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Potent Inhibition of Human Phosphodiesterase-5 by Icariin Derivatives


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.
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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.
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.
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.
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
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
and a pro-erectile effect on the rabbit corpus
F. hermonis has been studied for its effects on sexual
behavior in male and female rats;
C. cassia and “Epimedii Herba”
are components of Chinese herbal products patented for the
treatment of sexual dysfunction.
“Epimedii herba” is the
common name for the dried aerial parts of E. breVicornum,E.
sagittatum Maxim., or E. koreanum Nakai, collected in the
Among the extracts, only “Epimedii Herba” was active
against PDE5A1, for which the presence of icariin (1), the major
pharmacologically active constituent,
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,
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:
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
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.
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;
(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;
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;
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);
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.
PDE5A1 and PDE6C Enzyme Assays. PDE5A1 activity was
determined according to the method of Kincaid and Manganiello
minor modifications.
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,
and cell lysate
was stored at -80 °C. Total protein concentration was measured
according to Bradford.
cAMP-PDE activity was determined according to the method of
Kincaid and Manganiello
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.
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
(IC50 µM(SD)
(IC50 ratio)
(IC50 µM(SD)
(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
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Inhibition of Phosphodiesterase-5 by Icariin DeriVatiVes Journal of Natural Products,2008, Vol. 71, No. 9 1517
... Icariin has been tested in vitro to exhibit PDE5 inhibition leading many to believe that this is its mechanism of action and supporting the idea that icariin may be an herbal substitute for sildenafil. [7] How a PDE5 inhibitor works to create an erection was nicely summarized by David Rotella: -PDE5 is the primary cGMP-hydrolysing activity in human corpus-cavernosum tissue. Erection is largely a haemodynamic event, which is regulated by vascular tone and blood-flow balance in the penis. ...
... [8] The Icariin inhibition of PDE5 activity is much weaker than for sildenafil as shown by in vitro studies wherein PDE5 inhibition was 1/80th of that found with sildenafil. [7] Based on widely exaggerated stories and urban myths that have arisen around Icariin within the last few years, Icariin has become a popular herbal supplement, available online at many herbal suppliers. It appears that many re-sellers are buying Icariin powder from Chinese sources and repackaging the powder for sale as is, or after loading into capsules. ...
... -Potent Inhibition of Human Phosphodiesterase-5 by Icariin Derivatives.‖ [7] The report states: -Compound 1 [icariin] was 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.‖ While the paper can be commended for the head to head comparison of icariin analogs to sildenafil in an enzyme inhibition assay, the bottom line is that the data is based solely on inhibition rates on a phosphodiesterase-5 (PDE-5) enzyme inhibition assay, in vitro, far away from actual in vivo activity arising from oral ingestion of an herbal extract. ...
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Icariin, considered to be the active compound in the herb Epimedium (also known as Horny Goat Weed) has been shown to be possibly erectogenic in a variety of animal studies, leading to its popularity as an herbal supplement for ED even though there are essentially no human studies supporting its effectiveness.Icariin is highly insoluble, and many studies have additionally reported that Icariin is not absorbed in the gut due to a very low bioavailability. The purpose of this study was to solubilize Icariin in the formulation of a transdermal cream which would deliver the compound directly to the blood stream bypassing poor intestinal absorption; and by informal testing of the transdermal cream on volunteer subjects, to determine if the erectogenic effects of icariin could be improved. This study demonstrates that icariin, delivered via the transdermal route, failed to demonstrate any efficacy of icariin for ED, conforming to another recent study reporting no effect upon oral adminstration.
... It appears it can improve the function of organs including bones and the heart [73][74][75][76][77][78][79][80][81][82][83][84][85][86]. It is crystalline and stable at a low temperature (−20 • C) for approximately two years [87][88][89][90]. The chemical structure is depicted in Figure 2. The stock solutions of ICRN were usually made in DMF or DMSO (20 mg/mL). ...
... Due to low solubility and stability considerations in water, freshly prepared solutions in the buffer can be used. Usually, a stock solution in DMSO was diluted to increase the solubility in a buffer (the ratio of DMSO to PBS can be 1:10 at pH 7.2) a [87,88,90]. ...
... the heart [73][74][75][76][77][78][79][80][81][82][83][84][85][86]. It is crystalline and stable at a low temperature (−20 °C) for approx mately two years [87][88][89][90]. The chemical structure is depicted in Figure 2. The stock solu tions of ICRN were usually made in DMF or DMSO (20 mg/mL). ...
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Among scaffolds used in tissue engineering, natural biomaterials such as plant-based materials show a crucial role in cellular function due to their biocompatibility and chemical indicators. Because of environmentally friendly behavior and safety, green methods are so important in designing scaffolds. A key bioactive flavonoid of the Epimedium plant, Icariin (ICRN), has a broad range of applications in improving scaffolds as a constant and non-immunogenic material, and in stimulating the cell growth, differentiation of chondrocytes as well as differentiation of embryonic stem cells towards cardiomyocytes. Moreover, fusion of ICRN into the hydrogel scaffolds or chemical crosslinking can enhance the secretion of the collagen matrix and proteoglycan in bone and cartilage tissue engineering. To scrutinize, in various types of cancer cells, ICRN plays a decisive role through increasing cytochrome c secretion, Bax/Bcl2 ratio, poly (ADP-ribose) polymerase as well as caspase stimulations. Surprisingly, ICRN can induce apoptosis, reduce viability and inhibit proliferation of cancer cells, and repress tumorigenesis as well as metastasis. Moreover, cancer cells no longer grow by halting the cell cycle at two checkpoints, G0/G1 and G2/M, through the inhibition of NF-κB by ICRN. Besides, improving nephrotoxicity occurring due to cisplatin and inhibiting multidrug resistance are the other applications of this biomaterial.
... The characteristic chemical components of PFES are8-isopentenyl flavonoids, of which 6 compounds are high in content, including icariin, baohuoside I, epimedin A, B, C and 2-O"-rhamnosylicaridide II [10]. However, until now, it was only reported that icariin exhibited an IC 50 value of 5.9 μM against PDE5A1 in vitro [11], the 6 components have not been separated from PFES and the PDE5 inhibitory potencies have not been scrutinized. Moreover, there is few SAR study on flavonoids containing bulky substituents like isopentenyl at C8position as PDE5 inhibitors. ...
... In this study, for the first time, 6 main 8-isopentenyl flavonoids, Icariin: (1), 2-O"-rhamnosylicaridide II (2), Baohuoside I (3), Epimedin A, B, C (4-6) were separated from the PFES and identified by 1 H NMR, 13 C NMR and MS, and these compounds were detected PDE5A1 inhibitory activity with the novel HPLC assay. We also adapted a series of PDE5 inhibitors from different reports [11,13] to firstly establish CoMFA and CoMSIA models, aiming at finding the relationship between the biological activity and the key structural factors of 8-isopentenyl flavonoids from the models. Furthermore, The SAR results were verified by the cellular effects of the cGMP level and cytoplasmic Ca 2+ in CSMCs, and the mechanism on PKG/Ca 2+ signaling pathway was also studied. ...
... The potencies of compounds 1-6 and 16 were obtained by our in vitro activity assay combined with HPLC. The remains were adapted from two literature [11,13]. Although these compounds were taken from different sources, the compound 16 [11,13] showed similar values of inhibitory concentration (IC 50 ) with ours, 0.075, 0.010 and 0.014 (our result) μM, respectively. ...
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Background As known, inhibition of phosphodiesterase 5 (PDE5) has the therapeutic effect on male erectile dysfunction (ED), and the processed folium of Epimedium sagittatum Maxim. (PFES) characterized by 8-isopentenyl flavonoids is a famous herb for treating ED. However, the main flavonoids inhibitory activities, structure–activity relationship (SAR) and signaling pathway have been not systematically studied so that its pharmacodynamic mechanism is unclear. Methods We aimed to initially reveal the PFES efficacy mechanism for treating ED. For the first time, 6 main 8-isopentenyl flavonoids (1–6) from PFES were isolated and identified. Then based on HPLC detection, we proposed a novel method to screen inhibitors among them. We further analyze the three-dimensional quantitative structure–activity relationship (3D-QSAR) for those inhibitors. Results The results were verified by cellular effects of the screened flavonoids. Among 6 compounds, Icariin: (1), 2-Oʹʹrhamnosylicaridide II (2) and Baohuoside I (3) were identified with significant activities (IC 50 = 8.275, 3.233, 5.473 μM). Then 3D-QSAR studies showed that the replacement of C8 with bulky steric groups as isopentenyl, C3 with positive charge groups and C4' with a hydrogen bond acceptor substituent could increase inhibitory effects. In contrast, the substitution of C7 with bulky steric groups or hydrophilic groups tended to decrease the efficacies. And compounds 1, 2, 3 could increase cGMP level and decrease cytoplasmic Ca ²⁺ of rat corpus cavernosum smooth muscle cells (CCSMCs)by activating PKG. Conclusion 8-isopentenyl flavonoids could be the main pharmacodynamic substances of PFES in the treatment for ED, and some had significant PDE5A1 inhibitory activities so as to activate cGMP/PKG/Ca ²⁺ signaling pathway in CCSMCs, that was related to the substituents at the key sites such as C8, C3, C4ʹ and C7 in the characteristic compounds.
... The herb contains icariin, which enhances aromatase expression, thereby increasing estrogen biosynthesis [46]. An inhibitory effect of Epimedium on PDE-5 was also found, leading to an increase in the concentration of NO in the cavernous bodies [47]. However, the effects of Epimedium are known only from in vitro studies. ...
Since time immemorial, people have been trying to influence different aspects of their sexuality. They seek ways to increase sexual activity or sexual desire in themselves and their partners. In addition to resorting to all sorts of mystical rituals, people were looking for various effects from the use of natural raw materials. Some plant, animal, or fungal products have been shown to affect libido, sexual arousal, erectile function, orgasm or erogenous zone sensitivity. Such substances have been called "aphrodisiacs" in honor of the ancient Greek goddess of love - Aphrodite. Most often, aphrodisiacs were taken orally, but some were smoked while others applied to the genitals. In modern clinical practice, phosphodiesterase type 5 inhibitors are used. These are substances, which enhance erection and prevent detumescence in the presence of sexual arousal. Another group of drugs is selective serotonin reuptake inhibitors, which reduce sexual arousal and sexual afferent from the genitals, thus preventing the premature onset of orgasm in men and prolonging sexual intercourse. However, drugs from other pharmacological groups have not found widespread clinical application. Another issue now is a trend among people taking drugs of natural origin, therefore, all kinds of traditional aphrodisiacs are actively used to the present day. Very little is known about almost all of them. Clinical trials are in most cases limited to a few, often not randomized, studies. In this regard, it is very difficult to evaluate the adequate therapeutic and toxic doses of remedies. The situation is complicated by the fact that those few clinical studies were based on questionnaires, that is, the indicators taken into statistical calculations were extremely subjective. Moreover, it was uncertain whether all patients could adequately assess their dynamics in terms of parameters such as sexual satisfaction, or clearly distinguish between libido and sexual arousal. Since the majority of the studies were not blinded, a psychogenic influence on the results of the investigations could not be eliminated, which in the sexual area may be huge. It is worth emphasizing the toxicity of many traditional aphrodisiacs. Of course, there is a serious deficit in the spectrum of pharmacotherapy for sexual disorders. Perhaps further large, randomized, placebo-controlled trials would add some of the traditional aphrodisiacs or their modifications to the arsenal of the clinical specialist.
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Progressive up-regulation of β-catenin signaling is very common in the transformation of colorectal epithelium to colorectal cancer (CRC). Practical measures for opposing such signaling hence have potential for preventing or slowing such transformation. cAMP/PKA activity in colon epithelium, as stimulated by COX-2-generated prostaglandins and β2-adrenergic signaling, boosts β-catenin activity, whereas cGMP/PKG signaling has the opposite effect. Bacterial generation of short-chain fatty acids (as supported by unrefined high-carbohydrate diets, berberine, and probiotics), dietary calcium, daily aspirin, antioxidants opposing cox-2 induction, and nicotine avoidance, can suppress cAMP production in colonic epithelium, whereas cGMP can be boosted via linaclotides, PDE5 inhibitors such as sildenafil or icariin, and likely high-dose biotin. Selective activation of estrogen receptor-β by soy isoflavones, support of adequate vitamin D receptor activity with UV exposure or supplemental vitamin D, and inhibition of CK2 activity with flavanols such as quercetin, can also oppose β-catenin signaling in colorectal epithelium. Secondary bile acids, the colonic production of which can be diminished by low-fat diets and berberine, can up-regulate β-catenin activity by down-regulating farnesoid X receptor expression. Stimulation of PI3K/Akt via insulin, IGF-I, TLR4, and EGFR receptors boosts β-catenin levels via inhibition of glycogen synthase-3β; plant-based diets can down-regulate insulin and IGF-I levels, exercise training and leanness can keep insulin low, anthocyanins and their key metabolite ferulic acid have potential for opposing TLR4 signaling, and silibinin is a direct antagonist for EGFR. Partially hydrolyzed phytate can oppose growth factor-mediated down-regulation of β-catenin by inhibiting Akt activation. Multifactorial strategies for safely opposing β-catenin signaling can be complemented with measures that diminish colonic mutagenesis and DNA hypomethylation - such as avoidance of heme-rich meat and charred or processed meats, consumption of phase II-inductive foods and nutraceuticals (e.g., Crucifera), and assurance of adequate folate status.
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Background: Kidney-Yang deficiency syndrome (KDS) is a group of diseases related to hypothalamic-pituitary-adrenal (HPA) axis and sexual dysfunction. The folium of Epimedium brevicornu Maxim. (FEB) includes raw and prepared slices, named RFEB and PFEB, respectively. PFEB is traditionally believed to be good for tonifying kidney-Yang and improving sexual dysfunction. However, there are few studies comparing the pharmacological effects of RFEB and PFEB, and their underlying mechanisms. In this study, we aimed to compare the effects and safety of RFEB and PFEB on the HPA axis and sexual function. Additionally, the mechanisms of their roles in relation to the neuroendocrine-immune (NEI) network in the KDS model mice were explored. Methods: Male adult C57BL/6 mice were treated with corticosterone to establish a KDS mouse model, and RFEB and PFEB were administered intragastrically. Corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), testosterone levels and oxidative damage indexes were measured. The mRNA and protein levels of CRH and ACTH in hypothalamus and pituitary, endothelial nitric oxide synthase (eNOS) and phosphodiesterase 5 (PDE5) in corpus cavernosum were examined. TNFα, IL-6, NF-κB, eNOS and PDE5 were investigated in mouse corpus cavernosum. Results: Our results showed that PFEB was more effective than RFEB in increasing corticosterone-suppressed ACTH levels, enhancing CRH levels and cAMP/cGMP ratio, and reducing oxidative damage. In vivo , PFEB significantly increased eNOS and inhibited PDE5 expression in corpus cavernosum. PFEB showed stronger protective effect on normal spleen lymphocytes from apoptosis both in vitro and in vivo . Additionally, it noticeably inhibited the levels of inflammatory cytokines in corpus cavernosum. Both RFEB and PFEB were safe and did not cause any clinical signs of toxicity in mice at the dosage of 20 times dosages of that in the Chinese Pharmacopeia. Conclusion: We demonstrated that PFEB was better than RFEB at tonifying the kidney-Yang by comparing their effects on improving the NEI network, which includes the HPA axis, immune system and corpus cavernosum. This study revealed that PFEB could significantly improve the sexual function of KDS mice by regulating the HPA axis and activating the immune system through the NEI network.
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Testicular failure or male hypogonadism is an uncommon disorder in which the body does not synthesized enough of the hormone testosterone, which is important for male growth and development throughout puberty, or enough sperm, or both. This paper will review the symptoms, cause, diagnosis and possible management of the condition by modern science and herbs.
Introduction: Erectile dysfunction (ED) is one of the most common urologic problems in men worldwide, with an approximately high incidence rate, significantly affecting patients' quality of life and their sexual partners. Objectives: Due to the association of this disorder with essential diseases such as cardiovascular disease and diabetes, its prevention and treatment are vital for overall human physiologic and psychological health. Along with reviewing the history of treatment and current methods, we seek new approaches to curb this issue in the future. Methods: In this review, investigations were based on the focus of each section's content or conducted on an ad hoc basis. Searches were performed in Scopus and PubMed. Results: In recent years, many treatments for ED have been reported besides oral administration of phosphodiesterase 5 inhibitors such as sildenafil and tadalafil (approved by the Food and Drug Administration). Common oral medications, intracavernous injections, herbal therapies (eg, herbal phosphodiesterase 5 inhibitors), and topical/transdermal medications are routine ED treatment approaches. Moreover, some novel medications are innovative candidates for completing ED's treatment protocols: stem cell injection, low-intensity extracorporeal shock wave therapy, platelet-rich plasma injection, gene therapy, amniotic fluid matrices, rho-kinase inhibitors, melanocortin receptor antagonists, maxi-K channel activators (ie, large-conductance calcium-activated potassium channels), guanylate cyclase activators, and nitric oxide donors. Conclusion: Due to the importance of this complicated problem in men's society, a faster course of treatment trends toward new methods is needed to increase efficiency. Combining the mentioned treatments and attentively examining their efficacy through programmed clinical trials can be a big step toward solving this global problem.
An effort to identify novel active substance of the prepared folium of Epimedium sagittatum Maxim. (PFES) that was an important herb for male erectile dysfunction (ED) was taken. At present, phosphodiesterase-5A (PDE5A) is the most important target of new drugs for the treatment of ED. Therefore, the inhibition ingredients in PFES were systematically screened for the first time in this study. Eleven compounds, including eight new flavonoids and three prenylhydroquinones were isolated: sagittatosides DN (1-11), and their structures were elucidated by spectra and chemical analyses. Among them, a novel prenylflavonoid with oxyethyl group (1) was obtained and three prenylhydroquinones (9-11) were firstly isolated from Epimedium. All compounds were analyzed for the inhibition against PDE5A by molecular docking, and they all showed significant binding affinity as same as sildenafil. Their inhibitory activities were verified, and the results showed compound 6 had significant inhibition against PDE5A1. The isolation of new flavonoids and prenylhydroquinones with inhibitory activities of PDE5A from PFES implied that this herb might be a good source for the treatment of ED agents finding.
Prenylated flavonoids are the chemotaxonomic markers of genus Epimedium. Icariin, epimedin A, B and C are the main bioactive principles found in Epimedium spp. The chemical and pharmacological studies of a good number of prenylated flavonoids isolated from Epimedium species are reported in literature but their biosynthetic pathways have been explored to a limited extent only and remained ambiguous for a long time. In this review, an in‐depth and extensive coverage of Epimedium prenylated flavonoids is reported from 2012 to 2022 using SciFinder database for literature retreival. The review covers occurrence, synthesis, biosynthesis and quantification of prenylated flavonoids of genus Epimedium. Prenylated flavonoids are the chemotaxonomic markers of genus Epimedium. Icariin, epimedin A, B and C are the main bioactive principles found in Epimedium. The review covers occurrence, synthesis, biosynthesis and quantification of prenylated flavonoids of genus Epimedium.
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.
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.
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.
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.
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.
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.