Research Update Presentation_Diego Vasquez_04052024.pptx

Research update presentation for a natural products chemistry lab by an undergraduate biochemistry major student

1. Research Update Diego Arturo Vasquez Martinez

2. LC-MS Calibration Curve Training

3. Calibration Curve Preparation

4. Relative Intensity Graphs

5. MIC Bioassay Training

6. Bioassay training

7. Figure 1. Bioassay of S. aureus using berberine and vancomycin as a positive control Bioassay training using Berberine and S. aureus   1 2 3 4 5 6 7 8 9 10 11 12 A Berberine (300 µg/mL) Berberine (150 µg/mL) Berberine (75 µg/mL) Berberine (37.5 µg/mL) Bacteria B Berberine (18.8 µg/mL) Berberine (9.4 µg/mL) Berberine (4.7 µg/mL) Berberine (0 µg/mL) C Broth D Broth Positive C. (Vancomycin) Broth Negative C. DMSO E Broth Sterile F Berberine (300 µg/mL) Berberine (150 µg/mL) Berberine (75 µg/mL) Berberine (37.5 µg/mL) G Berberine (18.8 µg/mL) Berberine (9.4 µg/mL) Berberine (4.7 µg/mL) Berberine (0 µg/mL) H Broth Positive C. (Vancomycin) Broth Negative C. DMSO

8. MIC Results

9. Zanthoxylum clava-herculis L. (Rutaceae)

10. Z. clava-herculis Commonly known as ‘Hercules Club’ Member of the family Rutaceae Known to have some antimicrobial properties ¹ Found on the coastal plains of the Atlantic Ocean and Gulf of Mexico from southern Virginia to mid-Florida west to Texas ² Used by Native Americans as a toothache remedy ² Foto by Stang, David Foto by Brundage, Stephanie Gibbons, S., et al . Phytotherapy Research 2003 , 17 (3), 274–275. https://doi.org/10.1002/ptr.1112 . Zanthoxylum clava-herculis (Hercules Club, Pepperbark, Prickly Ash, Southern Prickly-ash, Southern Toothache Tree, Tickle Tongue, Toothache Tree) | North Carolina Extension Gardener Plant Toolbox . https://plants.ces.ncsu.edu/plants/zanthoxylum-clava-herculis/.

11. Zanthoxylum clava-herculis extraction

12. Figure 2. First separation of aqueous and chloroform layer Figure 3. Third separation of aqueous and chloroform layer

13. Z. clava-herculis MS analysis Hexane partition Commercial Crude extract Methanol Blank Chloroform partition Aqueous partition

14. Z. clava-herculis MS analysis Hexane partition Commercial Crude extract Methanol Blank Chloroform partition Aqueous partition

15. Matches with compounds found in Zanthoxylum genus Matches Found in LOTUS from Zanthoxylum species Matches Found in LOTUS from Zanthoxylum species Formula Mol. Wt. Exact Mass Name 1 C20H23NO4 341.4 342.17 2-[(2e)-4-hydroperoxy-4-methylpent-2-en-1-yl]-2,6-dimethylpyrano[3,2-c]quinolin-5-one 2 C20H23NO4 341.4 342.17 (12bs)-3,10,11-trimethoxy-7,8,12b,13-tetrahydro-5h-6-azatetraphen-4-ol 3 C20H23NO4 341.4 342.17 3,10,11-trimethoxy-7,8,12b,13-tetrahydro-5h-6-azatetraphen-4-ol 4 C20H23NO4 341.4 342.17 (9s)-3,4,15-trimethoxy-10-methyl-10-azatetracyclo[7.7.1.0²,⁷.0¹³,¹⁷]heptadeca-1(16),2(7),3,5,13(17),14-hexaen-16-ol 5 C20H23NO4 341.4 342.17 3,4,15-trimethoxy-10-methyl-10-azatetracyclo[7.7.1.0²,⁷.0¹³,¹⁷]heptadeca-1(16),2(7),3,5,13(17),14-hexaen-16-ol 6 C20H23NO4 341.4 342.17 (2r)-2-[(2e)-4-hydroperoxy-4-methylpent-2-en-1-yl]-2,6-dimethylpyrano[3,2-c]quinolin-5-one 7 C20H23NO4 341.4 342.17 2-(4-hydroperoxy-4-methylpent-2-en-1-yl)-2,6-dimethylpyrano[3,2-c]quinolin-5-one

16. Compound 1 Compound 7 Compound 6 Compound 2 Compound 3 Compound 4 Compound 5 Matches with compounds found in Zanthoxylum genus

17. Z. clava-herculis MS analysis Hexane partition Commercial Crude extract Methanol Blank Chloroform partition Aqueous partition

18. Possible compound Chelerythrine Molecular formula [C21H18NO4]+ Molecular weight 348.4 Exact weight 348.12 Antimicrobial activity reported¹ Gibbons, S., et al . Phytotherapy Research 2003 , 17 (3), 274–275. https://doi.org/10.1002/ptr.1112 .

19. Z. clava-herculis bioassay   1 2 3 4 5 6 7 8 9 10 11 12 A 0.705 0.71 0.676 0.707 0.743 0.738 0.728 0.736 0.747 0.899 0.959 1.314 B 0.792 0.824 0.825 0.615 0.663 0.635 0.675 0.657 0.678 0.773 0.788 0.923 C 0.05 0.05 0.05 0.855 0.777 0.739 0.754 0.797 0.806 0.679 0.686 0.752 D 0.049 0.049 0.05 0.05 0.05 0.049 0.749 0.649 0.049 0.049 0.049 0.614 E 0.05 0.655 0.051 0.049 0.634 0.049 0.706 0.051 0.05 0.051 0.05 0.051 F 0.982 0.081 0.089 0.085 0.081 0.077 0.078 0.077 0.072 0.122 1.382 0.128 G 0.128 0.125 0.112 0.066 0.065 0.064 0.053 0.053 0.053 0.05 0.049 0.049 H 0.049 0.05 0.05 0.05 0.05 0.049 0.05 0.049 0.05 0.049 0.062 0.049

20. Future Goals Isolate the compounds with mass 342.17, 348.124 and 296.165 using flash chromatography Determine the identity of the compounds found Test for any antimicrobial activity of the compounds

21. References Gibbons, S.; Leimkugel, J.; Oluwatuyi, M.; Heinrich, M. Activity of Zanthoxylum clava-herculis extracts against multi-drug resistant methicillin-resistant Staphylococcus aureus(mdr-MRSA). Phytotherapy Research 2003 , 17 (3), 274–275. https://doi.org/10.1002/ptr.1112 . Zanthoxylum clava-herculis (Hercules Club, Pepperbark, Prickly Ash, Southern Prickly-ash, Southern Toothache Tree, Tickle Tongue, Toothache Tree) | North Carolina Extension Gardener Plant Toolbox . https://plants.ces.ncsu.edu/plants/zanthoxylum-clava-herculis/.