New n-nonadecanoyl-β-sitosterol and other constituents from the stem-bark of Anacardium occidentale

Article abstract of DOI:10.1080/14786419.2019.1650353

A new steroidal ester bearing n-nonadecanoyl moiety (1) and a mixture of isomeric cerebrosides (2) along with two known compounds were isolated from the methanol extract of the stem-bark of Anacardium occidentale. The structure of the new steroidal ester was determined as 3-n-nonadecanoyl-β-sitosterol on the basis of modern spectroscopic techniques (IR, ESI-MS, HR-ESIMS, 1D and 2D NMR) and chemical degradation studies. The structures of the known compounds were identified as gallic acid and tanacetene by comparison of the spectroscopic data with those of reported data. The mixture of cerebrosides was confirmed based on the analysis of 1D and 2D NMR. These compounds were evaluated for cytotoxicity against human cancer cell lines A549, SCOV3 and rat normal cell line NRK49f.

Full text of DOI:10.1080/14786419.2019.1650353

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
New n-nonadecanoyl-β-sitosterol and other
constituents from the stem-bark of Anacardium
occidentale
Abdullahi Shehu, Mangala Gowri Ponnapalli, M. Mahboob, P. V. Prabhakar &
Gabriel Ademola Olatunji
To cite this article: Abdullahi Shehu, Mangala Gowri Ponnapalli, M. Mahboob, P. V.
Prabhakar & Gabriel Ademola Olatunji (2019): New n-nonadecanoyl-β-sitosterol and other
constituents from the stem-bark of Anacardiumꢀoccidentale, Natural Product Research, DOI:
10.1080/14786419.2019.1650353
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1650353
New n-nonadecanoyl-b-sitosterol and other constituents
from the stem-bark of Anacardium occidentale
Abdullahi Shehua, Mangala Gowri Ponnapallib, M. Mahboobc, P. V. Prabhakarc
and Gabriel Ademola Olatunjid
aDepartment of Chemistry, Faculty of Science, Federal University Lokoja, Lokoja, Kogi State, Nigeria;
bCentre for Natural Products and Traditional Knowledge, Indian Institute of Chemical Technology,
c
Hyderabad, India; Toxicology Unit, Biology Division, Indian Institute of Chemical Technology,
d
Hydrabad, India; Department of Industrial Chemistry, Faculty of Physical Science, University of
Ilorin, Ilorin, Nigeria
ABSTRACT
ARTICLE HISTORY
Received 22 October 2018
Accepted 24 July 2019
A new steroidal ester bearing n-nonadecanoyl moiety (1) and a
mixture of isomeric cerebrosides (2) along with two known com-
pounds were isolated from the methanol extract of the stem-bark
of Anacardium occidentale. The structure of the new steroidal
ester was determined as 3-n-nonadecanoyl-b-sitosterol on the
basis of modern spectroscopic techniques (IR, ESI-MS, HR-ESIMS,
1D and 2D NMR) and chemical degradation studies. The struc-
tures of the known compounds were identified as gallic acid and
tanacetene by comparison of the spectroscopic data with those
of reported data. The mixture of cerebrosides was confirmed
based on the analysis of 1D and 2D NMR. These compounds
were evaluated for cytotoxicity against human cancer cell lines
A549, SCOV3 and rat normal cell line NRK49f.
KEYWORDS
Anacardium occidentale;
3-n-nonadecanoyl-b-sitos-
terol; cerebroside;
cytotoxicity
1. Introduction
Anacardium occidentale Linn. (Anacardiaceae) is a tropical evergreen tree commonly
called cashew. It is native to Brazil and distributed throughout tropical countries like
Nigeria, Kenya, Tanzania and India. A. occidentale stem-bark extracts are used in some
CONTACT Abdullahi Shehu
abdusfa@gmail.com; Mangala Gowri Ponnapalli
mangala@iict.res.in;
pmgowri@yahoo.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1650353.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
A. SHEHU ET AL.
Figure 1. Compounds isolated from Anacardium occidentale.
part of Nigeria to treat diabetes and cardiovascular diseases (Eliakim-Ikechukwu et al.
2010; Okonkwo et al. 2010). Crude extracts of leaves, stem-bark, fruits and nut-shell
liquid have been reported by various researchers to exhibit wide spectrum of bio-
logical activities. The extracts have been shown to possess hypoglycaemic properties
(Abdullahi and Olatunji 2010), cytotoxic (Kubo et al. 2011; Santos et al. 2019), geno-
toxic effects (Barcelos et al. 2007), and tyrosinase inhibitory potential (Kubo et al.
1994). The hydroethanolic extract of the leaves of A. occidentale is reported to exhibit
pronounced effect on leukaemia cell lines compared to normal T-lymphocytes as well
as apoptic effect increased casp3 mRNA level expression in ALL cells (Santos et al.
2019). Further, the prior study of cashew kernel testa, an industrial waste from this
plant demonstrated that it is a rich source of industrially significant tannins by HPLC
(high performance liquid chromatography), TGA (thermo gravimetric analysis) and FTIR
(Fourier-transform Infrared spectroscopy) analysis (Viswanath et al. 2016). Previous
phytochemical investigations revealed the presence of polyphenols and tannins
(Trevisan et al. 2006; Viswanath et al. 2016), flavonoids, their glycosides (Edy et al.
2007) and sterols (Murthy et al. 1982; Alexander et al. 2004). Herein, we report the iso-
lation and structural elucidation of a new steroidal ester, a mixture of cerebrosides
and two known constituents from the stem-bark of Anacardium occidentale (Figure 1)
and their cytotoxicity evaluation against human cancer cell lines and a rat normal
cell line.
2. Results and discussion
Compound 1 was obtained as a white crystalline solid, mp 78.5 ꢀC. The ESIMS of com-
pound 1 displayed a pseudo-molecular ion [M þ Na]þ at m/z 717.60127, consistent
with the molecular formula of C48H86O2Na, indicating six degrees of unsaturation. The
IR spectrum of 1 showed absorption bands for ester carbonyl at 1729 cmꢁ1 and ole-
finic functionality at 1640 cmꢁ1. The analysis of the 1D NMR spectroscopic data indi-
cated that 1 was a steroidal ester structurally related to b-sitosterol. Its 1H NMR
spectrum (Table S1) exhibited characteristic signals for 18-Me at (d 0.69, s) and 19-Me

NATURAL PRODUCT RESEARCH
3
Table 1. IC50 values (mM) of compounds.
Cell lines
Compounds
A549
SCOV3
NRK-49f
1
3
4
1142.94
68.29
634.74
2.5
502.62
305.17
255.63
4.06
267.90
199.94
NA
Doxorubicin
6.2
NA: No activity.
at (d 1.02, s) of sterol nucleus in addition to three secondary methyls at (d 0.95, d,
J ¼ 6.5 Hz, 21-Me), (d 0.92, d, J ¼ 6.5 Hz, 26-Me), (0.81, d, J ¼ 6.5 Hz, 27-Me) and one pri-
mary methyl at (d 0.84, t, J ¼ 6.5 Hz, 29-Me). The diagnostic 6-H of the D5 sterol was
noticed at (d 5.37, br d, J ¼ 4.7 Hz) and deshielded oxygenated methine at (d 4.64, 1 H,
m), corresponded to an oxymethine attached to an ester carbonyl. Further, it showed
long chain terminal methyl at (d 0.88, t, J ¼ 6.9 Hz). Its 13C NMR spectrum
(Supplementary material Table S1) revealed resonances for 48 carbons which were dif-
ferentiated by DEPT and HSQC spectra into seven methyls, twenty eight methylenes,
nine methines and four quaternary carbons. It displayed the signals for an oxygenated
methine carbon at (d 73.7), a trisubstituted olefinic carbons at (d 122.6, 139.7). Four
spin systems involving protons H-19’/H-18’, H-2’/H-3’, H-2/H-3/H-4 and H-6/H-7 were
identified by the analysis of COSY spectrum (Supplementary material Table S2 and
Figure S1). Its HMBC spectrum (Supplementary material Table S2 and Figure S1) exhib-
ited correlations between H-4 and C-3, C-5 and C-6 confirmed the position of the oxy-
genated methine at C-3. Other HMBC correlations between H-6/C-7, C-8, 19 Me/C-1, C-
5, C-9 were allowed to locate the position of an olefin at C-5. The absence of NOESY
(Supplementary material Table S2 and Figure S2) correlations between ring junction
protons confirmed that compound 1 possesses trans ring fusion. The complete assign-
1
1
ment of H and 13C NMR spectra of 1 were done by H-1H COSY, HSQC and HMBC.
Alkaline hydrolysis of 1 afforded b-sitosterol and methyl ester of n-nonadecanoic acid,
which was further confirmed by IR and GC-MS analysis. Thus, the structure of 1 was
established as 3-n-nonadecanoyl-b-sitosterol.
The spectroscopic data of compound 2 (Supplementary material Tables S1 and S2)
are characteristic of a cerebrosides and similar to the one reported in the literature
(Kang et al. 1999). However, the data revealed that compound 2 is a mixture of iso-
meric cerebrosides as evident from the multiple signals in the olefinic, anomeric and
1
NH regions of the H NMR as well as different signals intensity ratios in the 13C NMR
spectrum. Thus, compound 2 is characterised as a mixture of isomeric cerebrosides.
1
Based on the physical and spectroscopic (MS, IR, H and 13C NMR) comparison with lit-
erature, the structures of the other known compounds were characterised as gallic
acid 3 and tanacetene 4 (Mahmood et al. 2002; Abri and Maleki 2016). Compounds 1,
3 and 4 were tested for their cytotoxicity activity on A 549 (lung adenocarcinoma),
SCOV3 (ovarian carcinoma) and NRK-49f (normal rat kidney fibroblast) cell lines using
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, compared
with doxorubicin as a positive control. The cell growth-inhibitory potencies of the
compounds, expressed as IC50 values as shown in Table 1. Compound 3 exhibited
moderate cytotoxicity activity (IC50 68.29 mM) against human lung adenocarcinoma

4
A. SHEHU ET AL.
(A549). This result lends support to the earlier report by Kubo et al. (2011) and
affirmed the role of phenolic compounds from A. occidentale as anti-cancer agents.
However, compounds 1 and 4 exhibited no significant antiproliferative effect against
the cancer cell lines achieving IC50 values >100 mM. The criteria for cytotoxicity for
crude extracts, as established by the U.S. National Cancer Institute (NCI), is an IC50
<
20 mM in the preliminary assay (Abdel-Hameed et al. 2012). The cytoxicity of com-
pound 3 was observed in a dose-dependent manner and the cell viability above
10 mM was significantly different (p < 0.05) from the control (Supplementary material
Figures S3 and S4).
3. Experimental
General experimental procedures
The IR spectra were recorded on a Nicolet-740 FT-IR Spectrometer (Thermo Scientific,
I.I.C.T., Hyderabad, India). The NMR spectra were recorded with Bruker Avance II
(600MHz), Bruker Avance (500 MHz), Varian Inova (400 MHz) and Bruker Avance
1
(300MHz) (Bruker, I.I.C.T., Hyderabad, India) for HNMR, and 150 MHz,125 MHz, 100 MHz,
75 MHz for 13CNMR in CDCl3,C5D5N and (CD3)2CO with tetramethylsilane as an internal
standard. Coupling constants are given in Hz. The ESI-MS data were recorded on an
Agilent 1100 MSD (Agilent Technologies, I.I.C.T., Hyderabad, India) with ESI SL Trap. The
HR-ESI-MS data were acquired on an Agilent 6510Q-T.O.F. (Agilent Technologies, I.I.C.T.,
Hyderabad, India and ESI probe. Thin-layer chromatography (TLC) was performed on
precoated silica gel GF254 plates (Merck & Co., I.I.C.T., India). The TLC plates were visual-
ised under UV light or by spraying with 5% H2SO4 in methanol and heating at 70ꢀC.
Plant material
The stem-barks of A. occidentale were collected at Olorunsogo Area in Ilorin, Kwara
State. They were identified and authenticated by Mr. Edward Bolu Ajayi at the
Herbarium of the Department of Plant Science, University of llorin, Nigeria. Voucher
Specimens (UIH 001/970) were deposited at the Department of Plant Science,
University of Ilorin, Nigeria.
Extraction and isolation
Air dried stem-bark of A. occidentale (1.8 kg) was coarsely powdered and percolated
with n-hexane, ethyl acetate and ethanol respectively. The ethanol extract (196 g) was
subjected to VLC on silica gel (230–400 mesh) and eluted with a mixture of CHCl3-
acetone-MeOH in order of increasing polarity yielding 28 fractions of 500 mL each. The
identical fractions were pooled based on the TLC profile. A total of eight main frac-
tions (ETF1–ETF8) were obtained (Jone & Kinghorn 2006). Repeated column chroma-
tography of fraction ETF2 (400 mg) afforded sub fractions, which was further subjected
to AgNO3-impregnated silica gel column chromatography CHCl3: hexane (1.5:8.5) fur-
nished compound 1 (15 mg). Fraction ETF6 was suspended in H2O (5 mL) and then
extracted with EtOAc. The EtOAc layers were concentrated under reduced pressure to

NATURAL PRODUCT RESEARCH
5
yield crude residue (10.4 g). A portion of the fraction (2.5 g) was subjected to further
purification on a silica gel (230–400 mesh) CC eluting with a mixture of MeOH/CHCl3
yielded ten fractions. Fraction 2 (5% MeOH/CHCl3) gave compound 2 (6 mg). The ethyl
acetate extract (204 g) was also subjected to VLC on silica gel (230–400 mesh) eluting
with a mixture of acetone-hexane of increasing polarity to afford a total of six fractions
(EAF1–EAF6). The more polar fraction (EAF5) afforded compound 3 (26 mg) while the
non-polar fraction (EAF1) gave compound 4 (5 mg).
Hydrolysis of compound 1
Compound degradation study was carried out to separate the steroid moiety from the
long chain saturated ester. Compound 1 (5 mg) was dissolved in a mixture of THF/H2O
(5 mL) v/v in a 25 mL round-bottomed flask and KOH (0.273 g) was added to the flask.
The reaction solution was refluxed for 12 h at 60 ꢀC. The mixture was extracted with
EtOAc (5x 5 mL) and dried over anhydrous Na2SO4. The product was purified by recrys-
tallization from MeOH to yield the basic part of the hydrolysed molecule 1a (2.3 mg).
The aqueous layer was acidified with 3% Con. HCl and then extracted with CHCl3 (3x
3 mL) to give the acidic part of the hydrolysed molecule. The CHCl3 extract was then
methylated by adding MeOH (3 mL), 3 drops conc. H2SO4 and the mixture was
refluxed for 12 h. The reaction mixture was then extracted with CHCl3 and the result-
ing methyl ester 1b (2.1 mg) was subjected to GC-MS & IR analyses.
Cytotoxicity assay (MTT assay)
Human tumour cell lines, A549 (lung adenocarcinoma), SKOV3 (ovarian carcinoma)
and normal cell line NRK-49F (normal rat kidney fibroblast) are seeded onto 96-well
micro titre plates at different concentrations and then incubated at 36.5 ꢀC in humidi-
fied CO2 5% incubator for 24 h. The growth medium was removed and maintenance
medium added 2 ml containing various concentrations of compounds 1, 3 and 4
whereas positive and negative control as standard drug (doxorubicin) and no standard
drug respectively and the plates were incubated for the next 24 h. After incubation,
medium was removed from the wells and 100 ml of maintenance medium þ 5 ml MTT
was added. Plates were incubated for 30–60 minutes, medium/MTT was removed and
insoluble product was dissolved in 50 ml DMSO. Finally, the absorbance was measured
at 540 nm. The 50% inhibitory concentration (IC50) value of the compounds was calcu-
lated (Ata-ur-Rahman et al. 2007).
3-nonadecanoyl-b-sitosterol (1): C48H86O2; white crystalline solid from a mixture of
15% EtOAc/n-hexane; Rf 0.5 (15% chloroform/n-hexane); m.p. 78.5 ꢀC; IR (KBr) mmax
1
2918, 2850, 1736, 1630, 1464, 1376, 1173, 765 cmꢁ1; H, 13C, and 2 D NMR data (see
Supplementary material Tables S1 and S2 and Figure S2); HR-ESIMS m/z 717.65200
[M þ Na]þ (calcd for C48H86O2Na 717.60028).

6
A. SHEHU ET AL.
4. Conclusion
The isolation and characterisation of a new steroidal ester are noteworthy. To the best
of our knowledge, this is the first time reported a b-sitosterol substituted at a C-3 car-
bon with a C19 long chain saturated aliphatic ester isolated from natural sources and
characterised as such. Although similar chain aliphatic ester with longer or shorter car-
bon chains have been isolated from many plants (Dupont et al. 1997; Dinda et al
2003), compound 1 differ from these owing to its odd number of carbon chains. The
existence of the other known compounds in Anacadiaceae family is not well known
and furthermore this is the first report of the compounds from Anacardium
occidentale.
Acknowledgement
The authors acknowledged Tertiary Education Trust Fund (TETFUND), Federal University Lokoja,
Nigeria and TWAS-CSIR Postgraduate fellowships given to Abdullahi Shehu and the Director, Dr.
S. Chandrasekhar, CSIR-Indian Institute of Chemical Technology, Hyderabad, India for constant
support and encouragement.
Disclosure statement
No potential conflict of interest was reported by the authors
Funding
This work was supported by Academy of Science for the Developing World [TWAS-CSIR
Postgraduate Fellowship 2014-2015] and Tertiary Education Trust Fund, FU Lokoja, Nigeria.
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