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Journal of the Chilean Chemical Society

versión On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.51 n.1 Concepción mar. 2006

http://dx.doi.org/10.4067/S0717-97072006000100002 

J. Chil. Chem. Soc., 51, Nº 1 (2006)

CONSTITUENTS OF HELENIUM ATACAMENSE Cabr.

GLAUCO MORALES *, PATRICIA SIERRA, ADRIÁN PAREDES, LUIS A. LOYOLA AND JORGE BORQUEZ

Laboratorio de Productos Naturales , Departamento de Química, Facultad de Ciencias Básicas, Universidad de Antofagasta, Casilla 170. Antofagasta, Chile.

E-mail:gmorales@uantof.cl


ABSTRACT

Constituents of Helenium atacamense Cabr were investigated following a preliminary screening of the biotoxicity against Artemia salina and the antitumoral activity of ethanol extract . Three compounds already identified were isolated from aerial parts of Helenium atacamense Cabr, and their structures were elucidated as helenalin, quercetin and 3-O-methylquercetin based on mass and various nuclear magnetic resonance techniques . Quercetin and 3- O- methylquercetin were tested by using TEAC Test.

Key words: Helenium atacamense Cabr, helenalin, quercetin and 3-O-methylquercetin . TEAC test.


 

INTRODUCTION

In the course of a continuing search for bioactive compounds from plant sources of Northern Chile, it was found that ethanol extract of Helenium atacamense Cabr. showed significant biotoxicity effects using the brine shrimp test (BST) and inhibitory activity against cells derived from lung carcinoma (A - 549) , breast carcinoma ( MCF - 7) and colon adenocarcinoma ( HT -29 ) carried in vitro. Consequently , a systematic study directed to the isolation of the active principles was undertaken . This paper is intended to present the fractionation of the active extract of Helenium atacamense Cabr., and the isolation of the sesquiterpenlactone : helenalin and the flavonoids: quercetin and 3- O- methylquercetin.

Helenium atacamense Cabr., locally known as " poquil" or "manzanilla cimarrona" , is one of the seven Helenium species growing in Chile (1), and no chemical investigation has previously been reported .

EXPERIMENTAL PART

General

Mps are uncorrected . Identities of compounds were established by IR, NMR and MS comparisons. 1H and 13 CNMR including 1D and 2D experiments were recorded at 300 MHz and 75 MHz , respectively, at Pontificia Universidad Católica de Chile . FTIR spectra were obtained in a Perkin Elmer 1600 spectrophotometer. MS spectra were registered on a Hewlett Packard 5995 spectrophotometer at IPNO Tenerife ,Canarias Island . Flash and medium pressure CC were carried out on silicagel .

Botanical Material

Helenium atacamense Cabr. ( Asteraceae ) was collected in Parque Nacional Llanos de Challe, III Region de Chile , (28º 13´ S ; 71º 04´ W). This plants grows in arid environments . Plant classification was confirmed by Professor Clodomiro Marticorena, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepcion, Chile. A voucher specimen is on deposit at the Herbario de la Universidad de Concepción .

In vitro antitumor activities

In vitro cytotoxicities against human solid tumor cells were measured in 7 - day MTT assays at Purdue Cancer Center , Cell Culture Laboratory of Purdue University, West Lafayette, Indiana, USA, for the A-549 ( lung carcinoma), MCF - 7 (human breast carcinoma) and HT - 29 (human colon adenocarcinoma). Adriamycin is always used as a positive cytotoxic control in the same runs (2)

Biotoxicicity Assays Against Artemia salina

The bioactivity of the ethanolic extract was monitored by the brine shrimp lethality test against Artemia salina Leach naupliis, according to previously described methods in three independent 36-h exposure experiments at 22-25 ºC . Artemia salina eggs were hatched in boiled, filtered seawater (3,4). Ten nauplii were used to test each dose of extract ( 1000, 100 and 10 mg/mL) . Survivors were counted with the aid of the a stereomicroscope, and the deaths at each dose level and control were determined. No deaths were observed after 36 hours of control. The lethal concentrations of ethanolic extracts resulting in 50% mortality of brine shrimp (LC50) and 95% confidence intervals were determined from the 36 hours counts and the dose - response data were transformed into a straight line by means of trendline fit linear regression analysis (MS Excel version 7); the LC50 was derived from the best-fit line obtained. HgCl2 was used as a positive control , and methanol ( 500 mL ) as solvent and as a negative control.

Extraction and Isolation of Natural Products

The air-dried leaves of Helenium atacamense Cabr., (1.5 Kg) were chopped and exhaustively extracted with EtOH (8 L) during one week at room temperature. The EtOH extract was then filtered and evaporated on a rotatory evaporator under reduced pressure to obtain a viscous mass of dark green extract. The dried EtOH residue(120 g ; brine shrimp LC50 150,5 mg/mL 123/176 mg/mL ) was partitioned between H2O y CHCl3 to give an inactive H2O-soluble residue and a brine shrimp toxic CHCl3-soluble residue (53 g; LC50 98,2 mg/mL 50/132 mg/mL .

Succesive column chromatographies of CHCl3 extract using silicagel and mixtures of CHCl3 - Me2CO with increasing polarity, rendered one crystalline colourless and two crystalline yellow products ( Compounds 1, 2 and 3 ) .

Compound 1, Helenalin . Colourless crystals, m.p. 172 -174º ( Acetone_Ethyl ether); [a]25D - 76º ( CHCl3 ; c 0 .84 ) ; IR ( KBr) 3440 - 3100, 2960 , 1758, 1696, 1653, 1457, 1382, 1235, 1301, 1234, 1157, 1068, 963, 812 cm -1 ; 1H NMR (300Mz, CDCl3 , TMS ): 0.98 ( s , H - 15), 1.25 (d , J = 6.7 Hz , H - 14), 1.82 (ddd , J = 15, 6.5, 2.5 Hz , H - 9a) , 2.07 (br ddq , J = 12, 6.5 , 4 Hz , H - 10) , 2.26 (ddd , J = 15 , 9 , 4 Hz , H - 9b) , 3.08 (ddd , J = 12 , 4.6 , 2 Hz , H - 1) , 3.57 (br dddd , J = 7.5 , 3, 3, 1.5 Hz , H - 7) 4.45 (br s H - 6) , 4.97 (ddd , J = 9 , 7.5 , 2.5 Hz, H - 8) , 5.80 (d , J = 3 Hz , H - 13') , 6.05 (dd , J = 3.7 , 2.8 Hz, H - 3) , 6.36 (d , J = 3 Hz , H - 13), 7.68 (dd , J = 6 , 1.6 Hz , H - 2) ; 13 C NMR ( 75 Mz, CDCl3 , TMS): 19.06( C - 15 ), 20.53 ( C - 14 ), 26.51( C - 10 ), 38.86 ( C - 9 ) , 51.26( C - 7) , 51.76 ( C - 1 ), 58.25( C - 5 ), 74.47 ( C - 6 ), 78.72 ( C - 8), 123.46 ( C - 13), 130.22( C - 3), 138.34( C - 11 ), 164.48( C - 2), 170.29( C - 12 ), 212.72( C - 4 ).

MS m/z (rel int.) 262.1197 [ M+ ], (9) , (calc. for C15 H18O4 , 262,121), 247 (8), 244(5) ,177(5), 152 (6), 151(20), 149 (5), 148(12), 138(16), 137(19), 125(11), 124(100).

Compound 2 . 3,5,7,3',4'_pentahydroxyflavone (quercetin) Yellow crystals, m.p. 149-151 º ( n-hexane- EtOAc). IR (KBr): 3407, 3220 -3095, 1660, 1562, 1520, 1463, 1460, 1382, 1210, 1200, 1132, 1109, 1039, 942, 879, 843, 705 cm-1.

1H NMR ( 300 MHz, CD3OD, TMS) d 6.22 ( d, J = 2 Hz, H-6 ), 6.43 ( d, J = 2 Hz,H-8) , 6.92 ( d, J = 8.5 Hz, H-5´) , 7.67 (dd, J = 2.1 , 8.5 Hz , H-6´), 7.77 (d, J = 2.1Hz, H-2´), 12.04 (1H , s ). 13C NMR ( 75 MHz, CDCl3, TMS) d 94.5(C - 8) , 99.5(C - 6), 104.2(C - 10) , 116.5(C - 5´), 116.1 ( C - 2´ ), 121.8(C - 6´) , 123.6 (C - 1´), 136.5(C - 3), 145.7(C - 3´) , 147.5(C - 2) , 148.1(C - 4´),156.7(C - 9) , 161.0(C - 5), 166.7 (C - 7), 176.5 ( C - 4).

MS m/z (rel int.) 302.2 [ M+ ], (100) , (C15 H10O7 ), 274 (11), 273(10), 245(10) ,229(10), 228 (9), 200(5), 153(17), 137 (23), 136(8).

Compound 3 5,7,3',4'-tetrahydroxy- 3 - methoxyflavone (3-O-methylquercetin) Yellow crystals, m.p. 280 - 282 º ( n-hexane - EtOAc). IR (KBr): 3438, 3220 -3095, 1656, 1612, 1576, 1496, 1460, 1379, 1277, 1163, 1109, 1039, 994, 954, 848, 705 cm-1.

1H NMR (300 MHz, CD3OD, TMS) d 3.87 (3H , s , OMe), 6.25 (1H , d, J = 2 Hz , H-6) , 6.48 (1H , d, J = 2 Hz ,H-8) , 7.00 (1H , d, J = 8.5 Hz, H-5´) , 7,59 (1H dd, J= 2, 8.5 Hz, H-6´), 7.72 (1H, d, J=2 Hz, H -2´), 12.04 (1H , s ).

13C NMR ( 75 MHz, CDCl3, TMS) d ) d 60.5 (OMe), 94.8(C - 8) , 99.7(C - 6), 106.2(C - 10) , 116.6(C - 2´), 116.7 (C - 5´ ), 123.4(C - 1´) , 122.5(C - 6´), 139.6(C - 3), 146.2(C - 3´) , 148.6(C - 4´) , 157.9(C - 2),162.3(C - 9) , 163.6(C - 5), 164.7(C - 7), 179.9 ( C - 4).MS m/z (rel int.) : 316.0477 [M+], (100) , (calc. for C16H12O7 , 316,0223), 315(30), 312(18), 302(12), 301(65), 300(21), 299(14), 298(51), 297(10), 288(6), 287(27), 286(6), 285(13), 282(9), 274(13), 273(67), 270(17), 257(15), 166(20), 138(30),134(14).

TEAC Test.

Quercetin and 3 - O - methylquercetin were tested by using the Trolox Equivalent Antioxidant Capacity (TEAC) assay . The TEAC value is based on the ability of the antioxidant to scavenge the radical cation 2,2´-azinobis (3-ethylbenzothiozoline-6-sulphonic acid diammonium salt) (ABTS.+) with spectrophotometric analysis (5). The ABTS.+ cation radical was produced by the reaction between 7 mM ABTS in H2O and 2.45 mM potassium persulfate , stored in the dark at room temperature for 12 h. The ABTS.+ solution was then diluted with phosphate buffered saline solution (PBS) (pH 7.4) , to an absorbance of 0.70 at 734 nm and equilibrated at 23ºC. Samples were diluted with methanol to produce solutions of 0.3, 0.5, 1, 1,5, 2 mM concentration. The reaction was initiated by the addition of 1 mL of diluted ABTS to 10 mL of each sample solution. Determinations were repeated three times for each sample solution. The percentage inhibition of absorbance at 734 nm was calculated for each concentration relative to a blank absorbance ( methanol) and plotted as a function of concentration compound or standard 6 - hydroxyl - 2,5,7,8 - tetramethylchroman - 2`- carboxylic acid (Trolox) (6)

RESULTS AND DISCUSSION

As a part of our interest in the isolation of bioactive compound from plants of Northern Chile,(7-10) the air-dried leaves of Helenium atacamense Cabr., were examined using the in-house brine shrimp (Artemia salina) lethality bioassay and the antitumoral test as a guide to fractionation. The antiproliferative activity of EtOH extract against three human solid tumor cell lines was determined by MTT assay , and their 50% growth inhibition (ED50 ) indicated moderate cytotoxicity to breast carcinoma cell line MCF - 7, showing ED50 5.23 mg/mL compared with adriamycin with ED50 0.5 mg/mL (11) , while the EtOH extract showed marginal cytotoxicity against colon adenocarcinoma cell line HT -29, ED50 15.8 mg/mL , and no activity against lung carcinoma cell line A - 549, ED50 >10 mg/mL Initial screening of the EtOH extract against BST (LC50 150,5 mg/mL , 123/176 mg/mL) and subsequent solvent partition CHCl3 - H2O of leaves of Helenium atacamense Cabr indicated that CHCl3 - residue was the most lethal to brine shrimp LC50 98,2 mg/mL 50/132 mg/mL. Successive column chromatographies of CHCl3 extract using silicagel and mixtures of CHCl3 - Me2CO with increasing polarity, rendered one crystalline colourless and two crystalline yellow products ( Compounds 1, 2 and 3 ) .

Compound 1 , Colourless crystals, m.p. 172 -174º ( Acetone_Ethyl ether), gave a molecular ion at m/z 262 with a molecular formula C 15H 18 O 4 . The nature of four oxygen atoms and of two C - C double bond may be interpreted from the IR bands at 3440 - 3100 (OH group), 1758 and 1653 (unsaturated g - lactone) and 1696 cm -1 ( cyclopentenone) . The 1H NMR spectrum indicates the presence of cyclopentenone as a pair of doublets at d 6.36 and 5.80 ( J = 3 Hz ) and an exocyclic methylene group as a pair of double doublets 7.68 and 6.05 (dd , J = 6 , 1.6 Hz ) . The IR , 1H and 13CNMR spectral data were very closely related to those of a sesquiterpen lactone type pseudoguaianolide with a cis - fused 7,8- a- methylene- g - lactone ring , closed to the C - 8 (12) . 13CNMR spectra of 1 exhibited signals from two olefinic carbon groups [ dc 164.48, 130.22, dc 138.34 , 123.46] , two methyl groups [ dc 19.06 and 20.53 ] one methylene group [ dc 38.86 ] , five methine groups [ dc 26.51, 51.26, 51.76, 74.47, 78.72 ], and three quaternary carbons [ dc 58.25, 170.29 and 212.72]. From DEPT, COSY, HMBC and HMQC experiments, the structure of Compound 1 was deduced as helenalin (13) .

Helenalin has received considerable attention in pharmacological research due to its wide spectrum of biological activity such as : potent antineoplastic, antiulcer activity , antimicrobial effect , anti-trypanosomal activity and anti-inflammatory activity (14-16).

The anti-inflammatory sesquiterpene lactone Helenalin inhibits the transcription factor NF-kB by directly targeting p65. NF-kB is a well-characterised protein responsible for regulating complex phenomena under certain physiological and pathological conditions. In unstimulated cells , NF-kB is present in the cytoplasm as an inactive heterodimer composed of two sub-units, p50 and p65 . The heterodimer is complexed with an inhibitory protein IkB-a preventing it from moving into the cell nucleus. When activated by certain inflammatory agents, specific protein kinases phosphorylate the IkB-a protein , causing its rapid degradation, and NF-kB becomes dissociated from IkB-a. Phosphorylated IkB is then rapidly degraded leading to the translation of NF-kB to the nucleus, where it binds to specific DNA sequence present in the prometers of numerous target genes.

Helenalin is the first anti-inflammatory agent shown to exert its effect by directly modifying the transcription factor NF-kB (17)

The EI - MS of Compound 2 , yellow crystals, exhibited an ion-molecular at m/z 302 for C15H10O7 , which is in accordance with a flavone containing five hydroxyl groups. The EI-MS of Compound 3 , yellow crystals, exhibited [ M+] at m/z 316 for C16H12O7. This data is consistent with a tetrahydroxymethoxyflavone. The HNMR, and NMR spectra for 3 were almost identical to those of 2 , with the exception of the presence of one methoxyl group appearing in both the proton and carbon spectra.

Compounds 2 and 3 , based on detailed spectroscopic analysis of UV, MS, NMR, and various 2DNMR techniques , were identified as 3,5,7,3',4'-pentahydroxyflavone (quercetin)(18) and 5,7,3',4'_tetrahydroxy- 3 - methoxyflavone (3-O-methylquercetin), respectively (19).

Evaluation of the antioxidative activity, conducted in vitro, by measurement of the Trolox equivalent antioxidant capacity values (TEAC), against ABTS using UV-Vis-spectrophotometric assays was carried out. This method measures the relative ability of antioxidant substances to scavenge the radical cation 2,2´-azinbo(3-ethylbenzothiazoline - 6 - sulfonate). As compared to a standard amount of the synthetic antioxidant Trolox (6-hydroxy-2.5,7,8-tetramethylchroman - 2- carboxylic acid ), an aqueous soluble vitamin E analogue. The TEAC value is defined as the concentration of standard Trolox with the same antioxidant capacity as a 1mM concentration of the antioxidant compound under investigation. The results showed that quercetin and 3 - O -methyl quercetin exhibited potent free - radical scavenging indicated by the TEAC value of 2.69 ± 0.09, and 1.50 ± 0.08, respectively .

Significant antioxidative behavior for quercetin and 3- O-methylquercetin can be reasonably attributed to the structural features of these molecules : presence of the heterocyclic C-ring composed of a 4 - keto group, 2,3 - double bond , 3-hydroxyl group , which it to form a "huge" p- bond that links the A and B ring for electron delocalization. The scavenging radicals activity is enhanced by a cathecol moiety in ring B and OH - groups at 5 and 7 position in the ring A.

Quercetin is a bioflavonoid that prevents oxidant injury and cell death by several mechanism, such as scavenging oxygen radicals protecting against lipid peroxidation and chelating metal ions. (20). Pre - treatment of quercetin may protect against ethanol- induced oxidative stress by directly quenching lipid peroxides and indirectly by enhancing the production of the endogenous antioxidant reduced form of gluthatione (GHS)(21)

Otherwise , 3-O- methylquercetin seems to have an anti-inflammatory effect , and to have a potential to inhibit bronchospasm in the treatment of asthma(22).

The evidence reported in the chemistry , biochemistry and pharmacy literature supports the view that flavonoids play a vital biological role, including the function of scavenging reactive oxygen species (23)

ACKNOWLEDGEMENTS

This work was supported by FONDECYT (Grant Nº 1040294). We thank Professor Clodomiro Marticorena, Universidad de Concepción, for the botanical identification efforts , and the staff of Cell Culture Laboratory, Purdue Cancer Center, Purdue University, West Laffayette ,Indiana (USA) for cytotoxicity assays of the human tumor cell lines.

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