Structure-activity relationships for naturally occurring coumarins as β-secretase inhibitor

Article abstract of DOI:10.1016/j.bmc.2011.12.002

The present study was demonstrated to evaluate the effects of naturally occurring coumarins (NOCs) including simple coumarins, furanocoumarins, and pyranocoumarins on the inhibition of β-secretase (BACE1) activity. Of 41 NOCs examined, some furanocoumarins inhibited BACE1 activity, but simple coumarins and pyranocoumarins did not affect. The most potent inhibitor was 5-geranyloxy-8-methoxypsoralen (31), which has an IC₅₀ value of 9.9 μM. Other furanocoumarin derivatives, for example, 8-geranyloxy-5- methoxypsoralen (35), 8-geranyloxypsoralen (24), and bergamottin (18) inhibited BACE1 activity, with the IC₅₀ values <25.0 μM. Analyses of the inhibition mechanism by Dixon plots and Cornish-Bowden plots showed that compounds 18, 31 and 35 were mixed-type inhibitor. The kinetics of inhibition of BACE1 by coumarins 24 was non-competitive inhibitors.

Full text of DOI:10.1016/j.bmc.2011.12.002

Bioorganic & Medicinal Chemistry 20 (2012) 784–788
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships for naturally occurring coumarins
as b-secretase inhibitor
⇑
Shinsuke Marumoto, Mitsuo Miyazawa
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Kowakae, Higashiosaka-shi, Osaka 577-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study was demonstrated to evaluate the effects of naturally occurring coumarins (NOCs)
including simple coumarins, furanocoumarins, and pyranocoumarins on the inhibition of b-secretase
(BACE1) activity. Of 41 NOCs examined, some furanocoumarins inhibited BACE1 activity, but simple
coumarins and pyranocoumarins did not affect. The most potent inhibitor was 5-geranyloxy-8-meth-
Received 12 October 2011
Revised 30 November 2011
Accepted 1 December 2011
Available online 8 December 2011
oxypsoralen (31), which has an IC50 value of 9.9
8-geranyloxy-5-methoxypsoralen (35), 8-geranyloxypsoralen (24), and bergamottin (18) inhibited
BACE1 activity, with the IC50 values <25.0 M. Analyses of the inhibition mechanism by Dixon plots
lM. Other furanocoumarin derivatives, for example,
Keywords:
Coumarin
Furanocoumarin
Pyranocoumarin
b-Secretase
l
and Cornish-Bowden plots showed that compounds 18, 31 and 35 were mixed-type inhibitor. The kinet-
ics of inhibition of BACE1 by coumarins 24 was non-competitive inhibitors.
Ó 2012 Published by Elsevier Ltd.
Structure–activity relationships
1. Introduction
(15), oxypeucedanin (16), imperatorin (21), (+)-byakangelicol
(33), and (+)-byakangelicine (34) from Angelica dahurica.4
Coumarins are widely distributed in nature and are found in all
parts of plants.1 These compounds are especially common in
grasses, orchids, citrus fruits, and legumes.1 Being so abundant in
nature, coumarins make up an important part of the human diet.
Based on chemical structure, they can be broadly classified as (a)
simple coumarins (e.g., coumarin, 1), (b) furanocoumarins of the lin-
ear (e.g., psoralen, 12) or angular (e.g., angelicin, 38) type, and (c)
pyranocoumarins of the linear (e.g., xanthyletin, 40) or angular
(e.g., seselin, 45) type.1 Simple coumarins are very widely distrib-
uted in the plant kingdom.1 Interestingly, citrus oils, in particular,
contain abundant amounts of both simple as well as furanocouma-
rins.2 Human are also exposed to furanocoumarins (e.g., bergapten,
14 and xanthotoxin, 20) in umbelliferous vegetables such as pars-
nips, celery, and parsley in substantial amounts.3 For example, pars-
nip root reportedly contains as much as 40 mg/kg of certain linear
furanocoumarins such as psoralen (12), bergapten (14), and xantho-
toxin (20), which are destroyed by normal cooking procedures (boil-
ing or microwave).3 In addition, in certain countries (e.g., China,
India, and Mexico) and in certain geographical areas within the Uni-
ted States (e.g., the Southwest) fresh coriander leaves (also known
as cilantro or Chinese parsley) are used extensively. The cilantro
leaves are used in soups, chutneys, and sauces and flavoring curries
and even wine. Recently, we reported the b-secretase (BACE1)
inhibitory activities of several furanocoumarins isoimperatorin
Alzheimer’s disease (AD) has become an increasingly severe
medical and social problem, due to the rapid growth of aging people
populations in industrialized countries, and even in some develop-
ing countries. The b-secretase (BACE1) has been recognized as a
valuable target for the treatment of AD. The BACE1 inhibitors have
potential to be developed as anti-dementia drugs. Nevertheless, all
drugs considered for AD must be able to cross the plasma mem-
brane, and most importantly the blood–brain barrier.5 Enzyme
inhibitors with therapeutic potential should preferably be smaller
than 700 Da, making large peptide-based inhibitors not viable as
drug candidates.6 Thus, the secondary metabolites of plants, which
have relatively low-molecular weights and high lipophilicity, may
offer possibilities for drugs against AD.6 There are only very few re-
ports on natural products-based BACE inhibitors.4,6–9
In the present study, a series of 41 kinds of NOCs were investi-
gated for their effects on inhibition of BACE1 activity, and we found
that some NOCs are strongly inhibitors of BACE1.
2. Results and discussion
2.1. Inhibition of BACE1 activity by 41 NOCs
A total of 41 naturally occurring coumarins (NOCs) from several
structural subclasses were investigated, including simple couma-
rins (1–11), linear-type furanocoumarins (12–14, 17–20, 22–32,
35–38), angular-type furanocoumarins (38 and 39), linear-type
pyranocoumarins (40–44), and angular-type pyranocoumarins
(45 and 46) (Fig. 1). Inhibition of BACE1 activity of five
⇑
Corresponding author. Tel.: +81 6 6721 2332; fax: +81 6 6727 2024.
E-mail address: miyazawa@apch.kindai.ac.jp (M. Miyazawa).
0968-0896/$ - see front matter Ó 2012 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2011.12.002

S. Marumoto, M. Miyazawa / Bioorg. Med. Chem. 20 (2012) 784–788
785
furanocoumarins (15, 16, 21, 33 and 34) was previously reported.4
From the regression curves of coumarin concentration against per-
centage of control activity, IC50 values of each coumarin were esti-
mated and presented in Table 1. Among the 41 compounds tested,
member from furanocoumarin group were most active, with 5-ger-
anyloxy-8-methoxypsoralen (31) being the most potent inhibitor
further study were bergamottin (18), 5-geranyloxy-8-methoxyp-
soralen (31), 8-geranyloxypsoralen (24), and 8-geranyloxy-5-
methoxypsoralen (35). The inhibition constants were determined
from Dixon plots,10 in which the inverse of enzyme activity is rep-
resented as a function of inhibitor concentration for at least two
different enzyme–substrate concentrations. For a more accurate
determination of this constant, Dixon plots were constructed using
at an IC50 value of 9.9
oxypsoralen (24), and 8-geranyloxy-5-methosypsoralen (35)
exhibited IC50s of 32.2, 20.4, and 11.1 M, respectively. Several
other compounds inhibited BACE1 to lesser extent, these included
phellopterin (32) from furanocoumarin group (IC50 of 143 M) as
well as kinidilin (30) with IC50 value of 344 M. All other couma-
rins, however, showed relatively less potent inhibitory effect of
BACE1 activity with IC50 values of more than 500 M.
lM. Similarly, bergamottin (18), 8-geranyl-
two different substrate concentrations 1.5 and 0.75 lM. Following
l
this methods, the extrapolated straight lines intersect in a single
point from which the inhibition constants were determined to be
Ki values of 18, 24, 31, and 35 with 34.8, 61.6, 102.7, and
l
l
55.0 lM, respectively. The Dixon plots is very useful determining
the inhibition parameters of coumarins to the activity of BACE1
activity but is not sufficient to fully elucidate the type of inhibition,
because the Dixon plots for mixed and competitive inhibition is
similar. The complementary plot, the Cornish-Bowden plots11 in
which the ratio of substrate concentration and enzyme activity is
plotted versus inhibitor concentration, allows the distinction be-
tween these two types of inhibition. This plots, by itself, is also
not sufficient to fully determine the type of inhibition because it
l
2.2. Inhibition mechanism of BACE1 by coumarins
Further investigations were undertaken to explore the inhibi-
tion mechanism of several coumarins compounds exhibiting high
inhibitory potency on BACE1 activity. Compounds for chosen for
Coumarins
HO
HO
MeO
MeO
O
O
O
O
HO
O
O
MeO
O
O
O
O
O
O
O
O
O
O
HO
O
O
coumarin (1)
umbeliferone (2)
methylumbeliferone (3)
O-prenylumbeliferone (4)
aurapten (5)
esculetol (6)
esculetin (7)
demethylsuberosin (8)
MeO
O
O
HO
O
O
MeO
O
O
suberosin (9)
osthenol (10)
osthol (11)
Furanocoumarins
O
O
OH
OH
OH
OMe
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
OMe
psoralen (12)
bergaptol (13)
bergapten (14)
isoimperatorin (15)
oxypeucedanin (16)
oxypeucedanin hydrate (17)
bergamottin (18)
xanthotoxol (19)
xanthotoxin (20)
OH
OMe
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
OH
OH
OH
O
OH
5,8-dihydroxypsoralen (25)
8-hydroxy-5-methoxypsoralen (26) 8-hydroxy-5-prenyloxypsoralen (27)
imperatorin (21)
heraclenin (22)
heraclenol (23)
O
O
O
OH
8-hydroxy-5-geranyloxypsoralen (28)
8-geranyloxypsoralen (24)
OMe
OMe
OMe
OMe
O
O
O
O
O
O
O
OH
O
O
O
OMe
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
marmesin (37)
O
O
O
O
O
O
O
OH
OH
O
O
O
OMe
OMe
OMe
isopimpinellin (29)
kinidilin (30)
5-geranyloxy-8-methoxypsoralen (31) phellopterin (32)
byakangelicol (33)
byakangelicine (34) 8-geranyl-5-methoxypsoralen (35)
cnidicin (36)
O
O
O
O
O
O
OH
columnbianetin (39)
angelicin (38)
Pyranocoumarins
O
O
O
O
HO
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
lomatin (46)
xanthyletin (40)
dihydroxanthyletin (41)
decursinol (42)
decursinol tiglate (43)
decursin (44)
seselin (45)
Figure 1. Structure of naturally occurring coumarins 1–46.

786
S. Marumoto, M. Miyazawa / Bioorg. Med. Chem. 20 (2012) 784–788
Table 1
Inhibition of BACE1 activity by NOCs
NOCs
IC50
(
lM)
NOCs
IC50 (lM)
Simple Coumarins
Coumarin (1)
Umbelliferone (2)
7-Methoxycoumarin (3)
7-Prenyloxycoumarin (4)
Aurapten (5)
>500 (8.1%)a
>500 (16.0%)
>500 (1.1%)
>500 (33.1%)
345.1 8.9
Esculetin (7)
Demethylsuberosin (8)
Suberosin (9)
Osthenol (10)
Osthol (11)
>500 (6.2%)
412.9 11.9
>500 (49.5%)
>500 (39.4%)
>500 (39.4%)
Esculetol (6)
>500 (9.8%)
Furanocoumarins
Psoralen (12)
Bergaptol (13)
Bergapten (14)
Isoimperatorin (15)
Oxypeucedanin (16)
Oxypeucedanin hydrate (17)
Bergamottin (18)
Xanthotoxol (19)
Xanthotoxin (20)
Imperatorin (21)
>500 (19.3%)
>500 (11.1%)
>500 (ꢀ6.0%)
244.2 8.2b
359.2 96b
>500 (30.7%)
32.2 40
>500 (32.8%)
>500 (ꢀ4.9%)
91.8 8.2b
8-Hydroxy-5-methoxypsoralen (26)
8-Hydroxy-5-prenyloxypsoralen (27)
5-Geranytoxy-8-hydroxypsoralen (28)
Isopimpnellin (29)
knidillin (30)
5-Geranytoxy-8-methoxypsoralen (31)
Phellopterin (32)
Byakangelicol (33)
Byakangelicine (34)
8-Geranyloxy-5-methoxypsoralen (35)
Cnidicin(36)
Marmesin (37)
>500 (8.2%)
>500 (42.1%)
>500 (38.6%)
>500 (12.8%)
3442 49
9.9 1.3
1431 1.5
104.9 2.4b
219.7 76b
11.1 1.1
Heraclenin (22)
Heraclenol (23)
8-Geranyloxypsoralen (24)
5,8-Dihydroxypsoralen (25)
443.6 20.1
>500 (17.2%)
20.4 1.0
>500 (1.4%)
>500 (2.9%)
>500 (9.5%)
>500 (20.8%)
Angelicin (38)
Columbianetin (39)
>500 (33.6%)
Pyranocoumarins
Xanthyletin (24)
Dihydroxanthyletin (41)
Decursinol (42)
>500 (37.2%)
>500 (13.5%)
>500 (7.6%)
>500 (18.8%)
Decursin (44)
Seselin (45)
Lomatin (46)
Referencec
>500 (26.8%)
>500 (14.0%)
>500 (12.8%)
0.2 0.01
Decursinol tiglate (43)
a
Inhibition (%) at 500
Ref. 4
lM.
b
c
Lys-Thr-Glu-glu-Ile-Ser-Glu-Val-Asn-(statine)-Val-Ala-Glu-Phe-OH was used as the positive control.
has the disadvantage of not always distinguishing between mixed
and uncompetitive inhibition. However, by analyzing the two plots
together, all types of inhibition can be characterized. Figure 2
showed the Dixon plots and the Cornish-Bowden plots for fur-
anocoumarins 18, 24, 31, and 35 studied in this work using two dif-
aurapten (5), with a geranyloxy group at the C7 position, exhib-
ited a moderate inhibitory effect, whereas umbelliferone (2), 7-
methoxycoumarin (3), and 7-prenyloxycoumarin (4) showed low-
er inhibitory effects. On the basis of this evidence, a geranyloxy
group at the C7 position of the simple coumarin skeleton seems
to be very important for activity. In the series of furanocoumarins
in Table 1, psoralen (12), having no additional substitutions on
the benzene ring, was found to have significantly lower activity.
The activities of isoimperatorin (15) and imperatorin (21), with
a prenyloxy group at the C5 or C8 position, showed increased
inhibitory effects, whereas in the latter, an additional methoxy
group at the C8 or C5 position seems to have no significant influ-
ence. This evidence was comparing the structure and activity of
15, 21, knidilin (30), and phellopterin (32). In addition, bergapten
(14) and xanthotoxin (20), with a methoxy group at the C5 or C8
position, showed no activity. Isoimpinellin (29), with the two
methoxy groups at both the C5 and C8 positions, had no inhibitory
activity to support of this inference. However, bergamottin (18)
and 8-geranyloxypsoralen (24), with a geranyloxy group at the
C5 or C8 position, were found to be significantly more active than
were prenyloxy-groups (15 and 21), and an additional methoxy
group at the C8 or C5 position seems to have significant influence.
Based on results, mentioned above, functional-substitutions on
the benzene ring selectively enhance or decrease inhibition of
BACE1 activity, and a geranyloxy group seems to be very impor-
tant for activity.
ferent concentrations of substrate, 1.5 and 0.75 lM. For the
compounds 18, 31, and 35 studied here, the plots showed mixed
inhibition. Because Ki > Ki0 or Ki < Ki0 (where Ki, the inhibition con-
stant, is the dissociation constant of the enzyme–inhibitor complex
and Ki0 is the dissociation constant of the enzyme–substrate–inhib-
itor complex), in both cases, the interaction is above the inhibitor
concentration axis in the Cornish-Bowden plots and below the
inhibitor axis in the Dixon plots. On the other hand, according to
Dixon and Cornish-Bowden plots, the inhibition kinetics of BACE1
by 24 are typical non-competitive, where Ki value are equal to Ki0
value and the interactions occurred on the 24 axis in both plots
(Fig. 2 and Table 2).
2.3. Structure–activity relationships of NOCs on BACE1
inhibitory activity
Coumarins have been reported to have several pharmacologi-
cal activities, such as antitumor and antiacethylcholinesterase
activities. Otherwise, in previous studies, furanocoumarins from
medicinal plant A. dahurica showed inhibition effects of BACE1.4
Therefore, in the present study, 41 kinds of naturally occurring
coumarins (NOCs) were screened for their effects on BACE1 activ-
ity. All coumarins in this experiment were screened for BACE1
In conclusion, our data suggest that dietary NOCs, particularly
geranyloxy appendage, effectively reach their target, BACE1, elicit-
ing site-specific inhibition. The IC50 values of our inhibitors,
although higher than peptide-derived transition state analog-
based BACE1 inhibitors are none-the-less still in the low micro-
molar range. Considering the development of therapeutic for AD,
chemical compounds must cross the blood–brain barrier (BBB)
and the plasma membranes. On the other hand, NOCs are relatively
inhibitory activity at a concentration of 500 lM. Then a series
of concentrations were used for an IC50 determination. Some
structure–activity relationships of coumarins can be deduced. Of
the simple coumarins in Table 1, coumarin (1), which is the fun-
damental skeleton lacking substitutions on the benzene ring,
showed a no inhibitory effect (8.1%) on BACE1. The activity of

S. Marumoto, M. Miyazawa / Bioorg. Med. Chem. 20 (2012) 784–788
787
concentrations used were 1.5
lM (j) and 0.75
l
M (
Figure 2. Dixon plots (panels A, C, E, and G) and CornNish-Bowden plots (panels B, D, F, and H) for inhibition of NOCs 18, 24, 31, and 35 on BACE1 activity. The BACE1 substrate
). The results are presented as means S.E.M. of triplicate experiments.
Table 2
Kinetics parameters for inhibition of BACE1 activity by compounds 18, 24, 31, and 34
3. Experimental procedure
Ki0
(l
M)
Type of inhibition
3.1. Chemicals
Compounds
Ki (l
M)
18
24
31
34
34.8
61.6
102.7
55.0
0.7
61.9
13.7
6.2
Mixed
Non-competitive
Mixed
A total of 46 coumarin derivatives were used for the inhibition
studiesof BACE1activity(Table 1). Coumarins;coumarin(1), umbel-
liferone (7-hydroxycoumarin, 2), 7-methoxycoumarin (3), 7-pre-
nyloxycoumarin (4), aurapten (7-geranyloxycoumarin, 5),
esculetol (6,7-dihydroxycoumarin, 6), esculetin (6,7-dimethoxy-
coumarin, 7), demethylsuberosin (7-hydroxy-6-prenylcoumarin,
8), suberosin (7-methoxy-6-prenylcoumarin, 9), osthenol (7-hydro-
xy-8-prenylcoumarin, 10), osthol (7-methoxy-8-prenylcoumarin,
11), furanocoumarins; psoralen (12), bergaptol (5-hydroxypsoralen,
Mixed
low-molecular-weight and high lipophilicity. Zhang et al. reported
that imperatorin (21) could pass through the BBB easily in rat.12
Given their enhanced lipophilic potency in cell, we believe that
our compounds 18, 24, 31, and 35 may be a possibility to prevent
Alzheimer’s disease.

788
S. Marumoto, M. Miyazawa / Bioorg. Med. Chem. 20 (2012) 784–788
13), bergapten (5-methoxypsoralen, 14), isoimperatorin (5-prenyl-
oxypsoralen, 15), oxypeucedanin (5-epoxyisopentenyloxypsoralen,
16), oxypeucedanin hydrate (5-(2,3-dihydroxy-3-methylbut-
oxy)psoralen, 17), bergamottin (5-geranyloxypsoralen, 18),
xanthotoxol (8-hydroxypsoralen, 19), xanthotoxin (8-methoxyp-
soralen, 20), imperatorin (8-prenyloxypsoralen, 21), heraclenin
(8-epoxyisopentenyloxypsoralen, 22), heraclenol (8-(2,3-dihy-
droxy-3-methylbutoxy)psoralen, 23), 8-geranyloxy psoralen (24),
5,8-dihydroxypsoralen (25), 8-hydrpxy-5-methoxy psoralen (26),
8-hydroxy-5-prenyloxypsoralen (27), 5-geranyloxy-8-methoxyps-
oraren (28), isopimpinellin (5,8-dimethoxypsoralen, 29), knidilin
(8-methoxy-5-prenyloxy psoralen, 30), 5-geranyloxy-8-methoxyp-
soralen (31), phellopterin (5-methoxy-8-prenyloxypsoralen, 32),
byakangelicol (5-epoxyisopentenyloxy-8-methoxypsoralen, 33),
(enzyme, sample solution, and substrate) after incubation, and S0
was the fluorescence of the tested samples at zero time. To allow
for the quenching effect of the samples, the sample solution was
added to the reaction mixture C, and any reduction in fluorescence
by the sample was then investigated. All data are the mean of three
experiments.
Supplementary data
Supplementary data (these data include synthesis and spectro-
scopic data of compounds 4, 5, 8–10, 13, 18, 19, 22–32, 35–37, 39,
and 42–46) associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2011.12.002.
byakangelicine
(5-methoxy-8-(2,3-dihydroxy-3-methylbutoxy)
psoralene, 34), 8-geranyloxy-5-methoxypsoralen (35), cnidicin
(5,8-diprenyloxypsoralen, 36), marmesin (37), angelicin (38),
columbianetin (39), pyranocoumarins; xanthyletin (40), dihydro-
xanthyletin (41), decursinol (42), decursinol tiglate (43), decursin
(44), seselin (45), lomatin (46). coumarins 1–3, 6, 7, 12, 14, 20, and
38 were obtained from Sigma Chemical Co. (St. Louis, MO), Wako
Pure Chemical (Osaka), or Tokyo Chemical Industry (Tokyo). All
other chemicals and reagents were of the highest purity commer-
cially available. Coumarins 11, 15, 16, 21, 33, and 34 were isolated
fromA. dahurica and Cnidium monnieri.4,13 Other NOCs were synthe-
sized as described previously in Supplementary data. 14–22 A BACE1
(recombinant human BACE1) assay kit was purchased from the Pan-
Vera Co. (United States).
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3.2. b-Secretase (BACE1) enzyme assay
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The b-secretase (BACE1) assay was carried out according to the
supplied manual with modifications.4 Briefly, a mixture of 10
l
l of
l of BACE1
of the substrate (750 nM Rh-EVNLDAEFK-
Quencher in 50 mM ammonium bicarbonate), and 10 l of sample
assay buffer (50 mM sodium acetate, pH 4.5), 10
(1.0 U/ml), 10
l
ll
l
dissolved in 30% DMSO was incubated for 60 min at room temper-
ature in the dark. The mixture was irradiated at 550 nm and the
emission intensity at 590 nm was recorded. The inhibition ratio
was obtained by the following equation:
16. Row, E. C.; Brown, S. A.; Stachulski, A. V.; Lennard, M. S. Org. Biomol. Chem.
2006, 4, 1604.
17. Abu-Mustafa, E. A.; El-Bay, F. K. A.; El-Khrisy, E. A. M.; Fayez, M. B. E. J.
Heterocycl. Chem. 1973, 10, 443.
18. Abu-Mustafa, Effat A.; El-Tawil, B. A. H.; Fayez, M. B. E. Indian J. Chem. 1967, 5,
283.
19. Kim, S.; Ko, H.; Son, S.; Shin, K. J.; Kim, D. J. Tetrahedron Lett. 2001, 42, 7641.
20. Gopalakrishnan, G.; Kasinath, V.; Singh, N. D. P.; Thirumurugan, R.; Raj, S.;
Shanmuga, S.; Shanmugam, G. Molecules 2000, 5, 880.
21. Nishita, Y.; Uesugi, K.; Satoh, Y.; Harayama, T. Yakugaku Zasshi 1998, 118, 599.
22. Murray, R. D. H.; Sutcliffe, M.; McCabe, P. H. Tetrahedron 1971, 27, 4901.
Inhibition ð%Þ ¼ ½1 ꢀ fðS ꢀ S0Þ=ðC ꢀ C0Þgꢁ ꢂ 100
ð1Þ
Where C was the fluorescence of the control (enzyme, buffer,
and substrate) after 60 min of incubation, C0 was the fluorescence
of control at zero time, S was the fluorescence of the tested samples