Inhibition of mushroom tyrosinase activity by orsellinates

Article abstract of DOI:10.1248/cpb.c17-00502

Several applications have been proposed for tyrosinase inhibitors in the pharmaceutical, food bioprocessing, and environmental industries. However, only a few compounds are known to serve as effective tyrosinase inhibitors. This study evaluated the tyrosinase-related activity of resorcinol (1), orcinol (2) lecanoric acid (3), and derivatives of this acid (4-15). Subjected to alcoholysis, lecanoric acid (3), a depside isolated from the lichen Parmotrema tinctorum, produces orsellinic acid (2,4-dihydroxy-6-methylbenzoic acid) (4) and orsellinates (2,4-dihydroxy-6-methyl benzoates) (5-15). At 0.50 mM, methyl (5), ethyl (6), n-propyl (7), tert-butyl (11), and n-cetyl orsellinates (15) acted as tyrosinase activators, whereas n-butyl (8), iso-propyl (9), sec-butyl (10), n-pentyl (12), n-hexyl (13), and n-octyl orsellinates (14) behaved as inhibitors. Tyrosinase inhibition rose with chain elongation-n-butyl (8) < n-pentyl (12) < n-hexyl (13) < n-octyl orsellinates (14)-suggesting that the enzyme site can accept an eight-carbon alkyl chain. A kinetic study of n-octyl orsellinate (14) revealed uncompetitive inhibition of tyrosinase, with an inhibition constant of 0.99 mM.

Full text of DOI:10.1248/cpb.c17-00502

Vol. 66, No. 1
Chem. Pharm. Bull. 66, 61–64 (2018)
61
Regular Article
Inhibition of Mushroom Tyrosinase Activity by Orsellinates
,a,b
Thiago Inácio Barros Lopes,* Roberta Gomes Coelho,a and Neli Kika Hondaa
a Instituto de Química, Universidade Federal de Mato Grosso do Sul; Campo Grande, MS 79070–900, Brazil: and
b Instituto Federal de Educação Ciência e Tecnologia de Mato Grosso do Sul; Aquidauana, MS 79200–000, Brazil.
Received June 21, 2017; accepted October 25, 2017
Several applications have been proposed for tyrosinase inhibitors in the pharmaceutical, food biopro-
cessing, and environmental industries. However, only a few compounds are known to serve as effective ty-
rosinase inhibitors. This study evaluated the tyrosinase-related activity of resorcinol (1), orcinol (2) lecanoric
acid (3), and derivatives of this acid (4–15). Subjected to alcoholysis, lecanoric acid (3), a depside isolated
from the lichen Parmotrema tinctorum, produces orsellinic acid (2,4-dihydroxy-6-methylbenzoic acid) (4)
and orsellinates (2,4-dihydroxy-6-methyl benzoates) (5–15). At 0.50mM, methyl (5), ethyl (6), n-propyl (7),
tert-butyl (11), and n-cetyl orsellinates (15) acted as tyrosinase activators, whereas n-butyl (8), iso-propyl
(9), sec-butyl (10), n-pentyl (12), n-hexyl (13), and n-octyl orsellinates (14) behaved as inhibitors. Tyrosinase
inhibition rose with chain elongation-n-butyl (8)<n-pentyl (12)<n-hexyl (13)<n-octyl orsellinates (14)-sug-
gesting that the enzyme site can accept an eight-carbon alkyl chain. A kinetic study of n-octyl orsellinate (14)
revealed uncompetitive inhibition of tyrosinase, with an inhibition constant of 0.99mM.
Key words tyrosinase; orsellinate; lecanoric acid; Parmotrema tinctorum
Tyrosinase (EC 1.14.18.1) is a copper enzyme that catalyzes microorganisms18) and Artemia salina,19) and also investigated
both the hydroxylation of monophenols to o-diphenols and for their antioxidant activity against 1,1-diphenyl-2-picrylhy-
the oxidation of o-diphenols to o-quinones.1,2) o-Quinones are drazyl (DPPH)20) and for antitumor properties.21)
highly reactive compounds that can polymerize spontaneously
The purpose of the present study was to investigate the
to form high-molecular-weight compounds, such as melanin. effect that resorcinol (1), orcinol (2), lecanoric acid (3), orsell-
In humans, melanin protects the skin against UV radiation, inic acid (4), and methyl (5), ethyl (6), n-propyl (7), n-butyl
but its overproduction can result in malignant melanoma.3) In (8), iso-propyl (9), sec-butyl (10), tert-butyl (11), n-pentyl (12),
foods, o-quinones can react with amino acids and/or proteins, n-hexyl (13), n-octyl (14), and n-cetyl orsellinates (15) exert
conferring enhanced brownish coloration and affecting the or- on tyrosinase activity. The inhibition mechanism involved in
ganoleptic properties and appearance of foodstuffs.4)
n-octyl orsellinate (14) was also investigated. To our knowl-
Several applications have been proposed for tyrosinase edge, this is the first report on the anti-tyrosinase behavior of
inhibitors in the pharmaceutical, food bioprocessing, and en- these compounds.
vironmental industries5)—e.g., for medicinal purposes, such
as in the treatment of neurodegenerative diseases,6) or to
Results and Discussion
improve the quality and shelf life of food products by inhib-
Tyrosinase Activation or Inhibition All compounds (Fig.
iting enzymatic browning.7) Tyrosinase inhibitors are often 1) were evaluated at 0.50mM and the results are expressed as
structurally analogous to phenolic substrates, which generally percent activation (A) or inhibition (I) (Table 1). The inhibi-
show competitive inhibition. A number of naturally occurring tory effect decreases with rising numerical values. Resorcinol
tyrosinase inhibitors have been described, mostly phenols and inhibited tyrosinase activity by 47.96%. Introducing a methyl
polyphenols,8) resorcinol derivatives,9–11) benzoic acid and pyr- group into position 5 of resorcinol to yield orcinol led to a
idine derivatives,12,13) gallate derivatives,14) and vanillin deriva- slight decrease in inhibition (50.79%). Khatib et al.10) reported
tives.15) However, only a few compounds are known to serve that tyrosinase inhibitors containing a resorcinol subunit are
as effective tyrosinase inhibitors, owing to high toxicity, low typically highly active, and 2,4-resorcinol derivatives are
activity, or economic factors. Potentially active compounds, significantly more potent than 3,5-substituted counterparts.
such as kojic acid and arbutin, have not shown the desired Introduction of a carboxyl into position 4 of orcinol to pro-
clinical effects.16)
duce orsellinic acid slightly diminished diphenolase inhibition
Recently, lichen extracts have been found to inhibit ty- to 63.35% an inhibitory level still higher than that of 82.69%
rosinase. Cladia aggregata, Cladonia dimorphoclada, Stereo- observed for lecanoric acid.
caulon ramulosum, and Stereocaulon microcarpum extracts
Modifying the alkyl chain of orsellinic acid had a pro-
have proven active against mushroom tyrosinase.17) In the nounced effect on the behavior of derivative 5–15 toward
present investigation, the activity of resorcinol (1), orcinol (2), mushroom tyrosinase. Orsellinates with small alkyl chains,
lecanoric acid (3), and derivatives of this acid (4–15) against such as methyl (110.10%), ethyl (114.49%), and n-propyl
mushroom tyrosinase was evaluated. Subjected to alcoholysis, (125.76%) orsellinates, activated the enzyme, enhancing its
lecanoric acid, a lichen depside isolated from Parmotrema diphenolase activity. In summary, orsellinates with small alkyl
tinctorum, yields orsellinic acid (2,4-dihydroxy-6-methyl- chains function as activators, and elongation of the alkyl chain
benzoic acid) (4) and orsellinates (2,4-dihydroxy-6-methyl causes an inhibitory activity to manifest, in contrast with al-
benzoates) (5–15). Orsellinates have been evaluated against kylgallates, which act as inhibitors regardless of alkyl chain
*To whom correspondence should be addressed. e-mail: inacio_thiago@hotmail.com
© 2018 The Pharmaceutical Society of Japan

62
Chem. Pharm. Bull.
Vol. 66, No. 1 (2018)
Fig. 1. Structures of the Compounds Investigated
et al.,9) who found 4-hexylresorcinol to be more active than
4-ethylresorcinol in preventing L-3,4-dihydroxyphenylalanine
(L-DOPA) oxidation to DOPA-quinone. However, the enhance-
ment of tyrosinase activity with increasing length of hydro-
carbon chains is expected to peak at a linear alkyl chain of
eight to ten carbons. Tyrosinase inhibition is likely related, at
least in part, to hydrophobic interactions with the enzyme’s
hydrophobic domain.24) This apparently holds true for orselli-
nates. Huang et al.13) suggested that the enzyme’s hydrophobic
site may well accept an alkyl chain of eight carbons, which
explains the enhanced activity observed for n-octyl orsellinate
(45.62%).
Table 1. Effect of Compounds 1–15 on Tyrosinase
Compound
Tyrosinase activity (%)
Resorcinol (1)a)
Orcinol (2)a)
Lecanoric acid (3)b)
Orsellinic acid (4)a)
Orsellinates
Methyl (5)a)
Ethyl (6)a)
n-Propyl (7)a)
n-Butyl (8)a)
47.96
50.79
82.69
63.35
110.10
114.49
125.76
90.59
iso-Propyl (9)a)
sec-Butyl (10)a)
tert-Butyl (11)a)
n-Pentyl (12)a)
n-Hexyl (13)a)
n-Octyl (14)b)
n-Cetyl (15)b)
88.06
Khatib et al.10) suggested that the increasing inhibitory
potency exhibited by 3-(2,4-dihydroxyphenyl) propionic es-
ters does not necessarily correlate with longer alkyl chains,
but may be related to the increasing volume of ester groups.
Similar results were obtained by Kubo et al.4) for esters of
gallic acid. This can explain the higher inhibition activity
observed for sec-butyl orsellinate (57.30%), relative to n-butyl
orsellinate (90.59%). Inhibiting activity was also observed for
iso-propyl orsellinate (88.06%), while the n-propyl counterpart
proved a tyrosinase activator (125.76%). Unexpected results,
57.30
111.05
65.92
58.55
45.62
110.04
a) 5% and b) 15% of DMSO in reaction mixtures. Tyrosinase activity compared
with a control reaction. For inhibitors, tyrosinase activity<100%; for activators, ty-
rosinase activity>100%.24)
size.4) The contrasting effects of alkyl chains in orsellinates however, were obtained for tert-butyl orsellinate (111.05%).
and in gallates can be related to differences in the position
The Mechanism of Diphenolase Inhibition by n-Octyl
of hydroxyl groups in orsellinates (2,4-dihydroxy-6-methyl Orsellinate Figure 2 depicts the behavior of diphenolase
benzoates) and gallates (3,4,5-trihydroxybenzoate) and to the activity in the presence of n-octyl orsellinate at various con-
presence of the methyl group in the latter. Previous reports centrations—a family of straight lines intercepting the origin.
have demonstrated a shift from activator to inhibitor, associ- Increasing inhibitor concentrations resulted in descending
ated with substituent modification.22) You et al. demonstrated slopes, indicating reversible inhibition of mushroom tyrosi-
that the introduction of a thiosemicarbazide group in amino- nase by this orsellinate.
acetophenones transforms them from activators into inhibi-
Double-reciprocal plots were used to investigate the mecha-
nisms of mushroom tyrosinase inhibition by the same com-
tors.23)
Among linear-chain orsellinates, inhibitory activity rose pound (Fig. 3). Plotting 1/V vs. 1/[S] yielded a family of paral-
with increasing number of carbons in the alkyl chain-namely, lel straight lines with equal slopes, indicating uncompetitive
n-butyl (90.59%)<n-pentyl (65.92%)<n-hexyl (58.55%)<n- inhibition.25) In uncompetitive inhibition, the inhibitor binds
octyl orsellinate (45.62%)-corroborating the findings of Huang at a different site from the substrate and combines with the
et al.,13) who observed that the inhibitory properties of p-alkyl enzyme–substrate complex (ES), but not with the free enzyme
benzoic acids were potentiated by increasing the length of (E).
hydrocarbon chains. A similar result was obtained by Jiménez
The equilibrium constant for binding with the enzyme–sub-

Vol. 66, No. 1 (2018)
Chem. Pharm. Bull.
63
strate–inhibitor complex (KESI) was obtained by regression
from 1/V vs. inhibitor concentration (Fig. 4). The inhibition
constant obtained for n-octyl orsellinate was 0.99mM.
Conclusion
The activity of orsellinates against mushroom tyrosinase
was investigated. The behavior of orsellinates differed from
those of orcinol and orsellinic acid. This difference was as-
cribed to the presence of an ester group ortho-positioned to
a methyl group, as well as to the presence of a hydroxyl in
the structure of orsellinates, as compared with orcinol, or to
esterification of the carboxyl group of orsellinic acid. Tested
at 0.50mM, methyl, ethyl, n-propyl, tert-butyl, and n-cetyl or-
sellinates activated mushroom tyrosinase, while n-butyl, iso-
propyl, sec-butyl, n-pentyl, n-hexyl, and n-octyl orsellinates
acted as inhibitors. Inhibition increased with chain elongation
(n-butyl<n-pentyl<n-hexyl<n-octyl orsellinates), suggesting
that the hydrophobic site of the enzyme can accept an alkyl
chain of eight carbons. The kinetic study of n-octyl orsellinate
revealed uncompetitive inhibition of tyrosinase, with an inhi-
bition constant of 0.99mM.
Fig. 2. Effect of n-Octyl Orsellinate (14) Concentration on Diphenolase
Activity of Mushroom Tyrosinase
Curves 1–4 (for 0, 0.05, 0.10, and 0.15mM n-octyl orsellinate, respectively) were
obtained using linear regression.
Experimental
General Procedures NMR spectra were recorded using
a Bruker DPX-300 spectrometer operating at 300.13MHz for
1H and 75.48MHz for 13C. Melting points were determined on
a Uniscience Melting Point apparatus without corrections. Si-
gel (Merck, 230–400 mesh) was used in the chromatography
column. TLC was performed on plates pre-coated with silica
gel 60F254 (Merck) and the spots were visualized with (a)
10% H2SO4 : methanol solution, followed by heating until the
appearance of spots and (b) p-anisaldehyde : sulfuric acid, fol-
lowed by reheating. Mushroom tyrosinase (6680U/mg) and L-
DOPA were purchased from Sigma Chemical (São Paulo, Bra-
zil). Resorcinol was obtained in analytical grade from Merck
(Darmstadt, Germany) and orcinol from Sigma (St. Louis,
MO, U.S.A.). Verification of purity grades was based on
melting points and NMR spectra. All aqueous solutions were
Fig. 3. Lineweaver–Burk Plots for Inhibition of Diphenolase Activity of
Mushroom Tyrosinase by n-Octyl Orsellinate (14)
Curves 1–4 (0, 0.05, 0.17, and 0.28mM n-octyl orsellinate, respectively) were prepared with deionized water. Phosphate buffer (Na2HPO4–
obtained using linear regression.
NaH2PO4) at 50.0mM, pH 6.8, was used in all reactions.
Preparation of Compounds Orsellinic acid (2,4-dihy-
droxy-6-methylbenzoic acid) (4) was obtained by hydrolyzing
lecanoric acid (3) isolated from a Parmotrema tinctorum spec-
imen. Alkyl orsellinates (2,4-dihydroxy-6-methyl benzoates)
(5–15) were prepared by reacting 200mg of lecanoric acid (3)
with 50mL of the corresponding alcohol at 40°C. The com-
pounds were purified by chromatography on a silica column
with chloroform and a chloroform:acetone gradient. Details of
the experimental procedure and spectral characterization for
compounds 1–11 can be found in Lopes et al.20)
Pentyl-2,4-dihydroxy-6-methyl benzoate (n-pentyl orselli-
1
nate) (12): H-NMR (acetone-d6) δ: 11.76 (1H, s, ArOH-2), 9.63
(1H, s, ArOH-4), 6.28 (1H, s, ArH-5), 6.24 (1H, s, ArH-3),
4.33 (2H, t, J=7.35Hz, CH2-1′), 2.48 (3H, s, ArCH3-8),
1.78 (2H, m, CH2-2′), 1.42 (2H, m, CH2-3′-4′), 0.92 (3H, m,
CH3-5′). 13C-NMR (acetone-d6) δ: 172.5 (C-7), 165.6 (C-4),
162.6 (C-2), 143.5 (C-6), 111.6 (C-5), 104.4 (C-1), 100.8 (C-3),
65.2 (C-1′), 29.1 (C-2′), 28.2 (C-3′), 28.1 (C-4′), 23.7 (C-8), and
Fig. 4. Plot of [S]/V vs. Concentration of n-Octyl Orsellinate (14), to
Determine the Enzyme–Substrate–Inhibitor Complex (KESI)
13.4 (C-5′). mp 58–59°C.
Hexyl-2,4-dihydroxy-6-methyl benzoate (n-hexyl orsel-
Curves 1–5 (0.12, 0.14, 0.21, 0.27 and 0.38mM L-DOPA, respectively) were ob-
1
linate) (13): H-NMR (acetone-d6) δ: 11.72 (1H, s, ArOH-2),
tained using linear regression.

64
Chem. Pharm. Bull.
Vol. 66, No. 1 (2018)
6.28 (1H, s, ArH-5), 6.23 (1H, s, ArH-3), 4.34 (2H, t,
Acknowledgments The authors wish to acknowledge the
J=7.35Hz, CH2-1′), 2.48 (3H, s, ArCH3-8), 1.79 (2H, m, financial support provided by Fundect-MS and Coordenadoria
CH2-2′), 1.47 (2H, m, CH2-3′) 1.35 (2H, m, CH2-4′-5′), 0.89 de Pesquisa da Pró-Reitoria de Pesquisa e Pós-Graduação
(3H, m, CH3-6′). 13C-NMR (acetone-d6) δ: 172.4 (C-7), 166.6 (CP-PROPP), Universidade Federal de Mato Grosso do Sul
(C-4), 163.1 (C-2), 144.0 (C-6), 112.0 (C-5), 105.0 (C-1), 101.3 (UFMS). T.I.B.L. appreciates the fellowship awarded by the
(C-3), 65.7 (C-1′), 31.8 (C-2′), 28.9 (C-3′), 26.2 (C-4′), 24.1 Brazilian Tutorial Education Program (PET).
(C-8), 22.8 (C-5′), and 13.9 (C-6′). mp 46–48°C.
Octyl-2,4-dihydroxy-6-methyl benzoate (n-octyl orsellinate)
Conflict of Interest The authors declare no conflict of
(14): 1H-NMR (acetone-d6) δ: 11.91 (1H, s, ArOH-2), 6.28 interest.
(1H, s, ArH-5), 6.23 (1H, s, ArH-3), 4.33 (2H, t, J=7.35Hz,
CH2-1′), 2.50 (3H, s, ArCH3-8), 1.77 (2H, m, CH2-2′), 1.42
(2H, m, CH2-3′), 1.31 (2H, m, CH2-4′-7′), 0.88 (3H, m,
CH3-8′). 13C-NMR (acetone-d6) δ: 171.9 (C-7), 165.4 (C-4),
160.2 (C-2), 144.0 (C-6), 111.3 (C-5), 105.8 (C-1), 101.3 (C-3),
65.6 (C-1′), 31.7 (C-2′), 29.2 (C-3′), 28.6 (C-4′), 26.2 (C-5′),
24.4 (C-8), 22.6 (C-6′-7′), and 14.10 (C-8′). mp 62–63°C.
Hexadecyl-2,4-dihydroxy-6-methyl benzoate (n-cetyl orsel-
References
1) Seo S. Y., Sharma V. K., Sharma N., J. Agric. Food Chem., 51,
2837–2853 (2003).
2) Olivares C., Jiménez-Cervantes C., Lozano J. A., Solano F., García-
Borrón J. C., Biochem. J., 354, 131–139 (2001).
3) Solano F., Briganti S., Picardo M., Ghanem G., Pigment Cell Res.,
19, 550–571 (2006).
4) Kubo I., Kinst-Hori I., Kubo Y., Yamagiwa Y., Kamikawa T., Hara-
guchi H., J. Agric. Food Chem., 48, 1393–1399 (2000).
5) Zaidi K. U., Ali A. S., Ali S. A., Naaz I., Biochem. Res. Int., 2014,
854687 (2014).
1
linate) (15): H-NMR (acetone-d6) δ: 11.86 (1H, s, ArOH-2),
6.27 (1H, s, ArH-5), 6.22 (1H, s, ArH-3), 4.32 (2H, t,
J=7.35Hz, CH2-1′), 2.50 (3H, s, ArCH3-8), 1.77 (2H, m,
CH2-2′), 1.62 (2H, m, CH2-3′), 1.25 (2H, m, CH2-4′-15′), 0.88
(3H, m, CH3-16′). 13C-NMR (acetone-d6) δ: 171.8 (C-7), 165.5
(C-4), 160.2 (C-2), 144.0 (C-6), 111.3 (C-5), 105.3 (C-1), 101.3
(C-3), 65.6 (C-1′), 32.0 (C-2′), 29.7 (C-3′), 29.4 (C-4′), 24.4
(C-8), 22.7 (C-5′-15′), and 14.13 (C-16′). mp 84–85°C.
Enzymatic Activity Inhibition of the diphenolase activ-
ity of mushroom tyrosinase was evaluated using L-DOPA as
the substrate.25) The test compounds were first dissolved in
dimethyl sulfoxide (DMSO) at 10.0mM for samples 1–2 and
4–14 and, because of lower solubility, at 3.3mM for samples 3
and 15. For analysis of samples 1–2 and 4–14, 0.1mL of sam-
6) Montine T. J., Underhill T. M., Valentine W. M., Graham D. G.,
Neurodegeneration, 4, 283–290 (1995).
7) Nerya O., Ben-Arie R., Luzzatto T., Musa R., Khativ S., Vaya J.,
Postharvest Biol. Technol., 39, 272–277 (2006).
8) Brahmi F., Hauchard D., Guendouze N., Madani K., Kiendrebeogo
M., Kamagaju L., Stévigny C., Chibane M., Duez P., Ind. Crops
Prod., 74, 722–730 (2015).
9) Jiménez M., García-Carmona F., J. Agric. Food Chem., 45, 2061–
2065 (1997).
10) Khatib S., Nerya O., Musa R., Tamir S., Peter T., Vaya J., J. Med.
Chem., 50, 2676–2681 (2007).
11) Dawley R. M., Flurkey W. H., J. Food Sci., 58, 609–610 (1993).
ple solution in DMSO was combined with 1.5mL of sodium 12) Masawat P., Harfield A., Namwong A., Food Chem., 184, 23–29
(2015).
phosphate buffer. For samples 3 and 15, 0.3mL of sample
13) Huang X. H., Chen Q. X., Wang Q., Song K. K., Wang J., Sha L.,
Guan X., Food Chem., 94, 1–6 (2006).
14) Lee C. W., Son E. M., Kim H. S., Xu P., Batmunkh T., Lee B. J.,
Koo K. A., Bioorg. Med. Chem. Lett., 17, 5462–5464 (2007).
15) Ashraf Z., Rafiq M., Seo S. Y., Babar M. M., Zaidi N. U. S. S.,
Bioorg. Med. Chem., 23, 5870–5880 (2015).
16) Briganti S., Camera E., Picardo M., Pigment Cell Res., 16, 101–110
solution in DMSO was combined with 1.2mL of the same
buffer. Each resulting solution was then mixed with 0.2mL
of L-DOPA (3.5mM) and 0.2mL of tyrosinase (10µg/mL). The
mixtures were homogenized and incubated for 5min at 30°C.
Absorbances were measured at 475nm to monitor the forma-
tion of dopachrome (ε=3700 M−1 cm−1) using a Spectrum 220
spectrophotometer. Tyrosinase activity was calculated by com-
paring with a control reaction:
(2003).
17) Honda N. K., Gonçalves K., Brandão L. F. G., Coelho R. G., Mi-
cheletti A. C., Spielmann A. A., Canêz L. S., Orbital-Electron. J.
Chemometr., 8, 181–188 (2016).
18) Gomes A. T., Smania A., Seidel C., Albino Smania E. D. F., Honda
N. K., Roese F. M., Muzzi R. M., Braz. J. Microbiol., 34, 194–196
(2003).
19) Gomes A. T., Honda N. K., Roese F. M., Muzzi R. M., Sauer L., Z.
Naturforsch. C, 61c, 653–657 (2006).
20) Lopes T. I. B., Coelho R. G., Yoshida N. C., Honda N. K., Chem.
Pharm. Bull., 56, 1551–1554 (2008).






(Asample − Acontrol
)
Tyrosinase activity%=
×100%
Bcontrol
where Asample is the absorbance of the sample reaction (con-
taining all reagents and the test compound), Acontrol is the
absorbance of the test compound, and Bcontrol is the absorbance
of the control reaction (containing all reagents, but no test
compound). For inhibitors, tyrosinase activity <100%; for ac-
tivators, tyrosinase activity >100%. All tests were performed
in triplicate.
21) Bogo D., Matos M. F. C., Honda N. K., Pontes E. C., Oguma P. M.,
Santos E. C. S., Carvalho J. E., Nomizo A., Z. Naturforsch C, 65c,
43–48 (2010).
22) Ferreira M. S., Pires D. A. T., Figueroa-Villar J. D., World J. Pharm.
Pharm. Sci., 4, 1705–1718 (2015).
23) You A., Zhou J., Song S., Zhu G., Song H., Yi W., Eur. J. Med.
Chem., 93, 255–262 (2015).
24) Chang T.-S., Int. J. Mol. Sci., 10, 2440–2475 (2009).
25) Qiu L., Chen Q. H., Zhuang J. X., Zhong X., Zhou J. J., Guo Y. J.,
Chen Q. X., Food Chem., 112, 609–613 (2009).
n-Octyl orsellinate was subjected to kinetic analysis to
determine inhibition type and the respective constant. The
Lineweaver–Burk equation in double-reciprocal form and
secondary replots were applied.25) The inhibition constant of
the uncompetitive inhibitor (KESI) was determined using the
graphical method.26)
26) Cornish-Bowden A., Biochem. J., 137, 143–144 (1974).