Supporting Information for Phytochemical Studies on Two Australian Anigozanthos Plant Species Rudi Hendra 1, Paul A. Keller 1* 1 School of Chemistry, University of Wollongong, NSW 2522, Australia Telephone: +61 2 4221 4692 Fax: +61 2 4221 4287 Email: keller@uow.edu.au S1
Table of Contents General Procedures... 4 Figure S1. HPLC profile of polar extracts from A. flavidus (a) and A. pulcherrimus (b) flowers... 5 Table S1. NMR spectroscopic data for compound 1... 6 Figure S2. 1 H-NMR spectrum of compound 1 (in methanol-d 4 )... 7 Figure S3. UV-Vis spectroscopy of compound 1... 7 Table S2. NMR spectroscopic data for compound 2... 8 Figure S4. 1 H-NMR spectrum of compound 2 (in methanol-d 4 )... 9 Figure S5. UV-Vis spectroscopy of compound 2... 9 Table S3. NMR spectroscopic data for compound 4... 10 Figure S6. 1 H-NMR spectrum of compound 4 (in methanol-d 4 )... 11 Figure S7. UV-Vis spectroscopy of compound 4... 11 Table S4. NMR spectroscopic data for compound 5... 12 Figure S8. 1 H-NMR spectrum of compound 5 (in methanol-d 4 )... 13 Figure S9. UV-Vis spectroscopy of compound 5... 13 Table S5. NMR spectroscopic data for compound 6... 14 Figure S10. 1 H-NMR spectrum of compound 6 (in methanol-d 4 )... 15 Figure S11. UV-Vis spectroscopy of compound 6... 15 Table S6. NMR spectroscopic data for compound 7... 16 Figure S12. 1 H-NMR spectrum of compound 7 (in methanol-d 4 )... 17 Figure S13. UV-Vis spectroscopy of compound 7... 17 Table S7. NMR spectroscopic data for compound 8... 18 Figure S14. 1 H-NMR spectrum of compound 8 (in methanol-d 4 )... 19 Figure S15. UV-Vis spectroscopy of compound 8... 19 Table S8. NMR spectroscopic data for compound 9... 20 Figure S16. 1 H-NMR spectrum of compound 9 (in methanol-d 4 )... 21 Figure S17. UV-Vis spectroscopy of compound 9... 21 Table S9. NMR spectroscopic data for compound 10... 22 Figure S18. 1 H-NMR spectrum of compound 10 (in methanol-d 4 )... 23 Figure S19. UV-Vis spectroscopy of compound 9... 23 Table S10. NMR spectroscopic data for compound 11... 24 Figure S20. 1 H-NMR spectrum of compound 11 (in methanol-d 4 )... 25 Figure S21. UV-Vis spectroscopy of compound 11... 25 S2
Table S11. NMR spectroscopic data for compound 13... 26 Figure S22. 1 H-NMR spectrum of compound 13 (in methanol-d 4 )... 27 Figure S22. UV-Vis spectroscopy of compound 13... 27 Characterisation of New Compounds... 28 Figure S23. LRESIMS from compound 3... 28 Figure S24. HRESIMS from compound 3... 29 Figure S25. UV-Vis spectroscopy of compound 3... 30 Figure S27. 13 C-NMR spectrum of compound 3 (in methanol-d 4 )... 31 Figure S28. HSQC spectrum of compound 3 (in methanol-d 4 )... 32 Figure S29. COSY spectrum of compound 3 (in methanol-d 4 )... 33 Figure S30. HMBC spectrum of compound 3 (in methanol-d 4 )... 34 Figure S31. HMBC spectrum of compound 3 (in methanol-d 4 ) (zoom)... 35 Figure S32. LRESIMS from compound 12... 36 Figure S33. HRESIMS from compound 12... 36 Figure S34. UV-Vis spectroscopy of compound 12.... 37 Figure S35. RP-HPLC profile from acid hydrolysis of 12 compared to authentic standard... 38 Figure S36. 1 H-NMR spectrum of compound 12 (in methanol-d 4 )... 39 Figure S37. 13 C-NMR spectrum of compound 12 (in methanol-d 4 )... 40 Figure S38. HSQC spectrum of compound 12 (in methanol-d 4 )... 41 Figure S39. COSY spectrum of compound 12 (in methanol-d 4 )... 42 Figure S40. NOESY spectrum of compound 12 (in methanol-d 4 )... 43 Figure S41. HMBC spectrum of compound 12 (in methanol-d 4 )... 44 Figure S42. GCMS Spectrum of Anigozanthos sp... 45 Table S12. GCMS Data of Anigozanthos sp.... 45 Table S13. Antibacterial Activity of methanol extracts and pure compounds obtained from A. rufus and A. pulcherrimus... 46 Figure S43. Colour solutions from compound 1 13 in methanol.... 47 References... 48 S3
General Procedures Optical Rotations for compound 3 and 12 was measured a 25 o C in methanol with a path length of 1.0 dm on a Jasco P-2000 Digital Polarimeter (l = 589 nm). Proton ( 1 H) and carbon ( 13 C) nuclear magnetic resonance (NMR) spectra were recorded at 500 and 125.7 MHz respectively on a Varian Unity Inova-500 MHz spectrometer, controlled by Varian VNMR software (version 6.1 revision C). NMR spectra were acquired in CD 3 OD with chemical shifts (δ) reported in parts per million (ppm) relative to CD 3 OD ( 1 H: δ = 3.31 ppm; 13 C: δ= 49.2 ppm). Coupling constants (J) are reported in Hertz (Hz). J values listed in 1 H NMR spectral data refer to coupling between hydrogen nuclei. Electrospray (ES) mass spectra were obtained on a LCMS-2010 EV (Shimadzu). Samples were injected as a solution in methanol HPLC grade. High Resolution (HR) ES mass spectrometry (MS) was performed on a Micromass QTOF2 Ultima Spectrometer. The HPLC profiles from both species were obtained using a Waters (Waters 1525 pump, Waters 2487 detector, controlled by Breeze software v3.30) with a Symmetry C 18 column (5 µm, 4.9 x 150 mm) with a Wakosil C 18 RS column (5 µm, 4.6 x 250 mm). All compounds were isolated by preparative HPLC using a Waters prep-lc system (LC-600 controller, 2489 detector, LC150 Pump, PD1 degasser) with a Waters reverse-phase OBD Sunfire TM C 18 column (5 µm, 19 x 150 mm) protected with a Waters Sunfire TM C 18 guard column (5 µm, 19 x 10 mm). To determine of sugar moiety from compound 12, a RP-HPLC (Shimadzu HPLC) system, coupled with an automatic sampler (Shimadzu SIL-10A XL) and a Sedere Sedex 60 LT Evaporative Light Scattering Detector (ELSD) with Prevail Carbohydrates ES column (250 x 4.6 i.d.; Alltech). All analytical HPLC samples were filtered through a Whatman syringe filter PTFE 0.45 µm, 4 mm and preparative HPLC samples were filtered through a Bonnet syringe filter 0.45 µm, 30 mm. A Büchi Rotary Evaporator (R-114/200) with a high vacuum pump was used for evaporation of solvents under reduced pressure at 40 ºC. S4
Figure S1. HPLC profile of polar extracts from A. flavidus (a) and A. pulcherrimus (b) flowers. AU 1.50 1.00 0.50 0.00 A 1 2 λ: 254 nm 8 3 4 7 5 10 9 12 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 Minutes 0.80 0.60 B λ: 254 nm AU 0.40 0.20 6 11 12 13 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 Minutes S5
Table S1. NMR spectroscopic data for compound 1 Cyanidin-3-rutinoside, red solid. UVmax (Me) (nm): 280 (7451); 519 (8905). ESIMS m/z: 595 [M+1] +. HRESIMS calculated for C 27 H 31 O 15 [M+H] + : 595.1752, found 595.1781 Position 0.1% TFA-d in Methanol-d 4 1 0.1% TFA-d in Methanol-d 4 δ H (J in Hz) δ C δ H (J in Hz) δ C 2 163.5 163.2 3 143.8 144.7 4 8.90, s 136.3 9.16, s 135.3 5 157.9 158.2 6 6.67, s 103.7 6.91, s 102.6 7 168.7 169.6 8 6.86, s 95.4 7.12, s 94.3 8a 156.4 157.9 4a 111.8 112.3 1' 120.1 120.3 2' 8.00, s 118.6 8.24, s 117.5 3' 145.9 146.5 4' 154.3 154.9 5' 7.00, d (8.7) 117.4 7.24, d (8.5) 116.5 6' 8.22, d (8.3) 128.4 8.49, d (9.0) 127.4 β-d-glucopyranose 1 5.25 d, (8.3) 103.6 5.51, d (8.0) 102.6 2 3.68 dd, (8.4, 8.0) 74.8 3.89-3.86, m 73.8 3 3.73 dd, (8.4, 8.0) 77.5 3.82-3.85, m 76.5 4 3.43-3.41, m 71.3 3.66-3.60, m 70.3 5 3.55 dd, (8.4, 8.0) 78.1 3.92-3.94, m 77.1 6 4.06 d, (11.0) 3.61-3.65, m 67.3 4.30, dd (11.0, 2.0) 4.05-4.00, m 66.9 α-l-rhamnopyranose 1 4.68 (s) 102.4 4.90 d, (8.0) 101.3 2 3.81-3.79, m 72.0 3.98-3.96, m 70.9 3 3.64-3.67, m 72.5 3.95-3.91, m 71.5 4 3.33-3.31, m 74.0 3.57-3.59, m 73.0 5 3.66-3.65 m 69.8 3.79-3.74, m 68.8 6 1.11 s 18.2 1.41 d, (6.0) 16.9 S6
Figure S2. 1 H-NMR spectrum of compound 1 (in methanol-d 4 ) HO 6 8 O 4 5' 1' 1'' O O HO 3'' 6'' O1''' O HO Figure S3. UV-Vis spectroscopy of compound 1 AU 1.50 1.00 280.4 519.1 0.50 0.00 S7
Table S2. NMR spectroscopic data for compound 2 Quercetin-3-rutinoside. Yellow solid. ESIMS m/z: 633 [M+Na] +, 609 [M+H] -. Position Methanol-d 4 2 Methanol-d 4 δ H (J in Hz) δ C δ H (J in Hz) δ C 2 159.3 158.5 3 135.6 135.6 4 179.4 179.4 5 162.9 162.9 6 6.20, s 99.9 6.21, d (2.0) 99.9 7 166.0 166.0 8 6.39, s 94.7 6.40, d (2.0) 94.9 8a 158.5 159.3 4a 105.6 105.6 1' 123.1 123.1 2' 7.67, s 117.7 7.66, d (2.1) 117.7 3' 145.8 145.8 4' 149.8 149.8 5' 6.88, d (8.4) 116.0 6.87, d (8.5) 116.1 6' 7.62, d (8.5) 123.6 7.62, dd (2.1, 8.5) 123.6 β-d-glucopyranose 1 5.10, d (7.5) 104.8 5.10, d (7.7) 104.7 2 3.68, dd (8.4, 8.0) 75.9 3.46, dd (7.7, 8.9) 75.7 3 3.73, dd (8.4, 8.0) 78.4 3.40, t (8.9) 78.2 4 3.43 3.41, m 71.5 3.26, t (8.9) 71.4 5 3.55, dd (8.4, 8.0) 77.3 3.32, ddd (1.2, 5.7, 8.6) 77.2 6 3.80, d (15.0) 3.40 3.45, m 68.7 3.80, dd (6.1, 11.0) 3.38, dd (6.1, 11.0) 68.5 α-l-rhamnopyranose 1 4.52, (s) 102.4 4.51, d (1.5) 102.4 2 3.81-3.79, m 72.2 3.62, dd (1.5, 3.4) 72.1 3 3.64-3.67, m 72.4 3.53, dd (3.4, 9.6) 72.2 4 3.33-3.31, m 74.0 3.27, t (9.6) 73.9 5 3.66-3.65, m 69.8 3.44, dq (6.2, 9.6) 69.7 6 1.12, d (6.2) 17.9 1.11, d (6.1) 17.8 S8
Figure S4. 1 H-NMR spectrum of compound 2 (in methanol-d 4 ) 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 200 250 300 350 400 450 500 Figure S5. UV-Vis spectroscopy of compound 2 S9
Table S3. NMR spectroscopic data for compound 4 Cyanidin-3-O-(6-O-p-coumaryl-O-β-D-glucopyranoside, red solid. UVmax (Me) (nm): 282 (8302); 313 (7052); 523 (6981). ESIMS m/z: 595 [M+1] +. HRESIMS calculated for C 30 H 27 O 13 [M+H] + : 595.1452, found 595.1461 Position 0.1% TFA-d in Methanol-d 4 3 0.1% TFA-d in Methanol-d 4 δ H (J in Hz) δ C δ H (J in Hz) δ C 2 164.5 162.1 3 145.1 146.0 4 8.91, s 136.8 8.92, s 136.8 5 159.1 158.1 6 6.53, s 103.3 6.69, d (1.8) 104.0 7 170.9 171.0 8 6.82, s 95.2 6.89, d (1.8) 95.0 8a 157.8 156.3 4a 113.0 113.1 1' 128.4 121.7 2' 8.00, d (2.2) 118.5 8.03, d (1.8) 119.9 3' 147.6 147.8 4' 155.8 156.3 5' 7.01, d (8.7) 117.4 7.02, d (8.7) 117.9 6' 8.24, dd (8.7, 2.2) 128.4 8.25, dd (1.8, 8.7) 129.4 β-d-glucopyranose 1 5.32, d (7.5) 103.1 4.67, d (7.1) 102.6 2 3.59, dd (7.5, 9.0) 78.0 4.06, dd (7.1, 8.5) 78.5 3 3.84, dd (9.0, 9.5) 76.1 3.82, dd (8.5, 2.7) 77.9 4 3.48, dd (9.5, 8.0) 71.8 3.94, d (2.7) 72.3 5 3.72, ddd (8.0, 6.5, 1.2) 74.8 3.62, ddd (8.0, 6.5, 1.2) 75.2 6 4.53, dd (1.2, 11.5), 64.5 4.39, dd (8.0, 12.0) 68.3 4.37, dd (6.5, 11.0) 4.24, d (12.0) p- Coumaryl 1 126.1 127.2 2 7.30, d (8.2) 131.4 6.88, d (1.6) 116.4 3 6.79, d (8.2) 116.8 6.78, dd (1.6, 8.0) 147.2 4 161.0 149.6 5 6.79, d (8.2) 116.8 6.68, d (8.0) 117.1 6 7.30, d (8.2) 131.4 123.4 α 6.22, d (16.0) 114.6 6.06, d (15.9) 114.5 β 7.44, d (16.0) 146.5 7.21, d (15.9) 146.6 carbonyl 169.1 168.9 S10
Figure S6. 1 H-NMR spectrum of compound 4 (in methanol-d 4 ) HO O O HO 1'' O 3'' 6'' O O Figure S7. UV-Vis spectroscopy of compound 4 AU 1.50 1.00 282.8 313.6 523.9 0.50 0.00 250.00 300.00 350.00 400.00 450.00 500.00 550.00 nm S11
Table S4. NMR spectroscopic data for compound 5 Kaempferol, yellow solid. UVmax (Me) (nm): 228 (13855); 267 (11997); 366 (10424). ESIMS m/z: 310 [M+Na] + Position Methanol-d 4 Methanol-d 2 4 δ H (J in Hz) δ C δ H (J in Hz) δ C 2 161.6 161.5 3 135.3 135.5 4 179.5 179.4 5 163.1 163.0 6 6.18, d (2.0) 99.8 6.21, d (2.0) 99.9 7 166.1 166.0 8 6.39, d (2.0) 94.5 6.40, d (2.0) 94.9 8a 158.7 158.6 4a 105.8 105.6 1' 122.8 122.8 2' 8.08, d (8.6) 132.6 8.05, d (8.6) 132.4 3' 6.90, d (8.6) 116.5 6.89, d (8.6) 116.1 4' 159.6 159.4 5' 6.90, d (8.6) 116.5 6.89, d (8.6) 116.1 6' 8.08, d (8.6) 132.7 8.05, d (8.6) 132.4 S12
Figure S8. 1 H-NMR spectrum of compound 5 (in methanol-d 4 ) 2 1.5 1 0.5 0 200 250 300 350 400 450 500 Figure S9. UV-Vis spectroscopy of compound 5 S13
Table S5. NMR spectroscopic data for compound 6 Kaempferol-3-rutinoside, yellow solid. UVmax (Me) (nm): 225 (12018); 268 (10675); 350 (9870). ESIMS, m/z:595 [M+1] +. HRESIMS: calculated for C 27 H 30 O 15 Na [M+Na] + : 617.1482, found 617.1484 Position CD 3 OD CD 3 OD 2 δ H (J in Hz) δ C δ H (J in Hz) δ C 2 161.6 161.4 3 135.3 135.5 4 179.5 179.4 5 163.1 163.0 6 6.20, d (2.1) 99.8 6.21, d (2.1) 99.9 7 166.1 166.0 8 6.39, d (2.1) 94.4 6.40, d (2.1) 94.9 8a 158.6 158.5 4a 105.7 105.5 1' 122.7 122.7 2' 8.06, d (8.4) 132.6 8.05,, d (8.5) 132.3 3' 6.89, d (8.5) 116.4 6.89, d (8.5) 116.1 4' 159.5 159.4 5' 6.89, d (8.5) 116.4 6.89, d (8.5) 116.1 6' 8.06, d (8.4) 132.6 8.05, d (8.5) 132.3 β-d-glucopyranose 1 5.12, d (7.0) 104.8 5.12, d (7.0) 104.5 2 3.43, dd (7.0, 8.5) 75.9 3.43, dd (7.7, 8.9) 75.7 3 3.40, dd (8.5, 9.0) 78.3 3.40, t (8.9) 78.1 4 3.24, dd (9.0, 8.5) 71.6 3.24, t (8.9) 71.4 5 3.32, ddd (8.5, 6.5, 1.5) 77.3 3.32, ddd (1.2, 6.1, 8.9) 77.2 6 3.80, dd (1.5, 10.5) 3.37, dd (10.5, 6.5) 68.7 3.80, dd (1.2, 11.0) 3.37, dd (6.1, 11.0) 68.5 α-l-rhamnopyranose 1 4.52, d (2.0) 102.5 4.51, d (1.5) 102.4 2 3.62, dd (2.0, 3.0) 72.2 3.62, dd (1.5, 3.5) 72.1 3 3.51, dd (3.0, 9.0) 72.4 3.51, dd (3.5, 9.6) 72.3 4 3.27, dd (9.0, 9.5) 74.0 3.27, t (9.6) 73.8 5 3.44, dd (9.5, 6.5) 69.9 3.44, dd (6.2, 9.6) 69.7 6 1.11, d (6.5) 18.1 1.11, d (6.2) 17.9 S14
Figure S10. 1 H-NMR spectrum of compound 6 (in methanol-d 4 ) HO O O O HO 1'' O 3'' 6'' O 1''' O HO 2 1.5 1 0.5 0 200 250 300 350 400 450 500 Series1 Figure S11. UV-Vis spectroscopy of compound 6 S15
Table S6. NMR spectroscopic data for compound 7 Apigenin-7-O-β-D-glucopyranoside, light yellow solid. ESIMS m/z: 469 [M+Na] +. Position Methanol-d 4 4,5 Methanol-d 4 δ H (J in Hz) δ C δ H (J in Hz) δ C 2 166.9 164.2 3 6.63, s 103.9 6.88, s 102.8 4 184.2 181.9 5 163.0 161.7 6 6.47, d (2.1) 101.3 6.46, d (2.0) 99.4 7 164.6 162.9 8 6.79, d (2.2) 96.1 6.85, d (2.0) 94.5 8a 158.9 156.9 4a 104.3 105.2 1' 123.1 120.6 2' 7.86, d (8.5) 129.8 7.96, d (9.0) 128.4 3' 117.2 6.95, d (9.0) 115.9 4' 162.8 161.0 5' 117.2 6.95, d (9.0) 115.9 6' 7.86, d (8.5) 129.8 7.96, d (9.0) 128.4 1'' 5.14, d (7.0) 101.5 5.16, d (7.0) 99.5 2'' 3.54 3.52, m 74.6 4.0 3.5, m 72.9 3'' 3.59 3.56, m 77.3 4.0 3.5, m 76.4 4'' 3.64, dd (10.6, 73.0 4.0 3.5, m 71.9 5.4) 5'' 4.11, d (9.6) 76.7 4.0 3.5, m 73.9 6'' 172.2 171.6 S16
Figure S12. 1 H-NMR spectrum of compound 7 (in methanol-d 4 ) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 200 250 300 350 400 450 500 Figure S13. UV-Vis spectroscopy of compound 7 S17
Table S7. NMR spectroscopic data for compound 8 Kaempferol-3-O-(6-O-p-coumaryl)-O-β-D-glucopyranoside. Yellow solid, ESIMS m/z: 593 [M-H] -, 617 [M+Na] + ; Position Methanol-d 4 6 Methanol-d 4 δ H (J in Hz) δ C δ H (J in Hz) δ C 2 161.6 158.8 3 132.4 135.5 4 179.7 179.6 5 163.8 163.2 6 6.14, s 100.2 6.13, d (2.0) 100.6 7 165.9 167.3 8 6.32, s 95.0 6.30, d (2.4) 95.3 8a 158.4 158.8 4a 105.7 105.6 1' 123.0 123.4 2' 8.06, d (8.5) 132.6 8.05, d (8.4) 132.3 3' 6.89, d (8.5) 116.4 6.89, d (8.4) 116.1 4' 159.5 159.4 5' 6.89, d (8.5) 116.4 6.89, d (8.4) 116.1 6' 8.06, d (8.5) 132.6 8.05, d (8.4) 132.3 β-d-glucopyranose 1 5.24, d (6.7) 104.1 5.26 d, (7.60) 104.2 2 3.48, dd (7.0, 8.9) 75.9 3.42-3.51, m 76.1 3 3.22, dd (9.0, 9.5) 78.2 3.42-3.51, m 78.3 4 3.34, dd (9.0, 8.0) 71.9 3.42-3.51, m 72.0 5 3.45, ddd (8.9, 6.5, 75.8 3.42-3.51, m 75.9 1.5) 6 4.18, dd (11.5, 6.8) 64.5 4.19, dd (11.5, 6.8) 64.6 4.30, dd (11.9, 2.2) 4.30, dd (11.9, 2.2) p- Coumaryl 1 126.1 127.2 2 7.30, d (8.2) 131.4 6.88, d (1.6) 116.4 3 6.79, d (8.2) 116.8 6.78, dd (1.6, 8.0) 147.2 4 141.0 149.6 5 6.79, d (8.2) 116.8 6.68, d (8.0) 117.1 6 7.30, d (8.2) 131.4 123.4 α 6.22, d (16.0) 114.6 6.06, d (15.9) 114.5 9 7.31, d (8.1) 131.2 7.32, d (8.4) 131.6 (C=O) 168.8 169.3 S18
Figure S14. 1 H-NMR spectrum of compound 8 (in methanol-d 4 ) 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 200 250 300 350 400 450 500 Figure S15. UV-Vis spectroscopy of compound 8 S19
Table S8. NMR spectroscopic data for compound 9 Quercetin-3-O-(6-O-p-coumaryl)-O-β-D-glucopyranoside. Yellow solid, ESIMS m/z: 609 [M-H] -, 633 [M+Na] + ; Position Methanol-d 4 6 Methanol-d 4 δ H (J in Hz) δ C δ H (J in Hz) δ C 2 161.6 158.8 3 132.4 135.5 4 179.7 179.6 5 163.8 163.2 6 6.14, s 100.2 6.13, d (2.0) 100.6 7 165.9 167.3 8 6.32, s 95.0 6.30, d (2.4) 95.3 8a 158.4 158.8 4a 105.7 105.6 1' 123.0 123.4 2' 6.80, d (8.1) 116.2 7.59, d (2.4) 115.0 3' 146.7 146.2 4' 146.8 146.9 5' 6.81, d (2.4) 116.3 6.81, d (8.4) 116.2 6' 7.99, d (8.3) 132.3 7.58, dd (8.4, 2.4) 123.4 β-d-glucopyranose 1 5.24, d (6.7) 104.1 5.26, d (7.60) 104.2 2 3.48, dd (7.0, 8.9) 75.9 3.42-3.51, m 76.1 3 3.22, dd (9.0, 9.5) 78.2 3.42-3.51, m 78.3 4 3.34, dd (9.0, 8.0) 71.9 3.42-3.51, m 72.0 5 3.45, ddd (8.9, 6.5, 1.5) 75.8 3.42-3.51, m 75.9 6 4.18, dd (11.5, 6.8) 64.5 4.19, dd (11.5, 6.8) 64.6 4.30, dd (11.9, 2.2) 4.30, dd (11.9, 2.2) p-coumaryl 2 6.07, d (15.9) 114.9 6.10, d (16.0) 117.6 3 7.40, d (15.8) 146.6 7.41, d (16.0) 131.5 4 161.4 161.5 5 7.31, d (8.1) 131.2 7.32, d (8.4) 131.6 6 6.84, d (8.3) 116.6 6.80, d (8.8) 117.6 7 141.2 140.2 8 6.84, d (8.3) 116.6 6.80, d (8.8) 117.6 9 7.31, d (8.1) 131.2 7.32, d (8.4) 131.6 (C=O) 168.8 169.3 S20
Figure S16. 1 H-NMR spectrum of compound 9 (in methanol-d 4 ) 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 200 250 300 350 400 450 500 Figure S17. UV-Vis spectroscopy of compound 9 S21
Table S9. NMR spectroscopic data for compound 10 Quercetin 3-O-β-D-glucopyranoside. Yellow solid. UVmax (Me) (nm): 228 (13855); 259 (13084); 359 (11830). ESIMS m/z: 465 [M+1 ]+ Position Methanol-d 4 2 Methanol-d 4 δ H (J in Hz) δ C δ H (J in Hz) δ C 2 158.2 156.6 3 135.6 135.2 4 179.7 175.9 5 163.2 161.0 6 6.21, d (2.1) 99.9 6.27, d (2.1) 97.8 7 165.9 164.1 8 6.41, d (2.1) 94.7 6.54, d, (2.1) 93.5 8a 159.0 156.8 4a 105.9 104.1 1' 122.2 122.7 2' 7.84, d (2.2) 116.0 7.74, d (2.1) 114.8 3' 149.1 147.3 4' 146.8 144.8 5' 6.87, d (8.6) 115.7 6.90, d (8.5) 114.6 6' 7.59, dd (8.5, 2.3) 122.0 7.64, dd (8.5, 2.2) 120.2 β-d-glucopyranose 1'' 5.16, d (7.5) 104.5 5.24, d (7.0) 104.1 2'' 3.47, dd (7.5, 9.0) 75.7 3.44, dd (7.5, 9.0) 75.7 3'' 3.42, dd (9.0, 9.5) 78.1 3.41, dd (9.0, 9.5) 78.2 4'' 3.34, dd (9.0, 8.5) 71.1 3.32, dd (9.0, 8.5) 71.4 5'' 3.25, ddd (8.5, 1.5, 6.5) 78.5 3.19, ddd (8.5, 1.5, 77.3 6'' 3.71, dd (1.5, 11.0) 3.58, dd (11.0, 6.5) 6.5) 62.6 3.68, dd (1.5, 11.0) 3.52, dd (11.0, 6.5) 68.6 S22
Figure S18. 1 H-NMR spectrum of compound 10 (in methanol-d 4 ) HO O O O HO 1'' O 3'' 6'' 2 1.5 1 0.5 0 200 250 300 350 400 450 500-0.5-1 Figure S19. UV-Vis spectroscopy of compound 9 S23
Table S10. NMR spectroscopic data for compound 11 Luteolin 7-O-β-D-glucopyranoside, Yellow solid. ESIMS m/z: 449 [M+1] Position Methanol-d 4 2 Methanol-d 4 δ H (J in Hz) δ C δ H (J in Hz) δ C 2 158.5 157.2 3 6.63, s 103.9 6.63, s 103.8 4 178.9 178.9 5 163.1 163.2 6 6.21, s 99.2 6.22, s 98.3 7 165.0 165.0 8 6.41, s 95.2 6.41, s 94.0 8a 159.1 157.2 4a 106.5 106.0 1' 122.2 122.2 2' 7.84, d (2.2) 116.0 7.74, d (2.1) 114.8 3' 149.1 147.3 4' 146.8 144.8 5' 6.87, d (8.6) 115.7 6.90, d (8.5) 114.6 6' 7.59, dd (8.5, 2.3) 122.0 7.64, dd (8.5, 2.2) 120.2 β-d-glucopyranose 1'' 5.14, d (6.5) 104.5 5.24, d (7.0) 109.5 2'' 3.47, dd (7.5, 9.0) 72.3 3.44, dd (7.5, 9.0) 82.1 3'' 3.42, dd (9.0, 9.5) 67.2 3.41, dd (9.0, 9.5) 78.3 4'' 3.34, dd (9.0, 8.5) 73.5 3.32, dd (9.0, 8.5) 88.0 5'' 3.25, ddd (8.5, 1.5, 6.5) 66.3 3.19, ddd (8.5, 1.5, 62.2 6'' 3.71, dd (1.5, 11.0) 3.58, dd (11.0, 6.5) 6.5) 62.3 3.68, dd (1.5, 11.0) 3.52, dd (11.0, 6.5) 68.6 S24
Figure S20. 1 H-NMR spectrum of compound 11 (in methanol-d 4 ) 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0-0.2200 250 300 350 400 450 500 Figure S21. UV-Vis spectroscopy of compound 11 S25
Table S11. NMR spectroscopic data for compound 13 Dihydroquercetin, A pale yellow solid. ESIMS m/z 305 [M-H] + Position Methanol-d 4 7 Methanol-d 4 δ H (J in Hz) δ C δ H (J in Hz) δ C 2 4.92, d (11.5) 85.2 5.01, d (11.5) 84.3 3 4.51, d (11.5) 73.7 4.60, d (11.5) 73.0 4 198.5 198.0 5 167.1 164.8 6 5.93, d (2.1) 97.4 5.98, s 97.0 7 169.6 167.8 8 5.89, d (2.1) 96.4 5.94, s 96.0 8a 163.6 164.0 4a 102.2 101.4 1' 129.8 129.6 2' 6.97, d (2.1) 116.0 7.06, d (2.1) 115.7 3' 146.5 145.6 4' 147.9 146.4 5' 6.81, d (8.1) 116.2 6.85, d (8.1) 115.7 6' 6.85, dd (8.1, 2.1) 121.0 6.90, dd (8.1, 2.1) 120.8 S26
Figure S22. 1 H-NMR spectrum of compound 13 (in methanol-d 4 ) 2 1.5 1 0.5 0 200 250 300 350 400 450 500-0.5-1 Figure S22. UV-Vis spectroscopy of compound 13 S27
Characterisation of New Compounds Figure S23. LRESIMS from compound 3 S28
Figure S24. HRESIMS from compound 3 S29
1.2 1 0.8 0.6 0.4 0.2 0 200-0.2 250 300 350 400 450 500-0.4 Figure S25. UV-Vis spectroscopy of compound 3 Figure S26. 1 H-NMR spectrum of compound 3 (in methanol-d 4 ) HO 4" 1" 3' 2' HO O O O 6 HO O 4 2 NH 2 S30
Figure S27. 13 C-NMR spectrum of compound 3 (in methanol-d 4 ) HO 4" 1" 3' 2' HO O O O 6 HO O 4 2 NH 2 2''/6'' 3''/5'' 7 1' 4'' 1 3' 1'' 2' 6 3 5 4 S31
Figure S28. HSQC spectrum of compound 3 (in methanol-d 4 ) HO 4" 1" 3' 2' HO O O O 6 HO O 4 2 NH 2 S32
Figure S29. COSY spectrum of compound 3 (in methanol-d 4 ) HO 4" 1" 3' 2' HO O O O 6 HO O 4 2 NH 2 S33
Figure S30. HMBC spectrum of compound 3 (in methanol-d 4 ) HO 4" 1" 3' 2' HO O O O 6 HO O 4 2 NH 2 S34
Figure S31. HMBC spectrum of compound 3 (in methanol-d 4 ) (zoom) S35
Figure S32. LRESIMS from compound 12 Figure S33. HRESIMS from compound 12 S36
0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 200 250 300 350 400 450 500 Figure S34. UV-Vis spectroscopy of compound 12. S37
B. Acid hydrolysis of 12 Figure S35. RP-HPLC profile from acid hydrolysis of 12 compared to authentic standard. RH-HPLC coupled with Evaporative Light Scattering Detector (ELSD) and separation was achieved using isocratic flow on a Prevail Carbohydrates ES Column. S38
Figure S36. 1 H-NMR spectrum of compound 12 (in methanol-d 4 ) S39
Figure S37. 13 C-NMR spectrum of compound 12 (in methanol-d 4 ) S40
Figure S38. HSQC spectrum of compound 12 (in methanol-d 4 ) 2'';3'';5'' 6'' α2 3'''/5''' 1'' 4 6' 4'' 2'''/6''' 3/5 β2 β1 2/6 α1 S41
Figure S39. COSY spectrum of compound 12 (in methanol-d 4 ) S42
Figure S40. NOESY spectrum of compound 12 (in methanol-d 4 ) O HO O 2' 2 4' A 1'' O O 5' 6' O 4'' B 5 6"' 5"' 1"' C 3"' S43
Figure S41. HMBC spectrum of compound 12 (in methanol-d 4 ) S44
Figure S42. GCMS Spectrum of Anigozanthos sp. Table S12. GCMS Data of Anigozanthos sp. NO. RT (min) Compound name 1 4.17 3,8-Dimethyldecane 2. 4.71 3-Mercapto-2-pyridinone 3. 6.19 (R)-4-Iodo-1,2-epoxybutane 4. 7.28 Neophytadiene 5. 8.14 Methylpalmitate 6. 10.19 (S)-4-Iodo-1,2-epoxybutane 7. 14.34 1,4-Bis(methoxymethyl)benzene S45
Table S13. Antibacterial Activity of methanol extracts and pure compounds obtained from A. rufus and A. pulcherrimus, showing percentage of inhibition (%) Compound Microorganism SA EC KP PA AB CA CN 1 8.81 1.44 13.93 16.30 35.87 1.13 ND 2 12.29 ND 14.76 13.42 12.16 ND ND 3 10.79 ND 9.56 10.63 38.88 1.40 ND 4 11.42 ND 16.91 14.22 11.57 1.26 ND 5 10.39 ND 12.03 11.32 17.06 4.92 ND 6 10.71 ND 9.54 9.31 31.58 8.37 ND 7 14.03 ND 15.80 10.34 16.62 3.06 ND 8 11.28 7.14 9.97 12.38 19.08 8.64 ND 10 13.50 ND 1.83 8.49 25.13 2.06 ND 11 14.36 ND ND 6.42 17.45 4.65 ND 12 10.75 ND 5.30 12.32 0.33 1.33 ND 13 7.10 5.14 11.99 15.99 21.29 0.66 ND Me extract of 14.94 ND 19.82 16.77 25.21 1.33 ND A. rufus Me extract A. pulcherrimus 11.09 ND 17.09 16.45 22.41 3.72 ND Staphylococcus aureus ATCC 43300 (MRSA), Eschericia coli ATCC 25922 (FDA control strain), Klebsiella penumoniae ATCC 700603 (MDA), Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, Candida albicans ATCC 90028, Cryptococcus neoformans ATCC 208821. ND: not inhibition detected. S46
Figure S43. Colour solutions from compound 1 13 in methanol. Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Compound 8 Compound 9 Compound 10 Compound 11 Compound 12 Compound 13 S47
References (1) Du, Q.; Zheng, J.; Xu, Y., J. Food Comp. Anal. 2008, 21, 390-395. (2) Kazuma, K.; Noda, N.; Suzuki, M., Phytochemistry 2003, 62, 229-237. (3) Qiu, F.; Luo, J.; Yao, S.; Ma, L.; Kong, L., J. Sep. Sci. 2009, 32, 2146-2151. (4) Vanhoenacker, G.; Van Rompaey, P.; De Keukeleire, D.; Sandra, P., Nat. Prod. Lett. 2002, 16, 57-63. (5) Han, X. H.; Hong, S. S.; Hwang, J. S.; Lee, M. K.; Hwang, B. Y.; Ro, J. S., Arch. Pharm. Res.2007, 30, 13-17. (6) Ren, X.; Shen, L.-l.; Muraoka, O.; Cheng, M., J. Carbohydr. Chem. 2011, 30, 119-131, Lavault, M.; Richomme, P., Chem. Nat. Compd. 2004, 40, 118-121. (7) Kiehlmann, E.; Szczepina, M. G., Cent. Eur. J. Chem. 2011, 9, 492-498. S48