Molecules 2012, 17, 12895-12909; doi:10.3390/molecules171112895
molecules
ISSN 1420-3049
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A icle
Seconda y Me aboli es om Two Species o Tolpis and
Thei Biological Ac i i ies
Jo ge T iana 1,*, Ma iana López 1, F ancisco Ja ie Pé ez 1, Milag os Rico 1, A oa López 1,
F ancisco Es é ez 2,3, Ma ía Te esa Ma e o 2, Ignacio B oua d 4 and F ancisco León 4,*
1 Depa amen o de Química, Unidad Asociada al CSIC, Uni e sidad de Las Palmas de G an Cana ia,
Campus de Ta i a, 35017, Las Palmas de G an Cana ia, Spain; E-Mails: mlope[email p o ec ed] (M.L.);
[email p o ec ed] (F.J.P.); m[email p o ec ed]c.es (M.R.); a [email protected] (A.L.)
2 Depa amen o de Bioquímica, Unidad Asociada al CSIC, Uni e sidad de Las Palmas de G an Cana ia,
Plaza D . Pas eu s/n, 35016, Las Palmas de G an Cana ia, Spain; E-Mails: es e ez@dbb .ulpgc.es
(F.E.); ma ia em[email p o ec ed]m (M.T.M.)
3 Fundación Cana ia Ins i u o Cana io de In es igación del Cánce , To e Agus ín A é alo-7ª Plan a
(PCTT-ULL) A da de la T inidad s/n 38204 San C is óbal de La Laguna-S/C de Tene i e, Spain
4 Ins i u o de P oduc os Na u ales y Ag obiología, Consejo Supe io de In es igaciones Cien í icas
(CSIC), A da. As o ísico F. Sánchez, 3, 38206 La Laguna, Spain; E-Mail: [email p o ec ed].es
* Au ho s o whom co espondence should be add essed; E-Mails: [email p o ec ed] (J.T.);
[email p o ec ed] (F.L.); Tel.: +34-922-256847 (ex . 224) (F.L.); Fax: +34-922-260135 (F.L.).
Recei ed: 27 Sep embe 2012; in e ised o m: 24 Oc obe 2012 / Accep ed: 26 Oc obe 2012 /
Published: 1 No embe 2012
Abs ac : Phy ochemical esea ch o wo Tolpis species, T. p ous ii and T. lagopoda, led
o he isola ion o h ee new compounds: 30-chlo o-3β-ace oxy-22α-hyd oxyl-20(21)-
a axas ene (1), 3β,22α-diace oxy-30-e hoxy-20(21)- a axas ene (2) and 3β,28-dihyd oxy-
11α-hyd ope oxy-12-u sene (3). The s uc u es o he new compounds we e elucida ed by
means o ex ensi e IR, NMR, and MS da a and by compa ison o da a epo ed in he
li e a u e. The in i o an ioxidan ac i i ies o he ex ac s we e assessed by he DPPH and
ABTS sca enging me hods. The cy o oxici y o se e al known compounds and i s
de i a i es was also assessed agains human myeloid leukemia K-562 and K-562/ADR
cell lines.
Keywo ds: As e aceae; Tolpis p ous ii; Tolpis lagopoda; u sane- i e penes;
a axas ane- i e penes
OPEN ACCESS
Molecules 2012, 17 12896
1. In oduc ion
The Tolpis genus (As e aceae: Cicho ioideae, Cicho iinae) consis s o some 20 species dis ibu ed
h oughou Eu ope, No h A ica, Cana y Islands, Cape Ve de and Asia, his genus being ep esen ed
in he Cana y Islands by a ound en species [1].
Al hough a wide- anging s udy o di e en species o Tolpis om he Cana y Islands and om
o he Maca onesian a chipelagos has been ca ied ou om he bo anical poin o iew [2,3], only one
phy ochemical epo has p e iously appea ed on he isola ion and s uc u al elucida ion o a oma ic
compounds, i e penes, and s e ols om T. webbi and T. spp [4].
As a pa o ou con inuing sea ch o no el, plan -de i ed biological agen s and ou sys ema ic
in es iga ion o he composi ion o Cana ian endemic plan s, he p esen wo k desc ibes he isola ion
and s uc u al elucida ion o he cons i uen s o he e hanolic ex ac s o he ae ial pa s o T. p ous ii
Pi a d in Pi a d and P ous and T. lagopoda C.Sm. ex Buch. The cons i uen s o hese ex ac s we e
pu i ied by CC, MPLC and p epa a i e TLC. The s uc u es o he known compounds we e con i med
by compa ison o hei spec oscopic da a wi h hose epo ed in he li e a u e.
F om T. p ous ii se en een compounds we e isola ed including wo a axan- i e penoid 30-chlo o-
3β-ace oxy-22α-hyd oxyl-20(21)- a axas ene 1, and 3β,22α-diace oxy-30-e hoxy-20(21)- a axas ene 2
a new u san- i e penoid 3β,28-dihyd oxy-11α-hyd ope oxy-12-u sene 3. F om T. lagopoda, eigh
compounds we e isola ed including a 3β,22α-diace oxy-30-e hoxy-20(21)- a axas ene 2 (Figu e 1).
Figu e 1. Compounds 1–3 isola ed o he T. p ous ii and T. lagopoda.
The high le el o sola adia ion and he high empe a u e p e ailing in he egion o he Cana y
Islands (opposi e he no hwes coas o A ica) o ce plan s o de elop de ence mechanisms agains
ul a iole adia ion and excessi e p oduc ion o ee adicals h ough he accumula ion o an ioxidan
me aboli es [5]. This p omp ed us o e alua e he an ioxidan ac i i y o he c ude ex ac o T. p ous ii
and T. lagopoda, and ha o some o he isola ed compounds. Fu he mo e, one o he mos impo an
mechanisms by which umo cells esis o cy o oxic e ec s o a a ie y o chemo he apeu ic d ugs is
o e exp ession o he human mul id ug esis ance (MDR1) gene and i s p oduc , P-glycop o ein [6].
He e we ha e e alua ed he e ec s o na u al compounds and de i a i es on he g ow h o he human
leukemia K-562 and he P-glycop o ein-o e exp essing K-562/ADR cell lines.
Molecules 2012, 17 12897
2. Resul s and Discussion
2.1. S uc u e Elucida ion o Compounds 1–3
The posi i e EIMS spec um o compound 1 showed a molecula ion a m/z 518/520 (3:1) in
ag eemen wi h he p esence o a chlo ine a om in he s uc u e, and wi h he o mula C32H51O3Cl o
his compound. In he same spec um, he ions a m/z 500/502 [M-H2O]+, 458/460 [M-CH3COOH]+
sugges ed ha his compound con ained a hyd oxyl and an ace yl g oup, espec i ely. Con i med by
he IR spec um wi h abso p ions o a hyd oxyl 3446 cm−1 and a ca bonyl 1730 cm−1 g oup. I s
HREIMS expe imen indica ed he molecula o mula C32H51O337Cl (calcd. o [M]+ 520.3497; ound
520.3497) and C32H51O335Cl (calcd. o [M]+ 518.3527; ound 518.3517). All spec al da a sugges ed
ha 1 was a pen acyclic i e pene wi h a isubs i u ed double bond in he E ing [7] wi h a
20(21)- a axas ane s uc u e.
The 1H and 13C-NMR (Table 1) spec a o 1 showed he p esence o he oxygena ed me hine p o on
a δH 3.35 (1H, d, J = 6.6 Hz) and an unusual chlo o a om a C-30 a δC 47.6 (Table 1). The s uc u e
elucida ion and NMR assignmen s we e he e o e based p ima ily on he esul s o COSY, HSQC,
HMBC, and NOESY expe imen s (Figu e 2).
Table 1. 1H- and 13C-NMR da a o compounds 1, 1a and 2 a.
1 1a 2
Posi ion H C H C H C
1 1.65 * 38.5 1.65 * 38.7 1.60 * 38.5
2 1.50 * 23.7 1.54 * 21.7 1.55 * 23.7
3 4.41 dd
(6.1,10.8)
81.0 4.43 dd
(6.4,10.0)
81.1 4.43 dd
(5.1 10.4)
81.0
4 - 38.3 - 38.0 - 37.8
5 0.75 * 55.4 0.74 * 55.6 0.75 * 55.4
6 1.45 *
1.35 *
18.2 1.44 *
1.34 *
18.0 1.30 * 18.2
7 1.35 * 34.2 1.34 * 34.4 1.35 * 34.2
8 - 41.2 - 41.3 - 41.1
9 1.25 * 50.3 1.26 * 50.6 1.25 * 50.4
10 - 37.1 - 37.3 - 37.1
11 1.20 * 21.6 1.21 * 21.4 1.20 * 21.6
12 1.60 *
1.20 *
27.6 1.60 * 27.7 1.58 * 27.5
13 0.95 * 38.6 0.95 * 1.66 * 38.6
14 - 42.3 - 42.4 - 42.3
15 1.72 *
1.05ddd
(2.5,4.0,13.05)
26.7 1.71 *
1.02 *
29.9 1.50 * 26.6
16 0.95 *
1.85 d (9.0,13.0)
29.7 0.95 * 29.9 1.60 * 29.9
17 - 37.8 - 37.4 - 37.2
18 1.45 * 40.5 1.44 * 41.5 1.45 * 41.5
Molecules 2012, 17 12898
Table 1. Con .
1 1a 2
Posi ion H C H C H C
19 2.00 (7.0) 31.6 2.03 b (7.0) 31.7 1.79 q (6.6) 32.3
20 - 144.9 - 146.5 - 147.7
21 5.89 d (6.5) 126.3 5.87 d (6.4) 123.1 5.75 d (6.3) 119.2
22 3.35 db (6.6) 73.3 4.51 d (6.4) 75.3 4.55 d (6.3) 75.3
23 0.78 s 28.0 0.79 s 28.1 0.81 s 28.0
24 0.77 s 16.6 0.78 s 16.7 0.80 s 16.5
25 0.82 s 16.4 0.82 s 16.7 0.81 s 16.4
26 0.99 s 16.1 0.98 s 16.2 0.96 s 16.1
27 0.93 s 14.7 0.91 s 14.7 0.92 s 14.6
28 0.63 s 17.8 0.70 s 17.9 0.70 s 18.2
29 0.99 d (6.5) 22.2 0.97 d (7.6) 22.1 0.96 d (6.6) 22.6
30 4.15 d (11.2)
3.89 d (11.2)
47.6 4.14 d (11.2)
3.89 d (11.6)
47.5 3.96 d (12.6)
3.72 d (12.6)
72.3
OH 3.14 m
OAc 1.97 s
21.4
171.4
1.97 s
1.98 s
21.3
21.3
171.1
171.1
1.97 s
1.98 s
21.3
21.3
171.1
171.1
OE 1.13 (6.9)
3.35 m
15.2
65.9
a δ in ppm and J (in Hz) a e in pa en heses. Reco ded in CDCl3 a 400 MHz and 125 MHz o 1H
and 13C-NMR, espec i ely. * o e lapped.
Figu e 2. Selec ed co ela ions o 1. Double-ended a ows indica e NOESY, and single
a ows indica e HMBC (C o H) co ela ions.
The mos impo an HMBC and NOESY co ela ions a e shown in Figu e 1. T ea men o 1 wi h
Ac2O-py idine ga e a diace yl de i a i e 1a o which i s HREIMS expe imen indica ed he molecula
o mula C34H53O437Cl (calcd. o [M]+ 562.3603; ound 562.3624) and C34H54O435Cl (calcd. o [M]+
560.3654; ound 560.3644). The da a implied he p esence o a double bond be ween C-20 and C-21,
he chlo o a om a C-30, and he ace yl g oup a C-3 and hyd oxyl g oup a C-22. H-22 was assigned
in β-o ien a ion on he basis o he coupling cons an wi h he inylic p o on a C-21 and c oss-peak
in he NOESY expe imen wi h he CH3-28. F om he abo e indings, he s uc u e o
Molecules 2012, 17 12899
30-chlo o-3β-ace oxy-22α-hyd oxyl-20(21)- a axas ene was assigned o 1, and i was named
chlo o olpidiol. To he bes o ou knowledge, his is he i s example o a pen acyclic i e pene o he
a axas ane-u sane se ies wi h a chlo o unc ionali y.
Compound 2 was ob ained as a colou less amo phous solid and i s molecula o mula was
de e mined by a HRESIMS expe imen as C36H58O5 (calcd. o [M+Na]+ 593.4182; ound 593.4176).
The IR spec um e ealed he abso p ion bands o a ca bonyl g oup (1732 cm−1) and double bond
(2872 cm−1). The 1H-NMR spec um (Table 1) exhibi ed six single me hyl g oups a δH 0.70, 0.80,
0.81 (6H), 0.92 and 0.96, a seconda y me hyl g oup a δH 0.96 (3H, d, J = 6.6 Hz) a ibu ed o C-29, a
inyl p o on a δH 5.75 (1H, d, J = 6.3 Hz), wo ace yl g oups a δH 1.97 s and 1.98 s, wo oxyme hine
signals a δH 4.43 (1H, dd, J = 5.1, 10.4 Hz) and 4.55 (1H, d, J = 6.3 Hz), an oxyme hylene signal a δH
3.96 (1H, d, J = 12.6 Hz) and 3.72 (1H, d, J = 12.6 Hz) and an e hoxy g oup a δH 3.35 (2H, m) and
1.13 (3H, , J = 6.9 Hz). The ex a ace yl signal a C-22 was obse ed, since he oxygena ed me hine
p o on o 1 a δH 3.35 (1H, d, J = 6.6 Hz) was displaced o δH 4.55 (1H, d, J = 6.3 Hz) in 2. Mo eo e ,
he halogena ed g oup was eplaced by a e hoxyl g oup a C-30 since he ca bon in 2 was displaced o
low ield a δC 72.3 (Table 1). The s uc u e o 2 was de e mined by a combina ion o COSY, DEPT,
HSQC, HMBC, and NOESY expe imen s. Based on he abo e da a, he new compound olpidiol A 2
was iden i ied as 3β,22α-diace oxy-30-e hoxy-20(21)- a axas ene.
Compound 3 was pu i ied as i s diace a e 3a by ea men wi h ace ic anhyd ide (Ac2O) in py idine,
3a was isola ed as a colou less amo phous solid and i s HRESIMS expe imen indica ed he molecula
o mula C34H54O6 (calcd. o [M+Na]+ 581.3818; ound 581.3801). The IR spec um e ealed he
abso p ion bands o ca bonyl 1728 cm−1, and hyd oxyl 3391 cm−1 g oups, he p esence o hese
g oups was con i med by he 1H and 13C-NMR spec a (Table 2). The 1H-NMR spec um o 3a showed
signals o i e e ia y me hyl g oups a δH 0.81(6H, b , s), 1.00, 1.02 and 1.11, and wo seconda y
me hyl g oups a δH 0.86 (3H, d, J = 6.4 Hz) and δH 0.88 (3H, d, J = 7.3 Hz) sugges ing a pen acyclic
i e pene wi h an u sane skele on. An ole inic p o on a δH 5.30 (1H, d, J = 3.1 Hz) was assigned o
H-12, wo oxygena ed me hines a δH 4.45 (1H, dd, J = 3.0, 9.6 Hz) and 4.46 (1H, dd, J = 5.0, 9.5 Hz)
co esponding o H-3 and H-11 espec i ely, he la e showing icinal co ela ion in he COSY
expe imen wi h he ole inic p o on H-12, while he p o on H-9 δH 1.81 (1H, d, J = 9.5 Hz) indica ed
he p esence o a hyd ope oxide a C-11. The p esence o an oxygena ed me hylene was con i med by
he signals a δH 3.56 (1H, d, J = 11.0 Hz) and 3.93 (1H, d, J = 11.0 Hz). The 13C-NMR (Table 2) and
DEPT da a indica ed he p esence o wo es e ca bonyl g oups, nine me hyl ca bons, nine alipha ic
me hylenes, wo ole inic ca bons, and se en me hine ca bons. Thus, he posi ion o ace yl g oups in
compound 3a was assigned by a HMBC co ela ion be ween he signal a δC 171.0 and ha a δH 4.45;
and he signals a δC 171.3 and δH 3.93. The coupling cons an be ween H-9 and H-11 (J = 9.5 Hz)
es ablished he α-o ien a ion o he hyd ope oxide a C-11. The EI-MS da a o 3a showed di ec loss o
H2O m/z 540 and H2O2 m/z 524, con i ming he p esence o he hyd ope oxide.
The s uc u e elucida ion and NMR assignmen s we e based p ima ily on he esul s o HSQC,
HMBC, and COSY expe imen s which allowed he comple e assignmen o all H- and C-a oms, and
he NOESY (Figu e 3) da a p o ided he con igu a ion o compound 3a. The e o e, he s uc u e o 3
was es ablished as 3β,28-dihyd oxy-11α-hyd ope oxy-12-u sene. To he bes o ou knowledge,
compound 3 is a no el i e penoid, which we named olpidiol B.

Molecules 2012, 17 12900
Table 2. 1H and 13C-NMR da a o compound 3a a.
Posi ion H C
1 0.85 m
2.08 d (3.5, 7.0) 39.4
2 1.58 * 23.7
3 4.45 dd (3.0,9.6) 80.6
4 - 38.0
5 0.88 m 55.3
6 1.45 *
1.31 * 18.1
7 1.45 m
1.25 m 33.3
8 - 43.2
9 1.81 d (9.5) 48.8
10 - 37.8
11 4.46 dd (5.0, 9.5) 81.6
12 5.30 d (3.1) 125.8
13 - 144.5
14 - 42.0
15 1.60 *
0.90 m 26.2
16 1.16 *
1.92 d (3.5,9.0) 23.3
17 - 36.9
18 1.45 m 53.7
19 1.35 m 39.0
20 1.28 m 39.3
21 1.40 m 30.4
22 1.31 m
1.52 d (3.0, 6.5) 35.5
23 0.81 s 28.2
24 0.81 s 16.7
25 1.02 s 16.8
26 1.00 s 18.0
27 1.11 s 22.2
28 3.56 d (11.0)
3.93 d (11.0) 71.0
29 0.86 d (6.4) 17.4
30 0.88 d (7.3) 21.3
OAc
1.97 s
1.97 s
21.0
21.3
171.0
171.3
a δ in ppm and J (in Hz) a e in pa en heses. Reco ded in CDCl3 a 400 MHz and 125 MHz o
1H- and 13C-NMR, espec i ely. * o e lapped.
Molecules 2012, 17 12901
Figu e 3. Selec ed co ela ions o 3a. Bond bolded indica e COSY, Double-ended a ows
indica e NOESY, and single a ows indica e HMBC (C o H) co ela ions.
Compound 1 could de i e om he known i e pene ace yl-p iloepoxide 4 [8,9] which was
iden i ied by us om T. p ous ii as an insepa able mix u e. T i e penes con aining an epoxide a he
Δ21-22 posi ion a e known and ha e been isola ed be o e om a Tolpis species [4]. Based on his,
we en isioned he o ma ion o compound 1 by chlo ina ion o he double bond, ollowed by
isome iza ion and β opening o he epoxide, and p o ona ion, main aining he α-o ien a ion a he C-22
obse ed in he p ecu so compound ace yl-p iloepoxide (Figu e 4).
Figu e 4. Ten a i e o ming p ocess o compound 1.
Al hough he numbe is ela i ely small, se e al halogena ed i e penes and o he highe e penes ha e
been desc ibed mainly om ma ine sou ces [10]. Howe e , he p esence o chlo ina ed i e penoids in
e es ial plan s is e y a e and jus ew cases ha e been epo ed [11–14]. Ini ially, compound 1 seemed
o be an a i ac o he isola ion p ocess, bu Chen e al. [14] p o ed ha o ob ain chlo ina ed compounds,
a chlo ine sou ce such as CHCl3 wi h HCl is necessa y. Du ing he isola ion p ocess, no chlo ina ed
sol en s we e used (see Expe imen al). In he ch oma og aphic sepa a ion, dichlo ome hane was used
in he p epa a i e TLC, which was no enough o in e ac wi h he possible p ecu so (p iloepoxide) o
1. Compound 2 was isola ed as a p esumed a i ac ; his compound was p obably ob ained om 1, by a
nucleophilic subs i u ion eac ion due o he use o ho E OH du ing he ex ac ion p ocess.
Addi ionally, om T. p ous ii 15 known compounds we e isola ed, including a oma ic compounds:
scopole in [15] aescule in [15] and apigenin [16]; he di e pene phy ene-1,2-diol [17] and he
i e penoids s igmas e ol [18], e gos e ol pe oxide [19], u solic acid [20], lupan-20(29)-ene-3,30-
diol [21], 21-hyd oxy a axas e ol [22], 11-oxo-β-amy in [23], 3-ace oxy-u s-12-ene-1,11-diol [24],
21,22-epoxy-20-hyd oxy-20(30)-dihyd o a axas e ol [4], 3-ace oxy-21,22-epoxy a axas an-
20α-ol [25], 22-oxo-20- a axas en-3-ol [9], -amy in [26]. F om T. lagopoda se en known
Molecules 2012, 17 12902
compounds we e isola ed, including a oma ic compounds: 2,4′-dihyd oxy-4-me hoxybenzophenone [4]
and i e penoids: s igmas e ol [18], e gos e ol pe oxide [19], a mix u e o 7-oxo-β-si os e ol and
7-oxo-s igmas e ol [26,27], u solic acid [20], and α-amy in [28]. Thei s uc u es we e con i med by
compa ison o hei spec al da a wi h hose epo ed in he li e a u e.
2.2. An ioxidan Ac i i ies
Na u al an ioxidan s ha a e p esen in plan s a e esponsible o inhibi ing o p e en ing he
dele e ious consequences o oxida i e s ess. In Table 3 he ela i e an ioxidan e iciency o bo h
Tolpis ex ac s agains he DPPH adical is shown. An ioxidan s supp ess he abso bance a 515 nm on
a ime scale dependen on he an ioxidan ac i i y o ex ac s. The RSA o he c ude ex ac o
T. p ous ii (59.6%) was highe han ha o T. lagopoda (41.4%). FRAP assay was used o s udy he
abili y o he an ioxidan s in he ex ac s o educe e ic i on o he e ous o m. The same beha iou
as o he DPPH assay was obse ed, T. lagopoda being less ac i e han T. p ous ii (4.1 and 18.1 µmol
o Fe(III) educed o Fe(II) pe g am o d y plan espec i ely) (Table 3). On he o he hand, he ee
adical sca enging and e ic educing powe assays e ealed ha aescule in (isola ed om T. p ous ii)
showed he highes an ioxidan ac i i ies as compa ed wi h hose o α- ocophe ol and BHA (Table 4).
Aescule in ga e a RSA alue o 100% wi h a 1/2 ( ime equi ed o 50% sca enging o DPPH adical
in he speci ied concen a ion o an ioxidan ) o 22.5 seconds, while BHA and α- ocophe ol showed
RSA o 21.9 and 17.7% espec i ely a e 20 min. Aescule in (a concen a ion 0.1 mg mL−1) showed
also highe an ioxidan ac i i ies han bo h ex ac s (a concen a ion 10 mg mL−1), because he ex ac s
a e complex mix u es ha include ac i e componen s a lowe le els. Mo eo e , he c ude ex ac s
end o ha e mo e in e e ing subs ances ha may in e ac wi h he an ioxidan s, dec easing hei
e ec i eness. The an ioxidan ac i i ies ound in his s udy indica ed ha aescule in, as well as bo h
ex ac s, a e ideal o use in he heal h ood indus y. Because o he high con en o aescule in ound
in he T. p ous ii ex ac (566.8 mg), his ex ac may be conside ed o be a na u al sou ce o aescule in
wi h di e se po en ial he apeu ic uses.
Table 3. An ioxidan ac i i y o c ude ex ac s de i ed om T. p ous ii and T. lagopoda.
Assays T. p ous ii T. lagopoda
RSA a 59.6 ± 0.4 41.4 ± 0.1
FRAP b 18.1 ± 0.4 4.1 ± 0.2
FRAP c 93 ± 2 41 ± 1
a % inhibi ion ± s anda d de ia ion o h ee measu emen s. b µmol o Fe(III) educed o Fe(II) pe
g am o d y plan ± s anda d de ia ion o h ee measu emen s. c µmol o Fe(III) educed o Fe(II)
pe g am o e hanolic esidue ± s anda d de ia ion o h ee measu emen s.
Table 4. An ioxidan ac i i y o aescule in, α- ocophe ol and bu yla ed hyd oxyanisol (BHA).
Assays Aescule in
0.1 mg mL−1
α-Tocophe ol
0.1 mg mL−1
BHA
0.1 mg mL−1
DPPH a 100 ± 0 17.7 ± 0.1 21.9 ± 0.6
FRAP b 9.4 ± 0.7 0.97 ± 0.03 3.13 ± 0.05
a % inhibi ion ± s anda d de ia ion o h ee measu emen s. b µmol o Fe(III) educed o Fe(II) pe
mg o compound ± s anda d de ia ion o h ee measu emen s.
Molecules 2012, 17 12903
2.3. Cy o oxic Ac i i y
K-562 and K-562/ADR cells which a e sensi i e o esis an o doxo ubicin, espec i ely, we e
incuba ed wi h he compounds shown in Table 5 o e alua e hei po en ial cy o oxici y. A e 72 h,
cell su i al was de e mined by he MTT assay and he IC50 alues a e summa ized in Table 5. Among
he di e en compounds u solic acid and 22-oxo-20- a axas en-3β-ol exhibi he s onges e ec s in
mi ochond ial educ ion o e azolium sal s o o mazan, while he u solic de i a i es and he
1,2-diace oxyphy ene exhibi he weakes e ec s. Fu he mo e, K-562 and K-562/ADR cells exhibi
compa able sensi i i y o compounds u solic acid and 22-oxo-20- a axas en-3β-ol (Table 5). These
esul s sugges ha he o e exp ession o he d ug e lux p o ein, P-glycop o ein does no con e
esis ance agains hese compounds.
Table 5. E ec s o some compounds and de i a i es isola ed om T. p ous ii and
T. lagopoda on he g ow h o he human leukemia cell lines.
Compound IC50 (M)
K562 K562/ADR
U solic acid 40.6 ± 3.6 49.2 ± 3.1
U solic acid me hyl es e 59.3 ± 15.5 64.0 ± 14.5
Ace yl u solic acid 99.5 ± 20.5 >100
Ace yl u solic acid me hyl es e >100 >100
Aescule in 63.2 ± 3.2 77.0 ± 5.1
Aescule in ace yl 68.6 ± 17.1 70.3 ± 19.2
Aescule in diace yl 62.3 ± 6.6 59.5 ± 4.5
11-Oxo-β-amy in >100 >100
22-Oxo-20- a axas en-3-ol 30.0 ± 10.0 43.0 ± 7.0
1,2-Diace oxyphy ene >100 >100
Cells we e cul u ed o 72 h and he IC50 alues we e calcula ed as desc ibed in he Expe imen al
sec ion. The da a shown ep esen he means ± SEM o 3–5 independen expe imen s wi h h ee
de e mina ions in each.
3. Expe imen al
3.1. Gene al Expe imen al P ocedu es
Op ical o a ions: Pe kin-Elme model 343 pola ime e . IR Spec a: B uke model IFS-55
spec opho ome e . 1H and 13C-NMR spec a: B uke model AMX-500 and AMX-400 spec ome e s
wi h s anda d pulse sequences, ope a ing a 500 and 400 MHz o 1H-, and 125 MHz o 13C-NMR,
CDCl3 was used as sol en and TMS as in e nal s anda d. EI–MS: Mic omass model Au ospec (70 eV)
spec ome e . The cons i uen s o he e hanolic ex ac s we e sepa a ed by g a i y column
ch oma og aphy, medium p essu e liquid ch oma og aphy (MPLC) and p epa a i e TLC. Column
ch oma og aphy (CC): silica gel SiO2; (70–230 mesh, Me ck), column ac ions we e moni o ed by
TLC (silica gel 60 F254), Medium p essu e column ch oma og aphy (MPLC): silica gel Me ck
(40–63 m). P ep. TLC: silica gel 60 PF254 + 366 pla es (20 × 20 cm, 1-mm hickness).
Rep oduced wi h pe mission o he copy igh owne . Fu he ep oduc ion p ohibi ed wi hou
pe mission.