Biocatalytic synthesis, structural elucidation, antioxidant capacity and tyrosinase inhibition activity of long chain fatty acid acylated derivatives of phloridzin and isoquercitrin

Article abstract of DOI:10.1016/j.bmc.2012.11.034

Our present investigation describes the regioselective enzymatic acylation of two series of acylated derivatives of phloridzin and isoquercitrin with six different long chain saturated, mono- and poly-unsaturated fatty acids. The biocatalytic synthesis wa

Full text of DOI:10.1016/j.bmc.2012.11.034

Bioorganic & Medicinal Chemistry 21 (2013) 684–692
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Biocatalytic synthesis, structural elucidation, antioxidant capacity and
tyrosinase inhibition activity of long chain fatty acid acylated derivatives
of phloridzin and isoquercitrin
⇑
Ziaullah, Khushwant S. Bhullar, Sumudu N. Warnakulasuriya, H. P. Vasantha Rupasinghe
Department of Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, Nova Scotia, Canada B2N 5E3
a r t i c l e i n f o
a b s t r a c t
Article history:
Our present investigation describes the regioselective enzymatic acylation of two series of acylated
derivatives of phloridzin and isoquercitrin with six different long chain saturated, mono- and poly-
unsaturated fatty acids. The biocatalytic synthesis was optimized to achieve 81–98% yields, using immo-
bilized lipase B, from Candida antarctica (Novozym 435Ò), in acetone at 45 °C. The synthesized esters have
been analyzed by 1H NMR, 13C NMR spectroscopy and evaluated for their antioxidant capacity and
tyrosinase inhibition, using in vitro assays. Among all the phloridzin and isoquercitrin derivatives, the
Received 25 September 2012
Revised 19 November 2012
Accepted 26 November 2012
Available online 5 December 2012
Keywords:
Flavonoid
Phloridzin
Isoquercitrin
greatest potential for inhibition of tyrosinase activity (p 60.05) was exhibited by the
ester of isoquercitrin.
a-linolenic acid
Ó 2012 Elsevier Ltd. All rights reserved.
Acylation
Candida antarctica lipase B
1H NMR
13C NMR spectroscopy
Antioxidant capacity
Tyrosinase
1. Introduction
tyrosinase and rhamnetin acts as a cofactor or substrate of tyrosi-
nase.8 This copper containing enzyme is involved in enzymatic
browning of plant tissues, formation of cuticle in insects and most
importantly, melanogenesis in humans.9 Tyrosinase triggers mela-
Flavonoids have been receiving a great deal of attention re-
cently, due to their valuable biological, pharmacological and
medicinal properties. These polyhydroxylated phenolic com-
pounds have antioxidant properties that include free radical termi-
nation and metal chelation.1 Flavonoids are also inhibitors of many
enzymes, such as cyclooxygenase and lipoxygenase, which can
lead to the formation of reactive oxygen species (ROS).2 In addition,
flavonoids have been discovered to have a variety of biological
activities, which include anti-inflammatory, anti-viral, antibiotic,
anti-spasmolytic, anti-allergenic, anti-diarrheic, anti-tumor, anti-
ulcer, oestrogenic and vasodilatory properties.2–7 The multiple
functionalities make the polyphenols distinguished chelators,
through their hydroxyl groups, to the metallic part of the enzymes
and thus, act as competitive inhibitors of many enzymes.
nogenesis by hydroxylation of
L-tyrosinase to L-3,4-dihydroxyphen-
ylalanine (
L
-DOPA) and its oxidation to dopaquinone.10 Tyrosinase is
involved in the treatment of dermatological disorders such as hyper-
pigmentation, melanoma, along with other skin disorders.11 Numer-
ous chemicals of natural origin, such as alkaloids,11 phenolics,12–14
chalcones,15 flavonoids,16 and tetraketones17 are promising
tyrosinase inhibitors. In this study we investigated the tyrosinase
inhibition of novel quercetin and phloridzin esters using
L-3,4-dihy-
droxyphenylalanine ( -DOPA) as the binding enzyme substrate.
L
These recognized health benefits have sparked the potential for
many industrial applications in food ingredients, cosmetics and
pharmaceuticals. However, these applications are significantly lim-
ited, due to the poor solubility of flavonoids in both hydrophobic
and hydrophilic media.18
Previous studies have shown that enzyme-catalyzed esterifica-
tion and transesterification of flavonoid glycosides have been suc-
cessful in increasing lipophilicity. With ideal reaction conditions,
relatively high conversion yields of the flavonoid esters have been
obtained.18–24 Herein, using long chain fatty acids as acyl donors
would have a wide range of health benefits. As described in earlier
So far, a number of studies have been carried out to identify nat-
ural inhibitorsof tyrosinase, as well as their syntheticanalogs. Flavo-
noids, which are one of the most investigated groups of plant
secondary metabolites, show tyrosinase inhibition. For example,
quercetin, kaempferol and morin act as competitive inhibitors of
⇑
Corresponding author. Tel.: +1 902 893 6623; fax: +1 902 893 1404.
E-mail address: vrupasinghe@dal.ca (H.P.V. Rupasinghe).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.034

Ziaullah et al. / Bioorg. Med. Chem. 21 (2013) 684–692
685
reports, they provide an important fuel source to humans, specifi-
cally to the heart and skeletal muscles.18 Besides, polyunsaturated
fatty acids are also responsible for the regulation of important bio-
logical functions, as they are the precursors of many metabolites.18
Germany), using a solvent mixture system of acetone/toluene
(60:40, few drops of acetic acid). The products were detected under
UV light, as well as by using anisaldehyde (4-methoxybenzalde-
hyde) as spray reagent, followed by heating at 110 °C. The acylated
products were purified by column chromatography, using silica gel
60 (0.040–0.063 mm; 230–400 mesh, Merck, Darmstadt, Germany)
and eluted with acetone toluene. The purity of the samples was
tested, using silver-ion thin-layer chromatography (Ag-TLC) as
well as NMR spectroscopy. TLC-plates were impregnated with
AgNO3 by immersion in a 10% (w/v) solution of AgNO3 in water/
methanol (95:5), under dark. After the plates were dried,
compounds were loaded onto them and developed with acetone–
toluene (6:4, v/v).The plates were then sprayed with 10% H2SO4
in methanol and heated at 110 °C. The purified compounds
appeared as dark black spots on the TLC plates.43
The
acids, have unique beneficial biological activities. Recently, anti-
tumour activities of -3 and -6 polyunsaturated acids have been
x-3 and x-6 polyunsaturated acids, which are essential fatty
x
x
discovered.25,26 The use of these fatty acids, in natural product
forms, is limited as they are prone to autooxidation, due to free
radicals. These free radicals can have deleterious effects on the hu-
man body, such as the formation of DNA lesions, loss of enzymatic
activities and potential for cell death.27 It would be advantageous
to combine the fatty acids with certain antioxidants that would
prevent the harmful effects of oxidation and preserve the health
benefits of the fatty acid. Recently, Salem et al.19 have found that
isoquercitrin esters increased antioxidant properties, including a
higher inhibition of xanthine oxidase, an enzyme that catalyzes
the production of superoxide radicals.
2.3. NMR and IR spectroscopic analysis
Chemical, enzymatic and chemo-enzymatic acylation of pheno-
lic compounds, with various acyl donors (C2–C18 chain length),
has been already reported.19,21,28–34 However, the enzymatic ap-
proach is preferred over the chemical acylation,24 due to its regi-
oselectivity and simple one-step reaction. Regioselective
reactions ensure that some hydroxyl groups remain on the flavo-
noid, preserving its antioxidant properties.19,21,28–42
The chemical structures of the acylated products were deter-
mined by 1H NMR and 13C NMR spectroscopic analysis in solvent
DMSO-d6 or MeOD, on Bruker AVANCE 300 MHz spectrometer
(Bruker Corp., Billerica, MA, USA). Chemical shifts were expressed
in d ppm, relative to an internal standard of tetramethylsilane
(TMS). The IR data was collected on Nicolet 6700 FT-IR spectrom-
eter, from Thermo Scientific (Ottawa, ON, Canada).
The aim of this study was to perform lipase-catalyzed acylation
of flavonoid glycosides, using very long chain unsaturated and sat-
urated fatty acids (C18–C22), with Lipase B (Novozym 435Ò) from
Candida antarctica as the catalytic enzyme. The focus of interest
was to investigate the amphipathic/amphiphilic properties, by
changing the types of acyl donor fatty acids. Specifically, modifica-
tions in the chain lengths and degrees of unsaturation, to synthe-
size a variety of flavonoid esters, with the long-term aim of
improving the bioavailability and biological activity. This current
study provides, for the first time, the detailed NMR structural
assignments, along with tyrosinase enzyme inhibition of the syn-
thesized esters of phloridzin and isoquercitrin.
2.4. The ferric reducing antioxidant power (FRAP) assay
The FRAP assay was performed according to the method de-
scribed by Rupasinghe et al.44 The working assay reagent (FRAP
solution) was prepared daily by mixing 300 mmol lꢀ1 acetate buf-
fer of pH 3.6, 10 mmol lꢀ1 TPTZ solution and 20 mmol lꢀ1 ferric
chloride in the ratio of 10:1:1. As the standard material, Trolox
solution was prepared by dissolving 0.025 g of Trolox in 100 ml
of 95% ethanol. The appropriate dilutions of Trolox solution was
carried out using 95% ethanol to obtain 50, 100, 200, 300, 400,
500, 1000
developed. All the sample compounds were dissolved in 95% etha-
nol to prepare desired concentrations. To perform the assay, 20
of blank, standard or sample was reacted with 180 l of FRAP solu-
l
mol lꢀ1 concentrations and calibration curve was
l
l
2. Materials and methods
2.1. Chemicals and reagents
l
tion in 96-well polystyrene plates after warming up to 37 °C. The
absorbance was measured at 595 nm after 10 min reaction time,
including 3 s shaking time using the FLUOstar OPTIMA plate reader
(BMG Labtech, Durham, NC, USA). Antioxidant capacity was calcu-
Phloridzin, isoquercitrin (quercetin-3-O-glucoside), oleic, stea-
ric, linolenic, linoleic, eicosapentanenoic, docosahexanoic acids
and Novozyme 435Ò, an immobilized lipase B from Candida antarc-
tica (with an activity of 10,000 propyl laurate units), 3 Å molecular
sieves, p-anisaldehyde, mushroom tyrosinase, phosphate buffer
lated as l
mol lꢀ1 Trolox equivalents of 1 mmol lꢀ1 of solution.
2.5. The oxygen radical absorbance capacity (ORAC) assay
and L-3,4-dihydroxyphenylalanine (L-DOPA) were purchased from
The ORAC assay was carried out using the method explained by
Rupasinghe et al.44 with slight modifications. The Trolox solution
and AAPH [2,20-azobis(2-amidino-propane) dihydrochloride] were
prepared daily immediate before the assay. The Trolox standard
solution was made using phosphate buffer and suitable dilutions
were carried out to develop the calibration curve. The buffer, stan-
Sigma–Aldrich (Mississauga, ON, Canada) while sodium acetate tri-
hydrate, ferric chloride, 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ), Trol-
ox, fluorescein, phosphate buffer, 2,2-diphenyl-1-picrylhydrazyl
(DPPH), spectrophotometric grade dimethyl sulfoxide, 2,20-azobis
(2-amidinopropane) dihydrochloride (AAPH) were purchased from
Sigma–Aldrich (St. Louise, MO, USA). Phosphorus (V) oxide (P2O5)
was purchased from Alfa Aesar (Ward Hill, MA, USA). Acetone (extra
dry), Acetic acid, hydrochloric acid, HPLC grade chloroform and
HPLC grade methanol were purchased from Fisher Scientific (Otta-
wa, ON, Canada). Ethanol anhydrous was obtained from Commercial
Alcohols (Montreal, QC, Canada). Round bottom 96-well micro-
plates were obtained from Corning Incorporated (Edison, NY, USA).
dard, or sample (35 ll) and 0.957 l ll)
mol lꢀ1 fluorescein (130
solutions were placed in the 96-well plates and pre-incubated at
37 °C for 10 min. The fluorescence was measured using FLUOstar
OPTIMA plate reader (BMG Labtech, Durham, NC, USA) after injec-
tion of 35 ll pre-warmed (37 °C) AAPH to each well in every 60 s
up to 3201 s. Antioxidant capacity was calculated as
l
mol lꢀ1
Trolox equivalents of 0.01 or 0.001 mmol lꢀ1 of solution.
2.2. TLC analysis
2.6. DPPH radical scavenging assay
The progress of reactions was monitored by thin-layer chroma-
tography (TLC) on silica gel 60 F254 plates from Merck (Darmstadt,
The DPPH radical scavenging assay was carried out as described
by Brand-Williams et al.45 with modifications. In solution, DPPH

686
Ziaullah et al. / Bioorg. Med. Chem. 21 (2013) 684–692
has a stable free radical and is a deep purple colour, with a charac-
teristic absorbance around 520 nm, but with the addition of an
antioxidant, the free radical is scavenged and DPPH becomes 2,2-
diphenyl-1-picryl hydrazine, a colourless compound. Thus, the
change in absorbance of a solution with DPPH with an added com-
pound, compared to solution of DPPH alone, is indicative of the
antioxidant activity of the compound. The reaction mixture con-
913, 737, 652; 1H NMR (DMSO-d6, 300 MHz): d 10.60 (s, 1H, ArOH),
9.18 (br s, 1H, ArOH), 7.06 (d, 2H, J = 8.4 Hz, H-2, H-6), 6.67 (d, 2H,
J = 8.4 Hz, H-3, H-5), 6.13 (d, 1H, 1.8 Hz, H-30), 5.98 (d, 1H,
J = 1.8 Hz, H-50), 5.46 (br s, 1H, OH), 5.37 (br s, 2H, 2OH), 5.04 (d,
1H, H-100), 4.35 (br d, 1H, J = 11.6 Hz, H-6a00), 4.14 (dd, 1H,
J = 11.6 Hz, 6.9 Hz, H-6b00), 3.65 (br t, J = 7.2 Hz, H-400), 3.46–3.20
(m, 6H, 2 ꢁ H , H-200, H-300, H-500, OH), 2.81 (br t, 2H, J = 7.5 Hz,
a
taining 40
ll of 0.2 mM DPPH solution and 40
ll from different
2 ꢁ Hb), 2.30 (t, 2H, J = 7.2 Hz, 2 ꢁ H-2000), 1.49 (m, 2H, 2 ꢁ H-3000),
1.25–1.19 (m, 28H, 2(CH2)), 0.87 (br t, 3H, J = 6.9 Hz, CH3); 13C
NMR (DMSO-d6, 75 MHz): d 205.34 (CO), 173.44 (OCO), 166.05
(C-40), 165.13 (C-60), 161.30 (C-20), 156.03 (C-4), 132.25 (C-1),
129.72 (C-2, C-6), 115.74 (C-3, C-5), 106.18 (C-10), 101.55 (C-100),
97.79 (C-30), 95.44 (C-50), 77.30 (C-300), 74.77 (C-500), 73.94 (C-200),
concentrations of test compounds or control in the solvent system
of dimethyl sulfoxide/chloroform/methanol in a ratio of 1:8:4 was
placed in 96-well plates following incubation for 30 min at 25 °C.
The absorbance was measured using FLUOstar OPTIMA plate read-
er (BMG Labtech, Durham, NC, USA) at 520 nm, at 0 s, and every
300 s until the reaction reached a plateau. The antiradical activities
of the compounds were expressed by their IC50 values, the concen-
tration required to scavenge 50% of the DPPH radical.
70.68 (C-400), 63.81 (C-600), 45.61 (C ), 34.17 (C-2000), 31.96 (Cb),
a
29.84, 29.34, 29.13 (C-4000, C-5000, C-6000, C-7000, C-8000, C-9000, C-10000, C-
11000, C-12000, C-13000, C-14000, C-15000, C-16000), 25.08 (C-3000), 22.73 (C-
17000), 14.53 (C-18000).
2.7. Tyrosinase inhibition assay
2.9.3. (6-{3,5-Dihydroxy-2-[3-(4-hydroxyphenyl)propanoyl]
phenoxy}-3,4,5-trihydroxytetra-hydro-2H-pyran-2-yl)methyl
(Z)-9-octadecenoate (3)
The tyrosinase inhibition assay was performed using L-DOPA, as
the substrate as recently described by Sirat et al.46 Tyrosinase inhi-
bition is depended upon the amount of dopachrome, produced in
the reaction mixture. Absorption was measured at 490 nm to cal-
culate the amount of dopachrome produced using 96-well clear
polystyrene plates and FLUOstar OPTIMA plate reader (BMG
Labtech, Durham, NC, USA). The tyrosinase inhibitory effect of
the compounds was measured by calculating the percent inhibi-
tion using the following equation:
Yield: 96%; light brownish yellow spongy solid; Rf: 0.56 (ace-
tone/toluene; 4:6: few drops of AcOH); IR (KBr) cmꢀ1: 3383,
2927, 2855, 2253, 1714, 1627, 1599, 1514, 1455, 1264, 1203,
1078, 908, 734, 650; 1H NMR (DMSO-d6, 300 MHz): d 11.82 (br s,
1H, ArOH), 10.63 (br s, 1H, ArOH), 9.18 (br s, 1H, OH), 7.09 (d,
2H, J = 8.7 Hz, H-2, H-6), 6.72 (d, 2H, J = 8.7 Hz, H-3, H-5), 6.17 (d,
1H, 1.8 Hz, H-30), 6.01 (s, 1H, H-50), 5.48–5.29 (m, 4H, H-9000, H-
10000), 5.04 (d, 1H, J = 6.9 Hz, H-100), 4.38 (br d, 1H, J = 11.4 Hz, H-
6a00), 4.18 (dd, 1H, J = 11.4 Hz, 6.6 Hz, H-6b00), 3.67 (br t, 1H,
absorbance ðblankÞ ꢀ absorbance ðsampleÞ
% Inhibition ¼
ꢁ 100
J = 7.8 Hz, H-400), 3.55–3.23 (m, 6H, 2 ꢁ H , H-200, H-300, H-500, OH),
absorbance ðblankÞ
a
2.84 (br t, 2H, J = 6.9 Hz, 2 ꢁ Hb), 2.32 (br t, 2H, J = 6.9 Hz, 2 ꢁ H-
2000), 2.01–1.98 (m, 4H, 2 ꢁ H-8000, 2 ꢁ H-11000), 1.54–1.50 (m, 2H,
2 ꢁ H-3000), 1.27–1.20 (m, 20H, 10(CH2)), 0.87 (br t, 3H, J = 6.9 Hz,
CH3); 13C NMR (DMSO-d6, 75 MHz): d 205.37 (CO), 173.41 (OCO),
166.13 (C-40), 165.15 (C-60), 161.34 (C-20), 156.06 (C-4), 132.30
(C-1), 130.30 (C-9000, C-10000), 129.73 (C-2, C-6), 115.78 (C-3, C-5),
106.25 (C-10), 101.63 (C-100), 97.86 (C-30), 95.50 (C-50), 77.37 (C-
300), 74.83 (C-500), 73.98 (C-200), 70.74 (C-400), 63.85 (C-600), 45.63
2.8. Statistical analysis
One way ANOVA was performed to analyse all data from antiox-
idant assays using MINITAB 16 and when there were significant
differences at P 60.05, the means were compared using Tukey’s
multiple means comparison test. All the analyses were performed
in triplicate.
(C ), 34.19 (C-2000), 31.97 (Cb), 29.83 (C-7000, C-12000), 29.54 (C-15000),
a
29.33 (C-4000, C-5000, C-14000), 29.20 (C-6000, C-13000, C-16000), 27.33 (C-
8000, C-11000), 25.10 (C-3000), 22.74 (C-17000), 14.50 (C-18000).
2.9. Biocatalytic synthesis
2.9.1. Procedure for the synthesis of saturated/unsaturated fatty
acid esters of phloridzin and isoquercitrin (2–7 and 9–14)
To flame dried 3 Å molecular sieves, in round bottom flask, was
added phloridzin (0.500 g; 1.15 mmol), stearic acid (1.62 g,
5.72 mmol), Novozyme 435Ò (1.30 g). It was followed by the addi-
tion of dry acetone (5 ml) and the mixture was stirred and heated
at 45 °C for 12–24 h. The progress of reaction was monitored by
thin layer chromatography (TLC), followed by staining with anisal-
dehyde spray reagent and then heating at 110 °C. After completion
of reaction, it was filtered, evaporated and passed through column
chromatography (acetone/toluene; 35:75 to 50:50) to get the pure
stearic acid ester (2) of phloridzin. All other reactions followed the
same procedure and produced esters 3–7 and 9–14, with yields in
the range of 81–98%. The pure compounds were then analyzed by
IR, 1H NMR and 13C NMR spectroscopy. All other compounds were
prepared by the same method.
2.9.4. (6-{3,5-Dihydroxy-2-[3-(4-hydroxyphenyl)propanoyl]
phenoxy}-3,4,5-trihydroxytetra-hydro-2H-pyran-2-yl)methyl
(9Z,12Z)-9,12-octadecadienoate (4)
Yield: 93%; light brownish yellow spongy solid; Rf: 0.57 (ace-
tone/toluene; 4:6: few drops of AcOH); IR (KBr) cmꢀ1: 3396,
2928, 2856, 2253, 1711, 1628, 1599, 1515, 1454, 1378, 1263,
1204, 1173, 1080, 907, 733, 650; 1H NMR (DMSO-d6, 300 MHz): d
10.62 (br s, 1H, ArOH), 9.16 (br s, 1H, ArOH), 7.08 (d, 2H,
J = 8.1 Hz, H-2, H-6), 6.70 (d, 2H, J = 8.1 Hz, H-3, H-5), 6.16 (d, 1H,
J = 1.2 Hz, H-30), 6.01(d, 1H, J = 1.2 Hz, H-50), 5.47 (br s, 1H, OH),
5.40–5.27 (m, 5H, H-9000, H-10000, H-12000, H-13000, OH), 5.04 (d, 1H,
J = 6.9 Hz, H-100), 4.37 (br d, 1H, J = 11.4 Hz, H-6a00), 4.18 (dd, 1H,
J = 11.4 Hz, 6.6 Hz, H-6b00), 3.66 (br t, 1H, J = 7.8 Hz, H-400), 3.57
(br s, 2 ꢁ OH), 3.48–3.22 (m, 5H, 2 ꢁ H , H-200, H-300, H-500), 2.83
a
(br t, 2H, J = 6.9 Hz, 2 ꢁ Hb), 2.74 (br t, 2H, J = 5.4 Hz, 2 ꢁ H-11000),
2.30 (br t, 2H, J = 7.5 Hz, 2 ꢁ H-2000), 2.15–1.99 (m, 4H, 2 ꢁ H-8000,
2 ꢁ H-14000), 1.53–1.48 (m, 2H, 2 ꢁ H-3000), 1.37–1.18 (m, 14H,
7(CH2)), 0.86 (br t, 3H, J = 6.6 Hz, CH3); 13C NMR (DMSO-d6,
75 MHz): d 205.42 (CO), 173.45 (OCO), 166.20 (C-40), 165.16
(C-60), 161.38 (C-20), 156.09 (C-4), 132.36 (C-1), 130.50 (C-9000,
C-13000), 129.75 (C-2, C-6), 128.48 (C-10000, C-12000), 115.84 (C-3, C-
5), 106.33 (C-10), 101.68 (C-100), 97.94 (C-30), 95.56 (C-50), 77.43
(C-300), 74.89 (C-500), 74.03 (C-200), 70.80 (C-400), 63.88 (C-600), 45.66
2.9.2. (6-{3,5-Dihydroxy-2-[3-(4-hydroxyphenyl)propanoyl]
phenoxy}-3,4,5-trihydroxytetra-hydro-2H-pyran-2-yl)methyl
stearate (2)
Yield: 98%; light yellow solid; Rf: 0.53 (acetone/toluene; 4:6:
few drops of AcOH); IR (KBr) cmꢀ1: 3383, 2944, 2832, 2523,
2247, 2043, 1719, 1631, 1591, 1512, 1450, 1269, 1113, 1028,

Ziaullah et al. / Bioorg. Med. Chem. 21 (2013) 684–692
687
(C ), 34.23 (C-2000), 31.64 (Cb), 29.95 (C-7000), 29.73 (C-15000), 29.44 (C-
1390, 1262, 1204, 1173, 1078, 907, 733, 650; 1H NMR (DMSO-d6,
300 MHz): d 10.62 (br s, 1H, OH), 9.15 (br s, 1H, OH), 7.06 (d, 2H,
J = 8.4 Hz, H-2, H-6), 6.68 (d, 2H, J = 8.4 Hz, H-3, H-5), 6.14 (d, 1H,
J = 1.5 Hz, H-30), 5.98 (d, 1H, J = 1.5 Hz, H-50), 5.46–5.25 (m, 14H,
H-4000, H-5000, H-7000, H-8000, H-10000, H-11000, H-13000, H-14000, H-16000, H-
17000, H-19000, H-20000, 2 ꢁ OH), 5.02 (d, 1H, J = 6.3 Hz, H-100), 4.38 (br
d, 1H, J = 12.0 Hz, H-6a00), 4.17 (dd, 1H, J = 12.0 Hz, 6.9 Hz, H-6b00),
a
4000), 29.23 (C-5000, C-6000, C-11000), 27.39 (C-8000, C-14000), 25.99 (C-16000),
25.13 (C-3000), 22.65 (C-17000), 14.47 (C-18000).
2.9.5. (6-{3,5-Dihydroxy-2-[3-(4-hydroxyphenyl)propanoyl]
phenoxy}-3,4,5-trihydroxytetra-hydro-2H-pyran-2-yl)methyl
(9Z,12Z,15Z)-9,12,15-octadecatrienoate (5)
Yield: 94%; light brownish yellow spongy solid; Rf: 0.52 (ace-
tone/toluene; 4:6: few drops of AcOH); IR (KBr) cm ꢀ1: 3373,
2944, 2831, 2520, 2247, 2043, 1708, 1449, 1365, 1270, 1228,
1112, 1028, 915, 736, 653; 1H NMR (DMSO-d6, 300 MHz): d 10.62
(br s, 1H, OH), 9.15 (br s, 1H, OH), 7.07 (d, 2H, J = 8.4 Hz, H-2, H-
6), 6.70 (d, 2H, J = 8.4 Hz, H-3, H-5), 6.15 (d, 1H, J = 1.2 Hz, H-30),
6.00(d, 1H, J = 1.8 Hz, H-50), 5.49–5.25 (m, 8H, H-9000, H-10000, H-
12000, H-13000, H-15000, H-16000, 2 ꢁ OH), 5.03 (d, 1H, J = 6.6 Hz, H-100),
4.36 (br d, 1H, J = 11.4 Hz, H-6a00), 4.16 (dd, 1H, J = 11.4 Hz,
7.2 Hz, H-6b00), 3.65 (br t, 1H, J = 8.4 Hz, H-400), 3.49–3.21 (m, 7H,
3.65 (br t, 1H, J = 8.4 Hz, H-400), 3.44–3.22 (m, 7H, 2 ꢁ H , H-200,
a
H-300, H-500, 2 ꢁ OH), 2.85–2.78 (m, 12H, 2 ꢁ Hb, 2 ꢁ H-6000, 2 ꢁ H-
9000, 2 ꢁ H-12000, 2 ꢁ H-15000, 2 ꢁ H-18000), 2.40–2.28 (m, 4H, 2 ꢁ H-
2000, 2 ꢁ H-3000), 2.15–2.00 (m, 2H, 2 ꢁ H-21000), 0.93 (br t, 3H,
J = 7.5 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz): d 205.27 (C@O),
174.86 (OC@O), 166.65 (C-40), 163.61 (C-60), 161.18 (C-20), 154.49
(C-4), 134.27 (C-1), 132.51 (C-20000), 130.22 (C-5000), 129.81 (C-2,
C-6), 129.15 (C-17000), 129,03 (C-14000), 128.89 (C-11000, C-16000),
128.59 (C-8000, C-10000), 128.40 (C-13000, C-7000), 128.00 (C-4000), 127.60
(C-19000), 115.52 (C-3, C-5), 107.53 (C-10), 101.32 (C-100), 99.51 (C-
30), 95.97 (C-50), 76.96 (C-300), 74.87 (C-500), 73.94 (C-200), 70.88 (C-
2 ꢁ H , H-200, H-300, H-500, 2 ꢁ OH), 2.84–2.77 (m, 6H, 2 ꢁ Hb,
a
2 ꢁ H-11000, 2 ꢁ H-14000), 2.30 (br t, 2H, J = 7.2 Hz, 2 ꢁ H-2000), 2.15–
1.98 (m, 4H, 2 ꢁ H-8000, 2 ꢁ H-17000), 1.52–1.48 (m, 2H, 2 ꢁ H-3000),
1.30–1.18 (m, 8H, 4(CH2)), 0.98 (br t, 3H, J = 7.5 Hz, CH3); 13C
NMR (DMSO-d6, 75 MHz): d 205.37 (CO), 173.43 (OCO), 166.08
(C-40), 165.12 (C-60), 161.31 (C-20), 156.04 (C-4), 132.25 (C-1, C-
16000), 130.66 (C-9000), 129.73 (C-2, C-6), 128.68 (C-10000, C-13000),
127.70 (C-12000), 115.78 (C-3, C-5), 106.27 (C-10), 101.60 (C-100),
97.85 (C-30), 95.49 (C-50), 77.35 (C-300), 74.82 (C-500), 73.97 (C-200),
400), 63.81 (C-600), 45.89 (C ), 34.57 (C-2000), 31.05 (C-18000), 30.21
a
(C-12000), 29.82 (Cb), 26.17 (C-21000, C-15000), 26.07 (C-9000), 23.08 (C-
6000), 20.94 (C-3000), 14.47 (C-22000).
2.9.8. (6-{[2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl]oxy}-4,5-dihy-droxytetrahydro-2H-pyran-2-
yl)methyl stearate (9)
Yield: 97%; greenish solid; Rf: 0.42 (acetone/toluene; 1:1: few
drops of AcOH); IR (KBr) cmꢀ1: 3449, 3271, 3070, 3007, 2926,
2854, 2559, 2250, 2124, 1997, 1830, 1767, 1655, 1454, 1366,
1308, 1203, 1172, 1057, 931, 822, 759, 731, 623; 1H NMR
(DMSO-d6, 300 MHz): d 10.63–8.98 (br s, 2H, ArOH), 7.56 (br s,
2H, H-20, H-60), 6.86 (br s, 1H, H-50), 6.41 (s, 1H, H-8), 6.22 (s, 1H,
H-6), 5.47–5.26 (m, 3H, H-100, 2OH), 4.20 (br d, 1H, J = 10.5 Hz, H-
6a00), 4.00 (br s, 1H, H-6b00), 3.55–3.20 (m, 5H, H-200, H-300, H-400,
H-500, OH), 2.00 (br s, 2H, H-2000, OH), 1.24–1.09 (m, 31H, 15xCH2,
OH), 0.83 (br. s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz): d 178.14
(CO), 173.09 (OCO), 165.09 (C-7), 162.09 (C-5), 157.16 (C-2, C-9),
149.25 (C-40), 145.59 (C-30), 133.97 (C-3), 122.19 (C-10), 121.95
(C-60), 116.98 (C-50), 115.90 (C-20), 104.66 (C-10), 101.73 (C-100),
99.47 (C-6), 94.21 (C-8), 77.26 (C-300), 75.01 (C-500), 74.79 (C-200),
71.01 (C-400), 63.78 (C-600), 34.00 (C-2000), 31.99 (C-16000), 29.73
29.37, 29.23, 29.06 (C-4000, C-5000, C-6000, C-7000, C-8000, C-9000, C-10000, C-
11000, C-12000, C-13000, C-14000, C-15000), 24.95 (C-3000), 22.74 (C-17000),
14.51 (C-18000).
70.73 (C-400), 63.83 (C-600), 45.62 (C ), 34.18 (C-2000), 31.22 (Cb),
a
29.89 (C-7000), 29.67 (C-5000), 29.17 (C-4000, C-6000), 27.36 (C-8000),
25.93 (C-11000), 25.85 (C-14000), 25.08 (C-3000), 20.72 (C-17000), 14.67
(C-18000).
2.9.6. (6-{3,5-Dihydroxy-2-[3-(4-hydroxyphenyl)propanoyl]
phenoxy}-3,4,5-trihydroxytetra-hydro-2H-pyran-2-yl)methyl
(5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-icosapentaenoate (6)47–49
Yield: 85%; light brownish yellow spongy solid; Rf: 0.57 (ace-
tone/toluene; 4:6: few drops of AcOH); IR (KBr) cm ꢀ1: 3386,
3013, 2964, 2931, 2836, 2253, 1710, 1628, 1599, 1514, 1451,
1366, 1261, 1204, 1079, 908, 733, 650; 1H NMR (DMSO-d6,
300 MHz): d 10.62 (br s, 1H, OH), 9.15 (br s, 1H, OH), 7.06 (d, 2H,
J = 8.4 Hz, H-2, H-6), 6.69 (d, 2H, J = 8.4 Hz, H-3, H-5), 6.15 (d, 1H,
J = 1.5 Hz, H-30), 5.99 (d, 1H, J = 1.5 Hz, H-50), 5.45–5.32 (m, 12H,
H-5000, H-6000, H-8000, H-9000, H-11000, H-12000, H-14000, H-15000, H-17000, H-
8000, 2 ꢁ OH), 5.03 (d, 1H, J = 6.6 Hz, H-100), 4.36 (br d, 1H,
J = 11.4 Hz, H-6a00), 4.15 (dd, 1H, J = 11.4 Hz, 6.6 Hz, H-6b00), 3.65
(br t, 1H, J = 8.4 Hz, H-400), 3.49–3.24 (m, 7H, 2 ꢁ H , H-200, H-300,
a
H-500, 2 ꢁ OH), 2.83–2.76 (m, 10H, 2 ꢁ Hb, 2 ꢁ H-7000, 2 ꢁ H-10000,
2 ꢁ H-13000, 2 ꢁ H-16000), 2.32 (br t, 2H, J = 7.5 Hz, 2 ꢁ H-2000), 2.15–
2.01 (m, 4H, 2 ꢁ H-4000, 2 ꢁ H-19000), 1.63–1.53 (m, 2H, 2 ꢁ H-3000),
0.94 (br t, 3H, J = 7.5 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz): d
205.02 (C=O), 174.69 (OC@O), 166.28 (C-40), 162.77 (C-60), 160.31
(C-20), 153.85 (C-4), 133.73 (C-1), 131.99 (C-17000), 129.28 (C-2, C-
6), 129.03 (C-6000), 128.62 (C-5000, C-14000), 128.36 (C-12000), 128.31
(C-8000), 128.12 (C-11000 or C-9000), 128.06 (C-11000 or C-9000), 127.86
(C-15000), 127.05 (C-18000), 115.52 (C-3, C-5), 106.77 (C-10), 100.63
(C-100), 98.72 (C-30), 95.06 (C-50), 76.90 (C-300), 74.17 (C-500), 73.36
2.9.9. (6-{[2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl]oxy}-3,4,5-trihy-droxytetrahydro-2H-pyran-2-
yl)methyl (Z)-9-octadecenoate (10)
Yield: 92%; greenish yellow spongy solid; Rf: 0.58 (acetone/tol-
uene; 1:1: few drops of AcOH); IR (KBr) cmꢀ1: 3373, 3065, 2947,
2836, 2497, 2251, 2121, 2042, 1898, 1763, 1640, 1596, 1560,
1459, 1448, 1229, 1165, 1026, 942, 739; 1H NMR (DMSO-d6,
300 MHz): d 11.45–9.01 (br s, 2H, ArOH), 7.53 (d, 2H, J = 9.0 Hz,
H-20, H-60), 6.85 (d, 1H, J = 9.0 Hz, H-50), 6.41 (d, 1H, J = 1.8 Hz, H-
8), 6.22 (d, 1H, J = 1.8 Hz, H-6), 5.48–5.25 (m, 5H, H-9000, H-10000,
H-100, 2OH), 4.20 (br.d, 1H, J = 11.7 Hz, H-6a00), 3.99 (dd, 1H,
J = 11.7 Hz, J = 7.2 Hz, H-6b00), 3.45–3.13 (m, 6H, H-200, H-300, H-400,
H-500, 2 ꢁ OH), 2.06–1.97 (m, 6H, 2 ꢁ H-2000, 2 ꢁ H-8000, 2 ꢁ H-11000),
1.27–1.09 (m, 23H, 11xCH2), 0.85 (br t, 3H, J = 6.6 Hz, CH3); 13C
NMR (DMSO-d6, 75 MHz): d 178.12 (CO), 173.05 (OCO), 164.99
(C-7), 162.06 (C-5), 157.12 (C-2, C-9), 149.21 (C-40), 145.56 (C-30),
133.92 (C-3), 130.29 (C-9000, C-10000), 122.16 (C-10), 121.93 (C-60),
116.95 (C-50), 115.88 (C-20), 104.65 (C-10), 101.65 (C-100), 99.42
(C-6), 94.18 (C-8), 77.23 (C-300), 74.98 (C-500), 74.76 (C-200), 70.98
(C-400), 63.75 (C-600), 33.96 (C-2000), 31.92 (C-16000), 29.79 (2C),
29.49, 29.29 (3C), 29.13, 29.03 (C-4000, C-5000, C-6000, C-7000, C-12000, C-
(C-200), 70.32 (C-400), 63.21 (C-600), 45.31 (C ), 33.55 (C-2000), 29.45
a
(Cb), 29.23 (C10000 or C-13000), 26.46 (C-4000), 25.61 (C-16000, C-7000, C-
13000 or C-10000), 24.66 (C-3000), 20.43 (C-19000), 13.97 (C-20000).
2.9.7. (6-{3,5-Dihydroxy-2-[3-(4-
hydroxyphenyl)propanoyl]phenoxy}-3,4,5-trihydroxytetra-
hydro-2H-pyran-2-yl)methyl (4Z,7Z,10Z,13Z,16Z,19Z)-
4,7,10,13,16,19-docosahexaenoate (7)47–49
Yield: 82%; light brownish yellow spongy solid; Rf: 0.58 (ace-
tone/toluene; 4:6: few drops of AcOH); IR (KBr) cmꢀ1: 3386,
3014, 2964, 2927, 2832, 2253, 1718, 1627, 1599, 1514, 1450,

688
Ziaullah et al. / Bioorg. Med. Chem. 21 (2013) 684–692
13000, C-14000, C-15000), 27.31 (C-8000, C-11000), 27.90 (C-3000), 22.69 (C-
17000), 14.49 (C-18000).
2 ꢁ H-16000), 2.14–1.85 (m, 6H, 2 ꢁ H-2000, 2 ꢁ H-4000, 2 ꢁ H-19000),
1.39–1.24 (m, 3H, 2 ꢁ H-3000, OH), 0.92 (br t, 3H, J = 7.8 Hz, CH3);
13C NMR (DMSO-d6, 75 MHz): d 178.16 (CO), 172.95 (OCO),
165.05 (C-7), 162.09 (C-5), 157.16 (C-2, C-9), 149.26 (C-40),
145.60 (C-30), 133.96 (C-3), 132.31 (C-18000), 129.61 (C-5000),
129.01, 128.89 128.79, 128.64, 128.47 (C-6000, C-8000, C-9000, C-11000,
C-12000, C-14000, C-15000, C-17000), 122.21 (C-10), 121.96 (C-60), 116.99
(C-50), 115.93 (C-20), 104.69 (C-10), 101.71 (C-100), 99.47 (C-6),
94.24 (C-8), 77.24 (C-300), 75.01 (C-500), 74.79 (C-200), 70.98 (C-400),
63.85 (C-600), 33.46 (C-2000), 26.63 (C-4000), 25.95 (C-7000, C-10000),
25.88 (C-16000, C-13000), 24.92 (C-3000), 20.74 (C-19000), 14.71 (C-18000).
2.9.10. (6-{[2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl]oxy}-3,4,5-tri-hydroxytetrahydro-2H-pyran-2-
yl)methyl (9Z,12Z)-9,12-octadecadienoate (11)
Yield: 94%; greenish yellow spongy solid; Rf: 0.59 (acetone/tol-
uene; 1:1: few drops of AcOH); IR (KBr) cmꢀ1: 3346, 2946, 2835,
2492, 2181, 2043, 1897, 1762, 1643, 1591, 1466, 1447, 1230,
1027, 941, 737, 700; 1H NMR (DMSO-d6, 300 MHz): d 11.46–8.97
(br s, 2H, ArOH), 7.58 (br s, 2H, H-20, H-60), 6.88 (br d, 1H,
J = 6.9 Hz, H-50), 6.43 (s, 1H, H-8), 6.24 (s, 1H, H-6), 5.50–5.37 (m,
7H, H-9000, H-10000, H-12000, H-13000, H-100, 2OH), 4.22 (br.d, 1H,
J = 10.5 Hz, H-6a00), 4.02 (dd, 1H, J = 10.5 Hz, J = 7.2 Hz, H-6b00),
3.55–3.20 (m, 6H, H-200, H-300, H-400, H-500, 2 ꢁ OH), 2.78 (br s, 2H,
2 ꢁ H-11000), 2.16–2.04 (m, 6H, 2 ꢁ H-2000, 2 ꢁ H-8000, 2 ꢁ H-14000),
1.29–1.11 (m, 17H, 8xCH2, OH), 0.88 (br s, 3H, CH3); 13C NMR
(DMSO-d6, 75 MHz): d 178.12 (CO), 173.05 (OCO), 165.01 (C-7),
162.06 (C-5), 157.13 (C-2, C-9), 149.22 (C-40), 145.58 (C-30),
133.91 (C-3), 130.49 (C-9000, C-10000), 128.45 (C-12000, C-13000),122.17
(C-10), 121.93 (C-60), 116.96 (C-50), 115.89 (C-20), 104.65 (C-10),
101.64 (C-100), 99.43 (C-6), 94.19 (C-8), 77.22 (C-300), 74.99 (C-500),
74.77 (C-200), 70.98 (C-400), 63.75 (C-600), 33.96 (C-2000), 31.57
(C-16000), 29.66 (C-15000), 29.38 (C-7000), 29.16 (C-5000), 29.03 C-4000,
C-6000), 27.34 (C-8000, C-14000), 25.94 (C-11000), 24.91 (C-3000), 22.59
(C-17000), 14.48 (C-18000).
2.9.13. (6-{[2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl]oxy}-3,4,5-tri-hydroxytetrahydro-2H-pyran-2-
yl)methyl (4Z,7Z,10Z,13Z,16Z,19Z)-4,7,10,13,16,19-docosa-
hexaenoate (14)47–49
Yield: 81%; greenish yellow spongy solid; Rf: 0.57 (acetone/tol-
uene; 1:1: few drops of AcOH); IR (KBr) cmꢀ1: 3373, 2944, 2913,
2835, 2678, 2499, 2251, 2182, 2045, 1890, 1767, 1651, 1592,
1466, 1447, 1383, 1229, 1027, 909, 732, 649; 1H NMR (DMSO-d6,
300 MHz):
d 11.61–8.96 (br s, 2H, ArOH), 7.52 (br d, 2H,
J = 8.1 Hz, H-20, H-60), 6.83 (d, 1H, J = 8.1 Hz, H-50), 6.38 (s, 1H, H-
8), 6.19 (s, 1H, H-6), 5.44–5.15 (m, 15H, H-400, H-500, H-700, H-800,
H-10000, H-11000, H-13000, H-14000, H-16000, H-17000, H-19000, H-20000, H-100,
2OH), 4.17 (br d, 1H, J = 11.4 Hz, H-6a00), 3.96 (dd, 1H, J = 11.4 Hz,
J = 6.6 Hz, H-6b00), 3.49 (br s, OH), 3.34–3.17 (m, 5H, H-200, H-300,
H-400, H-500, OH), 2.75–2.66 (m, 10H, 2 ꢁ H-6000, 2 ꢁ H-9000, 2 ꢁ H-
12000, 2 ꢁ H-15000, 2 ꢁ H-18000), 2.11–1.95 (m, 6H, 2 ꢁ H-2000, 2 ꢁ H-
3000, 2 ꢁ H-21000), 1.20 (br s, 1H, OH), 0.88 (t, 3H, J = 7.5 Hz, CH3);
13C NMR (DMSO-d6, 75 MHz): d 178.14 (CO), 172.53 (OCO),
165.07 (C-7), 162.07 (C-5), 157.16 (C-2, C-9), 149.26 (C-40),
145.60 (C-30), 134.00 (C-3), 132.30 (C-20000), 129.17 (C-4000),
128.88, 128.64, 128.44, 127.66 (C-5000, C-7000, C-8000, C-10000, C-11000,
C-13000,C-14000, C-16000, C-17000, C-19000), 122.20 (C-10), 121.95 (C-60),
117.01 (C-50), 115.92 (C-20), 104.68 (C-10), 101.81 (C-100), 99.49
(C-6), 94.27 (C-8), 77.24 (C-300), 74.96 (C-500), 74.79 (C-200), 70.94
(C-400), 63.94 (C-600), 33.94 (C-2000), 25.95 (C-6000, C-9000, C-12000,
C-15000, C-18000), 22.86 (C-3000), 20.73 (C-21000), 14.69 (C-18000).
2.9.11. (6-{[2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl]oxy}-3,4,5-tri-hydroxytetrahydro-2H-pyran-2-
yl)methyl (9Z,12Z,15Z)-9,12,15-octadecatrienoate (12)
Yield: 91%; greenish yellow spongy solid; Rf: 0.56 (acetone/tol-
uene; 1:1: few drops of AcOH); IR (KBr) cmꢀ1: 3346, 2946, 2835,
2492, 2180, 2049, 1895, 1765, 1645, 1594, 1464, 1447, 1230,
1027, 941, 736, 701; 1H NMR (DMSO-d6, 300 MHz): d 11.48–8.99
(br s, 2H, ArOH), 7.52 (br s, 2H, H-20, H-60), 6.82 (br d, 1H,
J = 8.7 Hz, H-50), 6.38 (s, 1H, H-8), 6.18 (s, 1H, H-6), 5.43–5.31 (m,
9H, H-9000, H-10000, H-12000, H-13000, H-15000, H-16000, H-100, 2OH), 4.16
(br.d, 1H, J = 11.4 Hz, H-6a00), 3.96 (dd, 1H, J = 11.4 Hz, J = 7.1 Hz,
H-6b00), 3.43–3.15 (m, 5H, H-200, H-300, H-400, H-500, OH), 2.84–2.71
(m, 4H, 2 ꢁ H-11000, 2 ꢁ H-14000), 2.11–1.98 (m, 6H, 2 ꢁ H-2000,
2 ꢁ H-8000, 2 ꢁ H-17000), 1.26–1.05 (m, 12H, 5xCH2, 2OH), 0.90 (br t,
3H, J = 7.2 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz): d 178.11 (CO),
173.03 (OCO), 164.95 (C-7), 162.04 (C-5), 157.10 (C-2, C-9),
149.19 (C-40), 145.55 (C-30), 133.91 (C-3), 132.22 (C-16000), 130.67
(C-9000), 128.65 (C-10000, C-12000), 128.18 (C-13000), 127.68 (C-15000),
122.15 (C-10), 121.91 (C-60), 116.94 (C-50), 115.87 (C-20), 104.64
(C-10), 101.63 (C-100), 99.40 (C-6), 94.16 (C-8), 77.21 (C-300), 74.96
(C-500), 74.74 (C-200), 70.96 (C-400), 63.73 (C-600), 33.93 (C-2000),
29.62 (C-7000), 29.12 (C-5000), 29.00 (C-4000, C-6000), 27.33 (C-8000),
25.90 (C-11000), 25.81 (C-14000), 24.87 (C-3000), 20.68 (C-17000), 14.65
(C-18000).
3. Results and discussion
3.1. Chemistry
Acylation of phloridzin and isoquercitrin with long chain fatty
acids were carried out at 45–50 °C in extra dry acetone along with
constant stirring. Briefly, defined quantities of flavonoids and fatty
acids were dissolved in acetone. Enzymatic reactions were initi-
ated by the addition of lipase (Novozyme 435Ò; with an activity
of 10,000 propyl laurate units). As the water content (<200 ppm)
is very important parameter in enzymatic catalysis in organic med-
ia. It was assumed that the activity of the enzyme could be im-
proved by reducing the hydration level of the different
components in the reaction medium. In the present investigation,
the highest efficiency was reached by drying the enzyme for 18–
20 h over P2O5 before use; besides, using flame dried molecular
sieves (3 Å) to remove any in situ generated water in the reaction
mixture (Schemes 1 and 2).
2.9.12. (6-{[2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-
chromen-3-yl]oxy}-3,4,5-tri-hydroxytetrahydro-2H-pyran-2-
yl)methyl (5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-icosapentaeno-ate
(13)47–49
Yield: 81%; greenish yellow spongy solid; Rf: 0.57 (acetone/tol-
uene; 1:1: few drops of AcOH); IR (KBr) cmꢀ1: 3346, 2946, 2835,
2490, 2180, 2044, 1893, 1768, 1647, 1591, 1466, 1446, 1231,
1027, 941, 737, 706; 1H NMR (DMSO-d6, 300 MHz): d 12.23–8.82
(br s, 2H, ArOH), 7.55 (br d, 2H, J = 8.5 Hz, H-20, H-60), 6.87 (d, 1H,
J = 8.5 Hz, H-50), 6.42 (s, 1H, H-8), 6.23 (s, 1H, H-6), 5.49–5.17 (m,
13H, H-500, H-600, H-800, H-9000, H-11000, H-12000, H-14000, H-15000, H-17000,
H-18000, H-100, 2OH), 4.20 (br.d, 1H, J = 11.4 Hz, H-6a00), 3.99 (dd,
1H, J = 11.4 Hz, J = 7.2 Hz, H-6b00), 3.49–3.14 (m, 6H, H-200, H-300,
H-400, 2 ꢁ OH), 2.82–2.71 (m, 8H, 2 ꢁ H-7000, 2 ꢁ H-10000, 2 ꢁ H-13000,
3.1.1. Effect on regioselectivity
The synthesized esters have been evaluated qualitatively by
using NMR spectroscopy. The 1H and 13C NMR assignment have
confirmed their structures. The location of the acylated site has
been well documented in previous studies for a range of acyl do-
nors and is in agreement with our studies.19,20,23 In our case it
was confirmed by comparing the downfield and upfield shifts be-
tween the newly synthesized products and the reported phloridzin

Ziaullah et al. / Bioorg. Med. Chem. 21 (2013) 684–692
689
and isoquercetin.19 13C NMR spectra showed that the acylation on
the 600-OH position led to an downfield shift of the C-600 signal by
approximately 3 ppm (ꢂ60.97 to ꢂ63.83 ppm) due to the reso-
nance effect towards carbonyl of the newly generated ester as well
as an upfield shift of the C-1000 (OCO) signal by approximately
3–4 ppm from their corresponding fatty acids47 (Fig. 1).
ated by long acyl chains of fatty acids. In this study, no significance
in such correlation was observed between the saturation number of
the fatty acids attached to the esters and their antioxidant capacity.
Although the radical scavenging ability of flavonoids were not com-
pletely diminished by esterification with fatty acids but rather the
high antioxidant capacity of these esters was significantly retained.
The isoquercitrin esters had a much greater antioxidant activity,
according to FRAP assay and DPPH assay which shows lower IC50
values than the phloridzin esters. This can be due to the high poten-
tial of radical scavenging ability possessed by isoquercitrin than
phloridzin. To our assumption, the incorporation of amphipathic/
amphiphilic properties in the flavonoid skeleton through esterifica-
tion would facilitate their interaction with membrane bound pro-
teins or receptors as well as enhance their absorption through cell
membrane thus increasing their bioavailability.
3.1.2. Effect of acyl chain length
A slight decrease in yield (compounds 6, 7 and 13, 14) was
observed with the increase in chain length which was possibly
because the longer chain length of fatty acids might have impact
on the active sites of enzyme.
3.1.3. Effect of molar ratios of substrate and acyl donor
The time course of enzymatic acylation of phloridzin and isoqu-
ercitrin was significantly affected by using different molar ratios of
substrate and acyl donor. The acyl donor concentration was
adjusted to have a flavonoid/acyl donor molar ratio of 1:5 for the
complete conversion of phloridzin and isoquercitrin into their
corresponding esters in shorter time.
3.3. Tyrosinase inhibition activity
The percent tyrosinase inhibition by the two novel series of fla-
vonoid esters were concentration dependent (Table 2). Of the two
precursors, the phloridzin (1) exhibited weak tyrosinase inhibition,
as compared to isoquercitrin (8). Among the series of phloridzin
3.2. Antioxidant capacity of flavonoid esters
and its esters (1–7), the
a-linolenic and stearic acid esters (2 and
For the two series of compounds the FRAP assay has resulted in
significantly higher (P 60.05) antioxidant capacity for the parent
compounds phloridzin (1) than their respective esters (2–7)
(Table 1). The phloridzin showed significantly higher (P 60.05) anti-
oxidant capacity than all of its esters in ORAC assay. Of the isoqu-
ercitrin series, the isoquercitrin (8) showed greater antioxidant
capacity in both FRAP and DPPH assays than their esters. Phloridzin
was found to have an IC50 value of 1.489 mmol lꢀ1, a value similar to
that was found by Yang et al.38 Isoquercitrin was found to have an
5) also exhibited weak tyrosinase inhibition. Similar to our present
results, Shoji et al.50, using B 16 mouse melanoma cells, also
showed that phloridzin was a weak tyrosinase inhibitor. However,
docosahexaenoic acid ester (7) of phloridzin exhibited the highest
tyrosinase inhibition (P 60.05) among all the tested esters of phlo-
ridzin (2–7).
In this study, it was discovered that 3 and 4 of phloridzin esters
showed stronger tyrosinase inhibition, with the increase in num-
ber of double bonds of fatty acids via structure-activity relation-
ships (SAR). The same trend of the increase in inhibitory
potential, with the increasing number of double bonds, has been
almost followed by compounds 6 and 7. However, as opposed to
isoquercitrin eicosapentaenoic acid and docosahexaenoic acid es-
ters (12 and 13, respectively), the eicosapentaenoic acid and doco-
sahexaenoic acid esters of phloridzin (6 and 7, respectively)
exhibited moderate to significant inhibition potency, which can
be explained based on the rationale of the structure of phloridzin.
The phloridzin backbone is more flexible (more conformational
isomers) in 3-dimensions, as compared to isoquercitrin, due to
IC50 value of 18 l
mol lꢀ1, which was very comparable to that found
by Shibano et al.39 The esters of isoquercitrin showed to have
poorer antioxidant capacity, and higher IC50 values than their par-
ent flavonoid, which was a consistent trend of acylated flavonoids
found in literature.19,41,42 Lue et al.42 reported the same for rutin
and its esters and it is explained that accessibility of hydroxyl
groups to the free radicals is obstructed by the steric hindrance cre-
OH
OH
the free rotation at C-
fatty acids (C20–C22) are attached to their skeleton, they result in
the orientation (through rotation at C- and C-b) of these flexible
molecules (2–7) in a way that some of their conformations may
help in better interaction with tyrosinase, in terms of adjusting
the positions of phenolic functionalities, as well as of the fatty acid
side chains. On the other hand, an increase in double bonds in the
side chains makes the fatty acid parts of the molecules conforma-
a and C-b (Fig. 1). Once the bulky unsaturated
HO
OH
OH
O
HO
OH
OH
O
a
a
+ H2O
RCOOH
O
O
HO
HO
HO
HO
O
O
OR
OH
2-7
1
tionally less flexible and hence, the p-electrons are frequently
available for some lipophilic interactions with the tyrosinase, in
addition to interaction of the polar polyphenolic functionalities of
these molecules with the binuclear copper metal of the enzyme.
OH
OH
OH
OH
HO
O
HO
O
Among the isoquercitrin series (8–14), the a-linolenic acid ester
(12) of isoquercitrin was the strongest tyrosinase inhibitor, at all
tested concentrations (P 60.05). It was followed by linoleic acid
(11), stearic acid (9), and oleic acid (10) esters of isoquercitrin,
respectively. The parent flavonoid, isoquercitrin (8), exhibited rea-
sonable tyrosinase activity at the tested concentration levels.
Considering the structure-activity relationship, it can be found
that the inhibition potential of the isoquercitrin series (8–14) in-
creases first, rises to its maximum (12) and then decreases (13
and 14) with the addition of double bonds and/or the chain length.
From the activity profile, it can be concluded that the presence of
double bonds is crucial for inhibition but the increase in chain
a
O
+ H2O
O
RCOOH
O
HO
OH
O
HO
OH
O
O
OH
OR
HO
HO
OH
OH
9-14
8
Scheme 1. Acylation of phloridzin and isoquercetrin using lipases. (a) Acetone, 3° A
molecular sieves, novozyme 435Ò, 45 °C, stirring, 24 h; R = oleic, stearic, linoleic,
linolenic, eicosapentaenoic (EPA), and docosahexaenoic acids or their esters.

690
Ziaullah et al. / Bioorg. Med. Chem. 21 (2013) 684–692
Yield
Compd.
2
Structure
Compd.
9
Structure
Yield
OH
OH
98 %
97 %
OH
OH
O
HO
HO
O
O
O
O
OH
HO
HO
HO
O
O
HO
OH
OH
O
O
O
O
OH
OH
3
96 %
10
92 %
94 %
OH
HO
OH
O
HO
O
O
O
O
OH
HO
HO
HO
O
O
HO
OH
OH
O
O
9''' 10'''
10'''
9'''
O
O
OH
OH
4
93 %
11
OH
HO
OH
O
HO
O
O
O
O
OH
HO
O
HO
HO
O
HO
OH
OH
O
O
O
O
OH
OH
5
94 %
85 %
12
91 %
81 %
OH
HO
OH
O
HO
O
O
O
O
OH
HO
HO
HO
O
O
HO
OH
OH
O
O
O
O
OH
OH
6
13
OH
HO
OH
HO
O
O
O
O
O
OH
HO
HO
HO
O
O
HO
OH
OH
O
O
O
O
OH
OH
7
82 %
14
81 %
OH
HO
OH
HO
O
O
O
O
O
OH
HO
HO
HO
O
O
HO
OH
OH
O
O
O
O
Scheme 2. Structures and percent yield of acylated fatty acid derivatives of phloridzin and isoquercetrin.
length, after a certain limit, prohibits the molecule from entering
the enzyme core, which can be seen in case of esters 13 and 14,
where the addition of double bonds is counterfeited by the size
of the chain lengths (C-20 and C-22, respectively).
Based on these results, it can be concluded that these fatty acid
esters of isoquercitrin and phloridzin are potential tyrosinase
inhibitors. The docosahexaenoic acid ester of phloridzin (7), fol-
lowed by
a-linoleic acid ester of isoquercitrin, (12), emerged as

Ziaullah et al. / Bioorg. Med. Chem. 21 (2013) 684–692
691
3'
6'
OH
OH
4'
5'
2'
3'
OH
4'
5'
5
2'
1'
HO
HO
6
OH
O
4
1
1'
8
β
2
O
HO
7
α
3
6'
1
3
2
6
O
O
1''
4
5
O
OH
2''
1''
O
2''
4''
HO
O
3''
HO
5''
3''
6''
5''
4''
HO
6''
OH
22'''
21'''
OH
22'''
21'''
O
1'''
1'''
O
18'''
15'''
3'''
6'''
9'''
12'''
18'''
15'''
3'''
6'''
9'''
12'''
O
O
7
14
Figure 1. Numbering of the esters of phloridzin (7) and isoquercitrin (14).
Table 1
Antioxidant capacity measured by FRAP, ORAC and DPPH radical assays for the two parent flavonoids and their modified esters
Compd no.
Compound
FRAPZ
(l
mol TE lꢀ1
)
ORACY
(l
mol TE lꢀ1
)
DPPH IC50 (l )
mol lꢀ1
1
2
3
4
5
6
7
8
Phloridzin (Pz)
166.3 14.6 f
88.5 6.1 g
90.2 6.6 g
96.0 8.1 g
87.6 7.2 g
120.2 2.6 e
26.0 2.3 h
76.1 6.9 f
1488.9 68.4 e
5265.3 228.4 i
9720.0 149.4 j
901.8 44.7 d
2873.6 144.8 h
1688.8 35.9 f
1907.7 96.4 g
17.9 1.7 a
42.0 3.6a
38.2 0.6 a
60.4 2.8 ab
97.9 4.7 b
36.95 2.1 a
146.5 1.8 c
Stearic acid ester of Pz
Oleic acid ester of Pz
Linoleic acid ester of Pz
61.2 2.6 g
57.8 3.0 g
34.9 4.3 h
64.7 7.7 fg
473.1 1.4 b
310.0 5.4 c
265.4 5.1 d
491.2 0.9 a
490.3 1.8 a
491.8 3.1a
488.9 4.6 a
a
-Linolenic acid ester of Pz
EPA ester of Pz
DHA ester of Pz
Isoquercitrin (Q3G)
Stearic acid ester of Q3G
Oleic acid ester of Q3G
Linoleic acid ester of Q3G
94.3 5.6 g
112.3 10.1 fg
1500.0 16.7 a
622.3 27.9 b
536.0 22.9 cd
340.4 18.9 e
613.8 62.0 b
569.4 18.4 bc
485.0 28.1 d
9
10
11
12
13
14
a
-Linolenic acid ester of Q3G
EPA ester of Q3G
DHA ester of Q3G
(Means standard deviation followed by the same letter within column are not significantly different, Tukey’s multiple means comparison test, P 60.05); Zlmol Trolox
equivalents (TE) lꢀ1 of 1 mmol lꢀ1 of solutions of all the test compounds; YFor phloridzin and its esters:
l
mol lꢀ1 Trolox equivalents of 0.01 mmol lꢀ1 of solution; For
Isoquercitrin and its esters:
acid.
l
mol lꢀ1 Trolox equivalents of 0.001 mmol lꢀ1 of solution; Pz: Phloridzin; Q3G: Isoquercitrin; EPA: Eicosapentaenoic acid; DHA: Docosahexaenoic
and/or skin care products, which can be useful in treatment of var-
ious dermatological disorders.
Table 2
Percentage inhibition of tyrosinase activity in vitro by flavonoid esters
Compd no.
Compound
Concentration (
100
l
M)
4. Conclusions
10
1000
1
2
3
4
5
6
7
8
Phloridzin (Pz)
0
0
0
0
0
0
0
16.56 j
17.17 j
31.85 i
66.96 e
12.53 l
46.25 h
91.21 a
54.55 g
69.20 d
62.44 f
80.58 c
85.55 b
7.06 m
32.53 i
Two series of long chain fatty acid esters of phloridzin and iso-
quercetin were synthesized and their structures were confirmed by
NMR analysis. The study explains how to increase the efficiency of
the acylation reactions by using the excess of acyl donors, as well
as by maintaining the extra dry conditions through the use of extra
dry acetone, flame dried molecular sieve, dried enzyme over P2O5
and keeping the reaction under inert atmosphere, which limits
the possibility of hydrolysis and drags the reaction forward. From
the bioactivity point of view, all the flavonoid esters retained sub-
stantial antioxidant capacity after the esterification. Although the
parent compounds have greater free radical scavenging ability
than their esters, the amphipathic/amphiphilic properties could
make them unique in terms of enhanced bioavailability and biolog-
ical activity. The chain lengths and the number of double bonds of
acyl donors, influenced the antioxidant capacity and ability to inhi-
bit tyrosinase in vitro, by the acylated derivatives of phloridzin and
Stearic acid ester of Pz
Oleic acid ester of Pz
Linoleic acid ester of Pz
a-Linolenic acid ester of Pz
EPA ester of Pz
16.59 i
20.53 h
28.16 g
0
19.59 h
DHA ester of Pz
25.71 d
0
0
38.89 b
32.07 c
55.78 a
0
62.09 c
40.59 f
56.44 d
47.54 e
73.31 b
78.77 a
0
Isoquercitrin (Q3G)
Stearic acid ester of Q3G
Oleic acid ester of Q3G
Linoleic acid ester of Q3G
9
10
11
12
13
14
a
-Linolenic acid ester of Q3G
EPA ester of Q3G
DHA ester Q3G
0
19.28 h
PZ: Phloridzin; Q3G: Isoquercitrin;
each column indicates that corresponding means are not significantly different.
Means separation by Tukey’s multiple means comparison test (P 60.05).
lM: Micromole; a–j: The same letter within
isoquercetin. The
a-linolenic acid ester of isoquercitrin (12) and
promising tyrosinase inhibitors, in vitro. These compounds exhib-
ited significantly higher tyrosinase inhibition (P 60.05) compared
to parent flavonoids, at tested concentrations (Table 2). The results
suggest that these flavonoid esters can serve as candidate mole-
cules for the design and synthesis of tyrosinase inhibitory drugs
docosahexaenoic acid ester of phloridzin (7) showed the greatest
tyrosinase inhibition (P 60.05) potential. Further investigations
are warranted to confirm their biological activities in vivo and to
investigate their possible candidacy for pharmaceutical and cos-
metic applications.

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Ziaullah et al. / Bioorg. Med. Chem. 21 (2013) 684–692
19. Salem, J. H.; Humeau, C.; Chevalot, I.; Harscoat-Schiavo, C.; Vanderesse, R.;
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We are thankful to the financial support of the Discovery Grant
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