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Research Article
Isolation and Biological Evaluation of Prenylated Flavonoids
from Maclura pomifera
Yerkebulan Orazbekov,1,2 Mohamed A. Ibrahim,1,3 Serjan Mombekov,4
Radhakrishnan Srivedavyasasri,1Ubaidilla Datkhayev,4Bauyrzhan Makhatov,2
Narayan D. Chaurasiya,1Babu L. Tekwani ,1,5 and Samir A. Ross 1,5
1National Center for Natural Products Research, University of Mississippi, University, MS 38677, USA
2South-Kazakhstan State Pharmaceutical Academy, Al-Farabi Square, Shymkent 160019, Kazakhstan
3Department of Chemistry of Natural Compounds, National Research Center, Dokki, Cairo 12622, Egypt
4Kazakh National Medical University, Almaty 050000, Kazakhstan
5Department of Biomolecular Science, University of Mississippi, University, MS 38677, USA
Correspondence should be addressed to Samir A. Ross; sross@olemiss.edu
Received 21 September 2017; Accepted 9 December 2017; Published 14 January 2018
Academic Editor: Armando Zarrelli
Copyright © Yerkebulan Orazbekov et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Phytochemical analysis of the ethanolic extract of Maclura pomifera fruits yielded four new compounds (I–IV)alongwith
eleven known compounds (V–XV). e crude extract exhibited signicant activity towards cannabinoid receptors (CB: .%
displacement; CB: .% displacement) and possibly allosteric interaction with 𝛿and 𝜇opioid receptors (−. and −.%
displacement, resp.). Compound Iwas found to be possibly allosteric for 𝜅and 𝜇opioid receptors (−. and −.% displacement,
resp.) and showed moderate activity (.% displacement) towards CB receptor. Compound II exhibited moderate activity towards
cannabinoid receptors CB and CB (. and .% displacement, resp.). e known compounds (V–VIII) exhibited prominent
activity towards cannabinoid receptors: pomiferin (V)(IC
50 of . and . 𝜇M for CB and CB, resp.), auriculasin (VI)(IC
50
of . 𝜇M for CB), warangalone (VII)(IC
50 of . and . 𝜇MforCBandCB,resp.),andosajin(VIII)(IC
50 of . and
. 𝜇M for CB and CB, resp.). e isolated compounds were also tested for inhibition of human monoamine oxidase-A and
monoamine oxidase-B enzymes activities, where all the tested compounds showed fewer inhibitory eects on MAO-A compared
to MAO-B activities: auriculasin (VI)(IC
50 of . and . 𝜇M for MAO-B and MAO-A, resp.).
1. Introduction
Maclura pomifera L. (Maclura aurantiaca Syn., Moraceae
family) is a native southwestern American plant commonly
known as Osage orange. Osage orange typically grows in
sunny areas and can grow in a wide range of soil condi-
tions []. Worldwide, various Maclura species are used in
folk medicine. Native Americans used M. pomifera for the
treatment of cancer []. In Bolivia, the plant sap is used
for the treatment of tooth pain, and the bark and leaves
are used for uterine hemorrhage []. Comanche Indians in
North America used the Osage orange roots decoction to
treat sore eyes []. M. pomifera and its components possess
several biological activities including cytotoxic, antitumor,
antibacterial, estrogenic, antifungal, antiviral, and antimalar-
ialactivities[–].Recently,isoavonesisolatedfromOsage
orange have been demonstrated to protect brain cells, or
neurons, from the toxic eect of amyloid beta peptide, which
is believed to be responsible for the degeneration of neurons
in Alzheimer’s disease patients. However, the mechanisms
by which isoavones block the toxicity of amyloid beta
peptide are unknown []. M.pomifera produces several
secondary metabolites belonging to dierent chemical classes
including prenylated avonoids. e prenylated avonoids
possess dierent biological activities such as antifungal,
antibacterial, antitumor, and antioxidant activities. e wide
range of bioactivities of these compounds is attributed to the
Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2018, Article ID 1370368, 8 pages
https://doi.org/10.1155/2018/1370368
Evidence-Based Complementary and Alternative Medicine
prenylation on the avonoids, which in turn increases their
lipophilicity and membrane permeability []. In this report,
we have examined M. pomifera growing in Kazakhstan which
has never been exposed to extensive phytochemical or bio-
logicalstudies.Wepresenttheisolationandcharacterization
of four new and eleven known metabolites from the fruits of
M. pomifera growinginKazakhstanandtheiraccompanying
cannabinoid, opioid, and MAO receptors activities.
2. Materials and Methods
2.1. Apparatus, Materials, and Chemicals. ABrukermodel
AMX NMR and NMR spectrometers operating
on a standard pulse system were used to acquire 1Hand
13C NMR and D spectra. e instruments ran at and
MHz for 1H while they ran at and MHz for
13C. CDCl3,DMSO-d6, and acetone-d6were used as NMR
solvents, and TMS was used as an internal standard. ESI-
MS data were recorded on ermo Orbitrap Fusion (ermo
Scientic).Sampleswereanalyzedinthenegativemodeof
ionization. Samples were directly infused at uL/min. Mass
was analyzed in Orbitrap (mass error on the instrument
< ppm). ESI-MS data were obtained on a Micromass Q-
Tof micromass spectrometer. FTMS-ESI was analyzed on
ermo Orbitrap Fusion (ermo Scientic). e sample
was analyzed in the negative mode of ionization. Mass was
analyzed in Orbitrap (mass error on the instrument <ppm).
TLC was performed on precoated silica gel GF plates
and Column Chromatography was performed on silica gel
(– mesh) and Sorbadex-LH (Sorbent Technologies,
Atlanta, GA, USA). e recombinant human monoamine
oxidase-A and monoamine oxidase-B enzymes were obtained
from BD Biosciences (Bedford, MA, USA). Kynuramine,
clorgyline, phenelzine, deprenyl, and DMSO were procured
from Sigma Chemical Company (St. Louis, MO, USA).
2.2. Plant Material. Fresh fruits of M. pomifera (L.) ( Kg)
were purchased from Shymkent, Kazakhstan, in October
. A voucher specimen of M. pomifera was identied by
Dr. Kulpan Orynbasarova and deposited at the Department
of Pharmacognosy and Chemistry, South-Kazakhstan State
Pharmaceutical Academy, Shymkent, Kazakhstan, with an
index number “MA-.”
2.3. Extraction and Isolation. Fresh fruits were cut into small
pieces and macerated with ethanol ( × L, h each)
at ∘C. e combined extracts were concentrated under
reduced pressure to yield crude extract (Kg). e extract
showed a yellow precipitate which was ltered and weighed
( g). Eighty grams of the precipitate was loaded on silica
gel and fractionated using DCM-MeOH gradient to yield
fractions (A–A). Fraction A ( g) was loaded on a
silica gel column, where the elution was completed using
DCM-MeOH gradient to yield stigmasterol (XIV,mg)
and 𝛽-sitosterol (XV, mg). Fraction A ( g) was loaded
on Sorbadex-LH and eluted with MeOH-H2Otoyield
pomiferin (V,mg)[],auriculasin(VI,mg)[],
warangalone (VII, mg) [], and osajin (VIII,mg)[].
Chromatographic purication of fraction A (. g)
on Sorbadex-LH using MeOH-H2O gradient yielded
compound I( mg), compound II ( mg), artocarpesin
(IX,mg)[],compoundIII ( mg), compound IV
( mg), kaempferol--O-𝛽-D-glucoside (XII,mg)[],
dihydrokaempferol--O-𝛽-D-glucoside (XIII, mg) [],
tonkinensisol (X, mg) [], and corchoionoside B (XI,
mg) []. e structures of the isolated compounds (Fig-
ure ) were established using NMR (D, D), IR, and mass
spectral data.
2.3.1. 3-(3,4-Dihydroxyphenyl)-5-hydroxy-10-(3-hydroxy-2-me-
thoxy-3-methylbutyl)-8,8-dimethylpyrano[3,2 -g]chromen-
4(8H)-one (I), Named Kazosajin I. IR (neat) cm−1:,
, , . HR-FTMS: m/z[M + Na]+calcd. for
C26H28NaO8: .; found: .. 1H NMR ( MHz,
DMSO-d, Supporting Information (available here)): Table .
13C NMR ( MHz, DMSO-d, Supporting Information
(available here)): Table .
2.3.2. 3-(3,4-Dihydroxyphenyl)-5-hydroxy-6-(2-hydroxy-3-
methylbut-3-enoyl)-8,8-dimethylpyrano[2,3-f]chromen-
4(8H)-one (II), Named Kazosajin II. IR (neat) cm−1:,
, , , cm−1.HR-FTMS:m/z[M −H]−calcd.
for C25H23O8: .; found: .. 1H NMR ( MHz,
DMSO-d, Supporting Information (available here)): Table .
13C NMR ( MHz, DMSO-d, Supporting Information
(available here)): Table .
2.3.3. 11-Hydroxy-7-(4-hydroxyphenyl)-2,2,10,10-tetramethyl-
11,12-dihydro-2H-dipyrano[2,3-f: 2,3-h]chromen-8(10H)-one
(III), Named Kazosajin III. IR (neat) cm−1: , , ,
, , , . HR-FTMS: m/z[M + H]+calcd. for
C25H25O6: .; found: .. 1H NMR ( MHz,
DMSO-d, Supporting Information (available here)): Table .
13C NMR ( MHz, DMSO-d, Supporting Information
(available here)): Table .
2.3.4. 3-(3,4-Dihydroxyphenyl)-10-((3,3-dimethyloxiran-2-
yl)(methoxy)methyl)-5-hydroxy-8,8-dimethylpyrano[3,2-
g]chromen-4(8H)-one (IV), Named Kazosajin IV. IR (neat)
cm−1: , , , , , . HR-FTMS: m/z
[M −H]−calcd. for C26H25O8: .; found: ..
1H NMR ( MHz, DMSO-d, Supporting Information
(available here)): Table . 13C NMR ( MHz, DMSO-d,
Supporting Information (available here)): Table .
2.4. Cannabinoid and Opioid Receptor Assay. e anities
of the total extracts and the isolated compounds towards
cannabinoid and opioid receptors were assessed according to
the published method [].
2.5. MAO-A and MAO-B Inhibition Assay. e inhibitory
eects of the chemical components of M. pomifera (L.) on
MAO-A and MAO-B were determined via the kynuramine
deamination assay, where it was adapted for -well plates
as described earlier [, ]. A xed concentration of the
Evidence-Based Complementary and Alternative Medicine
OO
O
OH
OH
(VII)
(I) (II) (III)
(IV)
O
OOH
OH
O
OH
(V)
O O
OOH
OH
OH
(VI)
(VIII)
O
O
OH
OH
O
(IX)
O
O
OH
HO
OH
OH
(X)
O
O
OH
O
OH
OH
(XI)
O
O
O
OH H
OH
OH
HO
OH
OO
O
OH
OH
OH
MeO
2
4
5
4a
8
8a
O
1
3
5
1
2
3
4
5 1
2
3
4 5
OO
O
OH
OMe
OH
OH
OH
2
4
5
4a
88a
1
3
5
1
2
3
4
5
2
1
3
4 5
O
O
OH
OH
O
OH
OH
2
4
5
4a
88a
O
1
3
5
1
2
3
5
4
1
2
3
4
5
O
O
O
OH
O
HO
2
4
5
4a
88a
1
3
5
2
3
4
5
1
1
2
3
4
5
F : Structures of the selected compounds from M. pomifera.
substrate and varying concentrations of the inhibitor were
used to determine the IC50 value at the point where %
inhibition of the catalytic activity of the enzyme occurred.
For MAO-A, the substrate (kynuramine) concentration of
𝜇Mwaschosen,sincethe𝐾𝑀value of substrate binding
reported previously was approximately 𝜇M[].𝐾𝑀is
the substrate concentration at half Vmax; therefore, 2×𝐾
𝑀
(2×40 = 80𝜇M) was selected for determining the IC50 values.
Similarly, for MAO-B, a substrate (kynuramine) concentra-
tion of 𝜇M was chosen. e assay was performed with the
addition of the inhibitor. Inhibition was calculated as percent
of the product formation compared to the corresponding
control (enzyme-substrate reaction) without the inhibitors.
e enzyme reactions were carried out in . M potassium
phosphate buer at pH .. Reaction mixtures contained
𝜇g/mL of MAO-A (. 𝜇Linbuer)and𝜇g/mL of
MAO-B (. 𝜇Linbuer).ecompoundsweredissolved
in DMSO and diluted in buer. e total reaction mixture
volume was 𝜇L, yielding a nal DMSO concentration of
.% in the reaction mixture. e reaction mixtures were
preincubated for min at ∘C followed by the addition
of MAO-A/MAO-B to initiate the reactions. e reaction
Evidence-Based Complementary and Alternative Medicine
T : 1HNMRdataofcompoundsI–IV.
Proton aCompound IaCompound II aCompound III bCompound IV
. (s) . (s) . (s) . (s)
. (m) . (s) . (d, .) . (s)
- - . (d, .) -
. (s) . (d, .) . (d, .) . (d, .)
. (m) . (d, .) . (d, .) . (d, .)
. (d, .) - . (dd, ., .), . (dd, ., .) . (d, .)
. (d, .) . (s) . (dd, ., .) . (d, .)
. (s) . (s), . (s) . (s) . (s)
. (s) . (s) . (s) . (s)
. (d, .) . (d, .) . (d, .) . (d, .)
. (m) . (d, .) . (d, .) . (d, .)
. (s) . (s) . (s) . (s)
. (s) . (s) . (s) . (s)
OMe . (s) - - . (s)
a
Data acquired at MHz. bData acquired at MHz.
T : 13 CNMRdataofcompoundsI–IV.
Carbon aCompound IaCompound II aCompound III bCompound IV
. . . .
. . . .
. . . .
a . . . .
. . . .
. . . .
. . . .
. . . .
a . . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
OMe . - - .
a
Data acquired at MHz. bData acquired at MHz.
mixtures were incubated for min at ∘Candstoppedby
the addition of 𝜇LofNNaOH.eformationof-
hydroxyquinoline was determined uorometrically by Spec-
tra Max M uorescence plate reader (Molecular Devices,
Sunnyvale, CA, USA) with an excitation and emission wave-
length of nm and nm, respectively, using the So Max
Pro program []. Appropriate controls were set up to check
the interference with the uorescence measurements. None
Evidence-Based Complementary and Alternative Medicine
of the tested fractions or compounds showed any interference
with the uorescence measurement. e determination of
IC50 valuesforinhibitionofMAO-AandMAO-Bbythe
M. pomifera (L.) compounds was performed using a xed
concentration of the substrate and varying the concentra-
tion of the inhibitor. M. pomifera compounds (. 𝜇Mto
𝜇M)andclorgyline(.𝜇Mto𝜇M) for MAO-A and
deprenyl (. 𝜇Mto𝜇M) for MAO-B were tested to
determine IC50 from the concentration dependent inhibition
curves using XL-Fit©soware.
3. Results and Discussion
3.1. Structural Elucidation. Compound Iwas obtained as a
yellow solid and exhibited a sodiated molecular ion peak in
HR-FTMS at m/z . corresponding to the molecular
formula C26H28O8Na (that calculated for C26H28O8Na is
.). 1H NMR ( MHz, DMSO-d) exhibited four
methyl singlets at 𝛿., ., ., and . which were
assigned to H-,
,
,and
.Asingletat𝛿.
was ascribed to the methoxy at C-.Adoubletat𝛿
. (. Hz) was assigned to methylene protons H-.A
multiplet integrating for one proton at 𝛿. was assigned
to an oxymethine proton at H- .Twodoubletsat𝛿.
(. Hz) and 𝛿. (. Hz) were assigned to H- and
methineprotons.Asingletat𝛿. and multiplet at 𝛿.
were assigned to H-,H-
,andH-
.Asingletat𝛿. was
assigned to oxygenated olenic proton H-.
e 13CNMRdataofI( MHz, DMSO-d) exhibited
carbon atoms. Four methyls at 𝛿., ., ., and .
were assigned to C-, C-,C-
, and C- .Carbonat𝛿
. is assigned to methoxy carbon at C-.Carbonat𝛿.
was attributed to C-. Two oxygenated carbon atoms at 𝛿
.and.wereassignedto
and .epeaksat𝛿.,
., ., ., ., and . were ascribed to C-, C-,
C-, C-a, C-,andC-
.epeakat𝛿. was assigned to
the carbonyl carbon at C-. e HMBC spectrum of Ishowed
key 3Jand 2Jcorrelations between the methoxy proton at 𝛿
. and the oxygenated methine carbon at C-,conrming
the position of the methoxy at C-. COSY correlations
have been noticed between H- to H- and H- to H- .
Hence, the structure of compound Iisdeducedtobe-(,-
dihydroxyphenyl)--hydroxy--(-hydroxy--methoxy--
methylbutyl)-,-dimethylpyrano[,-g]chromen-(H)-
one (I) and named Kazosajin I.
Compound II was obtained as a yellow solid and
exhibitedapeakinHR-FTMSatm/z . [M – H]−
corresponding to the molecular formula C25H23O8(that
calculated for C25H23O8is .). e 1H NMR ( M
Hz, CDCl3)showedsingletsat𝛿. integrating for
sixprotonsandat𝛿. integrating for three protons,
conrming the presence of three methyls at C-, C-,
and C-. A downeld singlet at 𝛿. (s) was ascribed to
the H-.Twosingletsat𝛿.and.wereassignedto
exomethylene protons H-.Twodoubletsat𝛿. (. Hz)
and.(.Hz)wereassignedtothetwoolenicprotons
at H- and H-.Twodoubletsat𝛿. (. Hz) and .
(. Hz) and a singlet at 𝛿. were assigned to trisubstituted
benzene ring protons at H-,H-
,andH-
.Anoxygenated
olenic proton H- appeared as a singlet at 𝛿.. 13CNMR
( MHz, CDCl3)ofII exhibited carbon atoms. It showed
three methyls at 𝛿., ., and . which were attributed
to C-, C-, and C-.Methinecarbonat𝛿. and
quaternary carbon at . were assigned to C- and C-.
e peaks at 𝛿., ., ., ., ., and . were
ascribed to the oxygenated carbon atoms at C-, C-, C-,
C-a, C-,andC-
.epeaksat𝛿. and . were
ascribed to carbonyls at C- and C-. e HMBC spectrum
of II showed 3Jand 2Jcorrelations between the H- and
C-, C- ,andC-
,andH-
with C- indicated the
presence of a prenylated side chain with 𝛼-hydroxy ketone.
Hence, the structure of compound II is deduced to be -(,-
dihydroxyphenyl)--hydroxy--(-hydroxy--methylbut--
enoyl)-,-dimethylpyrano[,-f]chromen-(H)-one (II)
and named Kazosajin II.
Compound III was obtained as a yellow solid and exhib-
ited a molecular ion peak in HR-FTMS at m/z . corre-
sponding to the molecular formula C25H25O6(that calculated
for C25H25O6is .). e spectral data of III is similar
to that of iso-osajin except for the hydroxylation at C-.1H
NMR ( M Hz, DMSO-d) showed ve singlets at 𝛿.,
., ., and . assigned to H-,H-
,H-
,andH-
.
Two r eson ances at 𝛿. (dd, ., . Hz) and 𝛿. (dd, .,
. Hz) are attributed to H2-.etripletat𝛿. (. Hz)
wasascribedtooxymethineprotonatH-
.etwodoublets
at 𝛿. (. Hz) and . (. Hz) are assigned to the olenic
protons H- and H- .etwodoubletsat𝛿. (.)
and . (.) were ascribed to four aromatic protons H-,
H-,H-
,andH-
on a p-disubstituted benzene ring. A
singlet at . was assigned to oxygenated olenic proton
H-.13C NMR ( MHz, DMSO-d) of III exhibited
carbon atoms. It showed four methyls at ., ., ., and
. attributed to C-, C-,C-
, and C- .Itshowed
three sp3oxygenated carbon atoms at 𝛿., ., and .
which were assigned to C-, C-, and C-,respectively.
It exhibited ve oxygenated aromatic carbon atoms at 𝛿.,
., ., ., and . which were assigned to C-,
C-, C-a, C-, and C-. e peak at 𝛿. was ascribed
to the carbonyl carbon at C-. e HMBC spectrum of III
showed 3Jand 2Jcorrelations between the methyl protons
at H- ,
to C-,and
indicating the presence of
a hydroxyl group at C-.Hence,thestructureofcom-
pound III is deduced to be -hydroxy--(-hydroxyphenyl)-
,,,-tetramethyl-,-dihydro-H-dipyrano[,-f: ,-
h]chromen-(H)-one (III) and named Kazosajin III.
Compound IV wasobtainedasayellowsolidand
exhibited a peak in HR-FTMS at m/z . [M −H]−
corresponding to the molecular formula C26H26O8(that
calculated for C26H25O8is .). 1HNMR(MHz,
CDCl3)showedfoursingletsat𝛿., ., ., and .
for the presence of four methyls at C-, C-,C-
,and
C-. A downeld singlet at 𝛿. was ascribed to the
methoxy protons at C-.Twodoubletsat𝛿. (. Hz)
and . (. Hz) were assigned to oxymethine protons H-
and H- and the two doublets at 𝛿. (. Hz) and .
(. Hz) were assigned to two olenic protons at H- and
H-. A singlet at 𝛿. and two doublets at 𝛿. (. Hz)
Evidence-Based Complementary and Alternative Medicine
T : Cannabinoid receptors activity of potential constituents (𝜇M) from M. pomifera.
Compound CB CB
% displacement IC50 (𝜇M) 𝐾𝑖 (𝜇M) % displacement IC50 (𝜇M) Ki (𝜇M)
Pomiferin (V) . . . . . .
Auricu lasin (VI) . . . - - -
Waran g a l o n e ( VII) . . . . . .
Osajin (VIII) . . . . . .
T : Inhibition of recombinant human monoamine oxidase-A and monoamine oxidase-B of I–VIII from M. pomifera.
Compounds Monoamine oxidase-A IC50
(𝜇M)
Monoamine oxidase-B
IC50 (𝜇M)
SI index
MAO-A/B
Kazosajin I74.33 ± 3.44 11.59 ± 1.39 .
Kazosajin II 72.03 ± 3.72 4.28 ±0.67 .
Kazosajin III > 7.16 ±1.15 -
Kazosajin IV 71.20 ± 2.61 17.66 ± 1.06 .
Pomiferin (V)> > -
Auricu lasin (VI)45.98 ± 4.48 1.91 ±0.32 .
Waran g a l o n e ( VII)> 5.69 ±0.38 -
Osajin (VIII)> > -
Clorgyline 0.0045 ± 0.0004 --
Deprenyl - 0.0326 ± 0.012 -
Notes.eresultsofIC
50 values are expressed as mean ±SD of triplicate obser vations.
and.(.Hz)wereassignedtothethreearomaticprotons
at H-,H-
,andH-
.AnoxygenatedolenicprotonH-
appeared as a singlet at 𝛿.. 13C NMR ( MHz, CDCl3)
of IV exhibited carbon atoms. It showed four methyls at
𝛿., ., ., and . attributed to C-, C-,C-
,
and C-.Downeldmethylcarbonat𝛿. was assigned
to the methoxy carbon at C-.efoursp
3oxygenated
carbon atoms at 𝛿., ., ., and . were assigned to
C-, C- , C-, and C-.epeaksat𝛿., .,
., ., ., and . were ascribed to the oxygenated
sp2carbon atoms at C-, C-, C-a, C-, C-,andC-
.
e peak at 𝛿. was ascribed to the carbonyl at C-.
e HMBC spectrum of IV showed 3Jand 2Jcorrelations
between the methyl protons at H- and to C- and
indicatingthepresenceofanepoxysystembetweenC-
and C- whichwasfurtherconrmedbyHRMS.Hence,
the structure of compound IV isdeducedtobe-(,-
dihydroxyphenyl)--((,-dimethyloxiran--yl)(methoxy)
methyl)--hydroxy-,-dimethylpyrano[,-g]chromen-
(H)-one (IV) and named Kazosajin IV.
Compounds V–XV wereisolatedandidentiedbycom-
paring their NMR data with the literature to be pomiferin
(V)[],auriculasin(VI)[],warangalone(VII)[],
osajin (VIII)[],artocarpesin(IX)[],tonkinensisol
(X) [], corchoionoside B (XI) [], kaempferol--O-𝛽-D-
glucoside (XII) [], dihydrokaempferol--O-𝛽-D-glucoside
(XIII) [], stigmasterol (XIV), and 𝛽-sitosterol (XV).
3.2. Cannabinoid and Opioid Receptors Assay. e anities
ofthetotalextractsandtheisolatedcompoundstowards
cannabinoid and opioid receptors were assessed using
CP- and Naloxone as controls for cannabinoid and
opioid receptors assays, respectively. e total extract showed
signicant activity towards cannabinoid receptor (CB:
.% displacement; CB: .% displacement), possibly
allosteric towards 𝛿and 𝜇opioid receptors (−. and −.%
displacement, resp.). e new compound Kazosajin Iwas
foundtobepossiblyanallostericcompoundin𝜅and 𝜇
opioid receptors (−. and −.% displacement, resp.).
Kazosajin II exhibited moderate activity towards cannabi-
noid receptors (CB: .% displacement; CB: .% dis-
placement). e known compounds—pomiferin (V) (CB:
.% displacement; CB: .% displacement), osajin
(VIII) (CB: .% displacement; CB: .% displacement),
and warangalone (VII) (CB: .% displacement; CB:
.% displacement)—exhibited prominent activity towards
cannabinoid receptors. e IC50 and Ki values for CB and
CB receptors active compounds are given in Table .
3.3. Determination of the Inhibitory Eect of M. pomifera (L.)
Compounds on Recombinant Human MAO-A and MAO-B.
e compounds isolated from M. pomifera (L.) fruits were
tested for their inhibitory eect against recombinant human
MAO isoforms (MAO-A and MAO-B) in vitro.eenzy-
matic activity of MAO-A and MAO -B was determined via
a uorescence based method []. All the tested compounds
showed fewer inhibitory eects on MAO-A compared to
MAO-B activities (Table ).
4. Conclusions
e Maclura pomifera total extract of the fruits showed sig-
nicant activity towards cannabinoid receptors and possibly
Evidence-Based Complementary and Alternative Medicine
allosteric interactions with 𝛿and 𝜇opioid receptors [].
Four new compounds (I–IV)alongwithelevenknowncom-
pounds (V–XV) were isolated and identied from the extract.
e new compound Kazosajin Iwas found to be possibly
allosteric towards 𝜅and 𝜇opioid receptors, while the new
compound Kazosajin II exhibited moderate activity towards
cannabinoid receptors CB and CB. Compounds V,VII,
and VIII exhibited prominent activity towards cannabinoid
receptors. All the isolated compounds from M. pomifera
(L.) fruits were tested for their inhibitory eect against
recombinant human MAO isoforms (MAO-A and MAO-B)
in vitro,wherefourcompounds(II,III,VI,andVII)showed
selective inhibition of MAO-B. All the tested compounds
showed fewer inhibitory eects on MAO-A compared to
MAO-B activities.
Disclosure
e content is solely the responsibility of the authors and does
not necessarily represent the ocial views of the National
Institute of General Medical Sciences or the National Insti-
tutesofHealth,USA.isworkhasbeenpresentedatGA
Conference, Basel, Switzerland [].
Conflicts of Interest
e authors declare no conicts of interest.
Acknowledgments
isprojectwassupportedbyKazakhstangovernmentand
the National Center for Natural Products Research, Uni-
versity of Mississippi, USA. e bioassays were conducted
at the in vitro neuropharmacological core supported by the
National Institute of General Medical Sciences (Award no.
PGM). e authors wish to thank Drs. Joshua Sharp
and Sandeep Misra for FTMS-ESI, Janet A. Lambert for
the CB/CB assays, and Dr. Mohamed Abo Elmagd for his
assistance with the laboratory work.
Supplementary Materials
D and D NMR and mass data for compounds I–IV;
this material is available free of charge via the Internet
through the journal’s website. Figure S: 1HNMRspectrum
for compound I(DMSO-𝑑, MHz). Figure S: 13CNMR
spectrum for compound I(DMSO-𝑑, MHz). Figure
S: DEPT spectrum for compound I(DMSO-𝑑, MHz).
Figure S: HSQC spectrum for compound I(DMSO-𝑑,
MHz). Figure S: HMBC spectrum for compound I
(DMSO-𝑑, MHz). Figure S: COSY spectrum for com-
pound I(DMSO-𝑑, MHz). Figure S: FTMS spectrum
for compound I.FigureS:1H NMR spectr um for compound
II (DMSO- d, MHz). Figure S: 13CNMRspectrumfor
compound II (DMSO-𝑑, MHz). Figure S: DEPT spec-
trum for compound II (DMSO-𝑑, MHz). Figure S:
HSQC spectrum for compound II (DMSO-𝑑, MHz).
Figure S: HMBC spectrum for compound II (DMSO-𝑑,
MHz). Figure S: FTMS spectrum for compound II.
Figure S: 1H NMR spectrum for compound III (DMSO-
𝑑, MHz). Figure S: 13C NMR spectrum for compound
III (DMSO-𝑑, MHz). Figure S: HSQC spectrum for
compound III (DMSO-𝑑, MHz). Figure S: HMBC
spectrum for compound III (DMSO-𝑑, MHz). Figure
S: FTMS spectrum for compound III.FigureS:1H
NMR spectrum for compound IV (DMSO-𝑑, MHz).
Figure S: 13C NMR spectrum for compound IV (DMSO-
𝑑, MHz). Figure S: DEPT spectrum for compound
IV (DMSO-𝑑, MHz). Figure S: HSQC spectrum for
compound IV (DMSO-𝑑, MHz). Figure S: HMBC
spectrum for compound IV (DMSO-𝑑, MHz). Fig-
ure S: COSY spectrum for compound IV (DMSO-𝑑,
MHz). Figure S: NOESY spectrum for compound
IV (DMSO-𝑑, MHz). Figure S: FTMS spectrum for
compound IV.(Supplementary Material)
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