1516 Journal of Natural Products, 2008, Vol. 71, No. 9
Dell’Agli et al.
Table 2. IC50 Values of 5 and Sildenafil on Human PDE6 and cAMP-PDE
PDE6C
(IC50 µM ( SD)
PDE6C/PDE5A1
(IC50 ratio)
cAMP-PDE
(IC50 µM ( SD)
cAMP-PDE/PDE5A1
compound
(IC50 ratio)
5
30.9 ( 2.6
0.16 ( 0.007
418
2.2
96.3 ( 12.9
27.5 ( 5.3
1301
367
sildenafil
Hz, H-2′, H-6′), 12.60 (s, 1H, OH-5); ESIMS (positive-ion mode) m/z
677 [M + H]+, 699 [M + Na]+.
Hz, H-2′, H-6′); ESIMS (positive-ion mode) m/z 479 [M + Na]+; anal.
C 65.82%, H 6.22%, calcd for C25H28O8, C 65.78%, H 6.18%.
Preparation of ꢀ-Anhydroicaritin (6). A solution of extract A, used
in the isolation of 1 (200 mg) in dioxane (25 mL), was added to 1 M
H2SO4 (12.5 mL) and refluxed for 24 h. After cooling, the reaction
mixture was adjusted to pH 7-8 with NaHCO3 and extracted with
EtOAc (3 × 50 mL). The organic phase was dried over anhydrous
Na2SO4 and evaporated under vacuum to afford ꢀ-anhydroicaritin as a
yellow powder (purity 96.5%; 105 mg, 23% yield of the dry extract);25
1H NMR, (DMSO-d6, 30 °C) δ 1.40 (6H, s, 14, CH3-15), 1.90 (2H, t,
H-12), 2.85 (2H t, H-11), 3.90 (3H, s, OCH3), 6.20 (1H, s, H-6), 7.15
(2H, d, J ) 8.9 Hz, H-3′, H-5′), 8.20 (2H, d, J ) 8.9 Hz, H-2′, H-6′),
9.57 (1H, s, OH-3), 12.20 (1H, s, OH-5); ESIMS (positive-ion mode)
m/z 369 [M + H]+.
Preparation of Icariside II (2). A solution of 1 (500 mg) in DMSO
(1 mL) was added dropwise for 48 h to a Na acetate-buffered
hydroalcoholic solution at 37 °C (0.25 M, pH 5.0, in EtOH/H2O, 30:
70) (50 mL) containing cellulase (210 mg). The suspension obtained
was stirred at 37 °C for 4 days. Then, a further amount of cellulase
(100 mg) was added, and the mixture was stirred under the same
conditions for a further 2 days. EtOH was then removed under vacuum
and the residue was diluted to 200 mL with H2O and extracted with
EtOAc (3 × 200 mL). The organic layer was dried over anhydrous
Na2SO4 and evaporated under reduced pressure to afford 2 (290 mg);
yield 76%; mp 208-210 °C;23 1H NMR (DMSO-d6, 300 MHz, 30 °C)
δ 0.90 (3H, s, rha CH3), 1.82 (3H, s, CH3-14), 1.87 (s, 3H, CH3-15),
3.05-3.60 (4H, m, rha protons, H-11), 3.85 (3H, s, OCH3), 4.22-4.24
(1H, m, rha proton), 4.55-4.80 (3H, m, sugar OH), 4.90 (1H, m, rha
proton), 5.20 (1H, t, J ) 6.8 Hz, H-12), 5.52 (1H, d, J ) 1.5 Hz, rha
proton), 6.37 (1H, s, H-6), 7.15 (2H, d, J ) 8.4 Hz, H-3′, H-5′), 7.83
(2H, d, J ) 8.4 Hz, H-2′, H-6′), 10.60 (1H, s, OH-7), 12.80 (1H, s,
OH-5); ESIMS (positive-ion mode) m/z 537 [M + Na]+.
Human Recombinant PDE5A1 Expression. Human recombinant
PDE5A1 was prepared by expression of the full-length cDNA of
PDE5A1 into COS-7 cells, as previously described.26
PDE5A1 and PDE6C Enzyme Assays. PDE5A1 activity was
determined according to the method of Kincaid and Manganiello27 with
minor modifications.28 Screening of plant extracts was performed at
50 µg/mL, whereas the individual compounds were tested at 10 µM.
PDE6C activity was evaluated under the same conditions used for
PDE5A1 activity, with 0.5 U enzyme/sample being used. Screening of
the individual compounds against PDE6C activity was performed at
concentrations 10-fold higher than each IC50 obtained against PDE5A1.
IC50 values were calculated using Graph Pad Prism 4 for sigmoidal
curves. Sildenafil was used as reference compound. Each result is the
mean ( SD of at least two experiments in triplicate.
Platelet Homogenate Preparation and Assay for cAMP-PDE
Activity. The blood fraction enriched in platelets, obtained from healthy
volunteers, was submitted to two centrifugations at 160g for 10 min at
room temperature. The pellet was removed, and platelet-rich plasma
(PRP) was centrifuged at 1000g for 15 min. The resulting pellet was
suspended in 10 mM Tris/HCl, pH 7.4 (2/5 of the initial volume). The
suspension was centrifuged at 1000g for 15 min and the pellet
suspended in the Tris/HCl buffer, pH 7.4 (1/12 of the initial volume).
All these steps were performed at 4 °C. Cells were disrupted by freezing
and thawing three times, obtaining the homogenate,29 and cell lysate
was stored at -80 °C. Total protein concentration was measured
according to Bradford.30
cAMP-PDE activity was determined according to the method of
Kincaid and Manganiello27 with minor modifications. Briefly, platelet
lysate (64 µg of protein/mL) was incubated with 0.5 µM cAMP and
63 nCi [3H]-cAMP suspended in 30 mM Tris-HCl, pH 7.4, 4 mM
MgCl2; final reaction volume was 250 µL. After 5 min of incubation
at 30 °C, the reaction was stopped with 0.1 N HCl. Samples were then
incubated for a further 4 min at 70 °C with AMP (5 mM) and cAMP
(5 mM), and the pH was adjusted to 7 on ice with 0.1 N NaOH. Samples
were then added with 50 µL of nucleotidase from Crotalus adamanteus
snake venom (1 mg/mL in Tris-HCl 0.1 M, pH 8.0) and incubated for
20 min at 37 °C. The reaction was stopped with 50 µL of 200 mM
NaEDTA containing 5 mM adenosine. The nucleoside formed during
the incubation was separated from the unreacted substrate by DEAE-
Sephadex A25 column chromatography. The eluted [3H]-adenosine was
counted in a ꢀ-scintillation counter. Compound 5 and sildenafil were
tested in a range of 1-250 µM, and IC50 values calculated using Graph
Pad Prism 4 for sigmoidal curves. Inhibition (%) by aminophylline
(100 µM) used as reference compound was 74.5 ( 1.3 (mean ( SD,
n ) 11). Each result is the mean ( SD of three experiments in triplicate.
Cytotoxicity Assay. Cellular toxicity was assessed using a 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) colo-
rimetric assay.31 Human skin fibroblasts were treated with increasing
concentrations (0.25-100 µM) of 5 for 24 h in DMEM-F12 supple-
mented with 10% heat-inactivated FBS, 1% penicillin, and 1%
L-glutamine. The medium was removed, and cells were incubated with
a solution containing MTT 0.5 mg/mL in PBS at 37 °C for 3 h. The
MTT solution was removed, the formazan was extracted with 2-pro-
Preparation of Icaritin (3). A solution of icariin (1) (526 mg) in
DMSO (1 mL) was added dropwise for 72 h to a Na acetate-buffered
hydroalcoholic solution at 37 °C (0.25 M, pH 5.0, in EtOH/H2O, 30:
70) (50 mL) containing naringinase (207 mg). The obtained suspension
was allowed to stir at 37 °C for 7 days. Then, a further amount of
naringinase (97 mg) was added and the mixture was stirred under the
same conditions for a further day. EtOH was removed by evaporation
and the aqueous suspension was filtered under vacuum and dried. The
residue obtained was washed with H2O and dried to give icaritin (3,
290 mg; purity 95%) as a yellow powder. The mother liquors were
diluted with H2O and extracted with EtOAc (2 × 200 mL). The organic
phase was dried over anhydrous Na2SO4 and evaporated under reduced
pressure to afford an additional amount of 3 (20 mg); quantitative yield,
mp 232-233 °C;24 1H NMR (CDCl3, 300 MHz, 30 °C) δ 1.78 (3H, s,
CH3-14), 1.87 (3H, s, CH3-15), 2.70 (2H, s, OH), 3.61 (2H, d, J ) 6.8
Hz, H-11), 3.89 (3H, s, OCH3), 5.36 (1H, t, J ) 6.8 Hz, H-12), 6.32
(1H, s, H-6), 7.04 (2H, d, J ) 8.4 Hz, H-3′, H-5′), 8.16 (2H, d, J ) 8.4
Hz, H-2′, H-6′); ESIMS (positive-ion mode) m/z 369 [M + H]+.
Preparation of 7-(2-Hydroxyethyl)-3-O-rhamnosylicariin (4). A
stirred suspension of 2 (200 mg, 0.39 mmol), 2-bromoethanol (50 mg,
0.43 mmol), and anhydrous K2CO3 (60 mg, 0.43 mmol) in dry acetone
(15 mL) was refluxed for 8 h. The hot reaction mixture was filtered,
and the solvent was evaporated under reduced pressure. The residue
was purified by flash chromatography on silica gel (EtOAc/CH3OH,
9.5:0.5) to give a yellow crystalline compound (4, 112 mg; purity
93.0%); 55% yield; mp 194-196 °C (EtOH); 1H NMR (acetone-d6 +
D2O, 300 MHz, 30 °C) δ 0.88 (3H, d, J ) 5.4 Hz, rha CH3), 1.64 (3H,
s, CH3-14), 1.75 (3H, s, CH3-15), 3.2-3.8 (3H, m, rha protons), 3.7
(2H, d, J ) 8.7, H-11), 3.91 (3H, s, OCH3), 4.21-4.24 (2H, m,
OCH2O), 4.40 (2H, t, J ) 6.0 Hz, CH2OH), 4.22-4.24 (1H, m, rha
proton), 5.25 (1H t, J ) 6.9 Hz, H-12), 5.52 (1H, d, J ) 1.5 Hz, rha
proton), 6.50 (1H, s, H-6), 7.14 (2H, d, J ) 6.9 Hz, H-3′, H-5′), 7.96
(2H, d, J ) 6.9 Hz, H-2′, H-6′); ESIMS (positive-ion mode) m/z 581
[M + Na]+; anal. C 62.31%, H 6.18%, calcd for C29H34O11, C 62.36%,
H 6.14%.
Preparation of 3,7-Bis(2-hydroxyethyl)icaritin (5). A stirred
suspension of 3 (250 mg, 0.7 mmol), 2-bromoethanol (210 mg, 1.7
mmol), and anhydrous K2CO3 (240 mg, 1.7 mmol) in dry acetone (75
mL) was refluxed for 8 h. The hot reaction mixture was filtered, and
the solvent was evaporated under reduced pressure. The residue was
purified by flash column chromatography on silica gel (CH2Cl2/acetone,
9:1) and crystallized from EtOH to give the desired compound as a
yellow crystalline powder (70 mg, purity 96.0%); 20.2% yield; mp
152-153 °C; 1H NMR (CDCl3, 300 MHz, 30 °C) δ 1.77 (3H, s, CH3-
14), 1.87 (3H, s, CH3-15), 3.61 (2H, d, J ) 6.8 Hz, H-11), 3.78-3.83
(2H, m, 7-OCH2O), 3.90 (3H, s, OCH3), 3.95-4.05 (4H, m, CH2OH),
4.15-4.22 (2H, m, OCH2O-3), 5.19 (1H, t, J ) 6.8 Hz, H-12), 6.32
(1H, s, H-6), 7.04 (2H, d, J ) 8.4 Hz, H-3′, H-5′), 8.16 (2H, d, J ) 8.4