New thiophene and flavonoid from tagetes minuta leaves growing in saudi arabia

Article abstract of DOI:10.3390/molecules19032819

Phytochemical investigation of the methanolic extract of Tagetes minuta L. (Asteraceae) leaves resulted in the isolation and identification of two new compounds: 5-methyl-2,2',5',2'',5'',2''',5''',2''''-quinquethiophene (1) and quercetagetin-6-O-(6-Ocaffe

Full text of DOI:10.3390/molecules19032819

Molecules 2014, 19, 2819-2828; doi:10.3390/molecules19032819
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
New Thiophene and Flavonoid from Tagetes minuta Leaves
Growing in Saudi Arabia
1
,
2
3
4
Nawal M. Al-Musayeib *, Gamal A. Mohamed , Sabrin R. M. Ibrahim and Samir A. Ross
1
Department of Pharmacognosy, Faculty of Pharmacy, King Saud University, Riyadh 11451,
Saudi Arabia
2
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch,
Assiut 71524, Egypt; E-Mail: gamals2001@yahoo.com
3
Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt;
E-Mail: sabrinshaur@gmail.com
4
National Center for Natural Products Research, Department of Pharmacognosy, School of Pharmacy,
The University of Mississippi, University, MS 38677, USA; E-Mail: sross@olemiss.edu
*
Author to whom correspondence should be addressed; E-Mail: nalmusayeib@ksu.edu.sa;
Tel./Fax: +966-11-228-2584.
Received: 28 January 2014; in revised form: 25 February 2014 / Accepted: 25 February 2014 /
Published: 4 March 2014
Abstract: Phytochemical investigation of the methanolic extract of Tagetes minuta L.
(
Asteraceae) leaves resulted in the isolation and identification of two new compounds:
-methyl-2,2',5',2'',5'',2''',5''',2''''-quinquethiophene (1) and quercetagetin-6-O-(6-O-
5
caffeoyl-β-D-glucopyranoside) (9), in addition to seven known compounds: quercetin-3,6-
dimethyl ether (2), quercetin-3-methyl ether (3), quercetin (4), axillarin-7-O-β-D-
glucopyranoside
(5),
quercetagetin-3,7-dimethoxy-6-O-β-D-glucopyranoside
(6),
quercetagetin-7-methoxy-6-O-β-D-glucopyranoside (7), and quercetagetin-6-O-β-D-
glucopyranoside (8). The compounds were identified by UV, IR, 1D, 2D NMR, and
HRESIMS spectral data. They showed significant antioxidant activity, comparable with
that of propyl gallate. Compounds 8 and 3 showed weak to moderate antileishmanial and
antimalarial activities, with IC50 values of 31.0 μg/mL and 4.37 μg/mL, respectively.
Keywords: Tagetes minuta; Asteraceae; thiophene; quercetagetin; antioxidant;
antimicrobial; antileishmanial; antimalarial

Molecules 2014, 19
. Introduction
The genus Tagetes (Asteraceae) is mainly native to the central and southern part of America. It
2820
1
consists of approximately 30 species [1]. Members of the genus Tagetes have a long history of human
use as beverages, condiments, ornamentals, and medicinal decoctions. Tagetes minuta L. has been
used as anthelmintic, diuretic, antispasmodic, and for treatment of stomach and intestinal diseases [2].
Tagetes oil is used as a flavor component in food products including cola and alcoholic beverages,
frozen dairy desserts, candy, baked goods, gelatins, puddings, condiments, and relishes [3]. The oil
has antibacterial [4], larvicidal [5], and insecticidal [6] activities. Previous phytochemical studies
of T. minuta L. led to the isolation of terpenes [2,7], flavonoids [8], thiophenes, and aromatic
compounds [9]. This article reports the isolation and characterization of two new compounds:
5
-methyl-2,2',5',2'',5'',2''',5''',2''''-quinquethiophene (1) and quercetagetin-6-O-(6-O-caffeoyl-β-D-gluco-
pyranoside) (9), together with seven known flavonoids (Figure 1).
Figure 1. Chemical structures of the isolated compounds 1–9.
2
. Results and Discussion
+
Compound 1 was isolated as brown needles. HRESIMS gave an [M+H] at m/z 427.6611 and
+
4
28.6613 [M+2H] , which is consistent with the molecular formula C21
H
14
S
5
, implying fifteen degrees
of unsaturation. The UV absorption maxima at 387 and 334 nm indicated the presence of
1
quinquethiophene moiety [10,11]. The H-NMR spectrum showed eleven protons signals at 
H
6.64–7.21
with coupling constants 5.5–3.5 Hz characteristic for 5-subsituted quinquethiophenes [11].
Additionally, the proton signal at  2.42 (3H, s) indicated the presence of a methyl group (Table 1).
H
1
3
The C-NMR spectrum exhibited twenty one carbon resonances. The multiplicities of the carbons in 1
were confirmed with DEPT and HSQC experiments, which showed one methyl, eleven methines, and
1
1
nine quaternary carbons. H- H COSY provided five spin systems for five thiophene rings (Figure 2).
The HMBC spectrum exhibited cross peaks from methyl protons at C-5 to C-4 and C-5. In HMBC
spectrum, cross-peaks from H-3 to C-2', H-3' to C-2, H-4' to C-2'', H-3'' to C-5', H-4'' to C-2''', H-3''' to
C-5'', H-4''' to C-2'''', and H-3'''' to C-5'''confirmed the connectivity of thiophene rings [11].

Molecules 2014, 19
2821
Accordingly, 1 was 5-methyl-2, 2',5', 2'',5'',2''',5''',2''''-quinquethiophene. Compound 1 was isolated for
the first time from natural origin.
Table 1. NMR spectral data for compound 1 (CDCl , 500 and 125 MHz).
3
No.
2

H
[mult., J (Hz)]

C
(mult.)
HMBC
-
133.5 (C)
125.9 (CH)
124.0 (CH)
137.3 (C)
-
3
6.93 d (3.5)
2, 4, 5
4
6.64 brs
2, 3, 5
5
-
-
2
3
4
5
'
'
'
'
-
134.2 (C)
-
6.97 d (3.5)
121.2 (CH)
122.3 (CH)
128. 9 (C)
134.7 (C)
2
6.96 d (3.5)
2', 3', 2''
-
-
2
3
4
5
''
''
''
''
-
-
6.98 d (3.5)
121.5 (CH)
122.3 (CH)
126.1 (C)
4''', 2'''
7.02 d (3.5)
2'''
-
-
2
3
4
5
'''
'''
'''
'''
-
135.1 (C)
-
4''', 5''', 2'''
2''', 2'''', 3''', 5'''
-
6.99 d (3.5)
125.8 (CH)
121.6 (CH)
132.8 (C)
7.12 d (3.5)
-
2
3
4
5
''''
''''
''''
''''
-
135.3 (C)
-
7.04 brs
122.5 (CH)
121.7 (CH)
123.0 (CH)
4'''', 5'''
2'''', 3''''
3'''', 4''''
4, 5
7.14 brd (3.5)
7.21 brd (5.5)
2.42 s
5
-CH
3
3
15.4 (CH )
1
1
Figure 2. H- H COSY and HMBC correlations of 1 and 9.
Compound 9 was isolated as a brown residue. It gave positive tests for flavonoids [12]. The
HRESIMS spectrum showed a pseudo-molecular ion peak at m/z 643.1223, consistent with a
molecular formula C30
26
H O16. The UV spectrum of 9 showed absorption bands at 275 and 365 nm
suggesting its flavonol nature [12]. IR spectrum showed absorption bands at 3,460 (OH), 2,976
−1
(
aromatic C-H), 1,668 (ester C=O), 1,608 (α,β-unsaturated C=O), and 1,058 (C-O) cm . Analysis of

Molecules 2014, 19
2822
the NMR spectra of 9 showed the presence of quercetagetin, trans-caffeoyl, and glucopyranosyl
+
moieties and confirmed by significant fragment ion peaks at m/z 480.0826 [M+H-caffeoyl] and m/z
+
1
3
18.0299 [M+H-(caffeoyl+hexose)] . The H-NMR spectrum revealed the presence of six singlets
signals at  6.59 (H-8), 8.50 (3-OH), 9.35 (3'-OH), 9.35 (4'-OH), 10.89 (7-OH), and 12.24 (5-OH).
H
H
Also, it showed three coupled protons at  6.88 (1H, d, J = 7.0 Hz, H-5'), 7.57 (1H, brd, J = 7.0 Hz,
H-6'), and 7.74 (1H, brs, H-2') for a tri-substituted B-ring (Table 2). Furthermore, an anomeric proton
signal at  5.02 (1H, d, J = 6.5 Hz, H-1'') indicated β-configuration of the glycosidic linkage [13]. In
H
addition, signals at 
1H, d, J = 6.8 Hz, H-5''') for a tri-substituted phenyl ring (ABX pattern) and two trans-coupled
olefinic protons at  7.43 (1H, d, J = 16.0 Hz, H-7''') and 6.23 (1H, d, J = 16.0 Hz, H-8''') indicating
H
6.96 (1H, d, J = 1.5 Hz, H-2'''), 6.93 (1H, dd, J = 6.8, 1.5 Hz, H-6''' ), and 6.77
(
H
1
3
the presence of trans caffeyol moiety (Table 2) [14–16] in which confirmed by C-NMR signals at 
13.4 (C-8'''), 115.3 (C-5'''), 115.7 (C-2'''), 120.5 (C-6'''), 145.3 (C-4'''), 145.5 (C-7'''), 148.3 (C-3'''),
and 166.5 (C-9''').
C
1
Table 2. NMR spectral data for compound 9 (DMSO-d
6
, 500 and 125 MHz).
No.
2

H
[mult., J (Hz)]

C
(mult.)
HMBC
-
148.1 (C)
135.6 (C)
176.1 (C)
151.5 (C)
129.6 (C)
151.4 (C)
93.5 (CH)
147.7 (C)
105.1 (C)
122.0 (C)
115.5 (CH)
145.0 (C)
147.5 (C)
115.3 (CH)
119.9 (CH)
100.9 (CH)
73.1 (CH)
75.7 (CH)
69.6 (CH)
77.2 (CH)
-
-
-
-
-
3
-
4
-
5
-
6
-
7
-
8
6. 59 s
6, 7, 10
9
-
-
1
0
-
-
1
'
'
'
'
'
'
-
-
2
7.74 brs
2, 4', 6'
3
4
5
6
-
-
-
-
6.88 d (7.0)
3', 6'
7.57 brd (7.0)
5.02 d (6.5)
2, 1', 4'
1
2
3
4
5
''
''
''
''
''
6
-
3.77 dd (7.0, 9.0)
3.86 m
-
3.23 dd (9.0, 9.5)
4.35 m
-
-
4
.41 dd (2.8, 12.0)
6
''
64.6 (CH2)
9'''
4
.30 dd (7.0, 12.0)
1
2
3
4
5
6
'''
'''
'''
'''
'''
'''
-
125.2 (C)
115.7 (CH)
148.3 (C)
-
6.96 d (1.5)
6''', 7'''
-
-
-
145.3 (C)
-
3'''
6.77 d (6.8)
115.3 (CH)
120.5 (CH)
6.93 dd (1.5, 6.8)
4''', 5''', 7''', 8'''

Molecules 2014, 19
2823
Table 2. Cont.
No.

H
[mult., J (Hz)]
7.43 d (16.0)
6.23 d (16.0)
-

C
(mult.)
HMBC
9'''
1''', 9'''
7
8
9
'''
'''
'''
145.5 (CH)
113.4 (CH)
166.5 (C)
-
-
-
-
-
-
5
7
-OH
-OH
12.24 s
10.89 s
9.35 s
9.35 s
-
-
-
-
-
3
4
'-OH
'-OH
3
-OH
8.50 s
13
The C-NMR spectrum displayed fifteen carbon signals were attributed to quercetagetin skeleton [16,17]
and six carbons for glucose. The multiplicity of each carbon was determined by HSQC experiment.
The glucose moiety was located at C-6 based on the HMBC cross peak of H-1'' at 
H
5.02 (1H, d, J =
6
.5 Hz) to C-6 ( 129.6) and further confirmed by its reaction with diagnostic shift reagents. In the
C
H
HMBC spectrum, the methylene protons at  4.41 (H-6''B) and 4.30 (H-6''A) correlated with the
caffeoyl carbonyl group at  166.5 suggesting the connectivity of caffeoyl moiety at C-6'' and
C
C
confirmed by the downfield shift of C-6'' ( 64.6). Acid hydrolysis of 9 afforded quercetagetin,
caffeic acid, and β-D-glucose. They were identified by co-chromatography with authentic samples
using (S5) [14]. Accordingly, 9 was identified as quercetagetin-6-O-(6-O-caffeoyl-β-D-glucopyranoside).
The other compounds were identified as quercetin-3,6-dimethyl ether (2) [18], quercetin-3-methyl
ether (3) [18], quercetin (4) [18], axillarin-7-O-β-D-glucopyranoside (5) [19], quercetagetin-3,7-
dimethoxy-6-O-β-D-glucopyranoside (6) [20], quercetagetin-7-methoxy-6-O-β-D-glucopyranoside (7) [20],
and quercetagetin-6-O-β-D-glucopyranoside (8) [20] by comparison of their physical and spectral data
with those in the literature. The antioxidant activity of the isolated compounds 2-9 was determined by
using a DPPH free radical scavenging system. The antioxidant percentage activity ranged from 91.6 to
6
8.3% (Table 3). The antioxidant effect of these compounds was related to the number of free phenolic
hydroxyl groups in the 3,4-dihydroxy form in their structures, which explains the close similarity of
their antioxidant activity. Absence or blocking of the hydroxyl groups by a methyl or glucose moiety
leads to a decrease of the antioxidant activity [21].
Table 3. Antioxidant activity of the isolated compounds.
Comp.
% Activity
81.1
2
3
4
5
6
7
8
9
82.4
91.6
68.3
69.1
71.3
83.0
89.1
Compounds 1–9 were evaluated for their antimicrobial, antimalarial and antileishmanial activities.
None of the isolated compounds 1–9 showed any antimicrobial activity. Compound 8 showed weak

Molecules 2014, 19
2824
antileishmanial activity with an IC 31.0 μg/mL. Compound 3 showed moderate antimalarial activity
5
0
against chloroquine sensitive (D6) clones of P. falciparum with an IC50 of 4.37 μg/mL.
3. Experimental
3.1. General Procedures
Melting points were measured on an Electrothermal 9100 Digital Melting Point apparatus
(
Electrothermal Engineering Ltd, Essex, England). Optical rotation was measured with a Perkin-Elmer
41 automatic polarimeter (Perkin-Elmer Inc, Massachusetts, MA, USA). HRESIMS was recorded on
2
a LTQ Orbitrap (ThermoFinnigan, Bremen, Germany) mass spectrometer. Low resolution mass
spectra were determined using a Finnigan MAT TSQ-7000 mass spectrometer. UV spectra were
recorded on a Shimadzu 1601 UV/VIS spectrophotometer (Kyoto, Japan). The IR spectra were
measured on a Shimadzu Infrared-400 spectrophotometer. 1D and 2D NMR spectra were recorded on
a Bruker Avance DRX 500 instrument (Bruker BioSpin, Massachusetts, MA, USA). Column
chromatography separations were performed on silica gel 60 (0.04–0.063 mm), RP (0.04–0.063 mm
1
8
Merck, Darmstadt, Germany), and Sephadex LH-20 (0.25–0.1 mm, Merck, Darmstadt, Germany).
TLC was performed on pre-coated plates with silica gel 60 F254 (0.2 mm, Merck). The solvent systems
used for TLC analyses were n-hexane/EtOAc (97:3, S1), CHCl
3 3
/MeOH (95:5, S2), CHCl /MeOH
(
90:10, S3), CHCl /MeOH (85:15, S4), and n-BuOH/HOAc/H O (4:1:5, S5).
3
2
3
.2. Plant Material
The leaves of Tagetes minuta L. (Asteraceae) were collected in June 2012 from Al-Baha, Saudi
Arabia. The plant was identified by Dr. A. A. Fayed, Prof. of Plant Taxonomy, Faculty of Science,
Assiut University, Egypt. A voucher specimen (TM-1-2012) was deposited at the herbarium of the
research center for medicinal, aromatic and poisonous plants, King Saud University.
3
.3. Extraction and Isolation
The air-dried powdered leaves (1.1 kg) were extracted with MeOH (4 × 5 L, 24 h each) at room
temperature. The combined extracts were concentrated under reduced pressure to afford a dark green
residue (30.8 g) which was suspended in distilled water (250 mL) then partitioned successively
between n-hexane (3 × 500 mL), EtOAc (3 × 500 mL), and n-BuOH (3 × 500 mL). Each fraction was
concentrated to give n-hexane (4.2 g), EtOAc (3.1 g), n-BuOH (2.6 g), and aqueous (17.8 g) fractions.
The n-hexane fraction (4.2 g) was subjected to vacuum liquid chromatography (VLC) using
a n-hexane-EtOAc gradient to afford four subfractions: H-1 to H-4. Subfraction H-1 (0.52 g) was
chromatographed over a silica gel column (100 g × 50 × 2 cm) using n-hexane/EtOAc (99:1 to 90:10)
to give 1 (17 mg, brown needles). The EtOAc fraction (3.1 g) was subjected to VLC using a CHCl
MeOH gradient, to afford four subfractions: E-1 to E-4. Subfraction E-1 (0.69 g) was chromatographed
over a silica gel column (100 g × 50 × 2 cm) using CHCl -MeOH gradients to give 2 (12 mg, yellow
3
-
3
needles) and 3 (17 mg, yellow needles). Subfraction E-2 (0.90 g) was similarly like subfraction E-1 to
give 4 (9 mg, yellow needles). Silica gel column chromatography of subfraction E-3 (0.51 g)
(
3
150 g × 50 × 3 cm) using CHCl -MeOH gradients yielded 5 (11 mg, yellow residue) and 6 (7 mg,

Molecules 2014, 19
2825
yellow residue). Subfraction E-4 (0.81 g) was chromatographed over a Sephadex LH-20 column
(
100 g × 50 × 3 cm) using MeOH as an eluent to give two subfractions: E-4A (295 mg) and E-4B
430 mg). Subfraction E-4B was subjected to RP column chromatography (100 g × 50 × 2 cm) using a
(
1
8
MeOH-H O gradient to afford 7 (16 mg, yellow residue). The n-BuOH fraction (2.6 g) was subjected
2
to Sephadex LH-20 column chromatography (100 g × 50 × 3 cm) using MeOH as an eluent to give three
subfractions: B-1 (611 mg), B-2 (355 mg), and B-3 (760 mg). Separately, subfractions B-2 and B-3, each
one was chromatographed over a RP column (40 g × 25 × 1 cm) using a MeOH-H O gradient to give
1
8
2
8
(13 mg, yellow residue) and 9 (11 mg, brown residue). The other subfractions were retained for
further investigation.
3.4. Spectral Data
5
-Methyl-2,2',5',2',5'',2''',5''',2''''-quinquethiophene (1). Brown needles (17 mg), m.p. 215–216 °C.
−1
R 0.86, silica gel 60 F (S1). UV (MeOH): max 334, 387 nm. IR (KBr): ν 2870, 1600 cm . NMR
data: see Table 1. HRESIMS: m/z 427.6611 (calcd for C H S , [M+H] , 427.6609); 428.6613 (calcd
f
254
max
+
2
1
15 5
+
16 5
for C21H S , [M+2H] , 428.6609).
Quercetagetin-6-O-(6-O-caffeoyl--D-glucopyranoside) (9). Brown residue (11 mg), R
f
0.76, silica gel
−176 (0.5, MeOH). UV (MeOH): λmax 275, 365 nm; NaOMe: 282, 405 nm; AlCl
95, 410 nm; AlCl /HCl: 293 388 nm; NaOAc: 296, 385 nm; NaOAc/H BO : 280, 385 nm. IR
6
2
0 F254 (S4). [α]
D
3
:
3
3
3
−1
(
KBr): νmax 3460, 2976, 1668, 1608, 1565, 1058 cm . NMR data: see Table 2. HRESIMS: m/z
+
6
43.1223 (calcd for C30
H
27
O16, [M+H] , 643.1221).
3
.5. Acid Hydrolysis of 9
Compound 9 (3 mg) was refluxed in 10 mL of 1 N HCl for 4 h. The aglycone was extracted with
CHCl . The sugar in the aqueous layer was identified by co-paper chromatography (PC) with authentic
materials using solvent system (S5) and aniline phthalate spray as detection reagent [14].
3
3.6. Antimicrobial Assay
All the isolated compounds 2–9 were tested for antimicrobial activity against Candida albicans
ATCC 90028, Candida glabrata ATCC90030, Candida krusei ATCC 6258, Asperigillus fumigates
ATCC 90906, methicillin-resistant Staphylococcus aureus ATCC 33591, Cryptococcus neoformans
ATCC 90113, Staphylococcus aureus ATCC 2921, Escherichia coli ATCC 35218,
Pseudomonus aeruginosa ATCC 27853, and Mycobacterium intracellulare ATCC 23068 as described
previously [22–24]. Ciprofloxacin and amphotericin B were used as positive standards.
3
.7. Antimalarial Assay
The isolated compounds were tested on chloroquine sensitive (D6, Sierraleon) and resistant (W2,
Indo-china) strains of Plasmodium falciparum using previously reported method [22,25]. Artemisinin
and chloroquine were included in each assay as anti-malarial drug controls.

Molecules 2014, 19
.8. Antileishmanial Assay
The anti-leishmanial activity of the isolated metabolites was tested in vitro against a culture of
2826
3
L. donovani promastigotes as previously outlined [26]. Pentamidine and amphoterecin B were used as
positive standards.
3
.9. Antioxidant Activity
The antioxidant activity of the isolated compounds 2–9 (20 μM) in DPPH solution (4 mg was
dissolved in HPLC MeOH 50 mL to obtain a concentration 80 μg/mL) was determined as previously
outlined [27–29].
4
. Conclusions
In conclusion, in this study nine compounds were isolated and elucidated from T. minuta L. two of
them (compounds 1 and 9) are new. The antioxidant, antimicrobial, antimalarial, and antileishmanial
activities of the isolated compounds were evaluated. They showed antioxidant activity ranging from
9
1.6% to 68.3%. Compound 8 showed weak antileishmanial activity with an IC 31.0 μg/mL, while
50
compound 3 showed moderate antimalarial activity against chloroquine sensitive (D6) clones of
P. falciparum with an IC50 4.37 μg/mL.
Acknowledgments
The authors are grateful to the research center for female scientific and medical colleges, deanship
of scientific research in King Saud University for the financial support and to Volker Brecht (Nuclear
Magnetics Resonance, Institute fuer Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität
Freiburg, Germany) for HRMS measurements. Also, the authors are thankful to Melissa Jacobs,
Shabana Khan, and Babu Tekwani for antimicrobial, anti-malarial, and anti-leishmanial testing.
Conflicts of Interest
The authors declare no conflict of interest
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Sample Availability: Samples of the isolated compounds are available from the authors.
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