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Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Bioactive isoquinoline alkaloids from Glaucium arabicumAhmed
Elbermawia, Amal Sallama,⁎, Hazem A. Ghabbourb,c, Mahmoud F.
Elsebaia,Mohamed F. Lahlouba, Hassan-Elrady A. Saadaa Department of
Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura
35516, EgyptbDepartment of Pharmaceutical Chemistry, College of
Pharmacy, King Saud University, Riyadh 11451, Saudi
ArabiacDepartment of Medicinal Chemistry, Faculty of Pharmacy,
Mansoura University, Mansoura 35516, Egypt
A R T I C L E I N F O
Keywords:Glaucium arabicumPapaveraceaeIsoquinoline
alkaloidsAraglaucine AAraglaucine BX-ray
A B S T R A C T
Phytochemical investigation of the aerial parts of Glaucium
arabicum Fresen. (Papaveraceae) led to the isolationof two
previously undescribed isoquinoline alkaloids araglaucine A, and
araglaucine B, together with sevenknown ones
1-[(3`,4`-dimethoxy-2`-methylcarboxy)benzoyl]-6,7-methylenedioxy
isoquinoline (araglaucine C),(7R,14S)-trans-N-methylcanadinium
nitrate, (R,S)-trans-N-methylstylopine, 14-hydroxy-N-methyl
canadine, 14-hydroxy-N-methyl stylopine, protopine,
norsanguinarine, as well as β-sitosterol, and β-sitosterol
3-O-β–D-glu-coside. Their structural elucidation was based on the
measurements of 1D, 2D NMR, HRESIMS, UV, IR and
X-raycrystallography. The compounds were evaluated for their
anti-melanogenesis activity using B16 melanoma celllines. Compound
(7R,14S)-trans-N-methylcanadinium nitrate exhibited a promising
melanin synthesis inhibitoryactivity (∼35%) at concentration 5
μg/ml (12.01 μM) with low cytotoxicity (∼12%).
1. Introduction
Genus Glaucium Mill. (Papaveraceae) includes about 23
speciesdistributed in Europe, Mediterranean region, southwest and
centralAsia. It is represented in Egypt by four species; G.
corniculatum L., G.flavum Cranz., G. grandiflorum Boiss., and G.
arabicum Fresen. (Boulos,2009; Täckholm, 1974). Glaucium arabicum
Fresen. (Papaveraceae) is awild herb endemic to Sinai Peninsula
where it is locally known asNo’maan or Ne’man. It grows wildly in
Palestine, Jordan, Iraq andLibya as well (Heywood, 1978). The
species of Glaucium have been usedin Iranian herbal medicine as
laxative, antidiabetic, hypnotic, anti-fungal and for treatment of
dermatitis (Morteza-Semnani et al., 2003).Glaucium arabicum is used
in the folk medicine of the Bedouins living inSinai for the
management of eye and skin infections (Khafagi andDewedar, 2000).
Plants belonging to the genus Glaucium are chemicallycharacterized
by their alkaloidal content especially isoquinoline alka-loids.
Many of the isolated alkaloids exhibited versatile biological
ac-tivities such as antitussive, antimicrobial, antispasmodic,
anti-hista-minic, anti-inflammatory, cytotoxic, anti-platelet
aggregation activitiesand in the treatment of intestinal disorders
(Shiomoto et al., 1991; Chiaet al., 2006; Grycová et al.,
2007).
Hyperpigmentation is a common harmless skin condition in
whichmelanocytes are stimulated by sunlight exposure, inflammation,
free
radicals and hormonal changes to overproduce melanin.
Therefore,seeking new natural compounds exhibiting melanin
synthesis inhibitoryactivity is the aim of many researches.
Although plants of genus Glaucium were extensively studied
fortheir alkaloid content and biological activities as
antimicrobial andsmooth muscle relaxant activities, there is a lack
of knowledge aboutthe alkaloids of the aerial parts of G. arabicum
growing in SinaiPeninsula and their antimelanogenesis activity.
Therefore, the aim ofthis work was to study the isolation &
structural elucidation of the al-kaloids of the aerial parts of G.
arabicum growing in Sinai. Additionally,the biological evaluation
of the isolated compounds regarding theirmelanin synthesis
inhibitory activity was studied.
2. Results and discussion
2.1. Identification of the isolated compounds
Using a combination of chromatographic techniques, eleven
com-pounds (1 - 11) were isolated from the methylene chloride
extract ofthe alkalinized aerial parts of G. arabicum. Their
structural elucidationswere performed using extensive
physicochemical and spectroscopicmethods including 1D-, 2D-NMR,
HRESI+MS, UV, IR, and X-ray crys-tallographic measurements.
Compounds 1 and 2 are new isoquinoline
https://doi.org/10.1016/j.phytol.2018.10.004Received 10 April
2018; Received in revised form 14 September 2018; Accepted 4
October 2018
⁎ Corresponding author.E-mail addresses: [email protected] (A.
Elbermawi), [email protected] (A. Sallam), [email protected]
(H.A. Ghabbour),
[email protected] (M.F. Elsebai), [email protected] (M.F.
Lahloub), [email protected] (H.-E.A. Saad).
Phytochemistry Letters 28 (2018) 139–144
Available online 18 October 20181874-3900/ © 2018 Published by
Elsevier Ltd on behalf of Phytochemical Society of Europe.
T
http://www.sciencedirect.com/science/journal/18743900https://www.elsevier.com/locate/phytolhttps://doi.org/10.1016/j.phytol.2018.10.004https://doi.org/10.1016/j.phytol.2018.10.004mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://doi.org/10.1016/j.phytol.2018.10.004http://crossmark.crossref.org/dialog/?doi=10.1016/j.phytol.2018.10.004&domain=pdf
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derivatives; compounds 3, 6, and 7 are reported in this study
from thefamily Papaveraceae for the first time; compounds 5 and
9–11 areisolated from G. arabicum for the first time.
Compound 1 (Fig. 1) was obtained as a yellow powder with a
mo-lecular formula C20H13NO7 on the basis of accurate mass
measurement(HRESI+MS, m/z 380.0756 [M+H]+, Figure S6) and the
number ofsignals in 1H, 13C NMR and HSQC spectra (Figures S1-S3).
Compound 1is composed of isoquinoline moiety connected via a
carbonyl bridge to asubstituted phenyl group. The 1H and 13C NMR
spectra showed onemethyl, two methylenes, six aromatic methines,
four oxygenated aro-matic quaternary carbons, two carbonyls and
five quaternary carbons.The 1H NMR spectrum exhibited two
characteristic methylene protonsOCH2O/C-6,7 and OCH2O/C-3`,4` at
δH/C 6.15/101.9 and 6.14/102.8,respectively. Their high down field
shift in the 1H NMR and 13C NMRspectra indicating their attachment
to two oxygen atoms producingmethylenedioxy groups (IR 1034 cm−1).
OCH2O/C-6,7 is connected tothe aromatic C-6 and C-7 via oxygen as
indicated by HMBC correlationsfrom OCH2O/C-6,7 (δH 6.15) to C-6 and
C-7 (δC 150.9 and 150.1, re-spectively) (Fig. 2, Figure S5). The 1H
NMR spectrum showed also twosinglet aromatic protons H-5 and H-8
and they have HMBC correlationswith both C-6 and C-7. Also H-5 and
H-8 having HMBC correlations tothe sp2 carbons C-4a and C-8a. The
1H NMR spectrum showed alsocharacteristic aromatic ortho coupled
protons at δH 8.31 (d, J=5.2)and δH 7.57 (d, J=5.2) for H-3 and
H-4, respectively, which wereconfirmed by COSY correlations between
them (Figure S4). H-3 hasHMBC correlation to the resonance peak at
δC 152.3 for C-1 through anN atom due to the chemical shifts of H-3
and C-1 at δH/C 8.31/140.2 andδC 152.3, respectively. The presence
of N is confirmed by the odd massnumber at 380.0756 [M+H]+ and by
measuring the 1H 15N HMBC(Figure S7) which showed a correlation at
57.3 ppm from both H-3 andH-4 to the N atom. The coupling constant
J=5.2 together with UVabsorption maxima at 241 and 334 nm are
characteristic for an iso-quinoline alkaloid (Rahman et al., 1992,
1995; Kim et al., 2010). The
aforementioned partial structure of compound 1 was identified as
asubstituted isoquinoline.
The second methylenedioxy protons OCH2O/C-3`,4`at δH/C
6.14/102.8 have HMBC correlations to the quaternary aromatic
carbons C-3`and C-4`. The 1H NMR spectrum also showed ortho coupled
aromaticmethine signals at δH/C 6.97/110.0 and 7.27/125.6 for CH-5`
and CH-6`, respectively, which was confirmed by COSY correlations
(FigureS4). The H-5` and H-6` showed HMBC correlations to the
quaternaryaromatic carbons C-1`/C-3` and C-2`/C-4`, respectively
indicating thepresence of tetra-substituted aromatic moiety.
The 1H and 13C NMR spectra showed the presence of a methyl
esterat δH/C 3.34/52.0 and a carbonyl moiety at δC 164.7/CO
representingCOOCH3/C-2` (IR 1700 cm−1) confirmed by HMBC
correlation be-tween δH 3.34 and CO. The methyl ester COOCH3
substitutes the aro-matic moiety at C-2` due to HMBC correlation
between H-6` andCOOCH3. The 13C-NMR spectrum showed a resonance
peak at δC 195.6(C-9) which is characteristic for a carbonyl group
(IR 1679 cm−1). Thiscarbonyl group is connected to the aromatic
moiety at carbon C-1` asshown by its HMBC correlation with δH 7.27
(H-6`). The carbonyl grouphas an sp2 carbon, attached to the
aromatic moiety at carbon C-1` andto the isoquinoline unit at C-1
and this carbonyl position was inagreement with the similar known
derivative compound 3 (Min et al.,2006).
From the above results, compound 1 was identified as 1-(3`,
4`-methylenedioxy-2`-methylcarboxybenzoyl)-6,7-methylenediox-yisoquinoline.
Compound 2 has a molecular formula of C20H16N2O6 which
wasdetermined from the [M+H-H2O]+ peak at m/z 363.0977 in
theHRESI+MS (Figure S12) and the number of protons and carbons in
the1D spectra (Figures S8 & S9). Compound 2 is unprecedented
one since itis composed of both isoquinoline and iso-indole
moieties. The iso-quinoline moiety of compound 2 has almost the
same spectroscopicdata as that of compound 1. Additionally compound
2 has an iso-indol-3`-one moiety attached directly to the
isoquinoline moiety, which wasconfirmed as follows. The 1H NMR
spectrum showed two resonancepeaks at δH 4.19 and δH 3.87 for two
methoxy protons and they areattached to the aromatic quaternary
carbons C-3` and C-4`, respectivelydue to the HMBC correlations to
the respective aromatic carbons. The1H NMR spectrum was
characterized also by the resonance peaks of theortho coupled
protons H-5` and H-6` (J=8.2). H-5` and H-6` havingHMBC
correlations (Figures 2 & S11) to C-1`, C-3` and C-4`, C-2`,
re-spectively creating the aromatic ring of the indole nucleus. The
13CNMR spectrum showed a resonance peak for the amide carbonyl
groupCO-11 at δc 168.2 and the 1H NMR spectrum showed a resonance
peakfor NH at δH 6.28. The amide carbonyl CO-11 is of lactam type
based onthe chemical shift of C-11 at 168.2 ppm (Elsebai et al.,
2011a andElsebai et al., 2011b, 2012. The lactam functionality was
further
Fig. 1. Structures of compounds 1 and 2.
Fig. 2. Key COSY and HMBC correlations for compounds 1 and
2.
A. Elbermawi et al. Phytochemistry Letters 28 (2018) 139–144
140
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confirmed by the IR absorption bands at 1692 cm−1 for lactam
car-bonyl group, and 3422 cm−1 for lactam NH (Silverstein et al.,
2005).The 13C NMR and HSQC spectra (Figures S9 & S10) showed a
resonancepeak at δC 85.0 which was assigned to the carbinol C-9.
The downfieldshift of the value of C-9 indicated its attachment to
an aromatic system.Therefore C-9 is attached to C-1`, this was
confirmed by HMBC corre-lation between the methine proton δH 6.72
(H-6`) with C-9. The che-mical shift value of C-1`(δC 142.7)
suggested its placement in the β-position to the carbonyl CO-11.
This was confirmed from HMBC cor-relation between H-5`and C-1`. The
remaining singlet proton resonatingat δH 6.01 in the 1H NMR
spectrum represents the proton of a hydroxylgroup. δC 85.0 (C-9) is
consistent with an sp3 carbon with an oxygenand nitrogen attached.
This indicates that the hydroxyl group exists as asubstituent on
C-9. This might explain the higher value of the chemicalshift of
this carbon and the removal of water producing the peak [M+H-H2O]+
in the HRESIMS spectrum at m/z 363.0977.
Compounds 1 and 2 are new natural compounds and they weregiven
the names araglaucines A and B.
Extensive spectroscopic analyses including 1D- and 2D-NMR,HRMS,
UV and IR of compounds 3-11 proved that they are as
following:compound 3 (Figures S13-S14) is the same as compound 1
except thatcompound 3 has two methoxy groups instead of methylene
dioxy.Compound 3 was reported before as
1-[(3`,4`-dimethoxy-2`-methyl-carboxy)benzoyl]-6,7-methylenedioxy
isoquinoline (Min et al., 2006)and it is designated in this study
as araglaucine C.
For compound 4, the extensive spectroscopic measurements
re-vealed that it is N-methylcanadinium nitrate and its absolute
config-uration was determined for the first time in this study to
be (7R,14S)-trans-N-methylcanadinium nitrate based on the X-ray
measurementsand comparison with the literature (Halim et al.,
1994). Compound 4was crystallized in the Triclinic, P1, a = 7.4653
(2) Å, b = 7.8998 (3)Å, c= 8.6677 (3) Å, α= 74.299 (1)°, β= 77.188
(1)°, γ= 88.025 (1)°,V=479.67 (3) Å3, Z=1. In compound 4,
C21H24NO4·NO3, fractionalatomic coordinates and isotropic or
equivalent isotropic displacementparameters (Å2) are present in
Table S17; the selected bond lengths,bond angles and torsion angles
are listed in Table S18; the crystal-lographic data and refinement
information are summarized in TableS20. The asymmetric unit
consisted of one independent molecule as acation with nitrate anion
as shown in Fig. 3. All the bond lengths andangles are in normal
ranges (Allen et al., 1987). In the crystal packing(Fig. S21)
molecules are linked via twelve intermolecular hydrogenbonds (Table
S19). Compound 4 contains two chiral centers at C-14 andat the
quaternary nitrogen. Their absolute configurations were de-termined
as (14S) and (7R), respectively, which were determined on thebasis
of the Flack parameter 0.01(9).
Compound 5 is the trans-N-methylstylopine based on
extensivespectroscopic data (Figures S22 and S23) and the
comparison with theliterature data (Iwasa et al., 1993). Compound 5
is the (7R,14S)-trans-
N-methylstylopine based on the chemical shift of C-13 at 28.4
ppm forthe trans isomers (Hussein et al., 1983) which was supported
by the X-ray measurements of compound 4 (δC for C-13 of compound
4=28.2)(Data S34).
Compound 6 is the same as 4 except there is an OH group
attachedto C-14 evidenced by the extensive spectroscopic
measurements in-cluding HRESIMS which revealed that 6 is the
hydroxyl-N-methyl ca-nadine (Tousek et al., 2005). For compound 7,
the extensive spectro-scopic measurements revealed that 7 is
14-hydroxy-N-methylstylopine.Compounds 6 and 7 were synthesized as
racemates upon adding HCl toprotopine (Tousek et al., 2005). Also
they were biosynthesized throughbiotransformation of
protoberberines (Iwasa et al., 1993) and withoutshowing any
spectroscopic data. The extensive spectroscopic measure-ments
revealed that compound 8 is protopine as in references (Halimet
al., 1994; Allen et al., 1987; Iwasa et al., 1993; Hussein et al.,
1983)and compound 9 is norsanguinarine as in reference (Tousek et
al.,2004). Compounds 10 and 11 were determined as β-sitosterol, and
β-sitosterol 3-O-β-D-glucoside based on comparison with TLC using
au-thentic samples and confirmed by IR spectra (Figures S32 &
S33).
2.2. Biological activity
Anti-melanogenesis activity using B16 melanoma cell line: The
isolatedcompounds were assayed using B16 melanoma cells in order to
evaluatetheir ability to inhibit melanin synthesis in B16 melanoma
cells andtheir effect on cell viability at their maximum solubility
(20 μg/ml).Results are shown in Fig. 4 and Table 2. The ability of
the testedcompounds to inhibit melanin formation in B16 melenoma
cells wasshown at various concentrations (Table 2).
Taking into consideration the cytotoxicity to the cells,
compound 4at concentration 5 μg/ml (12.01 μM) was the most active
compoundexhibiting melanin synthesis inhibition (∼35%) and at the
same timewith low cytotoxicity (∼12%). However, at concentration of
20 μg/ml,it exhibited higher melanin synthesis inhibition (∼60%)
but with re-latively high toxicity (∼35%) compared to the positive
control arbutin.At concentration 20 μg/ml, compounds 2 and 5 showed
moderate in-hibition of melanin synthesis but at the same time
showed a high cy-totoxic effect to the cells (Table 2). Compound 8
showed a moderatemelanin synthesis inhibition at all tested
concentrations (∼25% and20%) with very low cytotoxicity. No effect
on melanin synthesis in-hibition was recorded concerning the other
tested compounds. This isthe first time to investigate isoquinoline
alkaloids of Glaucium arabicumas potential drugs for the treatment
of hyperpigmentation conditions.(7R,14S)-trans-N-methylcanadinium
nitrate showed a promising mel-anin synthesis inhibitory activity
(∼35%) at concentration 5 μg/ml(12.01 μM) with low cytotoxicity
(∼12%).
Fig. 3. ORTEP diagram of compound 4. Displacement ellipsoids are
plotted at the 40% probability level for non-H atoms (the numbering
of different atoms is forcrystallographic data).
A. Elbermawi et al. Phytochemistry Letters 28 (2018) 139–144
141
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3. Conclusions
Phytochemical investigation of the aerial parts of Glaucium
arabicumFresen. (Papaveraceae) resulted in the isolation of two new
isoquinolinealkaloids designated as araglaucine A (1) and
araglaucine B (2), alongwith seven known ones
1-[(3`,4`-dimethoxy-2`-methylcarboxy)ben-zoyl]-6,7-methylenedioxy
isoquinoline (araglaucine C) (3), (7R,14S)-trans-N-methylcanadinium
nitrate (4), (7R,14S)-trans-N-methyl-stylopine (5),
14-hydroxy-N-methyl canadine (6), 14-hydroxy-N-methylstylopine (7),
protopine (8), norsanguinarine, (9), as well as β-sitosterol(10),
and β-sitosterol 3-O-β–D-glucoside (11). Moreover, the absolute
configuration of N-methylcanadinium nitrate is determined for
the firsttime in this study to be (7R,14S)-trans-N-methylcanadinium
nitratebased on the X-ray measurements.
4. Experimental
4.1. General experimental procedures
UV spectra (λ max) was carried out on UV–vis
spectrophotometer(Shimadzu 1601 PC, model TCC-240 A, Japan) using
spectroscopicmethanol. IR spectra (cm−1) was carried out on
Infra-red spectro-photometer, ThermoFisher Scientific, Nicolet 10
(USA) using KBr pel-lets. Accurate mass determinations were
performed on a Synapt G2HDMS mass spectrometer. Capillary voltage
3000 V and cone voltage20 V. Leu-enkaphaline was used as the lock
mass. The UPLC-MS systemwas operated with MassLynx 4.1 software.
NMR measurements wererecorded on a Bruker Avance 400 or Bruker
Avance 600 DPX spectro-meters.
4.2. X-ray measurements
Compound 4 was obtained as single crystals by slow
evaporationfrom ethanol solution of the pure compound at room
temperature. X-ray crystallographic data was collected using Bruker
APEX-II D8Venture diffractometer, equipped with graphite
monochromatic Cu Kαradiation, λ=1.54178 Å at 100 (2) K. Cell
refinement and data re-duction were carried out by Bruker SAINT.
SHELXT (Sheldrick, 2008)was used to solve structure. The final
refinement was carried out byfull-matrix least-squares techniques
with anisotropic thermal data fornonhydrogen atoms on F2. CCDC
1463769 contains the supplementarycrystallographic data for this
compound. These data can be obtainedfree of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html.
4.3. Reagents and media for cell line
Eagle’s Minimum Essential Medium (EMEM) was purchased fromNissui
Pharmaceutical (Tokyo, Japan). Fetal bovine serum (FBS) was
ob-tained from Gibco BRL (Tokyo, Japan). Thiazolyl blue tetrazolium
bro-mide (MTT) was purchased from Sigma (St. Louis, MO, USA). NaOH
andDMSO were purchased from Wako Pure Chemical Industries, Ltd
(Osaka,Japan). Other chemicals are of the highest grade
commercially available.
4.4. Cell line
A mouse B16 melanoma cell line was obtained from RIKEN CellBank.
The cells were maintained in EMEM supplemented with 10% (v/
Fig. 4. Effect of compounds isolated from Glaucium arabicum on
melanin synthesis in B16 melanoma cell. The values are represented
as the mean ± standarddeviation (± SD), n=3.
Table 1The 1H and 13C NMR spectroscopic data of compounds 1 and
2.
position araglaucine A (1) araglaucine B (2)
δc, type δH (Jin Hz)
δc, type δH (J in Hz)
1 152.3, C – 152.3, C –2 – – – –3 140.2, CH 8.31, d
(J=5.2)137.8,CH
8.34, d(J=5.2)
4 123.0, CH 7.57, d(J=5.2)
122.8, CH 7.59, d(J=5.2)
4a 135.7, C – 136.6,C –5 102.5, CH 7.11, s 101.2,CH 6.78, s6
150.9, C – 150.9, C –7 150.1, C – 147.9, C –8 102.6, CH 8.24, s
103.3,CH 7.09, s8a 124.3, C – 121.9, C –9 195.2, CO – 85.0, C –10 –
– – 6.28, brs, NH11 – – 168.2, C –1` 133.9, C – 142.7, C –2` 114.4,
C – 122.3, C –3` 147.1, C – 148.8, C –4` 150.9, C – 153.7, C –5`
110.0, CH 6.97, d
(J=8.0)117.4, CH 6.99, d
(J=8.2)6` 125.6, CH 7.27, d
(J=8.0)117.7, CH 6.72, d
(J=8.2)COOCH3 52.0 3.34, s – –COOCH3 164.7 – – –OCH3/C-3` – –
62.6 4.19, sOCH3/C-4` – – 56.6 3.87, sOCH2O/C-3`,4` 102.8 6.14, s –
–OCH2O/C-6,7
OH at position 9101.9–
6.15, s–
101.9–
5.98, s6.01, s
The NMR spectra were measured in CDCl3 (400MHz) for 1H NMR
and(100MHz) for 13C NMR. Chemical shift (δ) values are expressed in
ppm.Coupling constants (J) values in Hz.
A. Elbermawi et al. Phytochemistry Letters 28 (2018) 139–144
142
http://www.ccdc.cam.ac.uk/conts/retrieving.html
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v) fetal bovine serum (FBS), 100 μg/ml penicillin and 100
μg/mlstreptomycin. The cells were incubated at 37 °C in a
humidified atmo-sphere of 5% CO2.
4.5. Plant material
Glaucium arabicum Fresen. (Papaveraceae) herb was collected
onMay 2012 from the area of Deir El Raba and Saint Cathrine,
SinaiPeninsula, Egypt. The plant identity was confirmed by St.
CathrineHerbarium staff members. The aerial parts were separated
from theroots and the fresh collected parts were air dried in shade
at roomtemperature. A voucher specimen has been deposited at the
herbariumof Pharmacognosy Department, Faculty of Pharmacy,
MansouraUniversity given the code: GA 01 Mansoura-3.
4.6. Extraction and isolation
The air dried powdered aerial parts (1.5 kg) were defatted with
pet.ether at room temperature, then the defatted powdered aerial
partswere alkalinized with 25% NH4OH and left overnight, then
successivelyextracted with CH2Cl2 and MeOH. The methylene chloride
extract ofthe defatted alkalinized aerial parts of G. arabicum (40
g) was appliedonto the top of a silica gel column. The extract was
then gradient elutedwith pet. ether−EtOAc (100:0 to 0:100) then
EtOAc−MeOH (100:0to 0:100). The effluent was collected in 250ml
fractions, monitored byTLC then similar fractions were collected
into 10 groups (Gr. I till Gr.X)
Gr.III eluted with pet. ether− EtOAc (85:15, 188mg) was
furtherpurified on a silica gel column, eluted gradiently with
pet.ether−CH2Cl2. Subfractions eluted with pet. ether− CH2Cl2
(5:95)yielded compound 1 (5.2 mg).
Gr.V eluted with pet. ether− EtOAc (55:45, 198mg) was
re-chromatographed over reversed phase silica gel column Rp-18,
elutedgradiently with H2O−MeOH (50:50) to (0:100). Subfractions
elutedwith H2O−MeOH (40:60, 25mg), were further purified by PTLC
usingCH2Cl2−MeOH (14.5:0.5) as developing system to obtain compound
2(3 mg).
Groups I, II, IV &VI-X were purified using different
chromatographictechniques as normal phase, reversed phase, amino
silica and sephadexLH 20 yielding compounds 3-11.
Araglaucine A (1): fine yellow powder (5.2mg); UV (EtOH)λmax(log
ε) 241 (4.05), 334 (4.48) nm; IR νmax/cm−1 (ATR) 1700, 1679,1034,
1020; 1H NMR and 13C NMR data: see Table 1; HRESI+MS m/z[M+H]+ peak
at m/z 380.0756 (calcd for C20H14NO7, 380.0770)
Araglaucine B (2): white powder (3mg); UV (EtOH) λmax(log ε)240
(4.00), 336 (4.48) nm; IR νmax/cm−1 (ATR) 3422, 1692, 920;1HNMR and
13C NMR data: see Table 1; HRESI+MS m/z [M+H-H2O]+peak at 363.0977
(calcd for C20H16N2O6, 363.0981).
(7R,14S)-trans-N-methylcanadinium nitrate (4): isolated as
whiteneedles (22.4mg); UV (EtOH) λmax(log ε) 241 (4.23), 334
(4.18), 292 (sh)(3.52), 325 (3.41) nm; IR νmax/cm−1 (ATR); 1H NMR
and 13C NMR see
supplementary data; X-ray crystallographic structure: see Fig.
3; CCDC1463769,
http://www.ccdc.cam.ac.uk/conts/retrieving.html.
4.7. Biological activity
Anti-melanogenesis activity using B16 melanoma cell line. The
assaywas carried out according to reference (Ashour et al.,
2013).
Conflict of interest disclosure
The authors declare no conflict of interest.This research did
not receive any specific grant from funding
agencies in the public, commercial or non-for-profit
sectors.
Acknowledgements
The authors would like to extend their sincere appreciation to
theDeanship of Scientific Research at King Saud University for
funding thex-ray analysis, to Dr. Weaam Ebrahim for carrying out
some spectralanalyses, and to Dr. Ahmed Adel Ashour for his
valuable efforts incarrying out the antimelanogenesis assay.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in
theonline version, at
doi:https://doi.org/10.1016/j.phytol.2018.10.004.
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Comp. 5 μg/ml 10 μg/ml 20 μg/ml
MC CV MC CV MC CV
1 94.0 ± 9.4 103.4 ± 1.57 106.7 ± 9.9 90.8 ± 2.6 103.4 ± 11.2
94.2 ± 2.52 78.1 ± 2.8 96.8 ± 6.3 81.0 ± 4.5 82.8 ± 6.8 63.1 ± 11.5
54.8 ± 18.23 128.4 ± 12.2 100.9 ± 4.0 111.8 ± 6.7 106.1 ± 5.2 125.2
± 2.4 92.1 ± 0.94 65.4 ± 2.6 88.1 ± 2.2 66.0 ± 8.8 83.9 ± 3.7 39.9
± 1.3 64.4 ± 9.95 87.0 ± 5.3 26.5 ± 5.2 83.9 ± 12.4 25.9 ± 9.1 63.2
± 6.6 12.3 ± 0.68 77.5 ± 7.0 96.4 ± 3.6 76.0 ± 9.8 92.9 ± 2.4 80.7
± 7.7 97.4 ± 0.110 110.6 ± 15.7 100.7 ± 2.2 98.6 ± 8.7 100.1 ± 1.0
94.6 ± 1.3 93.9 ± 1.111 105.8 ± 10.9 104.0 ± 3.0 113.5 ± 5.9 111.5
± 5.6 109.3 ± 19.1 95.3 ± 2.0
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CV, cell viability (%). Arbutin was used as a positive control at
100 μg/ml, CV=75.5 ± 0.9,MC=49.5 ± 3.3.
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Bioactive isoquinoline alkaloids from Glaucium
arabicumIntroductionResults and discussionIdentification of the
isolated compoundsBiological activity
ConclusionsExperimentalGeneral experimental proceduresX-ray
measurementsReagents and media for cell lineCell linePlant
materialExtraction and isolationBiological activity
Conflict of interest disclosureAcknowledgementsSupplementary
dataReferences
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