cotton wool and then shook on a rotary shaker at 190-220 rpm for 24 hours. After 24 hours, the supernatant will be collected and the solvent will be evaporated to make the final volume one-fourth the original volume. The sample will be stored at 40C in airtight bottles (Harbone 1973).

Bacterial Strains—

Microorganisms will be obtained from a commercial supplier (atcc.org). Five microorganisms were selected due to human susceptibility to infection, while also being Level 1 Biohazards (which we are able to process at United Tribes Technical College Science Center). Of the five microorganisms, three are gram-positive bacteria, these include Bacillus cereus (ATCC11778), Staphylococcus epidermis (ATCC14990), and Micrococcus luteus (ATCC49732). The two other microorganisms are gram-negative bacteria and include Enterobacter aerogenes (ATCC13048), and Escherichia coli (ATCC25922). All the microorganisms will be maintained at 4oC on nutrient agar slants.

Media Preparation and Antibacterial Activity—

The antimicrobial assay will be performed using an agar well-diffusion method according to Bauer et al. (1966). For this method, six wells will be prepared in the plates using a cork-borer (0.85 cm). Into this well, 100 µl of the plant extract will be introduced. The plates will then be incubated overnight at 370C. Antimicrobial activity of the plant extract will be determined by measuring the diameter of the zone of bacterial-growth inhibition around each well. For each bacterial strain, controls will be maintained where pure solvents will be used instead of extract. This experiment will be replicated repeated three times and the mean values will be analyzed. A t-test will be used to determine differences between the antimicrobial activity of wild and cultivated Echinacea.

This project will be conducted in the labs of the Science & Technology Center at United Tribes Technical College during the spring 2016 semester.

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Author Biographies

I was born in Valley City, ND (lived there until I was 1), but I grew up in Bismarck, North Dakota. I did not have much research experience before participating in the INBRE NARCH program last summer and it helped me be more prepared for what was to come for my school project during the current year. My favorite courses are microbiology, anatomy and physiology, and any math classes (except geometry). My hobbies are playing in basketball leagues and tournaments, reading, and I also like to play some video games. I have one child, a son. He is 2 years old and his name is Josiah Wayne Jr. My plans after getting my BS degree in Environmental Science is to hopefully get into pharmacy school, or possibly graduate school, depending on how life goes. I really enjoy Environmental Science, which has surprised me because I was always most interested in medicine. I am thankful that I got a chance to be in the program at United Tribes and expand my horizons.

Levi Binstock is an instructor in the Tribal Environmental Science Department at United Tribes Technical College in Bismarck, North Dakota. He teaches courses in botany, soils, ecology, and geographic information systems. Levi is a graduate of North Dakota State University’s Animal and Range Science program, and also possesses a GIS Technician Certification from Bismarck State College. Before teaching at UTTC, he worked for both private consulting firms and for the USDA’s Natural Resources Conservation Service. Levi’s research interests are focused mainly on rangeland health and ecology, along with other agricultural topics and issues within the northern Great Plains. Originally from south-central North Dakota, Levi now lives in Bismarck with his wife, Brianne, and daughter, Lena.

Mandy Guinn has taught at United Tribes Technical College in Bismarck, ND for the past nine years. She teaches general courses such as Intro to Biology and Chemistry and advanced courses including Research Techniques and Laboratory Instrumentation. Over the past six years, she has found her passion, working with undergraduate students in array of field and lab settings, including in-class, externships, and internship programs. She managed a NASA internship program which provided opportunities for Tribal College students to work at NASA Ames Research Center in California and currently is the Director of the USDA-NIFA BATS II program at UTTC. She is also the outreach coordinator for the ND EPSCoR NATURE program at UTTC, which provides science academies for Native American K-12 students. Her research interests include the molecular ecology of bats and their economic role in North Dakota agriculture. She lives in Mandan with her husband, Jeremy, and their two built-in field assistants, Gavin (9) and Xavier (5).