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

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

3. 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, 172-188. Sen, Saikat and Raja Chakraborty: The Role of Antioxidants in Human. ACS Symposium Series. Vol 1083; 2011. 1-37 3

4. Introduction 4

5.  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, 1781-1786. R = H 7-epi-clusianone R = OH 18-hydroxy-7-epi-clusianone R = H Clusianone R = OH 18-hydroxy-clusianone Introduction 5

6. 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

7. Materials 7

8. 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

9. Antioxidant Activity 9 MTTFormazan Ascorbic Acid

10. Antioxidant Activity Results Crude ExtractConcentration (ppm)Absorbance (at 570 nm) Root MeOH6,2500.276 Root EtOAc6,2500.103 Aerial MeOH6,2500.341 Aerial Acetone6,2500.442 Aerial Hexane6,2500.143 10

11. 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, 1576- 1583. Matsuhisa, Michiko et al. Benzoylphloroglucinol Derivatives from Hypericum scabrum. J. Nat. Prod. 2002, 65, 290-294. Antioxidant Activity Results 11

12. 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 MeOH53.4-8.0 Root EtOAc54.8-2.0 Aerial MeOH52.7-2.5 Aerial Acetone50.0-1.5 Aerial Hexane56.5-- Chloramphenicol50.01.512.0 1) HBA-MeOH2) HBA-Hexane 3) HBA- Acetone4) HBR-ETOAc 5) HBR-MeOH6) DMSO - Control 7) Chloramphenicol - Control 12

13. 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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15. 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