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: Asbeder@mans.edu.eg (A. Elbermawi), asallam@mans.edu.eg (A. Sallam), hghabbour@mans.edu.eg (H.A. Ghabbour),

Elsebai72@mans.edu.eg (M.F. Elsebai), mfil@mans.edu.eg (M.F. Lahloub), hassan_elrady@mans.edu.eg (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:Asbeder@mans.edu.egmailto:asallam@mans.edu.egmailto:hghabbour@mans.edu.egmailto:Elsebai72@mans.edu.egmailto:mfil@mans.edu.egmailto:hassan_elrady@mans.edu.eghttps://doi.org/10.1016/j.phytol.2018.10.004http://crossmark.crossref.org/dialog/?doi=10.1016/j.phytol.2018.10.004&domain=pdf

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

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

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

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.

References

Allen, F.H., Kennard, O., Watson, D.G., Brammer, L., Orpen, a.G., Taylor, R., 1987. Tablesof bond lengths determined by X-ray and neutron diffraction. Part 1. Bond lengths inorganic compounds. J. Chem. Soc. Perkins Trans. II, 1–19.

Ashour, A., El-Sharkawy, S., Amer, M., Bar, F.A., Kondo, R., Shimizu, K., 2013. Melaninbiosynthesis inhibitory activity of compounds isolated from unused parts of Ammivisinaga. J. Cosmet. Dermatol. Sci. Appl. 3, 40–43.

Boulos, L., 2009. Flora of Egypt, Checklist. Al Hadara Publishing, Cairo, Egypt.Chia, Y., Chang, F., Wu, C., Teng, C., Chen, K., Wu, Y., 2006. Effect of isoquinoline al-

kaloids of different structural types on antiplatelet aggregation in vitro. Planta Med.72, 1238–1241.

Elsebai, M.F., Natesan, L., Kehraus, S., Mohamed, I.E., Schnakenburg, G., Sasse, F.,Shaaban, S., Gutschow, M., Konig, G.M., 2011a. HLE-inhibitory alkaloids with apolyketide skeleton from the marine-derived fungus Coniothyrium cereale. J. Nat.Prod. 74, 2282–2285.

Elsebai, M.F., Kehraus, S., Lindequist, U., Sasse, F., Shaaban, S., Guetschow, M., Josten,M., Sahl, H.-G., Koenig, G.M., 2011b. Antimicrobial phenalenone derivatives fromthe marine-derived fungus Coniothyrium cereale. Org. Biomol. Chem. 9, 802–808.

Elsebai, M.F., Nazir, M., Kehraus, S., Egereva, E., Ioset, K.N., Marcourt, L., Jeannerat, D.,Guetschow, M., Wolfender, J.-L., Koenig, G.M., 2012. Polyketide skeletons from theMarine alga-derived fungus Coniothyrium cereale. European J. Org. Chem. 2012,6197–6203.

Grycová, L., Dostál, J., Marek, R., 2007. Quaternary protoberberine alkaloids.Phytochemistry 68, 150–175.

Halim, A.F., Saad, H.-E.A., Hasish, N.E., 1994. Alkaloids of glaucium arabicum. MansouraJ. Pharm. Sci. 10, 265–274.

Heywood, V.H., 1978. The Flowering Plants of the World. Oxford University Press,London.

Table 2Effect of different compounds isolated from Glaucium arabicum on melanin content (MC) and cell viability (CV) of B16 melanoma cells.

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

Data presented as means ± SD (n= 3), MC, melanin content (%); CV, cell viability (%). Arbutin was used as a positive control at 100 μg/ml, CV=75.5 ± 0.9,MC=49.5 ± 3.3.

A. Elbermawi et al. Phytochemistry Letters 28 (2018) 139–144

143

http://www.ccdc.cam.ac.uk/conts/retrieving.htmlhttps://doi.org/10.1016/j.phytol.2018.10.004http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0005http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0005http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0005http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0010http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0010http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0010http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0015http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0020http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0020http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0020http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0025http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0025http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0025http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0025http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0030http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0030http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0030http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0035http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0035http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0035http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0035http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0040http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0040http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0045http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0045http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0050http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0050

Hussein, S.F., Cozler, B., Fajardo, V., Freyer, J.A., Shamma, M., 1983. The stereo-chemistry and 13C nmr spectra of protopinium salts. J. Nat. Prod. 46, 251–255.

Iwasa, K., Kamigauchi, O., Takao, N., Ushman, M., 1993. Stereochemical studies on thebiosynthesis of protoberberine, protopine, and benzophenanthridine alkaloids usingpapaveraceae plant cell cultures. J. Nat. Prod. 56, 2053–2067.

Khafagi, I.K., Dewedar, A., 2000. The efficiency of random versus ethno-directed researchin the evaluation of sinai medicinal plants for bioactive compounds. J.Ethnopharmacol. 71, 365–376.

Kim, K.H., Lee, I.K., Piao, C.J., Choi, S.U., Lee, J.H., Kim, Y.S., Lee, K.R., 2010.Benzylisoquinoline alkaloids from the tubers of Corydalis ternata and their cytotoxi-city. Bioorgan. Med. Chem. Lett. 20, 4487–4490.

Min, Y.D., Yang, M.C., Lee, K.H., Kim, K.R., Choi, S.U., Lee, K.R., 2006. Protoberberinealkaloids and their reversal activity of P-gp expressed multidrug resistance (MDR)from the rhizome of Coptis japonica makino. Arch. Pharm. Res. 29, 757–761.

Morteza-Semnani, K., Amin, G., Shidfar, M.R., Hadizadeh, H., Shafiee, A., 2003.Antifungal activity of the methanolic extract and alkaloids of glaucium oxylobum.Fitoterapia 74, 493–496.

Rahman, A.-U., Malik, S., Zaman, K., 1992. Nigellimine: a New isoquinoline alkaloid fromthe seeds of Nigella sativa. J. Nat. Prod. 55, 676–678.

Rahman, A.-U., Ahmed, S., Bhatti, M.K., Choudhary, M.I., 1995. Alkaloidal constituents ofFumaria indica. Phytochemistry 40, 593–596.

Sheldrick, G.M., 2008. A short history of SHELX. Acta Crystallogr. 64, 112–122.Shiomoto, H., Matsuda, H., Kubo, M., 1991. Effects of protopine on blood platelet ag-

gregation. III. Effect of protopine on the metabolic system. Chem. Pharm. Bull. 39,474–477.

Silverstein, R., Webster, F., Kiemle, D., 2005. Spectrometric Identification of OrganicCompounds, fifth ed. John Wiley & Sons: INC, New York.

Tousek, J., Dostal, J., Marek, R., 2004. Theoretical and experimental NMR chemical shiftsof norsanguinarine and norchelerythrine. J. Mol. Struct. 689, 115–120.

Tousek, J., Maliinkova, K., Dostal, J., Marek, R., 2005. Theoretical and experimental NMRstudy of protopine hydrochloride isomers. Magn. Reson. Chem. 43, 578–581.

Täckholm, V., 1974. Students҆ Flora of Egypt, second ed. Cairo University, CooperativePrinting Company Beirut.

A. Elbermawi et al. Phytochemistry Letters 28 (2018) 139–144

144

http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0055http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0055http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0060http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0060http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0060http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0065http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0065http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0065http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0070http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0070http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0070http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0075http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0075http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0075http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0080http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0080http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0080http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0085http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0085http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0090http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0090http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0095http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0100http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0100http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0100http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0105http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0105http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0110http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0110http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0115http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0115http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0120http://refhub.elsevier.com/S1874-3900(18)30217-9/sbref0120

本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源，

提供一站式文献检索和下载服务”的24 小时在线不限IP

图书馆。

图书馆致力于便利、促进学习与科研，提供最强文献下载服务。

图书馆导航：

图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具

http://www.xuebalib.com/cloud/http://www.xuebalib.com/http://www.xuebalib.com/cloud/http://www.xuebalib.com/http://www.xuebalib.com/vip.htmlhttp://www.xuebalib.com/db.phphttp://www.xuebalib.com/zixun/2014-08-15/44.htmlhttp://www.xuebalib.com/

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

学霸图书馆link:学霸图书馆