SUPPLEMENTARY MATERIAL
Evidence-based Medicinal Value of Rudbeckia hirta L. Flowers
Botros R. Michael a, Sahar R. Gedara a, Mohamed M. Amer a, Lesley Stevenson b,
and Atallah. F. Ahmed c,*
a
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University (MU), Mansoura, 35516 Egypt
b
Centre for Phytochemistry and Pharmacology, Southern Cross University (SCU), PO Box 157, Lismore NSW
2480, Australia
c
Department of Pharmacognosy, College of Pharmacy, King Saud University (KSU), P.O. Box 2457, Riyadh
11451, Kingdom of Saudi Arabia
*Corresponding author. Tel.: +866 14 677264; fax: +866 14 677245. E-mail address:
afahmed@ksu.edu.sa (A. F. Ahmed).
Evidence-based Medicinal Value of Rudbeckia hirta L. Flowers
Abstract: A phytochemical investigation on the 5-lipoxygenase (5-LOX) inhibitory
methanolic extract of Rudbeckia hirta L. flowers afforded ten phenolic metabolites,
including three phenolic acids, two phenolic acid esters, four flavonol glycosides, and a
trimethylated flavonol. The structures of the isolated metabolites were determined on
the basis of spectroscopic analyses and by comparison with literature data. Seven of
these metabolites were isolated for the first time from genus Rudbeckia. The in vitro 5lipoxygenase (5-LOX) inhibitory, immunomodulatory, and antioxidant (oxygen radical
absorbance capacity, ORAC) activities of the isolated compounds were evaluated and
the results provided a new scientific evidence for the ethnopharmacological use of the
herb in inflammatory conditions.
Keywords: antiinflammatory; antioxidant; immunomodulatory; 5-LOX inhibition; methylated
flavonols; ORAC; Rudbeckia hirta.
Experimental
1. General details
Optical rotation []D was determined using a Jasco P-1010 polarimeter (JASCO Perkin-Elmer,
MD, USA) fitted with a sodium lamp (589 nm). UV spectra were recorded on Hewlett-Packard
HP-8453 spectrophotometer (USA). 1D or 2D NMR spectra were run in C5D5N or CD3OD on a
Bruker Avance DRX 500 NMR spectrometer (Bruker. Biospin GmbH, Rheinstetten, Germany),
using tetramethylsilane (TMS) as internal standard. LR-APCI-MS and HR-APCI-TOF-MS
spectra were recorded by Kratos MS-25 mass spectrometer (Kratos Ltd., Manchester, UK).
Normal phase chromatography was performed using Si gel (70–230 mesh) (E-Merck, Darmstadt,
Germany). Thin layer chromatography (TLC) was performed on Si gel G60 F254 (E-Merck,
Darmstadt, Germany). Gilson preparative HPLC System (Gilson Inc., Middleton, WI, USA) was
used for preparative separation of compounds Separation was performed using either Alltech®
Alltima C18, 5µ, 150 x 22mm ID column (Alltech Associates, Inc., IL, USA) or Phenomenex®
Luna C18, 5µ, 150 x 21.2 mm ID column (Phenomenex Inc., CA, USA). The injected volume
was 1-2 mL at a concentration of 80-100 mg/mL, flow rate was adjusted to 10 mL/min, and
fractions were collected at 20-30s intervals. Fractions (3-5 mL) were combined according to their
similar LC-MS profile. Solvent system used in RP-HPLC was 0.05% trifluoroacetic acid
(TFA)/CH3CN 0.005% TFA/H2O [TA-TW] in different ratios for gradient and isocratic mode.
1. Plant material
Rudbeckia hirta L. herb was grown in the Medicinal Plant Garden at MU and was authenticated
by Prof. Dr. Ibrahim Mashaly, Faculty of Sciences, MU. A voucher sample (MUPHG07-2) was
deposited at Department of Pharmacognosy, Faculty of Pharmacy, MU. The fully grown flower
heads were collected, air-dried and finely powdered prior to the phytochemical investigation
2. Extraction and isolation
The powder of R. hirta flowers (2 kg) was exhaustively extracted with MeOH. A portion (50 g)
of the solvent-free methanolic extract (220 gm) was subjected to Si gel VLC which was
successively eluted by CHCl3, EtOAc, and MeOH to afford the correspondent fractions (8.71 g,
2.95 g, 36.08 g, respectively). The EtOAc fraction was subjected to repeated preparative RPHPLC-MS using TA-TW (1:9 to 9:1, gradient, 20 min, and then 9:1, isocratic, 5 min). Fractions
4-11 were combined based on their LC/MS profile and were further fractionated by preparative
RP-HPLC using TA-TW (0.5:9.5 to 4:6, gradient, 20 min, and then 9:1, isocratic, 5 min).
Subfractions 23, 37, and 38 afforded compounds 1 (11 mg), 2 (15 mg), and 8 (13 mg),
respectively. The MeOH fraction was also isolated by preparative RP-HPLC-MS using TA-TW
(1:9 to 9.5:0.5, gradient, 20 min, and then 9.5:0.5, isocratic, 5 min). Fractions 11 and 12 were
combined and further purified by preparative RP-HPLC using TA-TW (0:10 to 1.5:8.5, gradient,
40 min and then 9.5:0.5, isocratic, 10 min). Subfraction at tR 30 min afforded compound 3 (15
mg). Fractions 13 and 14 were pooled together and chromatographed in a similar way using TATW (0.5:9.5 to 2:8, gradient, 20 min and then 2:8, isocratic, 6 min). Subfractions 56 gave
compounds 4. Fractions 15-26 were combined and similarly chromatographed using TA-TW
(1:9 to 1:1, gradient, 20 min and then 1:1, isocratic, 6 min). Subfractions 40 yielded compound 5
(16 mg). Fractions 27-29 were combined and were further isolated by preparative RP-HPLC-MS
TA-TW (2.5:7.5, isocratic, 25 min). Subfractions 6 eluted and 12 afforded compound 7 (11 mg)
and 6 (11 mg), respectively. Combined fractions 31-36 were isolated in the same way as under
fractions 27-29 to yield compound 9 (9 mg). Fractions 49 eluted yielded compound 10 (11 mg).
2.1. Compound 6
Yellow needle crystals; m.p. (uncorrected) 251-253 oC; UV (MeOH, max): 263, 276 sh and 357
nm.; APCI-MS m/z [rel. int.]: 481 (100, [M+H]+), 318 (63, [M-C6H11O5+H]+); 1H NMR (500
MHz, pyridine-d5): H 8.62 (1H, d, J = 2.1 Hz, H-2`), 8.08 (1H, dd, J = 8.5, 2.1 Hz, H-6`), 7.41
(1H, d, J = 8.4 Hz, H-5`), 7.38 (1H, s, H-6), 5.94 (1H, d, J = 7.7 Hz, H-1``), 4.56 (1H, m, H6``a), 4.46 (1H, m, H-5``), 4.42 (1H, m, H-6``b), 4.38 (1H, m, H-2``), 4.35 (1H, m, H-4``), 4.23
(1H, m, H-3``). 13C NMR (125 MHz, pyridine-d5): C 178.1 (1C, qC, C-4), 153.7 (1C, qC, C-7),
149.8 (1C, qC, C-5), 149.1 (1C, qC, C-4’), 148.1 (1C, qC, C-2), 147.9 (1C, qC, C-9), 147.7 (1C,
qC, C-3’), 138.3 (1C, qC, C-3), 132.6 (1C, qC, C-8), 124.5 (1C, qC, C-1’), 121.8 (1C, CH, C-6’),
117.5 (1C, CH, C-5’), 95.6 (1C, CH, C-6), 117.4 (1C, CH, C-2’), 107.2 (1C, qC, C-10), 103.1
(1C, CH, C-1’’), 79.9 (1C, CH, C-3’’), 78.9 (1C, CH, C-5’’), 75.5 (1C, CH, C-2’’), 71.6 (1C,
CH, C-4’’), 62.9 (1C, CH2, C-6’’).
2.2. Compound 7
Yellow needle crystals; m.p. (uncorrected) 236-238 oC; UV (MeOH, max): 255, 277 sh and 360
nm.; APCI-MS m/z [rel. int.]: 481 (100, [M+H]+), 318 (75, [M-C6H11O5+H]+); 1H NMR (500
MHz, pyridine-d5): H 8.58 (1H, d, J = 2.1, H-2`), 8.04 (1H, dd, J = 8.3, 2.1 Hz, H-6’), 7.36 (1H,
d, J = 8.3 Hz, H-5`), 7.35 (1H, s, H-8), 5.91 (1H, d, J = 7.4 Hz, H-1``), 4.52 (1H, m, H-6``a),
4.36 (1H, m, H-6``b), 4.35 (1H, m, H-2``), 4.34 (1H, m, H-4``), 4.20 (1H, m, H-3``), 3.98 (1H,
m, H-5``). 13C NMR (125 MHz, pyridine-d5): C 177.5 (1C, qC, C-4), 153.1 (1C, qC, C-9), 149.2
(2C, qC, C-5 and C-7), 148.5 (1C, qC, C-2), 147.3 (1C, qC, C-4’), 147.1 (1C, qC, C-3’), 137.7
(1C, qC, C-3), 132.1 (1C, qC, C-6), 123.8 (1C, qC, C-1’), 121.2 (1C, CH, C-6’), 116.8 (1C, CH,
C-5’), 116.7 (1C, CH, C-2’), 106.6 (1C, qC, C-10), 102.5 (1C, CH, C-1’’), 95.1 (1C, CH, C-8),
79.3 (1C, CH, C-3’’), 78.5 (1C, CH, C-5’’), 74.6 (1C, CH, C-2’’), 71.2 (1C, CH, C-4’’), 62.3
(1C, CH2, C-6’’).
2.3. Compound 8
Pale yellow needle crystals; m.p. (uncorrected) 200-202 oC; UV (MeOH, max): 260 and 345 nm.;
APCI-MS m/z [rel. int.]: 493 (57, [M+H]+), 346 (100, [M-C6H11O4+H]+); 1H NMR (500 MHz,
methanol-d4): H 7.37 (1H, d, J = 2.1 Hz, H-2`), 7.34 (1H, dd, J = 2.1 Hz, 8.3, H-6`), 6.92 (1H,
dd, J = 8.3, 2.1 Hz, H-5`), 6.73 (1H, s, H-8), 5.37 (1H, br s, H-1``), 4.22 (1H, br s, H-2``), 3.96
(3H, s, 7- OCH3), 3.84 (3H, s, 6- OCH3), 3.75 (1H, dd, J = 9.4, 3.3 Hz, H-3``), 3.43 (1H, m, H-
5``), 3.34 (1H, dd, J = 9.4, 9.4 Hz, H-4``), 0.94 (3H, d, J = 6.0 Hz, H3-6``). 13C NMR (125 MHz,
methanol-d4): C 180.1 (1C, qC, C-4), 160.7 (1C, qC, C-7), 160.0 (1C, qC, C-2), 153.6 (1C, qC,
C-5), 154.2 (1C, qC, C-9), 150.1 (1C, qC, C-4’), 146.6 (1C, qC, C-3’), 136.4 (1C, qC, C-3),
133.6 (1C, qC, C-6), 123.1 (1C, CH, C-6’), 123.0 (1C, qC, C-1’), 117.2 (1C, CH, C-2’), 116.6
(1C, CH, C-5’), 107.4 (1C, qC, C-10), 103.7 (1C, CH, C-1’’), 92.2 (1C, CH, C-8), 73.4 (1C, CH,
C-4’’), 72.3 (1C, CH, C-3’’), 72.2 (1C, CH, C-5’’), 72.1 (1C, CH, C-2’’), 61.3 (1C, 6-OCH3),
57.1 (1C, 7-OCH3), 17.8 (1C, CH3, C-6’’).
2.4. Compound 9
Yellow needle crystals; m.p. (uncorrected) 260-262 oC; UV (MeOH, max): 260, 277 sh and 375
nm.; APCI-MS m/z [rel. int.]: 495 (100, [M+H]+), 464 (8, [M-OCH3+H]+), 332 (57, [MC6H11O5+H]+); 1H NMR (500 MHz, pyridine-d5): H 8.63 (1H, d, J = 1.2 Hz, H-2`), 8.08 (dd,
J=1.9, 8.2, H-6`), 7.39 (d, J=8.2, H-5`), 7.32 (1H, s, H-8), 5.89 (d, J=7.5, H-1``), 4.58 (1H, m, H6``a), 4.46 (1H, m, H-5``), 4.43 (m, H-2``), 4.40 (1H, m, H-6``b), 4.36 (1H, m, H-4``), 4.27 (1H,
m, H-3``), 4.08 (3H, s, 6-OCH3).
13
C NMR (125 MHz, pyridine-d5): C 178.2 (1C, qC, C-4),
158.0 (1C, qC, C-7), 153.7 (1C, qC, C-5), 152.8 (1C, qC, C-9), 150.7 (1C, qC, C-4’), 149.3 (1C,
qC, C-2), 147.8 (1C, qC, C-3’), 138.5 (1C, qC, C-3), 133.8 (1C, qC, C-6), 124.7 (1C, qC, C-1’),
121.9 (1C, CH, C-6’), 117.5 (1C, CH, C-5’), 117.4 (1C, CH, C-2’), 107.1 (1C, qC, C-10), 102.8
(1C, CH, C-1’’), 95.5 (1C, CH, C-8), 79.9 (1C, CH, C-3’’), 79.3 (1C, CH, C-5’’), 75.5 (1C, CH,
C-2’’), 71.9 (1C, CH, C-4’’), 63.4 (1C, CH2, C-6’’), 61.6 (1C, 6-OCH3).
2.5. Compound 10
Chrysosphenol-D (quercetagetin 3,6,7-trimethyl ether) (10): Pale yellow needle crystals; m.p.
(uncorrected) 233-235 oC; UV (MeOH, max): 262, 278 sh and 370 nm.; APCI-MS m/z [rel. int.]:
346 (100, [M-CH3+H]+); 1H NMR (500 MHz, methanol-d4): H 7.80 (1H, s, H-2`), 7.66 (d, J =
8.2 Hz, H-6`), 6.89 (1H, d, J = 8.2 Hz, H-5`), 6.72 (1H, s, H-8), 3.96 (3H, s, 7-OCH3), 3.83 (3H,
s, 6-OCH3), 3.65 (3H, s, 3-OCH3). 13C NMR (125 MHz, methanol-d4): C 177.6 (1C, qC, C-4),
160.5 (2C, qC, C-2 and C-7), 153.9 (1C, qC, C-9), 152.8 (1C, qC, C-5), 149.1 (1C, qC, C-4’),
146.4 (1C, qC, C-3’), 137.5 (1C, qC, C-3), 133.1 (1C, qC, C-6), 124.2 (1C, qC, C-1’), 121.9 (1C,
CH, C-6’), 116.9 (1C, CH, C-5’), 116.4 (1C, CH, C-2’), 105.9 (1C, qC, C-10), 91.9 (1C, CH, C8), 61.3 (1C, 6-OCH3), 56.9 (1C, 7-OCH3), 52.1 (1C, 3-OCH3).
3. In vitro biological assays
3.1. Lipoxygenase Inhibitor (5-LOX) Screening Assay
The arachidonate 5-LOX inhibitory activity of tested natural product (NP) samples was measured
with an enzymatic colorimetric method described by Gaffney (1996) using a diagnostic
lipoxygenase inhibitor screening assay kit (Cayman Chemical Co, MI, USA) following the
manufacturer’s protocol. Nordihydroguaiaretic acid (NADGA) (Cayman Chemical Co, MI, USA)
was used as a standard lipoxygenase inhibitor. Briefly, NADGA was dissolved in DMSO and
added into the assay system (in 96-well plate), which was initiated by adding of substrate
arachidonic acid (Cayman Chemical Co, MI, USA; catalogue), followed by shaking the 96-well
plate for 5 minutes, and terminated by chromogen (Cayman Chemical Co, MI, USA). The wells
added with assay solvent (DMSO), and 5-LOX standard served as vehicle control and positive
control respectively. Each tested NP sample was also added instead of NADGA in the screening
wells. Absorbance (A) was determined at 500 nm using a Wallac Victor2 plate reader, which is
correlated with lipoxygenase activity. Tests were carried out in duplicate. The percentage of
lipoxygenase inhibition was calculated using the following equation: Inhibition % = [(A0– A1) /
A0] x 100, where A0 was the absorbance of the control (without the tested sample) and A1 was the
absorbance (with the tested NP sample).
3.2. ATP-based Luminescence Cytotoxicity Assay
The cytotoxic activity of a NP sample was measured by the bioluminescent method of Cree and
Andreotti (1997). The assay is based on the production of light caused by the reaction of
liberated ATP from lysed cancer cells (mice lymphoblastoma P-388 cells, American Type
Culture Collection, ATCC) with added luciferase and D-luciferin. The emitted light intensity is
linearly related to the ATP concentration. The results were calculated as % inhibition = [(LcontrolLsample) / Lcontrol] x 100.
3.3. CellTiter 96® Non-Radioactive Lymphocyte Proliferation Assay
3.3.1. Blood collection
Normal blood samples of healthy, non-smoking donors (age of 30-40 years) recruited from SCU,
Lismore, NSW, Australia, were analyzed according to standard diagnostic laboratory procedures
at the Northern Rivers Pathology Service, Lismore Base Hospital, Lismore, NSW, Australia. All
procedures were approved by the SCU Human Research Ethics Committee and the University of
Queensland Medical Research Ethics Committee (Brisbane, QLD, Australia). Participants were
fully informed, and written consent was obtained. Study samples were collected in sterile BD
Vacutainer collection Tubes with lithium heparin anticoagulant (Becton Dickinson Systems, NJ,
USA) and processes within 6 h at the Center for Phytochemistry and Pharmacology, SCU,
Lismore, NSW, Australia.
3.3.2. Assay
The CellTiter 96® Non-Radioactive Lymphocyte Proliferation Assay is a colorimetric method
used to measure the ability of the tested material to stimulate or arrest metabolic activity of cells
through measurement of the dehydrogenase activity in active cells. MTS (3-[4,5-dimethylthiazol2-yl]-5[3-carboxymethoxyphenyl]-2-[4-sulfophen-yl]-2H-tetrazolium inner salt) with PMS
(phenazine methosulfate), is reduced to formazan in the presence of the prepared viable
lymphocyte (Riss and Moravec, 1992). Each of the standards: Echinacea purpurea extract (Ech,
Nature’s Own Sanofi-Aventis, QLD, Australia), tetramisole hydrochloride (TMS, SigmaAldrich, MO, USA), and dexamethasone phosphate disodium salt (DXS, Sigma-Aldrich, MO,
USA) and the tested samples in DMSO were serially diluted in the culture medium so that the
final concentration of DMSO in each well was kept less than 1%, and plated as 10 µl/well in
triplicates. A 10 µL of 200 µg/mL of the mitogen phytohaemagglutinin from Phaseolus vulgaris
(PHA, Sigma-Aldrich Chemicals, MO, USA) was added to each well except medium-only
control wells. The absorbance of reduced formazan, which is proportional to the number of
viable cells in culture, was measured at 490 nm using a Wallac Victor2 plate reader. The results
are calculated as a percentage lymphocyte proliferation relative to the solvent (DMSO) blank.
The lymphocyte proliferation % = [A(PBMCs + PHA + sample)  Ablank / A(PBMCs + PHA)  Ablank] x100.
3.4. Oxygen radical absorbance capacity (ORAC) assay
The ORAC assay is a kinetic assay measuring the antioxidant scavenging capacity against decay
of fluorescein (FL) fluorescence over time due to peroxyl radical generated by the thermal
breakdown of 2,2'-azobis[2-amidinopropane] dihydrochloride (AAPH) at 37°C. Trolox [6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid] serves as a positive control. The
antioxidant activity (ORAC value) of NP is calculated using linear regression between the Trolox
concentration and the net area under curve (AUC) of FL decay, and was expressed as Trolox
equivalent (TE) in micromole per g of NP.