Two Flavones from Salvia leriaefolia

Authors

Abstract

The methanolic extract of the aerial parts of Salvia leriaefolia (labiatae) afforded 5-hydroxy-4´, 6, 7-trimethoxy flavone (Salvigenin) and (5,4´-dihydroxy-7-methoxy flavone (Genkwanin). The structures of the isolated compounds were elucidated using 1 and 2 D-NMR, IR, UV and MS.

Keywords


Acute and Subchronic Toxicity?of Teucrium polium Total Extract in Rats
Iranian Journal of Pharmaceutical Research  (2009), 8 (3): 179-184
Received: January 2009
Accepted: March 2009

Copyright ? 2009 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services

Original Article

Two Flavones from Salvia leriaefolia

 

Seyyed Abdulmajid Ayatollahia,c, Asie Shojaiia,c*, Farzad Kobarfardb,c,
Mahdi Mohammadzadehd and Muhammad Iqbal Choudharye

aDepartment of pharmacognosy, School of Pharmacy, Shahid Beheshti University (M.C), Tehran, Iran. bDepartment of medicinal chemistry, School of Pharmacy, Shahid Beheshti University (M.C), Tehran, Iran. cPhytochemistry Research Center, Shahid Beheshti University (M.C), Tehran, Iran. dDepartment of toxicology and pharmacology, School of Pharmacy, Shahid Beheshti University (M.C), Tehran, Iran. eInternational center for chemical and biological sciences, H. E. J. Research Institute of Chemistry, Karachi University, Karachi, 75270, Pakistan.

 

Abstract

The methanolic extract of the aerial parts of Salvia leriaefolia (labiatae) afforded 5-hydroxy-4?, 6, 7-trimethoxy flavone (Salvigenin) and (5,4?-dihydroxy-7-methoxy flavone (Genkwanin). The structures of the isolated compounds were elucidated using 1 and 2 D-NMR, IR, UV and MS.

 

Keywords: Flavone; Salvigenin; Genkwanin; Salvia leriaefolia.

Introduction

Salvia is an important genus of the labiatae family consisting of over 900 species and is widely distributed in various regions of the world, namely, the Mediterranean area, South Africa, Central and South America, and South East Asia. These plants are perennial, rarely biennial, or annual, with attractive flowers in various colors (1). Over 58 species of this genus have been found in Iran, seventeen of which are endemic (2). These plants have been used in folk medicine all around the world for culinary purposes (3) as well as for their antibacterial, antioxidant, antidiabetic, antitumor and antituberculotic activities (1, 4). Previous chemical investigations on different species of Salvia have shown the presence of flavonoids, diterpenoids, triterpenoids, sesterterpenes, and essential oils (1, 3, 5) exhibiting antitumor, antimicrobial, cytotoxic and anti-inflammatory activities (1, 6). Flavonoids are widely distributed in species of Salvia, being mostly present as flavones, flavonols and their glycosides (3). To the best of our knowledge, except for a report on the isolation of a labdan diterpene from Salvia leriaefolia (7), no flavonoid has been reported of this species. In this article isolation of two flavones from this plant is described and their spectroscopic data have been discussed.

Experimental

The FT-IR spectra were recorded on a vector 22 instrument. The 1H-NMR was recorded on a Bruker AM400 and AMX 500 NMR (Avance) instruments using the UNIX data system at 400 and 500 MHz, respectively. The 13C-NMR spectrum was recorded at 100 and 125 MHz, respectively, using CDCl3 and CD3OD as solvents. 1H-13C HMBC and HMQC spectra were recorded as mentioned above. EI-MS spectra were recorded on a Finnigan MAT 312. Fab mass measurements were performed on Jeol JMS HX 110 mass spectrometer using glycerol as the matrix. HR-EIMS were carried out on Jeol JMS 600 mass spectrometer. Column chromatography was carried out on silica gel (M&N), 70-230 and 230-400 meshes. Compounds on the TLC was employed to detect compounds at 254 and 366 nm using ceric sulfate as spraying reagent.

 

Plant material

The aerial parts of Salvia leriaefolia were collected from Sabzewar, Province of Khorassan, in the north-east of Iran, at an altitude of 1400 m, in July 2006. A voucher specimen was deposited at the Herbarium of the Department of Botany, Shahid Beheshti University, Tehran, Iran.

 

Extraction and isolation

The fresh aerial parts of Salvia leriaefolia were dried in the shade for 3 weeks. The powdered materials (3 Kg) were extracted three times with methanol by maceration at room temperature. The solvent was evaporated under vacuum to give 300 g of extract. The obtained dry extract was suspended in H2O followed by extraction with hexane, CHCl3 and n-butanol for three times consecutively. The CHCl3 fraction was chromatographed on silica gel column (70-230 mesh) and eluted with varying portions of n-hexane, EtOAc and methanol to obtain fractions A-O. Two fractions (I, II) were selected from eluted fraction of n-hexane/EtOAc (7:3) (fraction F). Fraction IF was re-chromatographed on a silica gel column using 30% CHCl3/n-Hexane as eluent to obtain three fractions (IFa-IFc). Fraction IFb gave 15 mg of a yellow powder after evaporating the solvent. The compound had a melting point of 185?C and showed the following spectral characteristics:

Salvigenin (5-hydroxy-4?, 6, 7-trimethoxy flavone) (IFb):

UV max (EtOH): 274, 330 nm; IR (KBr) max: 1635 (C=O), 1600, 830 cm-1; EI-MS: m/z = 328 [M]+; 1H- NMR (400 MHz, CDCl3), δ ppm: 3.88, 3.9, 3.94 (s,OMe), 6.51 (s, 1H, H-8), 6.54 (s, 1H, H-3), 6.97 (d, 2H, J = 8.8 Hz, H-3?,5?), 7.79 (d, 2H, J = 8.8 Hz, H-2?,6?), 12.74 (s, OH); 13C-NMR (100 MHz, CDCl3), δ ppm: 55.47, 56.24, 60.77 (OMe), 90.5 (C-8a), 104(C-3), 106.4 (C-5), 114.44 (C-3?,5?), 123.43 (C-1?), 127.9 (C-2?,6?), 132.54 (C-7), 152.98 (C-6), 153.13 (C-4a), 158.65 (C-8), 162.55 (C-4?), 163.91 (C-2), 182.58 (C-4) (table 1). Fraction IIF was purified by preparative TLC with 1% MeOH/Hexan as eluent to obtain three fractions (IIFa-IIFc). Fraction IIFc gave 10 mg of a yellow powder after evaporating the solvent. The compound had a melting point of 285?C and showed the following spectral characteristics:

 

Genkwanin (5, 4?-dihydroxy-7-methoxy flavone) (IIFc) :

UV max (MeOH): 267, 300, 333 nm ; IR (KBr) :3270, 1660, 1600, 1340, 1200, 1180, 830 cm-1; EI-MS : m/z = 284 [M]+ ; 1H- NMR (500 MHz, CD3OD), δ ppm: 3.88 (s,OMe), 6.31 (d, J = 2 Hz, H-6), 6.53 (s, 1H, H-3), 6.63 (d, J = 2 Hz, H-8), 6.78 (d, J = 8.8, H-3?,5?), 7.78 (d, J = 8.8 Hz, H-2?,6?); 13C-NMR (125 MHz, CD3OD), δ ppm: 55.8 (ΟΜe), 93.3 (C-8), 99.1 (C-6), 103.4 (C-3), 105.9 (C-4a), 116.3 (C-3?,5?), 118.8 (C-1?), 129.2 (C-2?,6?), 157.7 (C-5), 159.2 (C-8a), 161.8 (C-4?), 167.2 (C-7), 167.8 (C-2), 183.8 (C-4) (table2).

Results and Discussion

The compound obtained from fraction IFb was identified as salvigenin and the one obtained from fraction IIFc was identified as genkwanin (figure 1) by interpretation of their MS, NMR and IR spectra as well as by comparison of their spectral data with those reported in the literatures (8-10). EI-MS and FAB MS [M+1]+ spectra of salvigenin confirmed the molecular weight at m/z 328(fragments at m/z 313,299,285,181,153). HR EI-MS showed the [M]+ at m/z 328.0956 in agreement with the molecular formula C18H16O6 (calcd. 328.0947) and indicated eleven degrees of unsaturation. The UV absorption maxima at 274 and 330 nm suggested the presence of a flavonoid moiety (11). The IR spectrum showed v max at 1635 (for C=O) and 830 cm-1 (for p-substituted phenyl ring). The 1H-NMR spectrum proved it to be a flavone (δ 6.54, 1H, s, H-3) (12). Three single peaks at δ=3.94, 3.9, 3.88, showed the position of methoxyl groups at C4?, C6, C7, respectively. A single peak at δ=6.51 assignable to H-8 and a pair of doublets at δ=7.79 and 6.97 (each 2H, J=8.8 Hz) assignable to the protons located at 2?,6?and 3?, 5?which was confirmed by 2D COSY. The single peak at δ=12.74, showed the position of hydroxyl group at C5. The 1H-13C HMBC spectrum confirmed the placement of the methoxyl groups at positions 6, 7 and 4? (Figure 2). Also H-3 (δ=6.54) showed correlation with C4, C2, C1? in HMBC, confirming the position of H-3 (Figures 3 and 4). The 13C-NMR spectra showed the presence of twelve aromatic carbons, which comprise of 5 methines[δ=158.65, 127.9 (2CH), 114.4(2CH)] and 7 quaternary carbons (table 1). All these data confirming the structure of IFb to be 5-hydroxy-4?, 6, 7 trimethoxyflavone (salvigenin). The EI-MS and FAB MS [M+1]+ spectra of genkwanin (II) confirmed the molecular weight at m/z 284. The UV absorption maxima at 267, 300, 333 nm (MeOH). HR EI-MS showed the [M]+ at m/z 284.0925 in agreement with the molecular formula C16H12O5. Flavone structure of genkwanin was revealed by comparison of 1H-NMR with salvigenin and the position of H-3 at δ = 6.53. The 1H-NMR spectrum exhibited one methoxy group (δ= 3.88, 3H, s), one set of meta coupled aromatic protons δ= 6.31(1H, d, J = 2.04 Hz) and δ= 6.63(1H, d, J = 2.04 Hz), two sets of ortho coupled aromatic hydrogens, δ= 7.78(2H, d , J = 8.8 Hz) and δ= 6.78 (2H, d, J= 8.8 Hz), and a non-coupled olefinic hydrogen at δ= 6.53 (1H, s) (table2). Supporting evidence for the structure of genkwanin was provided by HMBC spectral data, which confirmed the position of methoxyl group at C-7 (figure 3).Also H-3 showed cross peaks with C-1?, C-2, C-4, C-4a and H-8 showed HMBC correlation with C-8a, C-7, C-4a, C-6, confirming the position of these hydrogens (figure 6, 7). The 13C-NMR spectra showed the presence of twelve aromatic carbons, which comprise of 6 methines [δ= 93.3, 99.1, 129.2 (2CH), 116.3 (2CH)] and 6 quaternary carbons (table 2).

 

 

 

 

 

 

 

 

 

 

Some biological activities of salvigenin and genkwanin have been reported in the literature. In a study by Cottiglia,etal. genkwanin showed antimicrobial and antifungal activities (13). Kamatou, et al. have screened extracts of seventeen salvia species used in traditional medicine in South Africa for their ability to inhibit the in vitro growth/proliferation of Plasmodium falciparum (FCR-3 strain ) and the cytotoxic effects on three human cancer cells (breast adenocarcinoma, colon adenocarcinoma and glioblastoma). Salvia radula has displayed the most favorable activity. Two compounds have been subsequently isolated from the active fraction of S.radula and identified as betulafolientriol oxide and salvigenin. The IC50 value of 24.60 mg/mL has been reported for salvigenin against Plasmodium falciparum (14).

Brozic et al. have determined the inhibitory activity of some flavonoids on 17ß-hydroxy steroid dehydrogenase (17ß-HSD) type1 which converts estrone to estradiol. In this study genkwanin has demonstrated more than 80% inhibition of 17ß-HSD type1 activity at 6mM. 17ß-HSD type 1 represents an important target for the development of drugs for treatment of estrogen-dependent diseases ( 15 ).

Isolation and purification of other fractions of this plant is being carried out. Some of these fractions contain terpenoids or flavonoids that may be novel compounds.

References

        (1) Topcu G. Bioactive triterpenoids from Salvia species. J. Nat. Prod. (2006) 69: 482-487.
(2) Sefidkon F and Mirza M. Chemical composition of the essential oils of two Salvia species from Iran, Salvia virgata Jacq and Salvia syriaca. Flav. Frag. J. (1999) 14: 45-46.
(3) Liu Y and Foo Y. Polyphenols of Salvia. Phytochem. (2002) 59: 117-140.
(4) Ulubelen A. Cardioactive and antibacterial terpenoids from some Salvia species, Phytochem. (2003) 64: 395-399.
(5) Nikolova MT, Grayer RJ, Genova E and Porter EA. Exudate flavonoids from Bulgarian species of Salvia. Biochem. Sys. Ecol. (2006) 34: 360-364.
(6) Don M, Shen CC, Syu WJ, Ding YH and Sun CM. Cytotoxic and aromatic constituents from Salvia miltiorrhiza. Phytochem. (2006) 67: 497-503.
(7) Habibi Z, Eftekhar F, Samiee K and Rustaiyan A. Structure and antibacterial activity of a new labdan diterpenoid from Salvia leriaefolia. J. Nat. Prod. (2000) 63: 270-271.
(8) Bosabalidis A, Gabriel C and Niopas I. Flavone aglycones in glandular hairs of Origanum X intercedens. Phytochem. (1998) 49: 1549-1553.
(9) Youssef D and Frahm AW. Constituents of the Egyptian Centaurea scoparia; III. Phenolic constituents of the aerial parts. Planta Med. (1995) 61: 570-573.
(10) Wagner H and Mohan Chai V. 13C-NMR- spektren naturlich vorkommender flavonoide. Tetrahedron Lett. (1976) 21: 1799-1802.
(11) Andersen OM and Markham K R, Andersen M. Flavonoids; Chemistry, Biochemistry and Applications, CRC press, Taylor & Francis ( 2006) 39-42.
(12) Markham KR. Techniques of Flavonoid Identification. Academic Press, New York (1982) 75-78.
(13) Cottiglia F, Loy G, Garau D, Floris C, Casu M, Pompei R and Bonsignore L. Antimicrobial evaluation of coumarins and flavonoids from the stems of Daphne gnidium. Phytomedicine (2001) 8: 302-305.
(14) Kamatou GP, van Zyl RL, Davids H, van Vuuren SF, Lourens AC and Viljoen AM. Antimalarial and anticancer activities of selected South African Salvia species and isolated compounds from S. radula. South Afr. J. Bot. (2008) 74: 238-243.
(15) Brozic P, Kocbek P, Sova M, Kristl J, Martens S, Adamski J, Gobec S and Lanisnik Rizner T. Flavonoida and cinnamic acid derivatives as inhibitors of 17-β- hydroxysteroid dehydrogenase type 1.  Mol. Cell. Endocrinol. (2009) 301: 229-234.