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Journal of the Chilean Chemical Society

versión On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.48 n.2 Concepción jun. 2003

http://dx.doi.org/10.4067/S0717-97072003000200002 

J. Chil. Chem. Soc., 48, N 2 (2003)

SECONDARY METABOLITES FROM FOUR MEDICINAL
PLANTS FROM NORTHERN CHILE: ANTIMICROBIAL
ACTIVITY AND BIOTOXICITY AGAINST Artemia salina.

GLAUCO MORALES * , PATRICIA SIERRA, ARLETT MANCILLA, ADRIÁN PAREDES, LUIS A. LOYOLA, OSCAR GALLARDO AND JORGE BORQUEZ

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

( Received : June 20, 2001 ­ Accepted : October 15,2002 )

ABSTRACT

Antibacterial activity and biotoxicity against Artemia salina of chloroform and alcohol extracts and isolated products from four plants used in ethnomedicine in northern Chile is reported. Nine compounds already identified were isolated from aerial parts of Artemisia copa Phil., Acantholippia punensis Botta, Ephedra andina Poepp. ex C. A . Mey and Haplopappus rigidus Phil : 3,5 ­ dihydroxy ­ 6, 7, 3', 4' ­ tetramethoxyflavone, lupeol, b ­ amyrine , b ­ sitosterol, ephedrine, 2 ­ ethylhexanol phthalate , 18 ­ acetoxy ­ cis ­ cleroda ­ 3,13 Z ­ dien ­ 15 ­ oic acid, 5,4' ­ dihydroxy ­7­ methoxyflavanone and 3,5,7 ­ trihydroxy ­ 6, 4' ­ dimethoxyflavone.

Key Words: Artemisia copa Phil.(copa-copa), Acantholippia punensis Botta. (rica ­ rica) , Ephedra andina Poepp.ex C. A . Mey (pingo ­ pingo), Haplopappus rigidus Phil. (baylahuen) , medicinal plants, flavonoids, terpenoids, antimicrobial activity

INTRODUCTION

Andean High Plateau in Northern Chile (Puna Atacameña) between 3,000 to 4,200 m above sea level, is an ecoregion characterized by a very low relative humidity, cloudless skies during most of the year, daily large temperature extremes and between summer and winter, and a rainfall season in summer known as the Altiplano Winter. Small and distant villages are present in this particular ecological system. Ollague (2113' S, 6827' W) in the north and Socaire (2336' S, 6750' W) in the south, have a combined population of nearly 5,000 inhabitants1. This report provides information on the antimicrobial properties and toxicity against Artemia salina of alcohol and chloroform extracts and secondary metabolites isolated from four medicinal plants used by Atacameños communities 1-8 : Artemisia copa Phil., ( Asteraceae ), Acantholippia punensis Botta ( Verbenaceae), Ephedra andina Poepp.ex C. A . Mey ( Ephedraceae) and Haplopappus rigidus Phil ( Asteraceae).

Microbial resistance to antibiotics in use nowadays provides the need for the search of new compounds with potential effects against pathogenic bacteria 9-10. Infusions of Artemisia copa ( copa ­ copa) are used against abdominal, liver and kidneys pains. Acantholippia punensis ( rica ­ rica) infusions are used against colds, stomach and liver discomforts and to improve blood circulation. Ephedra andina ( pingo ­ pingo ) infusions are used against asthma, liver and urinary bladder discomforts and as antitussive and decongestant alternative. Haplopappus rigidus ( bailahuén ) infusions are used to prevent or as a cure of liver and gastrointestinal disorders and as a sexual stimulant.

EXPERIMENTAL PART

Botanical Material

Aerial parts of Artemisia copa Phil., ( Asteraceae ) , Acantholippia punensis Botta ( Verbenaceae) , Ephedra andina Poepp.ex C. A . Mey ( Ephedraceae) and Haplopappus rigidus Phil ( Asteraceae) were collected near Socaire (23 36´ S; 67 50´ W) at 3,500 m above sea level. Plants classification was confirmed by Professor Clodomiro Marticorena, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepcion, Chile. A sample of each plant was deposited at the Universidad de Concepcion Herbarium.

Extracts and Dry Residues Preparation

Alcohol and chloroform extracts were prepared by mixing 250 ml of EtOH-H2O (1:1) or chloroform with 100 g of dried, minced plants. The mixtures were boiled continously by reflux during 10 min. Dry residues from extracts were obtained by evaporation in a rotary evaporator, followed by lyophilization. In order to measure antimicrobial activity, extracts and pure products were prepared as 100 mg / mL and 10 mg / mL solutions in water : DMSO (9:1), respectively. For biotoxicity assays against Artemia salina, solutions were prepared to contain 1000 , 100 and 10 mg extract / mL or 300, 30 and 3 mg pure product / mL, in CH2Cl2 ­ EtOH (1:1).

Microorganisms

Gram-positive Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Bacillus subtilus (ATCC 6633), Gram-negative Acinetobacter baumanni (ATCC 19606), Salmonella typhi (ATCC 3492), Escherichia coli (ATCC 25922), Pseudomona aeruginosa (ATCC 27853 ) and the yeast Candida albicans (ATCC 10231) were grown at 37C for 24 h in a brain-heart infusion (Difco). A standard turbidity value of 0.5 (108 ufc / mL), using a McFarland tube, was obtained adding a sodium chloride solution if correction was necessary.

Preparation of Extract-soaked Disks

6-mm diameter filter paper disks (Whatman N 3) were soaked in 1 mL of water : DMSO solutions of either extracts or pure products in order to obtain a concentration of 100 mg extract per disk or 10 mg product per disk, respectively. Disks were dried overnight at 45 C under sterile conditions.

Antibacterial Activity Assays

Bacteria were inoculated on 9cm Petri dishes plates containing Mueller­Hinton agar and dried at 37C for 30 min, according to previously described procedures11-12 Filter paper disks containing the extracts were loaded on the agar and the bacteria plates were incubated at 37 C for 24 h. Antimicrobial activity was observed as inhibition zones. Results were expressed as follows : - no activity ; + ( 6 ­ 10 mm inhibition zone) ; ++ ( 11 ­ 20 mm inhibition zone) ; +++ ( 21 ­ 30 mm inhibition zone) . Ampicillin ( 10 mg / disk) and chloramfenicol ( 30 mg / disk) were used as control antibiotics in all plates and their inhibition zones against Escherichia coli were 17 and 18 mm, respectively.

Biotoxicicity Assays Against Artemia salina

Extracts toxicity was evaluated against Artemia salina Leach naupliis according to previously described methods in three independent 36-h exposure experiments, at 22-25 C in boiled, filtered seawater 13-14. LD50 was obtained using Probit Finney software.

Extraction and Isolation of Natural Products

Dry, minced aerial parts of Artemisia copa Phil., Acantholippia punensis Botta , Ephedra andina Poepp.ex C. A . Mey and Haplopappus rigidus were exhaustively extracted with a mixture of EP : MeOH : Et2O ( 1:1:1) (EME) during one week at room temperature. After solvent evaporation, extract F001 was obtained. Its partition with water and CH2Cl2 (1:1) provided an aqueous extract F002 and an organic extract F003. Partition of extract F003 between n - hexane and 90% aqueous MeOH (1:1) followed by solvent evaporation rendered a polar alcohol extract F005 and a non-polar extract F006.

Isolation and purification of natural products was carried out by succesive pressure column chromatographies using silicagel or Sephadex and mixtures of EP , EtOAc and CH2Cl2 with increasing polarity. Structure assignments of natural products was done based on their physical and spectroscopic properties. IR spectra were obtained in a Perkin Elmer 1600 spectrophotometer. NMR spectra were carried out on a Brucker AMX 300 and an AMX 400. MS spectra were registered on a Hewlett Packard 5995 spectrophotometer.

1,360 g of Artemisia copa Phil., ( Asteraceae )( copa ­ copa) rendered 289.6 g of F001 extract, 102.7 g of F003 extract, 82.5 g of F005 extract and 11.2 g of F006 extract. Extract F005 was subjected to succesive column chromatographies using silicagel and Sephadex with the appropiate solvents and it rendered a crystaline yellow product (Compound 1). Compound 1, 3,5 ­ dihydroxy ­ 6,7,3',4' ­ tetramethoxyflavone.- Yellow crystals . m.p. 210-212. IR ( KBr) 3440 - 3100, 2960 , 2940, 1680, 1620, 1560, 1540, 1465, 1420, 1385, 1235, 1210, 1200, 1180, 1010, 810 cm -1 ; 1H NMR ( 300Mz, CDCl3 , TMS ): ( d 3.85 ( 3H,s,OMe ), 3.91 ( 3H, s, OMe ), 3.98 ( 3H, s OMe ), 4.01 ( 3H, s OMe), 6.01 ( 1H, s, OH ), 6.52 ( 1H, s, H - 8) , 7.02 ( 1H, d, J = 8.5 Hz, H - 5' ) , 7.65 ( 1H, d, J = 8.5 Hz , H - 2' ), 7.73 ( 1H,s a , H - 6') , 12.65 (1H,s OH). 13 C NMR ( 75 Mz, CDCl3 , TMS): ( d 56.04 ( OMe at C -3' ), 56.23 ( OMe at C - 4' ), 60.07(OMe at C - 7 ), 60.79 ( OMe at C - 6 ), 90.23 ( C - 8 ), 104.80 ( C - 10 ), 110.84 ( C - 2' ) , 114.49 ( C - 5' ) , 122.37 ( C - 1' ), 122.51( C - 6' ), 138.61 ( C - 3 ), 146.26 ( C - 6 ), 148.25 ( C - 2, C - 3' ), 152.21( C - 4' ), 152.69( C - 5 ), 155.86( C - 7 ), 158.68( C - 9 ), 178.80( C - 4 ). MS( m/z) 374 ( [ M+ ], 100 ,C19 H18O8), 373 ( 27 ), 360 ( 16 ) , 359 ( 62 ), 355 ( 15 ) ,331 ( 13 ), 316 (5), 181 ( 6 ), 164 ( 7 ), 151 (12 ).

1,580 g of Acantholippia punensis Botta ( Verbenaceae)( rica ­ rica) rendered 190 g of F001extract. Its partition between CH2Cl2 and H2O rendered 112.8 g of F003 extract. The next partition rendered 32.5 g and 65.3 g of F005 and F006 extracts, respectively. Succesive column chromatographies of extract F006 using silicagel and mixtures of EP and EtOAc with increasing polarity, rendered two crystaline yellow products ( Compounds 2 and 3 ).

Compound 2, lupeol,: Colourless crystals, m.p. 211-213 ( EtOAc ­ hexane) ; [a]25D +23( CHCl3 ; c 0 .10) ; IR ( KBr) 3450, 3020, 2940, 1630, 1450, 1380, 1040, 880 cm -1 ; 1H NMR ( 300Mz, CDCl3 , TMS): d 0.76 ( 3H,s ), 0.79 ( 3H, s ), 0.84 ( 3H, s ), 0.96 ( 6H,s, ) , 1.03 ( 3H, s ) , 3.21 ( 1H, m ), 4.67 ( 2H,d ).

13 C NMR ( 75 Mz, CDCl3 , TMS): d 15.22 , 15.42 , 16.78 , 16.93 , 18.14 , 20.91 , 23.06 , 23.32 , 24.05 , 27.04 , 27.88 , 27.96 , 29.39, 30.52, 32.12, 32.85, 36.56, 36.79, 37.14, 41.88 , 47.40, 49.45, 52.58, 54.56 , 55.10, 78.69, 104.58, 125.21, 138.09, 152.86. MS ( m/z) 426 ( [ M+ ] ), 411, 408, 394, 218, 207, 189, 161, 147, 135, 121, 109 , 107, 97 , 83 , 69 , 57 , 55 , 43.

Compound 3, b - amyrine : Colourless crystals, m.p. 260, [a]25D +88 ( CHCl3) ; IR ( KBr) 3600, 3000, 2938, 2920, 1488, 1376, 1040, 1292, 1185, 1160, 963 cm ­1. 1H NMR ( 300Mz, CDCl3 , TMS): d 0.81( 6H,s ), 0.83 ( 3H, s ), 0.88( 3H, s ), 0.92 ( 3H,s, ) , 0.97 ( 3H, s ) , 1.02 (3H,s), 1.17 (3H , s), 3.21 ( 1H, t J = 7 Hz) , 5.14 ( 1H,t J = 3Hz ) . MS( m/z) 426 ( [ M+ ] ), 411, 219, 218 (100), 208, 207, 203, 190, 189, 175, 161, 147, 135, 121, 119 , 109, 107, 105, 95 , 69 .

3,033 g of Ephedra andina Poepp.ex C. A . Mey ( Ephedraceae) ( pingo ­ pingo) rendered 222 g of F001 extract followed by 148 g of F003 extract. Partition of F003 rendered 8,9 g of F005 extract and 135 g of F006 extract . Succesive column chromatographies of 41 g of F006 extract allowed the isolation and purification of Compound 4 . Another plant part was proccessed in search of alkaloids. 3,065 g de Ephedra andina were submerged in a CH2Cl2 ­ MeOH ­ NH3 ( 2:2:1) mixture during one week. Later and after an exhaustive MeOH extraction and solvent evaporation, 665 g of a dry residue were obtained. This extract was treated with a 10 % H2SO4 solution and the acid solution was extracted with CH2Cl2 to render 12 g of a residue. The remaining aqueous solution was alcalinized with NH3 and extracted with CH2Cl2 to render 7.53 g of a basic residue. Succesive column chromatographies in silicagel, Sephadex using CH2Cl2, EtOAc , MeOH and solvent mixtures, rendered a crystaline material and a liquid component named Compound 5 and Compound 6, respectively.

Compound 4, b - sitosterol : Colourless crystals, m.p. 137 ( EtOAc ­ hexane) ; 83.99% C, 12.15 % H ; [a]25D ­ 37( CHCl3 ; c 0 .50) ; IR ( KBr) 3440, 2960, 2940, 2850, 1640, 1465, 1385, 1055, 1030, 970, 810 cm -1 ; 1H NMR ( 300Mz, CDCl3 , TMS): d 0.70 ( 3H,s,Me ­ 18 ), 0.81 ( 3H,d, J = 7.0 Hz, Me ­ 27 ), 0.84 ( 3H,d, J = 7.0 Hz, Me ­ 26 ), 0.85 ( 3H,t, J = 7.0 Hz, Me ­ 29 ), 0.92 ( 3H,d, J = 6.6 Hz, Me ­ 21 ) , 1.01 ( 3H, s, Me ­ 19 ) , 3.52 ( 1H, m , H - 3a ), 5.36 ( 1H,d br, J = 5.3 Hz, H­ 6) . 13C NMR ( 75 Mz, CDCl3 , TMS): d 12.05 ( C ­ 18 ), 12.25 ( C ­ 29 ), 18.80 ( C ­ 26, C ­ 27 ), 18.90 ( C ­ 21 ), 19.40 ( C ­ 19 ), 21.50 ( C ­11 ), 22.78 ( C ­ 28 ) , 24.20 ( C ­ 15 ) , 25.84 ( C ­ 23 ), 28.34( C ­ 16 ), 31.50 ( C ­ 7, C ­ 8 ), 31.60 ( C ­ 2 ), 33.80 ( C ­ 22 ), 34.60( C ­ 25 ), 36.40( C ­ 20 ), 36.42( C ­ 10 ), 37.30( C ­ 1 ), 40.08( C ­ 12 ), 42.20( C ­ 4 ), 42.40 ( C ­ 13 ), 48.34( C ­ 24 ), 50.20( C ­ 9), 56.38( C ­ 17 ), 56.80( C ­ 14 ), 72.40( C ­ 3 ), 122.56( C ­ 6 ), 140.80( C ­ 5 ) MS( m/z) 414 ( [ M+ ] , 86 ,C29 H50O), 399 ( 31 ), 396 ( 45 ) , 381 ( 34 ), 354 ( 11 ) ,329 ( 91 ), 303 ( 72 ) , 255 ( 51 ), 213 ( 56 ) , 163 ( 50 ), 159 ( 70 ) , 147 ( 51 ), 145 ( 82 ) , 133 ( 62 ), 121 ( 48 ) , 119( 60 ), 107 ( 84 ) , 105 ( 78 ), 69 ( 60 ) , 55 ( 90 ), 43 ( 100 ).

Compound 5, ephedrine : Colourless crystals ; m.p. 35 ­ 40 C (EtOAc ­ hexane) ; 72.69% C, 9.15% H, 8.32 % ; [a]25D ­ 6.3( EtOH ; c 0 .60) ; IR ( KBr) 3480, 3320, 3100 ­ 2600, 1640, 1500, 1430, 1380, 1360, 1050, 1000,760 cm-1 . 1H NMR ( 300Mz, CDCl3 , TMS): d 0.80 ( 3H,d , J = 6.5 , Me ­ 2 ), 0.2.43 ( 3H, s , N ­ Me ), 2.75 ( 1H ,d d, J = 6.42, 4.07 Hz, H ­ 2 ), 4.72 ( 1H, d , J = 4.0 Hz, H ­ 1), 7.23 ­ 7.30 ( 5H,m ) ; 13C NMR ( 75 Mz, CDCl3 , TMS): d 14.53 q ( Me ­ 2 ), 32.60 q ( N ­ Me ), 60.85 d ( C ­ 2 ), 73.66 d ( C ­ 1 ), 126.54 d ( C ­ 4' ), 127.47( C ­3' , C ­ 5' ), 128.50 d ( C ­ 2' , C ­ 6' ) , 142.05( C ­ 1' ) . MS ( m/z) 165 ( [ M+ ], 0.3. C10 H15NO ), 147(2), 132 (2), 131 (2), 105(4), 91(3), 79(4), 77(12), 59(3), 58(100), 56(8), 51(7), 42(6), 30(10).

Compound 6, 2 ­ (ethylhexanol) phthalate: Colourless liquid ; 73.78% C, 10.37 % H, IR ( film) 2960 ­ 2865 , 1730, 1600, 1580, 1470, 1385, 1280, 1125, 1075, 760 cm-1 . 1H NMR ( 300Mz, CDCl3 , TMS): d 0.88 ­ 0.93 ( 12H, t , Me ), 1.36 ( 16H, m ), 1.69 ( 2H ,m ), 4.23 ( 4H, t , J =11.0 Hz ), 7.52 (2 H, d , J = 7.5, 1.5 Hz ) 7.70 (2 H, d , J = 7.5, 1.5 Hz ) . 13 C NMR ( 75 Mz, CDCl3 , TMS):d11.33 q, 14.41q , 23.36 t , 24.16 t, 29.31 t , 30.77 t, 39.14 d , 68.51 t , 129.18 d , 131.24 d , 168. 10 s . MS ( m/z) 390 ( [ M+ ], 2, C24H34O4 ), 279(11), 261 (4), 167 (40), 150 (10), 149 (100), 113 (14), 104 (8), 71 (21), 70 ( 18) , 59 ( 3).

4.3 Kg of Haplopappus rigidus Phil ( Asteraceae)( bailahuen) rendered 845 g of F001 and 625 g of F003 were obtained from its partition. After liquid ­ liquid distribution, 335 g of F005 and 240 g of F006 were obtained. Succesive chromatographic separations of F006 rendered Compound 7. Similarly, F005 rendered Compounds 8 and 9.

Compound 7, 18 ­ acetoxy ­ cis ­ clerode ­ 3,13 Z ­ dien ­ 15 ­ oic acid: uncolored crystals, m.p. 100 ­ 102.[a]D21 = - 201 (CHCl3;c 0.065) . IR (KBr): 3200 ­ 3000, 1710, 1680, 1640, 1380, 1270 cm-1 . 1H NMR ( 400 MHz, CDCl3, TMS) d 0.79 (3H, d, J = 6.7 Hz Me ­ 17), 0.80 (3H, s, Me ­ 20), 1.12 (3H, s, Me ­ 19), 1.27 ­ 1.43 (6H, m) , 1.63 (1H, m ), 2.07 (3H s, OAc) , 4.59 (2H , s ) , 5.69(1H), 5.71 (1H ). 13C NMR ( 75 MHz, CDCl3, TMS) d 15.82, 17.17, 17.31, 19.41, 21.07, 23.86, 28.73, 34.57, 34.69, 36.11, 36.31,37.17, 37.51, 40.26, 45.25, 66.45, 111.87, 129.10, 138.31, 164.22, 170.79, 171.71. MS ( m/z) : 302, 287, 227, 200, 189, 188, 187, 175, 173, 161, 159, 147, 145, 135, 134, 133, 132, 131, 123, 122, 120, 119, 117, 108, 107, 106, 105, 95, 92, 91, 90, 43 (100).

Compound 8, 5, 4' ­ dihydroxy ­ 7 ­ methoxyflavonone . Colorless crystals, m.p. 149-151 ( n-hexane- EtAOc). .[a]D21 = - 9.6 (CHCl3;c 0.400) . IR (KBr): 3529, 3454, 3261- 3154, 1623, 1571 , 1447, 1375, 1294, 1040, 840, 763 cm-1.

1H NMR ( 400 MHz, CDCl3, TMS) d 2.81 (1H, dd, J = 17 and 3.1 Hz), 3.10 ( 1H, dd J = 13 and 17 Hz), 3.82 (3H , s OMe), 5.37 (1H , dd, J = 3.1 and 13 Hz), 6.08 (2H , d, J = 7.23 Hz ) , 6.91 (2H , d, J = 8.5 Hz ) , 7.34 (2H , d, J = 8.5 Hz ), 12.04 (1H , s ). 13C NMR ( 75 MHz, CDCl3, TMS) d 43.59 ( C ­ 3 ), 56.09 (OMe) , 79.37 (C ­ 2 ), 94.67 ( C ­ 6) , 95.53 ( C ­ 8 ), 104.2 ( C ­ 9 ), 116.09 ( C ­ 3', C ­ 5' ), 128.38 ( C ­ 2', C ­ 6' ), 130.93 ( C ­ 1' ), 156.59 ( C ­ 4' ), 164.53 ( C ­ 9' ), 168.43 ( C ­ 7 ), 196.48 ( C ­ 4 ). MS ( m/z) : 286 ( 100), 269 (5), 243 (5) , 193 (32), 180 (35), 167 (95), 138 ( 23), 120 (35), 95 ( 15), 69 (7).

Compound 9 , 3,5,7 ­ trihydroxy ­ 6,4' ­ dimethoxyflavone , yellow crystals, m.p. 181 - 183 . IR (KBr): 3410 - 3330, 2980 ­ 2920 , 1635, 1565 , 1375, 1280 cm-1 . 1H NMR ( 400 MHz, CDCl3, TMS) d 11.99 (1H), 8.17(2H, d, 6.17) , 7.04 (2H, d, 6.17), 6.60 (1H , s) , 4.05 (3H,s) , 3 ,89 (3H, s).MS : ( m/z) 330 ([ M]+, 100), 315(14), 300 ( 39) , 287( 81), 165 (13), 135 (28).

RESULTS AND DISCUSSION

Shows the results obtained after the evaluation of the antimicrobial activity of four medicinal plants extracts from Northern Chile :Artemisia copa Phil., Acantholippia punensis Botta , Ephedra andina Poepp.ex C. A . Mey and Haplopappus rigidus Phil. and 3 isolated products.

Tabla 1. Antibacterisl activity of extracts and compounds isolated from 4 medicinal plants of Northern Chile

aS.a Staphylococcus aureus; E.f Enterococcus faecalis, B.s Bacillus subtilus, A.c Acinetobacter baumanni, S.t Salmonella typhi,E.c Escherichia coli, P.a Pseudomona aeruginosa, and C.a Candida albicans. Los resultados se expresan como: - no inhibición, + (6-10 mm), ++ (11-20mm), +++ (21-30 mm). Como control positivo se usó ampicilina (10 mg/ml) y cloramfenicol (30 mg/ml) frente a Escherichia coli.

Inhibition zone measurements using the filter paper disks method showed that chloroform and alcohol extracts have interesting effects against Gram ­ positive bacteria (Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Bacillus subtilus (ATCC 6633)). Their effects were negligible against Gram ­ negative bacteria (Acinetobacter baumanni (ATCC 19606), Salmonella typhi (ATCC 3492), Escherichia coli (ATCC 25922), Pseudomona aeruginosa (ATCC 27853)) and Candida albicans (ATCC 10231). Growth of Staphylococcus aureus and Bacillus subtilus was affected by the four plants extracts and the effect was much higher on Bacillus subtilus. Only extracts from Acantholippia punensis (rica ­ rica) showed inactivation of Enterococcus faecalis. Artemisia copa ( copa ­ copa) , used against stomach, liver and spleen discomforts, was active against 7 out of 8 strains used in this study, including Candida albicans. These results could be related to the used of these plants as a remedy against urinary and intestinal infections by the Atacameños people; however, isolated, purified products were inactive. Compound 6, (2 - ethyl) hexanol phthalate, showed the highest activity against S. aureus, B. subtilus and C.albicans. b - sitosterol ( Compound 4) and b - amyrine showed a marginal activity against E. coli.

Biotoxicity assays against Artemia salina nauplii were highly sensitive to the presence of substances with biological activity, as shown in Table II .

Biotoxicity of extracts and compounds isolated against Artemis salina

95% cinfidnece interval

The four plants extracts were highly toxic to Artemia nauplii and LD50 values ranged from 0.023 to 7.29 mg / mL. Ephedra andina ( pingo ­ pingo) extract was the most toxic. Only three compouds showed biotoxicity levels below 300 mg/mL and b -amyrine had the highest activity.

Compound 1 was isolated from Artemisia copa and its flavonoid nature was confirmed by its physical and spectroscopic properties. Its molecular formula, C 19H 18 O 8 , based on mass spectroscopy, is in agreement with the information inferred from the NMR spectra, that is, four methyl groups and two OH groups, one of them located at C ­ 5 ( d 12.65). The flavonol nature comes from the resonance values of C ­ 2 , C ­ 3 and C ­ 4 carbon atoms at ring C, d 148.25 , 138.61 and 178.80, respectively, in agreement with this flavonoid class. This information matches with that from 3,5 ­ dihydroxy ­ 6,7,3',4' ­ tetramethoxyflavone 15.

Acantholippia punensis ( rica ­ rica ) provided us with two isomeric, triterpenic natural products ( M+ 426). Compound 2 showed spectroscopic and physical properties identical to those described for 3b - hydroxylup- 20(29) ­ en , known as lupeol 16-17 . According to the available data, Compound 3 is a 3b - hydroxyolean ­ 12 - en known as b -amyrine 18.

From Ephedra andina ( pingo ­ pingo) we isolated three products . Compound 4, with a molecular formula C 29H50 O , showed physical and spectroscopic properties identical to those described for b - sitosterol ( 3b ­ stigmast ­ 5 ­ en ­ 3 ­ol ) 19 - 20 . Compounds 5 and 6 were obtained from an extraction protocol for alcaloids isolation. Compound 5 was identical in all properties to a sample of (­) ephedrine , a well known antitussive and decongestant . Compound 6 was an aromatic ester (C24H34 O4 , 1730, 1600 , 1580 cm-1). NMR spectra were central on structure determination since they showed a highly symmetrical molecule. The aromatic signals between d 7.70 and 7.52 ppm on the 1H NMR spectra have reazonable coupling constants for protons at the ortho- substituted ring. Signal at d 4.23 ppm is assigned to a methylene group geminal to the ester alcohol group. 2D NMR experiments allowed us to propose (2 - ethyl) hexanol phthalate as its molecular formula. This compound has been recently isolated as a natural product from Cassia auriculata 21.

Crystaline Compound 7 was isolated from Haplopappus rigidus (bailahuén) along other previously reported compounds: rigiduside (13 ­ O ­ b ­ xylopiranosil ­ 13 ­ epimanool) 22, rigidusol (13 ­ hydroxy ­18 ­ acetoxy ­ cis ­ clerode ­ 3,14 ­ dien) and desacetylrigidusol ( 13,18 ­ dihydroxy ­ cis ­ clerode ­ 3,14 ­ dien) 23. Its physical and spectroscopic properties indicated the presence of an ester and an acid group, both a, b ­ unsaturated, which coincided with those described for 18 ­ acetoxy ­ cis ­ clerode ­ 3,13 Z ­ dien ­ 15 ­ oic acid , a product we have isolated from Croton chilensis and its structure was confirmed by X ­ rays crystalographic techniques24.

Properties of Compound 8 indicated it could be a flavonoid. Its uncolored crystals, optical activity and NMR spectra suggest a flavonone structrure and, considering the whole information available, its structure would be 5,4' ­ dihydroxy ­ 7 ­ metoxyflavanone 25.

Finally, properties of Compound 9 are in agreement with those from 3,5,7 ­ trihydroxy ­ 4',6 ­ dimethoxyflavone 26 .

ACKNOWLEDGEMENTS

Funds for this work were provided by FONDECYT (Project N 1980391) and the DGI de la Universidad de Antofagasta (Project PROIM 2000). We thank Professor Clodomiro Marticorena, Universidad de Concepción, for his botanical identification efforts.

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