Antioxidant Activity of Hypericum brachyphyllum Tung Pham, Anita Rahman, Omar E. Christian McNeese State University Lake Charles, LA 1

Outline  Introduction  Antioxidants  Hypericum brachyphyllum  Objectives  Antioxidant Activity  Antibacterial Activity  Future Works 2

Introduction  Importance of antioxidants:  Reactive oxygen species (ROS) - cancer, hypertension, neurodegenerative diseases, and aging  Antioxidants: prevent free radicals (chemically unstable and highly reactive) from harming healthy tissues Fig. 1 – How antioxidants work Fig. 2 – Antioxidant doing their job Sayre, Lawrence M., George Perry, and Mark A. Smith: Oxidative Stress and Neurotoxicity. Chem. Res. Toxicol. 2008, 21, Sen, Saikat and Raja Chakraborty: The Role of Antioxidants in Human. ACS Symposium Series. Vol 1083;

Introduction 4

 Hypericum brachyphyllum (coastal plains St. John’s Wort):  Used by many Native American tribes to treat a variety of conditions  Hypericum genus: polyphenolic compounds (prenylated benzophenones) - antioxidant capacity Christian, Omar E.; Stewart McLean, William F. Reynolds, and Helen Jacobs: Prenylated Benzophenones from Hypericum hypericoides. Natural Product Communications. 2008, 3, R = H 7-epi-clusianone R = OH 18-hydroxy-7-epi-clusianone R = H Clusianone R = OH 18-hydroxy-clusianone Introduction 5

Objectives  Evaluate antioxidant activity of Hypericum brachyphyllum aerial part and root extracts  using MTT assay (3(4,5-dimethylthiazole-2-yl)-2,5- diphenyltetrazolium bromide)  Evaluate antibacterial activity of crude extracts 6

Materials 7

Antioxidant Activity Methods:  MTT was dissolved in DMSO - 30 mg/mL  Ascorbic acid (1 mg/mL to 30 mg/mL) was reacted with MTT to obtain a standard curve.  Ascorbic acid in the solutions reacted completely with MTT to form formazan which absorbed at 570 nm.  Excess MTT did not interfere with the absorption of formazan. 8

Antioxidant Activity 9 MTTFormazan Ascorbic Acid

Antioxidant Activity Results Crude ExtractConcentration (ppm)Absorbance (at 570 nm) Root MeOH6, Root EtOAc6, Aerial MeOH6, Aerial Acetone6, Aerial Hexane6,

Results and Discussion:  Acetone extract of aerial parts displayed the most potent activity  Hypericum genus is a rich source of benzophenones.  Antioxidant potential of benzophenones is well known. Dimitrina Zheleva-Dimitrova; Paraskev Nedialkov; Ulrich Girreser and Gerassim Kitanov. Benzophenones and Flavonoids from Hypericum Maculatum and Their Antioxidant Activities. Natural Product Research. 2012, 26, Matsuhisa, Michiko et al. Benzoylphloroglucinol Derivatives from Hypericum scabrum. J. Nat. Prod. 2002, 65, Antioxidant Activity Results 11

Antibacterial Activity  Results and Discussion  Inhibited on the Gram-positive bacteria Staphylococcus aureus.  The most potent: methanolic extract of the roots  The known metabolites of hyperibone A to D inhibit MRSA in S. aureus. Zone of Inhibition (mm) Crude ExtractConcentration (mg/mL)E. coli (Gram-)S. aureus (Gram+) Root MeOH Root EtOAc Aerial MeOH Aerial Acetone Aerial Hexane Chloramphenicol ) HBA-MeOH2) HBA-Hexane 3) HBA- Acetone4) HBR-ETOAc 5) HBR-MeOH6) DMSO - Control 7) Chloramphenicol - Control 12

Future Works  Continue bioassay-directed purification of acetone extract from aerial parts  Separation and examination of methanol root extract for antibacterial activity H. brachyphyllum Aerial Parts Acetone Extract H. brachyphyllum Root Methanol Extract 13

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Acknowledgement We would like to thank  Dr. Mark Merchant for his help and the use of his lab for running the antibacterial bioassay  Office of Research Services and Sponsored Programs  McNeese State University 15