ArticlePDF Available

Potent Inhibition of Human Phosphodiesterase-5 by Icariin Derivatives

Authors:

Abstract and Figures

Plant extracts traditionally used for male impotence (Tribulus terrestris, Ferula hermonis, Epimedium brevicornum, Cinnamomum cassia), and the individual compounds cinnamaldehyde, ferutinin, and icariin, were screened against phosphodiesterase-5A1 (PDE5A1) activity. Human recombinant PDE5A1 was used as the enzyme source. Only E. brevicornum extract (80% inhibition at 50 microg/mL) and its active principle icariin (1) (IC50 5.9 microM) were active. To improve its inhibitory activity, 1 was subjected to various structural modifications. Thus, 3,7-bis(2-hydroxyethyl)icaritin (5), where both sugars in 1 were replaced with hydroxyethyl residues, potently inhibited PDE5A1 with an IC50 very close to that of sildenafil (IC50 75 vs 74 nM). Thus, 5 was 80 times more potent than 1, and its selectivity versus phosphodiesterase-6 (PDE6) and cyclic adenosine monophosphate-phosphodiesterase (cAMP-PDE) was much higher in comparison with sildenafil. The improved pharmacodynamic profile and lack of cytotoxicity on human fibroblasts make compound 5 a promising candidate for further development.
Content may be subject to copyright.
Potent Inhibition of Human Phosphodiesterase-5 by Icariin Derivatives
Mario Dell’Agli,*
,†
Germana V. Galli,
Esther Dal Cero,
Federica Belluti,
Riccardo Matera,
§
Elisa Zironi,
§
Giampiero Pagliuca,
§
and Enrica Bosisio
Department of Pharmacological Sciences, UniVersity of Milan, Via Balzaretti 9, 20133, Milan, Italy, Department of Pharmaceutical Sciences,
UniVersity of Bologna, Via Belmeloro 6, 40126, Bologna, Italy, and Department of Veterinary Public Health and Animal Pathology, UniVersity
of Bologna, Via Tolara di Sopra 30, 40064 Ozzano Emilia (BO), Italy
ReceiVed January 23, 2008
Plant extracts traditionally used for male impotence (Tribulus terrestris, Ferula hermonis, Epimedium breVicornum,
Cinnamomum cassia), and the individual compounds cinnamaldehyde, ferutinin, and icariin, were screened against
phosphodiesterase-5A1 (PDE5A1) activity. Human recombinant PDE5A1 was used as the enzyme source. Only E.
breVicornum extract (80% inhibition at 50 µg/mL) and its active principle icariin (1) (IC50 5.9 µM) were active. To
improve its inhibitory activity, 1was subjected to various structural modifications. Thus, 3,7-bis(2-hydroxyethyl)icaritin
(5), where both sugars in 1were replaced with hydroxyethyl residues, potently inhibited PDE5A1 with an IC50 very
close to that of sildenafil (IC50 75 vs 74 nM). Thus, 5was 80 times more potent than 1, and its selectivity versus
phosphodiesterase-6 (PDE6) and cyclic adenosine monophosphate-phosphodiesterase (cAMP-PDE) was much higher
in comparison with sildenafil. The improved pharmacodynamic profile and lack of cytotoxicity on human fibroblasts
make compound 5a promising candidate for further development.
The inability to achieve or maintain an erection sufficient for
satisfactory sexual function is an increasing problem with a
considerable impact on interpersonal relationships and quality of
life for men.
1
During erection, nitric oxide is released from the
axon terminals of the parasympathetic nerves and diffuses into
smooth muscle cells of the arterial walls of the corpus cavernosum.
The consequent activation of guanyl cyclase, converting guanosine
triphosphate (GTP) into cyclic guanosine monophosphate (cGMP),
causes smooth muscle relaxation, leading to dilation and increased
influx of blood into the penile tissue. The trapping of blood in the
penis results in an erection.
2
Selective inhibitors of cGMP-
phosphodiesterase-5 (PDE5) such as sildenafil (Viagra), tadalafil,
and vardenafil are currently used for erectile dysfunction (ED).
However, several adverse effects have been recorded in clinical
trials, including priapism and visual disturbances.
3
Furthermore,
therapy with PDE5 inhibitors is cost-effective. Thus, the search
for new compounds of this type for drug development could be
worthwhile. A variety of natural plant products, including berberine,
forskolin, papaverine, and yohimbine, are claimed to be useful for
improving sexual performance. Extracts from Lepidium meyenii
Walp. (maca), Panax ginseng C.A. Meyer, Ginkgo biloba L., Ferula
hermonis Boiss., and many other herbal remedies, alone or in
combination, have been promoted for the treatment of sexual
problems.
4,5
With the aim of looking for new leads for selective
PDE5 inhibitors, plant extracts and their putative active principles
were selected for screening against human PDE5 activity in vitro.
Our attention focused on Tribulus terrestris L., Ferula hermonis,
Epimedium breVicornum Maxim., and Cinnamomum cassia L.,
since these extracts are claimed traditionally to improve sexual
performance. T. terrestris caused vasodilating and antihypertensive
effects in rats
6
and a pro-erectile effect on the rabbit corpus
cavernosum;
7
F. hermonis has been studied for its effects on sexual
behavior in male and female rats;
8,9
C. cassia and “Epimedii Herba”
are components of Chinese herbal products patented for the
treatment of sexual dysfunction.
10-14
“Epimedii herba” is the
common name for the dried aerial parts of E. breVicornum,E.
sagittatum Maxim., or E. koreanum Nakai, collected in the
summer.
15,16
Among the extracts, only “Epimedii Herba” was active
against PDE5A1, for which the presence of icariin (1), the major
pharmacologically active constituent,
17-19
was considered a lead
compound for chemical modifications in order to improve inhibitory
activity. Modifications applied at the hydroxyl groups at C-3, C-7,
and C-8 included partial or complete removal of the sugar moieties,
partial or complete sugar replacement with a hydroxyethyl residue,
and cyclization of the prenyl group (Scheme 1). All compounds
produced (1-6) were tested for PDE5A1 activity. Also, selectivity
versus human retina PDE6C and human platelet cAMP-PDE, and
cytotoxicity on human fibroblasts were investigated.
Results and Discussion
The activity of plant extracts and individual compounds against
human recombinant PDE5A1 is shown in Figure S1 (Supporting
Information). Cinnamaldehyde, icariin (1), and ferutinin were
considered as the putative active principles of C. cassia,E.
breVicornum, and F. hermonis, respectively, since the compounds
represent the most abundant secondary metabolites of those species.
Only E. breVicornum and icariin (1) strongly inhibited PDE5A1
(-80% and -72%, respectively), whereas the other test materials
were much less active (-15 to -23%). Inhibition by cinnamalde-
hyde (-16%) and ferutinin (-7%) was not significant. The
medicinal plants tested in the present study had a reputation for
aphrodisiac effects and therefore represented the start of a screening
program to search for compounds to be developed as a new natural
drug alternative to sildenafil. The observation that only E. breVi-
cornum and its active principle 1inhibited PDE5 in a significant
manner, in agreement with previous results,
17-20
suggests that the
other plant extracts may interfere with erectile function through
mechanisms other than PDE5 inhibition.
Compound 1was a good PDE5 inhibitor (IC50 of 5.9 µM), but
required improvement in order to have equivalent potency to
sildenafil, which gave an IC50 of 75 nM. The inhibitory effects of
icariin derivatives 2-6on PDE5A1 is shown in Table 1. Since
aglycons might be expected to possess higher activity than the
corresponding glycosides, the first general structural modification
to 1was the removal of one or both of the sugar moieties at the
hydroxyl groups at positions C-3 and C-7 of the flavone scaffold.
Enzymatic hydrolysis of 1with cellulase and naringinase allowed
the partial or total removal of the sugar moieties, respectively,
* To whom correspondence should be addressed. Tel: +39-02-50318345.
Fax: +39-02-50318391. E-mail: mario.dellagli@unimi.it.
University of Milano.
Department of Pharmaceutical Sciences, University of Bologna.
§
Department of Veterinary Public Health and Animal Pathology,
University of Bologna.
J. Nat. Prod. 2008, 71, 1513–1517 1513
10.1021/np800049y CCC: $40.75 2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/09/2008
affording the known compounds 2and 3. Indeed, the removal of
the glucose at the hydroxyl group in C-7, thus furnishing icariside
II (2), improved drastically the enzyme inhibition, attaining an IC50
value on the nanomolar order (IC50 156 nM). Conversely, icaritin
(3), where both sugars were removed, was only around 3-fold more
potent than 1(IC50 2.2 µM).
To investigate if the prenyl moiety is essential for enzyme
inhibition activity, β-anhydroicaritin (6) was tested. The cyclization
Scheme 1. Outline of the Synthetic Route Followed for the Synthesis of Icariin (1) Derivatives
a
a
Reagents and conditions: (a) cellulase, 37 °C, Na acetate pH )5 buffered hydroalcoholic solution, 6 days; (b) naringinase, 37 °C, Na acetate pH )5 buffered
hydroalcoholic solution, 11 days; (c) H2SO4, dioxane, reflux, 24 h; (d) 2-bromoethanol, K2CO3, acetone, reflux, 8 h.
Chart 1
1514 Journal of Natural Products,2008, Vol. 71, No. 9 Dell’Agli et al.
led to a dramatic drop in inhibitory activity. The IC50 value for 6
was 45.5 µM, indicating that a free prenyl group at position C-8 is
important for enzyme inhibition. To confirm that the prenyl group
is required for enzyme inhibition, the 8-prenyl derivatives of
naringenin (8-PN), quercetin (8-PQ), and apigenin (8-PA) were
tested and their activity compared to that of the corresponding free
flavonoid. As shown in Figure S2 (Supporting Information), all
prenylflavonoids inhibited PDE5A1 with the following order of
potency: 8-PQ (IC50 0.70 (0.10 µM) >8-PA (IC50 1.29 (0.11
µM) .8-PN (IC50 16.23 (1.16 µM). Quercetin, apigenin, and
naringenin (10 µM) showed 23%, 12%, and 6% inhibition,
respectively, much lower than the corresponding prenyl derivatives.
Data from the literature confirm the importance of the prenyl group:
sophoflavescenol, a prenylated flavonol from Sophora flaVescens
Ait. (Leguminosae), and osthole, a prenyl coumarin from Angelica
pubescens Maxim., are two additional examples of PDE-5 inhibitors
in the class of prenylated phenolic compounds.
21,22
The last modification to 1was the replacement of one or both
sugar moieties with the hydroxyethyl side chain, representing a
simplification of the sugar residue. The substitution for Glc by a
hydroxyethyl group at C-7 gave 7-(2-hydroxyethyl)-3-O-rhamno-
sylicariin (4), which was less potent than 2(IC50 363 vs 156 nM,
respectively). When both hydroxyls at C-3 and C-7 were derivatized
with hydroxyethyl moieties, as in 3,7-bis(2-hydroxyethyl)icaritin
(5), PDE5A1 was potently inhibited, with an IC50 of 74 nM, almost
identical to that of sildenafil (75 nM).
The selectivity against human PDE5A1 was investigated by
testing all compounds against human PDE6C, at concentrations 10-
fold higher than their PDE5A1 IC50 values. Compounds 1and 4
inhibited PDE6 activity (45% and 86%, respectively), while 2,3,
5, and 6were inactive. For 5, the best PDE5 inhibitor among the
icariin derivatives tested, concentration inhibition curves for PDE6
and cAMP-PDE were performed. The results were compared with
those obtained for sildenafil (Table 2). The PDE6C/PDE5 IC50 ratio
was 418 for 5and 2.2 for sildenafil, while the cAMP-PDE/PDE5
IC50 ratio was 1300 for 5and 367 for sildenafil. These results
indicate that the selectivity of 5for PDE5 was improved with
respect to that of sildenafil. Compound 5was not cytotoxic for
human fibroblasts even at the highest concentration tested (100 µM).
Thus, the inhibitory potency of 5was 80-fold higher than that of
the parent compound icariin (1). Its selectivity and lack of
cytotoxicity make 5a candidate worthy of further study.
Experimental Section
General Experimental Procedures. Melting points were determined
in open glass capillaries using a Bu¨chi apparatus and are uncorrected.
Nuclear magnetic resonance (1H NMR) spectra were recorded on a
Varian VXR 200 or Varian VXR 300 spectrometer equipped with
VNMR software. Chemical shifts (δ) are reported in ppm with
tetramethylsilane (TMS) as the internal standard, and spin multiplicities
are given as s (singlet), d (doublet), t (triplet), q (quartet), dd (double
doublet), dt (double triplet), m (multiplet), or br (broad). ESIMS were
obtained on a Finnigan MAT LCQ ion trap mass spectrometer or Waters
Micromass ZQ 4000 apparatus equipped with a Microsoft Windows
NT data system and an ESI interface. HPLC-MS analysis was carried
out with a Waters 600 MS liquid chromatograph equipped with an
Agilent Zorbax SB C18 column (4.6 mm ×2.5 cm) held at 35 °C and
a Waters 486 tunable detector set at 289 nm. Analytical conditions
were as follows: elution gradient 0.01% trifluoroacetic acid in CH3CN
(A) and 0.01% trifluoroacetic acid (v/v) in water (B) eluting in gradient
mode starting from 10% (A) up to 60% (A) in 40 min at a flow rate of
1.0 mL/min.
HPLC-UV analysis was carried out with a Kontron 325 pump/system
controller equipped with a Merck-Hitachi UV-vis detector set to 278
nm. The analyses were performed on Phenomenex Luna RP C18 (3
µm, 4.6 mm ×1.5 cm) columns. Analytical conditions were as follows:
elution gradient CH3CN (A) and 0.01% trifluoroacetic acid (v/v) in
water (B) according to the following profile: 0-60 min, 15-100% A,
85-0% B; flow rate 1.0 mL/min.
All solvents and reagents were obtained from commercial sources
and used without further purification unless otherwise noted. Reaction
courses and product mixtures were routinely monitored by TLC and
HPLC. TLC was carried out on precoated silica gel F254 (Merck) plates
or on silica gel 60 (Merck) plates (visualizing developed chromatograms
by spraying plates with 20% CH2O/H2SO4followed by heating at 100
°C for 3 min). Column chromatography was carried out with silica gel
(Kieselgel 40, 0.040-0.063 mm; Merck) using the flash technique. For
the semisynthetic derivatives, yields are reported after chromatographic
purification and crystallization.
Dulbecco’s modified Eagle’s medium, trypsin, protease inhibitors,
naringinase (from Penicillium decumbens, 596 units/g solid; β-glu-
cosidase activity: 69 units/g solid), and all chemical reagents for cell
culture were purchased from Sigma Aldrich (Milan, Italy). Cellulase
(from Aspergillus niger) was from Fluka (Milan, Italy). Penicillin,
streptomycin, and L-glutamine were from GIBCO (Grand Island, NY);
fetal calf serum was provided by Mascia Brunelli SpA (Milan, Italy).
The COS-7 cell line was purchased from ATCC (Manassas, VA).
Superfect reagent for transient transfections was obtained from Qiagen
GmbH (Hilden, Germany). The expression plasmid pcDNA3 containing
the full-length cDNA of PDE5A1 was a kind gift of Prof. C. S. Lin
(Department of Urology, University of California, San Francisco, CA).
Human recombinant PDE6C, cloned from the human retina and
expressed in S. frugiperda insect cells using a baculovirus expression
system, was purchased from Scottish Biomedical (Glasgow, UK). [3H]-
cGMP and [3H]-cAMP were from Amersham Pharmacia Biotech
(Amersham Place, Little Chalfont, Buckinghamshire, UK). DEAE-
Sephadex A25 was from Pharmacia (Uppsala, Sweden). cGMP, cAMP,
AMP, and Crotalus adamanteus snake venom were purchased from
Sigma Aldrich. Sildenafil was provided by Sequoia Research Products
(Oxford, UK). Cinnamaldehyde and ferutinin were supplied by Indena
Spa (Milan, Italy). 8-Prenylnaringenin, 8-prenylquercetin, and 8-pre-
nylapigenin (purity >98%) were donated by Prof. Giovanni Appendino
(Universita` del Piemonte Orientale, Italy).
Plant Material. T. terrestris L. dried extract (44% furostanolic
saponins) was from Farmbio Ltd. (Sofia, Bulgaria); the ethanolic extract
of the aerial parts of E. breVicornum Maxim. (20.9% icariin) was from
Chengdu Wagott Natural Products Co. Ltd., Xian City, People’s
Republic of China. The root methanolic extract from F. hermonis Boiss.
(26.3% ferutinin) and C. cassia L. oil extract (73.4% cinnamaldehyde)
were supplied by Indena Spa (Milan, Italy). Plant material was identified
against a crude drug standard and/or authoritative literature source by
a suitable qualified person. A voucher of each plant is kept at the
botanical laboratory of the company. Extracts were quantified by HPLC,
and the chromatographic profiles are shown in Figures S3-S6
(Supporting Information).
Extraction and Isolation of Icariin (1). A dried extract of E.
sagittatum as a greenish-brown residue (4 g) was dissolved in a mixture
of CH3OH/H2O (1:1) (200 mL). The solution was stirred for 20 min
and then washed with CH2Cl2(3 ×80 mL). Methanol was evaporated
under vacuum, and the remaining aqueous solution was diluted with
water to 400 mL. The solution was extracted with EtOAc (5 ×400
mL), and the organic phase was taken to dryness. The extract was
resuspended with CH2Cl2(200 mL) and filtered under vacuum to yield
1.08 g of extract (A), from which icariin (1) was purified by
precipitation with methanol (50 mL) as a yellow powder (purity 95.3%)
(530 mg; 13% yield on the dry extract); mp 224-226 °C;
22
1H NMR
(DMSO-d6, 300 MHz, 30 °C) δ0.80 (3H, d, J)5.4 Hz, rha CH3),
1.60 (3H, s, CH3-14), 1.70 (3H, s, CH3-15), 3.05-3.20 (4H, m, H-11
and sugar protons), 3.40-3.80 (7H, m, sugar protons), 3.87 (3H, s,
OCH3), 4.00 (1H, m, sugar proton), 4.55-4.78 (3H, m, OH), 4.85-5.22
(6H, m, sugar protons and OH), 5.30 (1H, t, J)6.9 Hz, H-12), 6.60
(s, 1H, H-6), 7.15 (2H, d, J)8.4 Hz, H-3, H-5), 7.85 (2H, d, J)8.4
Table 1. IC50 Values of Icariin Derivatives and Sildenafil on
Human PDE5A1
compound PDE5A1 (IC50 µM(SD)
15.9 (1.1
20.16 (0.02
32.2 (0.09
40.36 (0.06
50.074 (0.007
645.5 (4.6
sildenafil 0.075 (0.004
Inhibition of Phosphodiesterase-5 by Icariin DeriVatiVes Journal of Natural Products,2008, Vol. 71, No. 9 1515
Hz, H-2, H-6), 12.60 (s, 1H, OH-5); ESIMS (positive-ion mode) m/z
677 [M +H]+, 699 [M +Na]+.
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
Na2SO4and evaporated under reduced pressure to afford 2(290 mg);
yield 76%; mp 208-210 °C;
23
1H NMR (DMSO-d6, 300 MHz, 30 °C)
δ0.90 (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/z537 [M +Na]+.
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 Na2SO4and 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) δ1.78 (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/z369 [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) δ0.88 (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/z581
[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) δ1.77 (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
Hz, H-2, H-6); ESIMS (positive-ion mode) m/z479 [M +Na]+;anal.
C 65.82%, H 6.22%, calcd for C25H28O8, C 65.78%, H 6.18%.
Preparation of β-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 NaHCO3and extracted with
EtOAc (3 ×50 mL). The organic phase was dried over anhydrous
Na2SO4and evaporated under vacuum to afford β-anhydroicaritin as a
yellow powder (purity 96.5%; 105 mg, 23% yield of the dry extract);
25
1H NMR, (DMSO-d6,30°C) δ1.40 (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/z369 [M +H]+.
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 Manganiello
27
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 160gfor 10 min at
room temperature. The pellet was removed, and platelet-rich plasma
(PRP) was centrifuged at 1000gfor 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 1000gfor 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 Manganiello
27
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 β-scintillation counter. Compound 5and 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 5for 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 °Cfor3h.The
MTT solution was removed, the formazan was extracted with 2-pro-
Table 2. IC50 Values of 5and Sildenafil on Human PDE6 and cAMP-PDE
compound
PDE6C
(IC50 µM(SD)
PDE6C/PDE5A1
(IC50 ratio)
cAMP-PDE
(IC50 µM(SD)
cAMP-PDE/PDE5A1
(IC50 ratio)
530.9 (2.6 418 96.3 (12.9 1301
sildenafil 0.16 (0.007 2.2 27.5 (5.3 367
1516 Journal of Natural Products,2008, Vol. 71, No. 9 Dell’Agli et al.
panol/DMSO (9:1; 500 µL/well) for 15 min at 37 °C, and aliquots of
100 µL were read on a plate reader (Bio-Rad Laboratories) at 560 nm.
Acknowledgment. The authors gratefully acknowledge Prof. C. S.
Lin for the supply of PDE5A1 cDNA, and Prof. G. Appendino for the
8-prenyl flavonoids.
Supporting Information Available: Figures showing the HPLC
traces of the extracts under study and the effects of plant extracts and
pure compounds on the inhibition of PDE5A1. This information is
available free of charge via the Internet at http://pubs.acs.org.
References and Notes
(1) Anonymous. J. Am. Med. Assoc. 1993,123,23-27.
(2) Drewes, S. E.; George, J.; Khan, F. Phytochemistry 2003,62, 1019–
25.
(3) Boyce, E. G.; Umland, E. M. Clin. Ther. 2001,23, 2–23.
(4) McKay, D. Altern. Med. ReV.2004,9, 4–16.
(5) Colman-Saizarbitoria, T.; Boutros, P.; Amesty, A.; Bahsas, A.;
Mathison, Y.; Garrido, M. D.; Israel, A. J. Ethnopharmacol. 2006,
106, 327–332.
(6) Phillips, O. A.; Mathew, K. T.; Oriowo, M. A. J. Ethnopharmacol.
2006,104, 351–5.
(7) Adaikan, P. G.; Gauthaman, K.; Prasad, R. N.; Ng, S. C. Ann. Acad.
Med. Singapore 2000,29, 22–26.
(8) Zanoli, P.; Zavatti, M.; Rivasi, M.; Baraldi, M. Physiol. BehaV.2005,
86, 69–74.
(9) Zanoli, P.; Rivasi, M.; Zavatti, M.; Brusiani, F.; Vezzalini, F.; Baraldi,
M. Int. J. Impot. Res 2005,17, 513–518.
(10) Zhang, J.; Li, H.; Fang, L.; Zhou, Z.; Ai, J. Peop. Rep. China Patent
1814119, 2006.
(11) Chen, F.; Wang, D. Peop. Rep. China Patent 1927326, 2007.
(12) Zhang, W. Peop. Rep. China Patent 1903244, 2007.
(13) Zhao, Z. A. Peop. Rep. China Patent 1903310, 2007.
(14) Tu, C.; Liao, N. Peop. Rep. China Patent 1872244, 2006.
(15) Kim, J.-H.; Mun, Y.-J.; Im, S.-J.; Han, J.-H.; Lee, H.-S.; Woo, W.-H.
Int. Immunopharmacol. 2001,1, 935–944.
(16) He, W.; Sun, H.; Yang, B.; Zhang, D.; Kabelitz, D. Arzneim.-Forsch.
1995,45, 910–913.
(17) Xin, Z. C.; Kim, E. K.; Lin, C. S.; Liu, W. J.; Tian, L.; Yuan, Y. M.;
Fu, J. Asian J. Androl. 2003,5, 15–18.
(18) Liu, W. J.; Xin, Z. C.; Xin, H.; Yuan, Y. M.; Tian, L.; Guo, Y. L.
Asian J. Androl. 2005,7, 381–388.
(19) Ning, H.; Xin, Z. C.; Lin, G.; Banie, L.; Lue, T. F.; Lin, C. S. Urology
2006,68, 1350–1354.
(20) Chiu, J. H.; Chen, K. K.; Chien, T. M.; Chen, C. C.; Wang, J. Y.;
Lui, W. Y.; Wu, C. W. Int. J. Impot. Res. 2006,18, 335–342.
(21) Shin, H. J.; Kim, H. J.; Kwak, J. H.; Chun, H. O.; Kim, J. H.; Park,
H.; Kim, D. H.; Lee, Y. S. Bioorg. Med. Chem. Lett. 2002,12, 2313–
2316.
(22) Teng, C. M.; Lin, C. H.; Ko, F. N.; Wu, T. S.; Huang, T. F. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 1994,349, 202–208.
(23) Mizuno, M.; Hanioka, S.; Suzuki, N.; Iinuma, M.; Tanaka, T.; Liu,
X.; Min, Z. Phytochemistry 1987,26, 861–863.
(24) Mizuno, M.; Iinuma, M.; Tanaka, T.; Sakakibara, N.; Fujikawa, T.;
Hanioka, S.; Ishida, Y.; Liu, X.; Murata, H. Phytochemistry 1988,
27, 3645–3647.
(25) Kang, S. S.; Kang, Y. J.; Lee, M. W. J. Nat. Prod. 1991,54, 542–
546.
(26) Lin, C. S.; Lau, A.; Tu, R.; Lue, T. F. Biochem. Biophys. Res. Commun.
2000,268, 628–635.
(27) Kincaid, R. L.; Manganiello, V. C. Methods Enzymol. 1988,159, 457–
470.
(28) Dell’Agli, M.; Galli, G. V.; Vrhovsek, U.; Mattivi, F.; Bosisio, E. J.
Agric. Food Chem. 2005,53, 1960–1965.
(29) Giovanazzi, S.; Accomazzo, M. R.; Letari, O.; Oliva, D.; Nicosia, S.
Biochem. J. 1997,325 (Part 1), 71–77.
(30) Bradford, M. M. Anal. Biochem. 1976,72, 248–254.
(31) Denizot, F.; Lang, R. J. Immunol. Methods 1986,89, 271–277.
NP800049Y
Inhibition of Phosphodiesterase-5 by Icariin DeriVatiVes Journal of Natural Products,2008, Vol. 71, No. 9 1517
... Fluorescence polarization has been widely applied as a high-throughput screen to identify inhibitors from a large library [36]. Traditionally, PDEs have been targeted alone by fluorescence assays [37] or using radioactive-labeled cyclic nucleotides such as [ 3 H], [ 14 C], or [ 32 P] cAMP/cGMP as substrates [37][38][39] and cell-based assays [40]. Alternatively, luciferase-luciferin coupled detection of ATP levels in cAMP signaling pathway has been developed to quantify the PDE activity by luminescence. ...
... Fluorescence polarization has been widely applied as a high-throughput screen to identify inhibitors from a large library [36]. Traditionally, PDEs have been targeted alone by fluorescence assays [37] or using radioactive-labeled cyclic nucleotides such as [ 3 H], [ 14 C], or [ 32 P] cAMP/cGMP as substrates [37][38][39] and cell-based assays [40]. Alternatively, luciferase-luciferin coupled detection of ATP levels in cAMP signaling pathway has been developed to quantify the PDE activity by luminescence. ...
Article
Full-text available
Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE–PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE–PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems—cAMP-specific PDE8–PKAR, cGMP-specific PDE5–PKG, and dual-specificity RegA–RD complexes—and ranked inhibitors according to their inhibition potency. Targeting PDE–PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.
... Fluorescence polarization has been widely applied as a high-throughput screen to identify inhibitors from a large library [37]. Traditionally, PDEs have been targeted alone by fluorescence assays [38], or using radioactive-labeled cyclic nucleotides such as [ 3 H], [ 14 C] or [ 32 P] cAMP/cGMP as substrates [38][39][40], cell-based assays [41]. Alternatively, luciferase-luciferin coupled detection of ATP levels in cAMP signaling pathway has been developed to quantify the PDE activity by luminescence. ...
... Fluorescence polarization has been widely applied as a high-throughput screen to identify inhibitors from a large library [37]. Traditionally, PDEs have been targeted alone by fluorescence assays [38], or using radioactive-labeled cyclic nucleotides such as [ 3 H], [ 14 C] or [ 32 P] cAMP/cGMP as substrates [38][39][40], cell-based assays [41]. Alternatively, luciferase-luciferin coupled detection of ATP levels in cAMP signaling pathway has been developed to quantify the PDE activity by luminescence. ...
Preprint
Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (Protein Kinases, PK) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PK generates an expanded active site which enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PK, and aid in signal termination. PDEs are important drug targets and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targets PDE-PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay has been adapted to identify inhibitors that block cyclic nucleotide pockets in PDE-PK complexes in one mode, and disrupt protein-protein interactions between PDEs and cyclic nucleotide activating protein kinases in a second mode. We tested this approach with three different systems: cAMP-specific PDE8-PKAR, cGMP-specific PDE5-PKG and dual-specificity RegA-RD complexes and ranked inhibitors according to their inhibition potency. Targeting PDE-PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.
... Interest in drugs derived from plant-based extraction processes has increased 8 . Several herbal remedies claim efficacy for ED including Panax ginseng C.A.Mey., Lepidium meyenii Walp., Ferula hermonis Boiss., and Ginkgo biloba L., [9][10][11][12][13] . The orchid Eulophia macrobulbon (E.C. ...
Article
Full-text available
Eulophia macrobulbon (E.C.Parish & Rchb.f.) Hook.f. contains a natural PDE5A1 inhibitor, phenanthrene, 1-(4'-hydroxybenzyl)-4,8- dimethoxyphenanthrene-2,7-diol (HDP), a potential agent for the treatment of erectile dysfunction. The aim of this study was to improve the extraction efficiency of HDP from E. macrobulbon by using a more environmentally friendly extraction method, subcritical liquid dimethyl ether extraction (sDME), instead of classical solvent extraction (CSE) and ultrasound-assisted extraction (UAE). The efficiency and quality of the extracts obtained were evaluated using the following criteria: %process yield; solvent amount; extraction time; temperature; %HDP content by LC–MS, bioactivity as inhibition of phosphodiesterase-5A1 (PDE5A1) by radio-enzymatic assay; and chemical profiles by LC-QTOF-MS. sDME provided the highest content of HDP in the extract at 4.47%, much higher than the use of ethanol (0.4–0.5%), ethyl acetate (1.2–1.7%), or dichloromethane (0.7–1.4%). The process yield for sDME (1.5–2.7%) was similar to or lower than the other solvents (0.9–17%), but as long as the process yield is not prohibitively low, the concentration is a more important measure for clinical use. The optimal conditions for sDME extraction were: Extraction time, 40 min; 200% water as co-solvent; sample-to-solvent ratio of 1:8; temperature, 35 °C. Phenanthrene aglycone and glycoside derivatives were the major constituents of the sDME extracts and lesser amounts of phenolic compounds and sugars. The inhibition of PDE5A1 by sDME (IC50 0.67 ± 0.22 µg/ml) was tenfold more potent than ethanolic extract and other extraction methods, suggesting a high probability of clinical efficacy. Thus, sDME was a more efficient, faster, solvent-saving and environmentally friendly extraction method and more selective for phenanthrene when extracted from E. macrobulbon.
... As reported in the literature, Ica exerts a direct effect as a PDE5a inhibitor [73][74][75], which was further confirmed in our experiments. Indeed, we showed a PDE5a inhibitory activity of Ica 1 μM which becomes statistically significant at 5 μM when compared to the positive control and to the known PDE5a inhibitor sildenafil. ...
Article
Full-text available
Doxorubicin (Doxo) is a widely used antineoplastic drug which often induces cardiomyopathy, leading to congestive heart failure through the intramyocardial production of reactive oxygen species (ROS). Icariin (Ica) is a flavonoid isolated from Epimedii Herba (Berberidaceae). Some reports on the pharmacological activity of Ica explained its antioxidant and cardioprotective effects. The aim of our study was to assess the protective activities of Ica against Doxo-detrimental effects on rat heart-tissue derived embryonic cardiac myoblasts (H9c2 cells) and to identify, at least in part, the molecular mechanisms involved. Our results showed that pretreatment of H9c2 cells with 1 μM and 5 μM of Ica, prior to Doxo exposure, resulted in an improvement in cell viability; a reduction in ROS generation; the prevention of mitochondrial dysfunction, and mPTP opening. Furthermore; for the first time, we identified one feasible molecular mechanism through which Ica could exerts its cardioprotective effects. Indeed, our data showed a significant reduction in Caveolin-1(Cav-1) expression levels and a specific inhibitory effect on phosphodiesterase 5 (PDE5a) activity; improving mitochondrial function compared to Doxo-treated cells. Besides; Ica significantly prevented apoptotic cell death and downregulated the main pro-autophagic marker Beclin-1 and LC3 lipidation rate, restoring physiological levels of activation of the protective autophagic process. These results suggest that Ica might have beneficial cardioprotective effects in attenuating cardiotoxicity in patients requiring anthracycline chemotherapy through the inhibition of oxidative stress and, in particular, through the modulation of Cav-1 expression levels and the involvement of PDE5a activity; thereby leading to cardiac cell survival.
... Icaritin (Scheme 1A), a prenylflavonoid derivative from the Epimedium genus that has been historically used in Traditional Chinese Medicine, is considered to exhibit various potential pharmacological and biological activities such as anti-cancer (in Phase III clinical trial [1]), anti-inflammation [2], anti-osteoporosis [3], and treatment of erectile dysfunction [4]. Due to its rigid structure, the aqueous solubility of icaritin is less than 1 μg/mL. ...
Article
Full-text available
The purpose of this study was to develop mixed polymeric micelles with high drug loading capacity to improve the oral bioavailability of icaritin with Soluplus® and Poloxamer 407 using a creative acid-base shift (ABS) method, which exhibits the advantages of exclusion of organic solvents, high drug loading and ease of scaling-up. The feasibility of the ABS method was successfully demonstrated by studies of icaritin-loaded polymeric micelles (IPMs). The prepared IPMs were characterized to have a spherical shape with a size of 72.74 ± 0.51 nm, and 13.18% drug loading content. In vitro release tests confirmed the faster release of icaritin from IPMs compared to an oil suspension. Furthermore, bioavailability of icaritin in IPMs in beagle dogs displayed a 14.9-fold increase when compared with the oil suspension. Transcellular transport studies of IPMs across Caco-2 cell monolayers confirmed that the IPMs were endocytosed in their intact forms through macropinocytosis, clathrin-, and caveolae-mediated pathways. In conclusion, the results suggested that the mixed micelles of Soluplus® and Poloxamer 407 could be a feasible drug delivery system to enhance oral bioavailability of icaritin, and the ABS method might be a promising technology for the preparation of polymeric micelles to encapsulate poorly water-soluble weakly acidic and alkaline drugs.
... As a novel anti-cancer molecule, icaritin has been shown to suppress hepatocellular carcinoma (HCC) initiation and malignant growth through the Interleukin-6/Janus-activated kinases 2/Signal transducer and activator of transcription 3 pathway [1]. In addition, icaritin has been found to ameliorate osteoporosis via estrogen-receptor-dependent and -independent pathways [2] as well as to treat erectile dysfunction by inhibiting human phosphodiesterase-5 [3]. ...
Article
Full-text available
Icaritin is a promising anti-hepatoma drug that is currently being tested in a phase-III clinical trial. A novel combination of amorphization and nanonization was used to enhance the oral bioavailability of icaritin. Amorphous icaritin nanoparticles (AINs) were prepared by a reactive precipitation technique (RPT). Fourier transform infrared spectrometry was used to investigate the mechanism underlying the formation of amorphous nanoparticles. AINs were characterized via scanning electron microscopy, X-ray powder diffraction, and differential scanning calorimetry. Our prepared AINs were also evaluated for their dissolution rates in vitro and oral bioavailability. The resultant nanosized AINs (64 nm) were amorphous and exhibited a higher dissolution rate than that derived from a previous oil-suspension formulation. Fourier transform infrared spectroscopy (FTIR) revealed that the C=O groups from the hydrophilic chain of polymers and the OH groups from icaritin formed hydrogen bonds that inhibited AIN crystallization and aggregation. Furthermore, an oral administration assay in beagle dogs showed that Cmax and AUClast of the dried AINs formulation were 3.3-fold and 4.5-fold higher than those of the oil-suspension preparation (p < 0.01), respectively. Our results demonstrate that the preparation of amorphous drug nanoparticles via our RPT may be a promising technique for improving the oral bioavailability of poorly water-soluble drugs.
Article
Background: Radical prostatectomy (RP) and radiation treatment are standard options for localized prostate cancer. Even though nerve-sparing techniques have been increasingly utilized in RP, erectile dysfunction (ED) due to neuropraxia remains a frequent complication. Erectile function recovery rates after RP remain unsatisfactory, and many men still suffer despite the availability of various therapies. Objective: This systematic review aims to summarize the current treatments for post-RP-ED, assess the underlying pathological mechanisms, and emphasize promising therapeutic strategies based on the evidence from basic research. Method: Evaluation and review of articles on the relevant topic published between 2010 and 2021, which are indexed and listed in the PubMed database. Results: Phosphodiesterase type 5 inhibitors, intracavernosal and intraurethral injections, vacuum erection devices, pelvic muscle training, and surgical procedures are utilized for penile rehabilitation. Clinical trials evaluating the efficacy of erectogenic drugs in this setting are conflicting and far from being conclusive. The use of androgen deprivation therapy in certain scenarios after RP further exacerbates the already problematic situation and emphasizes the need for effective treatment strategies. Conclusion: This article is a detailed overview focusing on the pathophysiology and mechanism of the nerve injury developed during RP and a compilation of various strategies to induce cavernous nerve regeneration to improve erectile function (EF). These strategies include stem cell therapy, gene therapy, growth factors, low-intensity extracorporeal shockwave therapy, immunophilins, and various pharmacological approaches that have induced improvements in EF in experimental models of cavernous nerve injury. Many of the mentioned strategies can improve EF following RP if transformed into clinically applicable safe, and effective techniques with reproducible outcomes.
Preprint
Full-text available
Eulophia macrobulbon (E.C.Parish & Rchb.f.) Hook.f. contains a natural PDE5A1 inhibitor, the phenanthrene, 1-(4'-hydroxybenzyl)-4,8- dimethoxyphenanthrene-2,7-diol (HDP) a potential treatment for erectile dysfunction. This investigation aimed to improve extraction efficiency of HDP from E. macrobulbon by using greener extraction methodology, subcritical fluid dimethyl ether extraction (sDME) rather than classical solvent extraction (CSE) and ultrasound-assisted extraction (UAE). The efficiency and quality of obtained extracts were evaluated by: %process yield; solvent amount; extraction period; temperature; %HDP content by LC-MS assay, bioactivity as inhibition of phosphodiesterase-5A1 (PDE5A1) by radio-enzymatic assay; and chemical profiles by LC-QTOF-MS analysis. sDME yielded the highest content of HDP in the extract at 4.47%, much higher than using ethanol (0.4-0.5%), ethyl acetate (1.2-1.7%), or dichloromethane (0.7-1.4%). Process yield for sDME (1.5-2.7%) was similar or less than that observed with other solvents (0.9-17%), but providing that process yield is not prohibitively low, concentration is a more important metric for clinical application. Optimal sDME extraction conditions were: extraction period, 40 mins; 200% water as a cosolvent; sample-to-solvent ratio of 1:8; temperature, 35°C. Phenanthrene aglycone and glycoside derivatives were major constituents in sDME extracts and lesser amounts of phenolic compounds and sugars. Inhibition of PDE5A1 by sDME (IC 50 0.67±0.22 µg/mL) was 10-fold more potent than the ethanolic extract and other extraction methods, suggesting a high likelihood of clinical efficacy. Thus, sDME was more efficient, faster, solvent-sparing, greener extraction methodology and more selective for phenanthrene when extracted from E. macrobulbon .
Article
Microbial transformation is an important tool to perform selective conversion of compounds to derivatives which are difficult to produce synthetically. In order to obtain icariside II and icaritin, the active components in Herba Epimedii in vivo, biotransformation studies using microbes as biocatalysts were carried out. Icariside II (2) and icaritin (3) were produced through biotransformation of icariin (1) using the fungi Hormoconis resinae and Mortierella ramanniana var. angulispora in 98% and 92% yields, respectively. In the subsequent transformation studies, 2 was deglycosylated to form 3 by Gliocladium deliquescens, whereas 3 was further converted to a novel compound icaritin-3-O-β-d-glucopyranoside (4) and previously known icaritin-3,7-O-β-d-diglucopyranoside (5) by Mucor hiemalis. Biological evaluation of these compounds using MTT assay exhibited potent cytotoxic activities against human cancer cell lines A549, A375P, and MCF-7, with icariin being the most active, indicating that glycosylation plays a role in the cytotoxic activity.
Chapter
Erectile dysfunction (ED) is defined as an inability to achieve or maintain penile erection sufficiently enough for sexual performance. Sexual function involves an interplay between psychological, vascular, neurological, endocrine functions; thus the various etiologies of ED can be divided into psychosocial or organic causes. Current mainstays of treatment for ED are centered around enhancing levels of nitric oxide at the nerve terminal with pharmacologic agents such as phosphodiesterase inhibitors, followed by more invasive therapies such as intracavernosal injections and penile prosthesis. The market for supplements for ED has been growing in popularity and has warranted further investigation into mechanisms of action and efficacy. This chapter discusses several supplements that have been marketed as therapies for ED, their efficacies in clinical trials, as well as their mechanism of action on the biochemical level, safety profiles, and adverse effects.
Article
Article
Two new flavonol glycosides, together with epimedoside A{2}, icariin {3}, and ikarisoside A {5}, have been isolated from the underground parts of Epimedium koreanum and characterized as 2''-O-rhamnosy ikarisoside A {1} and 2''-O-rhamnosyl icarisid II{4} by chemical and spectral data.
Article
Three new flavonol glycosides, designated sagittatosides A, B and C, were isolated from the aerial parts of Epimedium sagittatum in addition to epimedins A, B and C. Their structures were established by spectroscopic methods.
Article
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
This chapter describes the assay developed in laboratory that yields quantitative recovery of product with very low background. The reaction catalyzed by cyclic nucleotide phosphodiesterases involves the breaking of a high-energy phosphodiester bond in a metaldependent, hydrolytic process. For most enzymes, the preferred metal cofactors are divalent cations such as Mg2+ or Mn2+, although other metals, such as Co2+, Ni2+, and Zn2+, can support lower activity; Ca2+, by itself, is virtually ineffective. The pH optimum for all cyclic nucleotide phosphodiesterases appears to be between 7.0 and 8.0. The release of a substantial amount of energy accompanies the reaction, but no direct link between this event and another energy-requiring reaction has been found. Of the many methods developed for assay of phosphodiesterase, most employ a radiolabeled cyclic nucleotide substrate. In addition, a phosphodiesterase assay using a fluorescent cAMP analog is described in the chapter, with specific emphasis on its value for measurement of activity at high enzyme concentrations.
Article
A convenient way to estimate the number of viable cells growing in microtitre tray wells is to use a colorimetric assay and an automatic microplate scanning spectrophotometer. One such assay, developed by Mosmann, depends on the reduction by living cells of tetrazolium salt, MTT, to form a blue formazan product. However the original technique has several technical limitations, namely a less than optimal sensitivity, a variable background due to protein precipitation on adding an organic solvent to dissolve the blue formazan product, and a low solubility of the product. These problems have been overcome by the following modifications: avoidance of serum in the incubation medium, thus overcoming precipitation problems in the organic solvent; avoidance of phenol red in the incubation medium, thus avoiding the use of acid in the final solvent which altered the spectral properties of the formazan; elimination of the medium containing MTT after the reaction and subsequent use of pure propanol or ethanol to rapidly solubilize the formazan; use of a higher concentration of MTT; use of half-area microtitre trays to increase the spectrophotometer readings from a given amount of formazan; use of a more judicious reference wavelength in a dual wavelength spectrophotometer. With these modifications the reliability and sensitivity of the test have been increased to the point where it can in many cases replace the [3H]thymidine uptake assay to measure cell proliferation or survival in growth factor or cytotoxicity assays. Examples of its use in IL-2 assays are given.
Article
The effect of osthole, isolated from Angelica pubescens, on the contraction of guinea-pig trachea was studied. Osthole (25-100 mumol/l), theophylline (10-1000 mumol/l) and higher concentrations of nifedipine (0.1-100 mumol/l) suppressed the contraction response curves of tracheal smooth muscle caused by carbachol, prostaglandin F2 alpha (PGF2 alpha), U46619 (thromboxane A2 analogue) and leukotriene C4 (LTC4) in a concentration-dependent manner. The contraction caused by high K+ (120 mmol/l) and cumulative concentrations of CaCl2 (0.03-3 mmol/l) was also inhibited concentration-dependently by osthole (25-100 mumol/l), theophylline (10-1000 mumol/l) and lower concentrations of nifedipine (0.01-0.1 mumol/l). The relaxant actions of osthole were not affected by propranolol (1 mumol/l), glibenclamide (10 mumol/l) or removal of tracheal epithelium. Osthole (100 mumol/l) was still effective in causing tracheal relaxation in the presence of nifedipine (1 mumol/l). In Ca(2+)-free- and EGTA (0.2 mmol/l)-containing medium, the relaxing effect of osthole was more potent than in normal Krebs solution. Osthole (25 and 50 mumol/l) caused 2.9 and 6.5, or 3.0 and 5.6 fold, respectively, increase in potency of forskolin or sodium nitroprusside in causing tracheal relaxation but did not affect that by cromakalim. Osthole (50 mumol/l) enhanced the increase in tissue cAMP and cGMP levels induced by forskolin and sodium nitroprusside, respectively, and in higher concentrations (100 and 250 mumol/l), itself increased markedly tissue cAMP and cGMP contents. Osthole (10-250 mol/l) inhibited the activity of cAMP and cGMP phosphodiesterases in a concentration-dependent manner. It is concluded that osthole exerts a non-specific relaxant effect on the trachealis by inhibiting the cAMP and cGMP phosphodiesterases.