Tagenols A and B: New lipoxygenase inhibitor flavonols from Tagetes minuta

Article abstract of DOI:10.1016/j.phytol.2016.04.004

Phytochemical investigation of the methanolic extract of Tagetes minuta L. (Asteraceae) aerial parts afforded two new flavonol derivatives: tagenol A (quercetagetin-8-hydroxy-3,6,4′-trimethy ether) (1) and tagenol B (quercetagetin-8-hydroxy-3-methoxy-6-O-β-d-glucopyranoside) (4), together with three known flavonoids: quercetin-3,6-dimethyl ether (2), quercetin-3-methyl ether (3), and axillarin-7-O-β-d-glucopyranoside (5). Their structures were identified by UV, IR, 1D (¹H and ¹³C), 2D (¹H-¹H COSY, HSQC, and HMBC) NMR, and HRESIMS spectral data as well as comparison with literature data. The isolated compounds were evaluated for their lipoxygenase inhibitory and antioxidant activities. Compounds 1-5 displayed moderate lipoxygenase inhibitory activities with IC₅₀ values of 12.2, 9.4, 7.1, 5.8, and 4.2 μM, respectively compared to indomethacin (IC₅₀ 0.9 μM). Moreover, they displayed promising antioxidant activities with IC₅₀ values of 14.9, 14.3, 13.5, 12.7, and 11.2 μM, respectively using DPPH assay compared to propyl gallate (IC₅₀ 7.38 μM).

Full text of DOI:10.1016/j.phytol.2016.04.004

Phytochemistry Letters 16 (2016) 141–145
Contents lists available at ScienceDirect
Phytochemistry Letters
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Tagenols A and B: New lipoxygenase inhibitor flavonols from Tagetes
minuta
Gamal A. Mohameda,b,
*
a
Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch 71524, Assiut, Egypt
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Phytochemical investigation of the methanolic extract of Tagetes minuta L. (Asteraceae) aerial parts
afforded two new flavonol derivatives: tagenol A (quercetagetin-8-hydroxy-3,6,40-trimethy ether) (1)
Received 5 February 2016
Received in revised form 25 March 2016
Accepted 6 April 2016
and tagenol B (quercetagetin-8-hydroxy-3-methoxy-6-O-
known flavonoids: quercetin-3,6-dimethyl ether (2), quercetin-3-methyl ether (3), and axillarin-7-O-
-glucopyranoside (5). Their structures were identified by UV, IR, 1D (1H and 13C), 2D (1H–1H COSY,
b-D-glucopyranoside) (4), together with three
Available online xxx
b-D
Keywords:
Tagetes minuta
Tagenol
Flavonol
Lipoxygenase inhibitor
Antioxidant
HSQC, and HMBC) NMR, and HRESIMS spectral data as well as comparison with literature data. The
isolated compounds were evaluated for their lipoxygenase inhibitory and antioxidant activities.
Compounds 1–5 displayed moderate lipoxygenase inhibitory activities with IC50 values of 12.2, 9.4, 7.1,
5.8, and 4.2
promising antioxidant activities with IC50 values of 14.9, 14.3, 13.5, 12.7, and 11.2
DPPH assay compared to propyl gallate (IC50 7.38 M).
m
M, respectively compared to indomethacin (IC50 0.9
m
M). Moreover, they displayed
m
M, respectively using
m
ã 2016 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
et al., 2010; Gillij et al., 2008). It has been shown to possess wide
range of biological properties such as
a-amylase inhibitor,
The genus Tagetes belonging to family Asteraceae, includes
about 56 species (Soule, 1993). Several species of this genus have
been investigated as possible sources of different chemical and
biochemical compounds of high pharmaceutical and nutritional
values (Vasudevan et al., 1997; Shahzadi et al., 2010). They have
been used as a source of essential oil for the flavoring in the food
industries. Also, the powders and extracts of Tagetes are rich in the
orange-yellow carotenoid and used as a food colorant in foods:
mayonnaises, pasta, vegetable oil, margarine, salad dressing, baked
goods, confectionery, dairy products, ice cream, yogurt, citrus
juice, and mustard (Al-Musayeib et al., 2014; Nerio et al., 2010;
Leung,1980). Tagetes minuta L. commonly known as marigold, is an
annual perennial herb. It is widely grown from temperate to
tropical regions of the world in a wider range of climatic conditions
(Shahzadi and Shah, 2015). It has been used as a remedy for several
medical aliments such as colds, respiratory inflammations,
stomach problem, chest and skin infections, coughs, catarrh,
wounds, cuts, calluses, and bunions (Maity et al., 2011; Rahimi
antimicrobial, anti-spasmodic, anti-parasitic, anti-septic, insecti-
cidal, sedative, anti-inflammatory, and acaricidal (Ibrahim et al.,
2015; Andreotti et al., 2013; Shahzadi et al., 2010; Tereschuk et al.,
1997). Previous chemical investigations of T. minuta growing in
Saudi Arabia revealed the presence of thiophenes and flavonoids
(Ibrahim et al., 2015; Al-Musayeib et al., 2014). In the present study,
the air-dried powdered aerial parts were exhaustively extracted
with MeOH. The total MeOH extract was dissolved in H2O and
fractionated with n-hexane, EtOAc, and n-BuOH. The EtOAc
fraction was subjected to silica gel, Sephadex LH-20, and RP18
column chromatography to yield two new flavonol derivatives:
tagenols A (1) and B (4), along with quercetin-3,6-dimethyl ether
(2), quercetin-3-methyl ether (3), and axillarin-7-O-b-Dglucopyr-
anoside (5) (Fig. 1). Herein, the isolation and structure elucidation
as well as lipoxygenase inhibitory and antioxidant activities of the
isolated compounds have been reported.
2. Results and discussion
Compound 1 was obtained as yellow amorphous powder and
gave positive tests for flavonoids (Mabry et al., 1970). Its molecular
formula was determined as C18H16O9, on the basis of the HRESIMS
pseudo-molecular ion peak at m/z 377.0879 (calcd for C18H17O9,
* Corresponding author at: Department of Natural Products and Alternative
Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi
Arabia.
E-mail address: gamals2001@yahoo.com (G.A. Mohamed).
http://dx.doi.org/10.1016/j.phytol.2016.04.004
1874-3900/ã 2016 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

142
G.A. Mohamed / Phytochemistry Letters 16 (2016) 141–145
OH
OH
OCH3
OH
OH
OH
HO
O
O
HO
O
O
H3CO
OCH3
OH
H3CO
OCH3
OH
2
1
OH
O
OH
OH
OH
HO
O
O
O
HO
O
O
HO
HO
OCH3
OH
OH
O
OCH3
4
OH
OH
3
OH
OH
HO
O
O
O
HO
HO
O
OH
OH
OH
5
Fig. 1. Structures of the isolated compounds 1–5.
[M + H]+, 377.0873), together with 13C NMR and HSQC spectral
data. The UV spectrum bands at 262 and 352 nm suggested 1 to
have flavonol skeleton (Mabry et al., 1970). The IR spectrum
The HMBC cross peaks of 3-OCH3 to C-3, 6-OCH3 to C-6, and 40-
OCH3 to C-40 established their placement at C-3, C-6, and C-40. On
the basis of the above evidences, the structure of 1 was determined
as quercetagetin-8-hydroxy-3,6,40-trimethy ether and named
tagenol A.
Compound 4 was isolated as yellow amorphous powder. The
compound gave positive reaction for the flavonoids (Mabry et al.,
1970). The HRESIMS spectrum showed a pseudo-molecular ion
peak at m/z 511.1091 [M + H]+, which compatible with the
molecular formula C22H22O14. The presence of flavonol moiety
was deduced from the UV absorption maxima at 264 and 348 nm
(Mabry et al., 1970). Compound 4 was 134 mass units and one
degree of unsaturation more than 1. The UV and NMR spectral data
of 4 were similar to those of 1 except the signals for 6- and 40-OCH3
groups at dH 3.79/dC 62.9 (6-OCH3) and 3.96/dC 58.3 (40-OCH3) in 1
were not present. Instead, a signal of an anomeric proton at dH 5.20
(1H, d, J = 7.0 Hz, H-100) with a coupling constant characteristic of a
showed absorption bands at 3405 (OH), 1658 (a,b-unsaturated
CO), and 1608 (aromatic ring) cmꢀ1 (Silverstein and Webster,
1998). The 13C NMR data displayed resonances for 18 carbons,
including one carbonyl (dC 178.0), 3 methoxys, 3 methines, and
9 oxygen-bonded quaternary carbons. The 1H NMR spectrum
showed resonances for three coupled aromatic protons at dH 7.67
(d, J = 1.5 Hz, H-20), 7.55 (dd, J = 8.5, 1.5 Hz, H-60), and 6.88 (d,
J = 8.5 Hz, H-50). The HSQC spectrum correlated these signals to the
carbons resonating at dC 115.0, 120.0, and 115.5, respectively,
suggesting the presence of 1,3,4-tri-substituted ring B (Williams
and Harborne, 1994). This was confirmed by the observed HMBC
cross peaks of H-20 and H-50 to C-10, C-30, and C-40 and H-60 to C-20
and C-40. The connectivity of ring B at C-2 of ring C was established
by the 3J HMBC cross peaks of H-20 and H-60 to C-2. The two singlet
signals at dH 12.58 and 10.89 were assigned to 5-chelated OH and
7-OH groups, respectively (Table 1) (Agrawal, 1989; Harborne,
1988). Their positions were established based on the HMBC cross
peaks of 5-OH to C-5, C-6, and C-10 and 7-OH to C-6, C-7, and C-8
(Fig. 2). The bathochromic shift of the UV absorption bands; 60
(band I) and 34 (band II) nm upon addition of AlCl3 and NaOAc,
respectively, confirmed the presence of 5- and 7-OH groups,
respectively. In addition, signals for three methoxy groups at dH
3.96 (6H, s, 3,40-OCH3) and 3.76 (3H, s, 6-OCH3) were observed in
the 1H NMR spectrum. They showed HSQC cross peaks with the
carbons at dC 62.9 (6-OCH3), 60.0 (3-OCH3), and 58.3 (40-OCH3).
b
-configuration and additional sugar protons at dH 5.01–3.21,
indicated the presence of -glucopyranose moiety (Agrawal 1992).
b
This was confirmed by the 13C NMR signals at dC 100.9 (C-100), 73.1
(C-200), 75.7 (C-300), 69.6 (C-400), 77.2 (C-500), and 62.6 (C-600) and the
HRESIMS fragment ion peak at m/z 348.0471 [M + H-Glu]. In the
HMBC, the cross peak of H-100 to C-6 (dC 108.6) suggested the
attachment of the glucose moiety at C-6 (Fig. 2). Furthermore,
three hydroxyl group signals at dH 9.35 (s, 30-OH), 9.38 (s, 40-OH),
and 8.53 (s, 8-OH) were observed. In the HMBC, the cross peaks of
5-OH to C-5, C-6, and C-10, 7-OH to C-7 and C-8, 30-OH to C-30, 40-
OH to C-40, and 8-OH to C-7 and C-8 established the position of

G.A. Mohamed / Phytochemistry Letters 16 (2016) 141–145
143
Table 1
NMR Data of compounds 1 and 4 (DMSO-d6, 500 and 125 MHz).
No.
1
4
dH [mult., J (Hz)]
dC (mult.)
HMBC
dH [mult., J (Hz)]
dC (mult.)
HMBC
2
3
4
5
6
7
8
9
10
1‘
2‘
3‘
4‘
5‘
6‘
1“
–
157.2 (C)
139.9 (C)
178.0 (C)
145.6 (C)
132.3 (C)
142.9 (C)
130.8 (C)
139.3 (C)
106.5 (C)
121.9 (C)
115.0 (CH)
146.9 (C)
149.7 (C)
115.5 (CH)
120.0 (CH)
–
–
–
155.6 (C)
139.6 (C)
177.1 (C)
145.3 (C)
108.6 (C)
142.6 (C)
130.0 (C)
137.6 (C)
106.1 (C)
122.0 (C)
115.3 (CH)
146.5 (C)
147.7 (C)
117.2 (CH)
119.9 (CH)
100.9 (CH)
73.1 (CH)
75.7 (CH)
69.6 (CH)
77.2 (CH)
62.6 (CH2)
58.9 (CH3)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
7.67 d (1.5)
–
2, 1‘, 3‘, 4‘
7.72 brs
–
2, 4‘, 3‘, 6‘
–
–
–
–
6.88 d (8.5)
7.55 dd (8.5, 1.5)
1‘, 3‘, 4‘
2, 2‘, 4‘
6.93 d (8.0)
7.55 d (8.0)
5.20 d (7.0)
3.21-5.01 m
1‘, 3‘, 4‘
2, 2‘, 4‘
6
1“
5“
2“
3“
6“
–
–
2“
3“
4“
5“
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
6“
–
–
–
–
3-OCH3
6-OCH3
4‘-OCH3
5-OH
7-OH
3‘-OH
4‘-OH
8-OH
3.96 s
3.76 s
3.96 s
12.58 s
10.89 s
–
60.0 (CH3)
62.9 (CH3)
58.3 (CH3)
–
3
6
4‘
3.94 s
–
3
–
–
–
–
5, 6, 10
6, 7, 8
–
–
–
12.22 s
9.74 s
9.35 s
9.38 s
8.53 s
–
5, 6, 10
7, 8
3‘
4‘
7, 8
–
–
–
–
–
–
–
–
–
–
hydroxyl groups at C-5, C-7, C-8, C-30, and C-40. This was confirmed
by UV spectral data with diagnostic shift reagents. The presence of
3-OCH3 group was established by the signals at dH 3.94/dC
58.9 and further confirmed by its HMBC cross peak to C-3 (dC
139.6). Upon the hydrolysis of 4, quercetagetin-8-hydroxy-3-
methyl ether and glucose were obtained. The glucose moiety was
identified by co-chromatography alongside with authentic sample.
On the basis of the abovementioned data, the structure of tagenol
B was assigned as quercetagetin-8-hydroxy-3-methoxy-6-O-b-D-
glucopyranoside.
The following known compounds were identified through the
analysis of the spectroscopic data (1D, 2D NMR, and MS) and
comparison of their data with those in the literature: quercetin-
3,6-dimethyl ether (2) (Andersen and Markham, 2006; Williams
and Harborne, 1994), quercetin-3-methyl ether (3) (Andersen and
Markham, 2006; Williams and Harborne, 1994), and axillarin-7-O-
b-D
-glucopyranoside (5) (Schmeda-Hirschmanna et al., 2004).
Compounds 1–5 displayed moderate lipoxygenase inhibitory
activity. Their IC50 values were found to be 12.2, 9.4, 7.1, 5.8, and
4.2 M, respectively compared to indomethacin (IC50 0.9 M) used
m
m
as positive control (Table 2). In addition, they showed promising
antioxidant activity with IC50 values of 14.9, 14.3, 13.5, 12.7, and
11.2 mM, respectively compared to propyl gallate (IC50 7.38 mM)
using DPPH assay. These results were in accordance with previous
studies that attributed the anti-inflammatory and antioxidant
potentials of flavonoids to the presence of C-2,3 double bond, 3-
hydroxyl group, and catechol group at ring B (Lago et al., 2014;
Pietta 2000; Dugas et al., 2000; Kim et al., 1998). Moreover, the
double bond between C-2/C-3 induces coplanarity between rings A
Table 2
Lipoxygenase-5 inhibitory and antioxidant activities results.
Compound
LOX-5 inhibitor
IC50
M), ꢁ S.D
Antioxidant activity
IC50
M), ꢁ S.D
(
m
(m
1
2
3
4
12.2 ꢁ 0.41
9.4 ꢁ 0.67
7.1 ꢁ 0.11
5.8 ꢁ 0.14
4.2 ꢁ 0.10
0.9 ꢁ 0.07
–
14.9 ꢁ 0.11
14.3 ꢁ 0.16
13.5 ꢁ 0.75
12.7 ꢁ 0.9
11.2 ꢁ 0.13
–
5
Indomethacin
Propyl gallate
7.38 ꢁ 0.05
Fig. 2. Important HMBC correlations of compounds 1 and 4.

144
G.A. Mohamed / Phytochemistry Letters 16 (2016) 141–145
and C, favoring the interaction of the flavonoid with the enzymatic
site receptor (Lattig et al., 2007). Kim et al. (2004) reported that the
presence of hydroxyl groups at C-5 and C-7 (ring A) and C-3‘ and C-
4‘ (ring B) positions is an important feature for the anti-
inflammatory action. However, their glycosylation reduces the
activity (Kim et al., 2004).
(6.7 mg, yellow powder) and 3 (11.5 mg, yellow amorphous
powder). Sub-fraction TME-7 (108.9 mg) was subjected to silica
gel column (50 g ꢃ 50 ꢃ 2 cm) using CHCl3:MeOH gradient to get
impure 4 and 5. Their purification was achieved using RP18 column
eluting with MeOH:H2O gradient to give 4 (5.6 mg, yellow
amorphous powder) and 5 (13.1 mg, yellow amorphous powder).
The other sub-fractions were retained for further investigation.
3. Experimental
3.4. Spectral data
3.1. General experimental procedures
3.4.1. Quercetagetin-8-hydroxy-3,6,40-trimethy ether (1)
Yellow amorphous powder (4.2 mg); UV lmax MeOH (nm): 352,
262; NaOCH3: 363, 301 sh, 267; A1C13: 412, 302; +HC1: 370, 272;
NaOAc: 364, 296; +H3BO3: 376, 294; IR (KBr) nmax: 3405, 2989,
1658, 1608, 1056 cmꢀ1; NMR see Table 1; HRESIMS m/z: 377.0879
(calcd for C18H17O9, [M + H]+, 377.0873).
Optical rotation was measured on a JASCO digital polarimeter.
Ultraviolet (UV) spectra were measured on a Hitachi 300 spectro-
photometer. The IR spectra were measured on a Shimadzu
Infrared-400 spectrophotometer (Kyoto, Japan). ESIMS spectra
were obtained with a LCQ DECA mass spectrometer (ThermoFin-
nigan, Bremen, Germany) coupled to an Agilent 1100HPLC system
equipped with a photodiode array detector. HRESIMS was recorded
on LTQ Orbitrap mass spectrometer (ThermoFinnigan, Bremen,
Germany). 1D and 2D NMR spectra were recorded on Bruker
Avance DRX 500 MHz spectrometer. For column chromatography,
silica gel (0.063–0.200 mm, Merck, Darmstadt, Germany), Sepha-
dex LH-20 (0.25–0.1 mm, Sigma-Aldrich), and RP18 (0.04–
0.063 mm Merck, Darmstadt, Germany) were used. Pre-coated
silica gel 60 F254 plates (0.2 mm, Merck, Darmstadt, Germany) were
used for thin-layer chromatography. Six mL standard extraction
3.4.2. Quercetin-3,6-dimethyl ether (2)
Yellow amorphous powder (6.7 mg); 1H NMR (500 MHz, DMSO-
d6): dH 6.52 (1H, s, H-8), 7.67 (1H, d, J = 1.5 Hz, H-20), 6.88 (1H, d,
J = 8.5 Hz, H-50), 7.55 (1H, dd, J = 8.5, 1.5 Hz, H-60), 3.96 (3H, s, 3-
OCH3), 3.76 (3H, s, 3-OCH3),12.58 (1H, s, 5-OH),10.89 (1H, s, 7-OH),
9.34 (1H, s, 30-OH), 9.63 (1H, s, 4-OH); 13C NMR (125 MHz, DMSO-
d6): dC 147.7 (C-2), 135.4 (C-3), 176.0 (C-4), 151.7 (C-5), 130.8 (C-6),
157.2 (C-7), 93.6 (C-8), 151.3 (C-9), 103.3 (C-10), 121.9 (C-10), 115.0
(C-20), 146.9 (C-30), 145.0 (C-40), 115.5 (C-50), 120.0 (C-60), 60.0 (3-
OCH3), 62.9 (6-OCH3); HRESIMS m/z: 347.0762 (calcd for C17H15O8,
[M + H]+, 347.0767).
tube (RP18, 40–63 mm, Merck, Darmstadt, Germany) was used for
compounds purification. The compounds were detected by UV
absorption at lmax 255 and 366 nm, exposure to NH3 vapor
followed by spraying with p-anisaldehyde:H2SO4 reagent and
heating at 110 ꢂC for 1–2 min. Linoleic acid, 5-lipoxygenase kits,
indomethacin, 2,20-diphenylpicrylhydrazyl, and propyl gallate
were purchased from Sigma-Aldrich (St. Louis, MO, USA).
3.4.3. Quercetin-3-methy ether (3)
Yellow amorphous powder (11.5 mg); 1H NMR (500 MHz,
DMSO-d6): dH 6.20 (1H, brs, H-6), 6.44 (1H, brs, H-8), 7.68 (1H,
brs, H-20), 6.89 (1H, d, J = 8.5 Hz, H-50), 7.54 (1H, brd, J = 8.5 Hz, H-60),
3.76 (3H, s, 3-OCH3), 12.49 (1H, s, 5-OH), 10.89 (1H, s, 7-OH), 9.37
(1H, s, 30-OH), 9.66 (1H, s, 40-OH); 13C NMR (125 MHz, DMSO-d6):
dC 156.1 (C-2), 135.7 (C-3), 175.8 (C-4), 160.6 (C-5), 98.2 (C-6), 163.9
(C-7), 93.3 (C-8), 156.1 (C-9), 102.9 (C-10), 121.9 (C-10), 115.0 (C-20),
147.7 (C-30), 145.0 (C-40), 115.6 (C50), 119.9 (C60), 59.9 (3-OCH3);
HRESIMS m/z: 317.0668 (calcd for C16H13O7, [M + H]+, 317.0661).
3.2. Plant material
The plant sample was collected in March 2014 from Al-Baha city,
Saudi Arabia. The plant was kindly identified by a taxonomist at the
Department of Natural products and Alternative Medicine, King
Abdulaziz University, Saudi Arabia, in addition to its morphological
featuresandthelibrarydatabase(Collenette,1999).Itwas confirmed
by Dr. Nahed Morad, Faculty of Science, King Abdulaziz University,
Saudi Arabia. Avoucher specimen (TM-11-2014) was archived at the
Department of Natural Products and Alternative Medicine herbari-
um, King Abdulaziz University, Saudi Arabia.
3.4.4. Quercetagetin-8-hydroxy-3-methoxy-6-O-
b-D-
glucopyranoside (4)
Yellow amorphous powder (5.6 mg); [a]D
25 + 59.08 (c 0.5,
MeOH); UV lmax MeOH (nm): 348, 264; NaOCH3: 402, 274;
A1C13: 416, 308; +HC1: 377, 280; NaOAc: 367, 291; +H3B03: 388,
295; IR (KBr) nmax: 3436, 3989, 1647, 1052 cmꢀ1; NMR see Table 1;
HRESIMS m/z: 511.1091 (calcd for C22H23O14, [M + H]+, 511.1088).
3.3. Extraction and isolation
The air-dried powdered aerial parts (0.9 kg) were macerated
with MeOH (2 ꢃ 3 L). The total methanolic extract was concentrat-
ed under vacuum (22.6 g). The later was mixed with 100 mL
distilled water and fractionated successively between n-hexane
(3 ꢃ 500 mL), EtOAc (3 ꢃ 500 mL), and n-BuOH (3 ꢃ 500 mL). Each
fraction was concentrated to give n-hexane (3.7 g), EtOAc (2.3 g), n-
BuOH (2.1 g), and aqueous (11.2 g) fractions. The EtOAc fraction
(2.3 g) was chromatographed over a Sephadex LH-20 column
(100 g ꢃ 50 ꢃ 3 cm) using MeOH as an eluent to obtain nine sub-
fractions: TME-1 to TME-9. Sub-fraction TME-2 (71.2 mg) was
chromatographed on silica gel column (40 g ꢃ 50 ꢃ 2 cm) and
eluted with CHCl3:MeOH gradient to give impure 1. Purification of
1 was carried out on reversed phase silica gel extraction tube using
H2O with gradual increase of acetonitrile to yield 1 (4.2 mg, yellow
amorphous powder). Silica gel column (50 g ꢃ 50 ꢃ 2 cm) of sub-
fraction TME-4 (115.4 mg) using CHCl3:MeOH gradient elution
gave impure 2 and 3 which were purified on reversed phase silica
gel extraction tube using H2O:acetonitrile gradient to yield 2
3.4.5. Quercetagetin-6-glucose (5)
Yellow amorphous powder (13.1 mg); 1H NMR (500 MHz,
DMSO-d6): dH 6.89 (1H, s, H-8), 7.72 (1H, brs, H-20), 6.94 (1H, d,
J = 8.5 Hz, H-50), 7.54 (1H, brd, J = 8.5 Hz, H-60), 5.02 (1H, d, J = 7.0 Hz,
H-10), 3.21-5.44 (m, sugar protons), 12.24 (1H, s, 5-OH), 9.68 (1H, s,
7-OH), 9.33 (1H, s, 30-OH), 9.39 (1H, s, 40-OH), 8.48 (1H, s, 3-OH);
13C NMR (125 MHz, DMSO-d6): dC 148.1 (C-2), 135.6 (C-3), 176.1 (C-
4), 145.3 (C-5), 129.6 (C-6), 151.5 (C-7), 93.5 (C-8), 147.5 (C-9), 105.1
(C-10), 122.0 (C-10), 115.3 (C-20), 145.0 (C-30), 147.7 (C-40), 115.5 (C-
50), 119.9 (C-60), 100.8 (C-100), 73.1 (C-200), 75.7 (C-300), 69.6 (C400),
77.2 (C-500), 60.6 (C-600); HRESIMS m/z: 481.0987 (calcd for
C
21H21O13, [M + H]+, 481.0982).
3.5. Acid hydrolysis
A solution of the isolated glycoside 4 (3 mg in 5 mL MeOH) was
treated with 3% H2SO4 (1.5 mL) and heated at 100 ꢂC for 1 h. The
aglycone was extracted with EtOAc, concentrated under reduced

G.A. Mohamed / Phytochemistry Letters 16 (2016) 141–145
145
pressure, and purified on Sephadex LH-20 column using MeOH.
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4. Conclusion
Two new flavonols (1 and 4), along with three known
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Appendix A. Supplementary data
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phytol.2016.04.004.
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