New Thiophene and Flavonoid from Tagetes minuta Leaves Growing in Saudi Arabia

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-O-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.


No.
  The 13 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 ( C 129.6) and further confirmed by its reaction with diagnostic shift reagents. In the HMBC spectrum, the methylene protons at  H 4.41 (H-6''B) and 4.30 (H-6''A) correlated with the caffeoyl carbonyl group at  C 166.5 suggesting the connectivity of caffeoyl moiety at C-6'' and confirmed by the downfield shift of C-6'' ( C 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).

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 241 automatic polarimeter (Perkin-Elmer Inc, Massachusetts, MA, USA). HRESIMS was recorded on 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 18

Plant Material
The leaves of Tagetes

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 3 . 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].

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.

Antileishmanial Assay
The anti-leishmanial activity of the isolated metabolites was tested in vitro against a culture of L. donovani promastigotes as previously outlined [26]. Pentamidine and amphoterecin B were used as positive standards.

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 91.6% to 68.3%. Compound 8 showed weak antileishmanial activity with an IC 50 31.0 μg/mL, while compound 3 showed moderate antimalarial activity against chloroquine sensitive (D6) clones of P. falciparum with an IC 50 4.37 μg/mL.