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Oklahoma Native Plant Record, Volume 15, Number 1, December 78 Oklahoma Native Plant Record Volume 15, December 2015 ANTIFUNGAL ACTIVITY IN EXTRACTS OF PLANTS FROM SOUTHWESTERN OKLAHOMA AGAINST ASPERGILLUS FLAVUS Tahzeeba Frisby Cynthia Thompson Department of Biological Sciences Angela Gibbons Cameron University Kristen Gonzalez 2800 W. Gore Blvd. Renea Lawler Lawton, OK 73505 Victoria Boudiette [email protected] Breanna Jones Tabitha Garner Cameron University Students: Ezekiel Oetinger Brooke Armstrong Kateri Gebhart Chelsey Morin Oluwatoyin Kayode Susan Pustejovskey Sarah McLaughlin Victoria Vanderslice Patrick McAnerney Jackie Harper Jared Stokes Paul Copeland Key words: antifungal, Aspergillus flavus, medicinal plant, undergraduate research ABSTRACT The use of medicinal plants has been an integral part of human civilization since antiquity. Naturally occurring pesticidal compounds are synthesized by the plant defense system, which includes antimicrobial proteins and lower molecular weight natural products. In this study, plants were collected from southwestern Oklahoma, and plant tissues were extracted and assayed for antifungal activity against Aspergillus flavus, a mycotoxin producing fungus. Out of the 84 plant tissue extracts tested, 40 extracts exhibited complete to very strong inhibition of fungal growth. Extracts were dialyzed in Tris buffer using 3,500 molecular weight cut-off dialysis membrane to remove low molecular weight compounds. After dialysis, the majority of the plant extracts lost antifungal activity against A. flavus. Four plant extracts, however, retained complete activity. The source plants of these four extracts were identified as belonging to Asparagaceae. Three of the extracts came from three different plants of the genus Allium. The fourth extract was from Camassia scilloides. INTRODUCTION the power of plants to cure ailments and used 300 different plant species to heal his The history of plant use for medicinal patients. Pedanious Dioscorides (50–70 purpose is as old as human civilization. Six AD) assembled De Materia Medica where he thousand year old excavated clay slabs from described comprehensive use, preparation, early Sumerian civilizations revealed recipes side effects, and cultivation of 600 plants for drug preparation using over 250 (Sumner 2000). There are examples from different plants (Petrovska 2012). every culture about healing abilities of Additionally, an Egyptian scroll dated about plants. Modern research indicates that the 1500 BP mentioned more than 850 plant majority of ethnobotanical claims are valid based medicines (Petrovska 2012). and correspond with our current knowledge Hippocrates (460–377 BP) also believed in of plant-derived compounds. Benefits of Tahzeeba Frisby https://doi.org/10.22488/okstate.17.100115 Oklahoma Native Plant Record 79 Volume 15, December 2015 traditional plants have been explored via Once identified, the active compounds have scientific research, which led to the to be purified and characterized. Many of discovery of many valuable drugs for the these natural compounds are structurally modern world. Examples include reserpine complex. Therefore, to go from discovery from Rauwolfia serpentina (Indian snake root), to high throughput commercial production vincristine from Catharanthus roseus can be technically challenging and time (Madagascar periwinkle), artimisinin from consuming. Some of these issues have Artemisia annua, (sweet sagewort), capsaicin contributed to the decline of plant-based from Capsicum annuum (chili pepper), drug discovery (Gupta et al. 2005). morphine from Papaver somniferum (poppy), However, over the last few decades, the atropine from Atropa belladonna (deadly night world has watched the reemergence of shade), silymarin from Silybum marianum infectious diseases once thought to be (milk thistle), and ephedrine from Ephedra eradicated (Gupta et al. 2005; Lam 2007; sinica (Chinese ephedra) (Farnsworth et al. Petrovska 2012). At the same time, 1985; Houghton 1995; Sumner 2000; Gupta, incidences of pest and pathogen resistance et al. 2005; Goutam 2015). against antimicrobial products have Plants provide a rich source for the increased in alarming numbers. There is also discovery of new drugs. A number of an increase in the prevalence of multidrug bioactive compounds can be isolated from resistant bacterial pathogens. In light of different parts of a single plant. Azadirachta these facts, there is renewed interest in indica (neem) is one such plant. Neem plant looking into nature’s wealth for newer and is known as the ‘village dispensary’ in India better medicines. because of its wide spectrum of biological Plants are a storehouse of naturally activities and no-cost availability due to the occurring pesticidal compounds that are widespread growth of this plant in the molecularly diverse. Plants have developed region (Arora et al. 2008; Asif 2013). Over an arsenal of defense mechanisms from 135 bioactive compounds have been protection against pests and pathogens. As a isolated from different parts of this plant. result, different and unique chemicals Some are well known to exhibit antiviral, compounds are synthesized by plants. These antifungal, antibacterial, anti-insect, and diverse molecules in plants are under antitumor activity. There are over 250,000 constant evolutionary selection. This makes plant species in the world, but only 6% have the plant kingdom a continuously wealthy been screened for biological activity. source for finding new antimicrobial According to Farnsworth et al. (1985), there compounds (Hossain 1999). are 119 plant-derived drugs used today all The compounds that are synthesized by over the world, and all of those came from plant defense systems, either to prevent less than 90 plant species. The possibility of pathogen attack or to destroy invading finding novel compounds from plants that pathogens, include proteins and lower can be exploited for medicinal use is molecular weight natural products. Defense- enormous. related proteins produced by plants include Although the origin of many life-saving hydroxyproline-rich glycoproteins; glycine- modern medicines came from natural rich proteins; amylase inhibitors; proteinase sources, tremendous achievements in inhibitors; toxic proteins such as lectins and synthetic chemistry have made it possible thionins; hydrolases such as chitinases and for pharmaceutical companies to design and β-1,3-glucanases; anti-microbial peptides introduce drugs at a faster pace. To find such as defensins; and other cysteine-rich medicinal compounds from a plant source, a proteins (Hossain 1999). Lower molecular large number of plants need to be screened. weight natural products include various Tahzeeba Frisby 80 Oklahoma Native Plant Record Volume 15, December 2015 alkaloids, tannins, flavonoids, terpenes, etc. MATERIALS AND METHODS (Goutam 2015). Southwestern Oklahoma has a rich Collection of Plant Materials history in the use of medicinal plants. Forty-seven species of plants were Jordan et al. (2006) documented the use of collected from four different locations in over 100 species of vascular plants by the southwestern Oklahoma (Table 1). These Plains Apache tribe. Thirty-nine of those locations include Medicine Park, East species were used in rituals and for Lawton, Stephens County, and Anadarko. medicinal purposes. According to the study, Plants were collected based on out of the 105 documented species used by ethnobotanical information (Jordan et al. the tribe, 98 are native to southwestern 2006) as well as field observation. Oklahoma and occur throughout the Therefore, not all of the collected plants western U. S. and Great Plains (Jordan et al. have known medicinal use. The field 2006). Students from the Cameron observations of healthy plants growing in University Biology Department collected the midst of plants infested by pests and plants from four different locations in pathogens could indicate defense related southwestern Oklahoma. Aqueous crude compounds protecting these plants. Such and dialyzed extracts from collected plants field observations were a part of the were screened for antifungal activity against collection process. On location, the Aspergillus flavus, which was chosen because collected materials were photographed, this fungus produces carcinogenic bagged, and kept in ice. Once transported to the laboratory, the plant materials were mycotoxins known as aflotoxins. This study o was part of an assignment for a medicinal placed in -80 C for long term storage. plants class. In this report we present the result of the study. Table1 List of plants screened for antifungal activity against Aspergillus flavus PCN, Plant Collection Number. AF, Stephens County; MP, Medicine Park; CSL, East Lawton; AN, Anadarko. R, root; L, leaf; B, bulb; Fl, flower; Yl, young seedling. NI, not identified. Approximate latitudes and longitudes of the locations are: Lawton (N34.069424, W98.417781); Medicine Park (N 34.733270, W 98.483923); Anadarko (N35.069203, W96.265657); Stephens County (N34.36, W98.23). Extract. # PCN # Scientific Name Common Name Family 1 AF#19 L NI 7 AF#19R 2 AF#10R Yucca glauca Nutt. Soapweed Agavaceae 4 AF#10L 37 AF#10F 3 AF#13L Artemisia sp. Asteraceae Tahzeeba Frisby Oklahoma Native Plant Record 81 Volume 15, December 2015 5 AF#18L NI NI NI 25 AF#18L 6 AF#1L Ulmus sp. Elm seedling Ulmaceae 8 AF#17L Callirhoe involucrata (Torr. & A. Purple Poppy Malvaceae Gray) A. Gray 18 AF#17R 9 AF#17Fl 10 AF#15F Castilleja indivisa Engelm Indian Paint Brush Orobanchaceae 15 AF#15R 11 AF#9L Rumex crispus L. Curly Dock Polygonaceae
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