Officinalioside, a new lignan glucoside from Borago officinalis L.

Article abstract of DOI:10.1080/14786419.2015.1088540

A new lignan glucoside, officinalioside (1), was isolated from n-BuOH fraction of the aerial parts of Borago officinalis L., together with four known compounds: actinidioionoside (2), roseoside (3), crotalionoside C (4) and kaempferol 3-O-β-D-galactopyranoside (5). The structure of the new compound was established by means of spectroscopic and chemical analyses. Compounds 1 and 2 showed a moderate DPPH radical scavenging activity (IC₅₀: 52.6 ± 1.70 and 41.3 ± 0.25 M, respectively) comparable with that of the standard trolox (16.6 ± 2.2 M) without any significant cytotoxicity towards human cell line A549 (IC₅₀ > 100 M).

Full text of DOI:10.1080/14786419.2015.1088540

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Officinalioside, a new lignan glucoside from
Borago officinalis L.
Mamdouh Nabil Samy, Ashraf Nageeb El-Sayed Hamed, Sachiko Sugimoto,
Hideaki Otsuka, Mohamed Salah Kamel & Katsuyoshi Matsunami
To cite this article: Mamdouh Nabil Samy, Ashraf Nageeb El-Sayed Hamed, Sachiko
Sugimoto, Hideaki Otsuka, Mohamed Salah Kamel & Katsuyoshi Matsunami (2015):
Officinalioside, a new lignan glucoside from Borago officinalis L., Natural Product Research,
DOI: 10.1080/14786419.2015.1088540
To link to this article: http://dx.doi.org/10.1080/14786419.2015.1088540
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Date: 20 September 2015, At: 13:22

Natural Product research, 2015
http://dx.doi.org/10.1080/14786419.2015.1088540
Officinalioside, a new lignan glucoside from Borago officinalis L.
Mamdouh Nabil Samya,b, Ashraf Nageeb El-Sayed Hamedb, Sachiko Sugimotoa,
Hideaki Otsukac, Mohamed Salah Kamelb and Katsuyoshi Matsunamia
adepartment of Pharmacognosy, Graduate school of Biomedical & health sciences, hiroshima university,
hiroshima, Japan; bFaculty of Pharmacy, department of Pharmacognosy, Minia university, Minia, egypt;
cFaculty of Pharmacy, department of Natural Product chemistry, YasudaWomen’s university, hiroshima, Japan
ABSTRACT
ARTICLE HISTORY
received 20 april 2015
accepted 2 august 2015
A new lignan glucoside, officinalioside (1), was isolated from
n-BuOH fraction of the aerial parts of Borago officinalis L., together
with four known compounds: actinidioionoside (2), roseoside (3),
crotalionoside C (4) and kaempferol 3-O-β-D-galactopyranoside (5).
The structure of the new compound was established by means of
spectroscopic and chemical analyses. Compounds 1 and 2 showed
a moderate DPPH radical scavenging activity (IC50: 52.6 1.70 and
41.3 0.25 μM, respectively) comparable with that of the standard
trolox (16.6 2.2 μM) without any significant cytotoxicity towards
human cell line A549 (IC50 > 100 μM).
KEYWORDS
Borago officinalis;
Boraginaceae; lignan;
officinalioside; dPPh;
cytotoxicity
1. Introduction
Borago officinalis L. (Boraginaceae), commonly known as bee bread, borage or starflower, is
native to the Mediterranean region and North Africa, and cultivated in Iran, Turkey, Spain,
Asia Minor and India as an ornamental plant and to repel insects. B. officinalis is traditionally
used to treat respiratory, cardiovascular and gastrointestinal diseases (Gilani et al. 2007;
Bianchi 2015), and borage juice and tea are used to treat influenza, colds, injuries and ulcers
CONTACT Katsuyoshi Matsunami
matunami@hiroshima-u.ac.jp
supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2015.1088540.
© 2015 taylor & Francis

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M. N. SAMy ET AL.
(Leos-Rivas et al. 2011; Farhadi et al. 2012; Kaskoos et al. 2012). Borage is commercially cul-
tivated for borage seed oil which is rich in gamma-linolenic acid and is used as a dietary or
food supplement (Morteza et al. 2015). Recently, pyrrolizidine alkaloids were isolated from
this plant (Asadi-Samani et al. 2014), and the seasonal variation of antioxidant activity of
essential oil was evaluated (Salem et al. 2014). The present study reports the isolation and
structural elucidation of a new lignan glucoside (1), together with three known megastig-
mane glucosides (2–4) and one known flavonoid galactoside (5) (Figure 1) in addition to the
evaluation of their cytotoxicity and DPPH radical scavenging activity.
2. Results and discussion
The n-BuOH fraction of a 95% EtOH extract of B. officinalis was subjected to silica gel and
octadecylsilanised (ODS) column chromatography (CC) and high-performance liquid chro-
matography (HPLC) to give one new lignan glucoside, officinalioside (1), in addition to four
known compounds (2–5): actinidioionoside (2) (Samy et al. 2014a), (6S,9R)-roseoside (3)
(Samy et al. 2014b), crotalionoside C (4) (Shitamoto et al. 2010) and kaempferol 3-O-β-D-
galactopyranoside (5) (Lu & Foo 1999).
Compound 1, [α]D24 −8.14, was obtained as an amorphous powder having the molecular
formula C28H36O14 as determined by the positive-ion mode HR-ESI-MS. The IR spectrum of 1
showed bands at 3360 and 1699 cm−1 corresponding to hydroxy and carbonyl group absorp-
tions, respectively. The coupling constant of an anomeric proton signal at δH 6.08 (1H, d,
J = 7.8 Hz), the chemical shift values of six carbon signals (δC 104.4, 79.4, 78.8, 76.4, 71.9 and
62.9) and the HPLC analysis of hydrolysate of 1 using an optical rotation detector indicated
the presence of a β-D-glucopyranosyl. Two singlet proton signals at δH 7.11 (2H, s) and 7.68
(2H, s) and two methoxy groups at δH 3.73(6H, s) and 3.79(6H, s) indicated the presence of
two symmetrically substituted benzene rings. The 13C NMR data of the aglycone moiety were
similar to those of (7′S,8S,8′R)-4,4′-dihydroxy-3,3′,5,5′-tetramethoxy-7′,9-epoxylignan-9′-ol-
7-one (Xiong et al. 2011), which were confirmed by the COSy correlations between H-8′/H-7′
as well as H-9/H-8/H-8′/H-9′ and HMBC correlations between H-7′/C-1′, H-8/C-7, H-2,6/C-7,
3,5-OCH3/C-3,5 and 3′,5′-OCH3/C-3′,5′. In addition, the HMBC cross peak of the anomeric
proton of the glucopyranosyl moiety (δH 6.08) with C-4 (δC 140.8) confirmed the sugar linkage
to the hydroxy group at the C-4 position of the lignan skeleton. The relative configuration
of 1 was determined by the phase-sensitive ROESy. The correlations between H-8 and H-7′
indicated the same orientation for these protons. And the relatively large coupling constant
of H-7′ (J = 8.2 Hz) suggested the axial-like trans orientation for H-8′, which was further con-
firmed by the correlations of H-8/H-9′ and H-7′/H-9′ without the correlation of H-8/H-8′ and
H-7/H-8′ with the aid of the MM2 calculation model. The absolute configuration of 1 was
established by the analysis of its CD spectrum. The cotton effects (positive at 320 and nega-
tive at 283 nm) caused by the exciton coupling of the benzoyl and benzene chromophores
suggested that the compound had the 7′R,8R,8′S configuration which is opposite to that of
(7′S,8S,8′R)-4,4′-dihydroxy-3,3′,5,5′-tetramethoxy-7′,9-epoxylignan-9′-ol-7-one (Xiong et al.
2011). Thus, compound 1 was identified as (7′R,8R,8′S)-4,4′-dihydroxy-3,3′,5,5′-tetramethoxy-
7’,9-epoxylignan-9′-ol-7-one 4-O-β-D-glucopyranoside, named officinalioside.
Compounds 1–5 were examined for their 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging activity and tumor cell growth inhibitory activity towards A549 by means of the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. None of the tested

NATuRAL PRODuCT RESEARCH
3
Figure 1. structures of the isolated compounds 1–5.
compounds (1‒5) showed any significant activity towards A549 (IC50 > 100 μM). Compounds 1
and 2 showed moderate DPPH radical scavenging activity (IC50: 52.6 1.70 and 41.3 0.25 μM,
respectively) comparable with that of the standard trolox (16.6 2.2 μM).
3. Experimental
3.1. General experimental procedures
Optical rotation data were measured on a JASCO P-1030 polarimeter. CD, IR and uV spec-
tra were obtained on a Jasco J-720, Horiba FT-710 Fourier transform infrared and Jasco
V-520 uV/vis spectrophotometer, respectively. 1H- and 13C-NMR spectra were recorded on
a Bruker AVANCE 600 MHz spectrometer (Bruker Biospin, Tokyo, Japan). An HR-ESI mass
spectrum was taken on a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, MA,
uSA). Silica gel CC was performed on silica gel 60 [(E. Merck, Darmstardt Germany), 70‒230
mesh]. Reversed-phase [octadecylsilanised silica gel (ODS)] open CC (RPCC) was performed
on Cosmosil 75C18-OPN (Nacalai Tesque, Kyoto, Japan) (Φ = 2 cm, L = 40 cm, 10 g fractions
being collected). HPLC was performed on an ODS column [Inertsil ODS-3; GL Science, Tokyo,
Japan; (Φ = 10 mm, L = 25 cm, flow rate: 2.0 mL/min)] using a refractive index and/or a uV
detector. Sugar analysis was performed with an amino column (Shodex Asahipak NH2P-50

4
M. N. SAMy ET AL.
4E (4.6 × 250 mm), CH3CN–H2O (3:1), 1 mL/min) and a chiral detector OR-2090plus (JASCO
corporation, Tokyo, Japan). Pre-coated silica gel 60 F254 plates (E. Merck; 0.25 mm in thickness)
were used for TLC analyses, visualised by spraying with a 3% H2SO4 solution in EtOH and
heating to around 150 °C on a hotplate. A human lung cancer cell line, A549, was obtained
from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, National Institute
of Biomedical Innovation.
3.2. Plant material
The aerial parts of B. officinalis L. were collected in April 2013 from the Faculty of Agriculture,
Minia university, Minia, Egypt and propagated in the experimental station of the Faculty of
Pharmacy, Minia university. The plant was identified by Prof. Mahmoud A.H. Abdo, Director
of Floriculture Nursery (Aromatic and Medicinal plants), Faculty of Agriculture, Minia
university, Minia, Egypt. A voucher specimen of the plant is deposited in the Herbarium of
Pharmacognosy Department, Faculty of Pharmacy, Minia university, Egypt (Mn-Ph-Cog-008).
3.3. Extraction and isolation
The air-dried powdered aerial parts (5.0 kg) of B. officinalis were extracted with 95% EtOH
(5 L × 5) until exhaustion and then concentrated under reduced pressure to yield a viscous
gummy material (226.6 g). This residue was dissolved in 500 mL of water and defatted with
n-hexane (1 L × 5). The aqueous layer was evaporated to remove traces of organic solvent,
and then extracted with EtOAc and n-BuOH, successively (1 L × 5 each). The EtOAc and
n-BuOH fractions were concentrated under reduced pressure to give 24.0 and 22.0 g of res-
idue, respectively. The remaining aqueous layer was concentrated to furnish a water-soluble
fraction (123.0 g).
The n-BuOH fraction (22.0 g) was subjected to silica gel chromatography (600 g),
(Ф = 60 mm, L = 45 cm), using a CHCl3–MeOH gradient system with 200 mL fractions being
collected and similar fractions combined to yield nine fractions. Fraction V (4.0 g) was purified
on RPCC, affording thirteen fractions. The first fraction V-1 (135.0 mg) was purified by HPLC
(30% MeOH) to produce 2 (22.2 mg) and 3 (14.2 mg). The fourth fraction V-4 (256.8 mg) was
purified by HPLC (30% MeOH) to give 1 (14.24 mg) and 4 (8.8 mg). The tenth fraction V-10
afforded compound 5 (39.2 mg).
Officinalioside (1): White amorphous powder; [α]D24 −8.14 (c = 0.97, MeOH); uV λmax (MeOH)
nm (log ε): 255 (4.63), 310 (4.66), 360 (4.74), 380 (4.68); CD (MeOH) nm (Δε): 283 (−0.14), 320
(+0.53), IR (film) νmax 3360, 2938, 2882, 1699, 1649, 1557, 1540, 1511, 1456, 1418, 1120, 1068,
671 cm−1; 1H NMR (600 MHz, pyridine-d5): 3.19 (1H, m, H-8′), 3.73 (6H, s, 3′,5′-OCH3), 3.79 (6H,
s, 3,5-OCH3), 3.99 (1H, m, H-3″), 4.09 (1H, m, H-9′a), 4.13 (1H, m, H-9′b), 4.29 (1H, m, H-4″),
4.30 (1H, m, H-6″b), 4.35 (1H, m, H-2″), 4.34 (1H, m, H-5″), 4.39 (1H, m, H-6″a), 4.46 (1H, m,
H-9b), 4.65 (1H, m, H-9a), 4.69 (1H, m, H-8), 5.15 (1H, d, J = 8.2 Hz, H-7′), 6.08 (1H, d, J = 7.8 Hz,
H-1″), 7.11 (2H, s, H-2′, 6′), 7.68 (2H, s, H-2, 6) and 13C NMR (150 MHz, pyridine-d5): 50.3 (C-8),
55.3 (C-8′), 56.7 (3′, 5′-OCH3), 57.0 (3, 5-OCH3), 61.0 (C-9′), 62.9 (C-6″), 71.2 (C-9), 71.9 (C-4″),
76.4 (C-2″), 78.8 (C-5″), 79.4 (C-3″), 85.0 (C-7′), 104.4 (C-1″), 105.7 (C-2′, 6′), 108.1 (C-2, 6),
132.7 (C-1′), 132.9 (C-1), 137.7 (C-4′), 140.8 (C-4), 149.6 (C-3′, 5′), 153.8 (C-3, 5), 199.4 (C-7);
HR-ESI-MS (positive-ion mode) m/z: 619.1990 [M + Na]+ (Calcd for C28H36O14Na: 619.1997).

NATuRAL PRODuCT RESEARCH
5
3.4. Analysis of the sugar moiety
About 1 mg of compound 1 was hydrolysed with 1 M HCl (1.0 mL) at 80 °C for 2 h. The
reaction mixture was neutralised with Amberlite IRA96SB (OH−), then partitioned with an
equal amount of EtOAc (1.0 mL) and the water layer was analysed for its sugar component.
The sugar was determined by HPLC on an amino column [Shodex Asahipak NH2P-50 4E
(4.6 × 250 mm), CH3CN–H2O (3:1), 1 mL/min], using a chiral detector (JASCO OR-2090plus), in
comparison with authentic sugar (D-glucose). Compound 1 gave the peak for D-(+)-glucose
at a retention time of 8.32 min.
3.5. DPPH radical scavenging activity
The test compounds dissolved in DMSO were diluted with MeOH (100 μL) in 96-well micr-
otitre plates and the absorbance of the compounds was measured at 515 nm as Abackground
.
Then, a 200 μM DPPH solution (100 μL) was added to each well, followed by incubation at
room temperature for 30 min. The absorbance was measured again as Asample
.
The % inhibition was calculated using the following equation:
% inhibition = [1 − (Asample − Abackground)∕(Acontrol − Abackground)] × 100
where Acontrol is the absorbance of the control reaction mixture containing vehicle and all
reagents except for the test compound. IC50 was determined as the concentration of sample
required to inhibit the formation of the DPPH radical by 50% (Samy et al. 2014c).
3.6. Human cancer cell growth inhibition assay
This assay was performed using a human lung cancer cell line (A549) and the viability was
estimated by means of the colorimetric MTT assay. Dulbecco’s modified Eagle’s medium
supplemented with 10% heat-inactivated FBS and 100 μg/mL kanamycin was used as the
cell culture medium. The test compounds were dissolved in DMSO and then added to the
wells of 96-well microtitre plates to a final concentration of 1%. A549 cells (5 × 103 cells/well)
were cultured in a 5% CO2 incubator at 37 °C for 72 h. MTT solution was added to each well
and the plates were incubated for a further 1.5 h. The formazan precipitates were dissolved in
DMSO and the optical density value for each well was measured at 540 nm with a microplate
reader. Doxorubicin was used as a positive control.
The cell growth inhibition was calculated using the following equation:
% inhibition = [1 − (Asample − Ablank)∕(Acontrol − Ablank)] × 100
where Acontrol is the absorbance of the control reaction mixture containing DMSO and all
reagents except for the test compound. IC50 was determined as the concentration of sample
required to inhibit the formation of MTT formazan by 50% (Samy et al. 2014c).
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1, S2, andTable S1.

6
M. N. SAMy ET AL.
Acknowledgements
The authors are grateful for access to the superconducting NMR instrument, uV and ESI-MS at the
Analytical Center of Molecular Medicine, the Analysis Center of Life Science and the Natural Science
Center for Basic Research and Development of the Graduate School of Biomedical and Health Sciences,
Hiroshima university.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by JSPS KAKENHI [grant number 26460122]. It was also supported by Ministry
of Higher Education of Egypt through the Scientific Mission System.
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