A new indole alkaloid, antioxidant and antibacterial activities of crude extracts from Saccocalyx satureioides

Article abstract of DOI:10.1080/14786419.2018.1519817

The new acylated indole alkaloid glucoside indole-3-carboxylic acid-(6'-O-caffeoyl)-β-D-glucoside 1 has been isolated from the ethyl acetate (EtOAC) extract of Saccocalyx satureioides Coss. & Dur. (Lamiaceae) together with eight known secondary metabolites 2-9. Two indoles 2 and 3, five methylated flavone aglycones 4-8 and one monoterpene glucoside 9 were reported for the first time in the genus Saccocalyx. The structural elucidation of these compounds was accomplished by spectroscopic methods including 1 D (¹H and ¹³C) and 2 D (COSY, HSQC and HMBC) NMR techniques, and mass spectrometry, and by comparison with literature data. Light petroleum, EtOAc, chloroform and n-butanol (n-BuOH) extracts of S. Satureioides were screened for their antioxidant activity using DPPH radical scavenging and β-carotene bleaching methods. The antibacterial activity of these extracts indicates that n-BuOH and EtOAc extracts possess the strongest activity.

Full text of DOI:10.1080/14786419.2018.1519817

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
A new indole alkaloid, antioxidant and
antibacterial activities of crude extracts from
Saccocalyx satureioides
Hayat Kherkhache, Imane Benabdelaziz, Artur M. S. Silva, Mokhtar Boualem
Lahrech, Mokhtar Benalia & Hamada Haba
To cite this article: Hayat Kherkhache, Imane Benabdelaziz, Artur M. S. Silva, Mokhtar Boualem
Lahrech, Mokhtar Benalia & Hamada Haba (2018): A new indole alkaloid, antioxidant and
antibacterial activities of crude extracts from Saccocalyxꢀsatureioides, Natural Product Research,
DOI: 10.1080/14786419.2018.1519817
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2018.1519817
A new indole alkaloid, antioxidant and antibacterial
activities of crude extracts from Saccocalyx satureioides
Hayat Kherkhachea, Imane Benabdelazizb, Artur M. S. Silvac
,
Mokhtar Boualem Lahrecha, Mokhtar Benaliad and Hamada Habab
a
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Laboratoire de Chimie Organique et Substances Naturelles, Universite de Djelfa, Djelfa, Algerie;
b
0
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Departement de Chimie, Laboratoire de Chimie et Chimie de l Environnement (L.C.C.E), Faculte des
c
ꢁ
ꢀ
ꢀ
Sciences de la Matiere, Universite de Batna-1, Batna, Algerie; Department of Chemistry & QOPNA,
University of Aveiro, Aveiro, Portugal; Departement de Chimie Industrielle, Laboratoire de Chimie,
Universite de Laghouat, Laghouat, Algerie
d
ꢀ
ꢀ
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ABSTRACT
ARTICLE HISTORY
Received 12 April 2018
Accepted 2 September 2018
The new acylated indole alkaloid glucoside indole-3-carboxylic
acid-(6’-O-caffeoyl)-b-D-glucoside 1 has been isolated from the
ethyl acetate (EtOAC) extract of Saccocalyx satureioides Coss. &
Dur. (Lamiaceae) together with eight known secondary metabo-
lites 2-9. Two indoles 2 and 3, five methylated flavone aglycones
4-8 and one monoterpene glucoside 9 were reported for the first
time in the genus Saccocalyx. The structural elucidation of these
compounds was accomplished by spectroscopic methods includ-
ing 1 D (1H and 13C) and 2 D (COSY, HSQC and HMBC) NMR tech-
niques, and mass spectrometry, and by comparison with literature
data. Light petroleum, EtOAc, chloroform and n-butanol (n-BuOH)
extracts of S. Satureioides were screened for their antioxidant
activity using DPPH radical scavenging and b-carotene bleaching
methods. The antibacterial activity of these extracts indicates that
n-BuOH and EtOAc extracts possess the strongest activity.
KEYWORDS
Saccocalyx satureioides;
Lamiaceae; indole;
antioxidant activity;
antibacterial activity; NMR
CONTACT Hamada Haba
haba.hamada@yahoo.fr, hamada.haba@univ-batna.dz
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1519817
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
H. KHERKHACHE ET AL.
1. Introduction
Saccocalyx satureioides Coss. & Dur., an endemic species of Algeria, is a small aromatic
shrub growing in Sahara septentrional and belonging to the family Lamiaceae (Quezel
and Santa 1963; Ozenda 2004). It has attracted a great attention due to its traditional
medicinal usage for gastric disorders and spasms (Biondi et al. 2006). The essential oil
of S. satureioides has been extensively investigated and revealed the presence of
numerous oil components, which possess interesting biological activities (Biondi et al.
2006; Laouer et al. 2006; Bendahou et al. 2008). However, a literature survey reveals
that there is only one study describing the isolation and structural elucidation of
phytochemical components of this plant (Mohamadi et al. 2015).
The TLC profile of the different extracts (light petroleum, CHCl3, EtOAc and n-BuOH)
obtained from S. satureioides revealed the especial richness of EtOAc extract in phyto-
chemicals compared to the other extracts, and this is the reason why our phytochem-
ical study is focused on this extract.
This study led to the isolation and identification of one new acylated indole alkaloid
glucoside 1 and eight known compounds, including two indoles 2 and 3, five methy-
lated flavone aglycones 4-8 and one monoterpene glucoside 9. Since several isolated
compounds such as indole derivatives and methylated flavones were reported to have
an interesting antibacterial and antioxidant activities, we decided to assess these bio-
activities against the light petroleum, CHCl3, EtOAc and n-BuOH extracts obtained
from this species.
2. Results and discussion
2.1. Chemicals constituents of EtOAc extract
The multistep chromatographic separations and purifications (VLC, CC and TLC) were
applied on EtOAc extract of the aerial parts of S. satureioides allowing the isolation of
indole-3-carboxylic acid-(6’-O-caffeoyl)-b-D-glucoside 1, a new indole alkaloid deriva-
tive (Figure 1), together with eight known secondary metabolites 2-9. The structures
of the isolated compounds 1-9 were elucidated using 1 D and 2 D NMR analysis,
HRESI-MS spectroscopy and comparison with literature data. The known products
were identified as: indole-3-carboxylic acid-b-D-glucoside 2, indole-3-carboxylic acid 3
(Hagemeier et al. 2001), luteolin-7-methyl ether 4 (Saewan et al. 2011), velutin 5 (Kraut
et al. 1995), conzalitosin 6 (Salan et al. 2001), 4’,5,6-trihydroxy-3’,7-dimethoxyflavones
7 (Intekhab and Aslam 2011), thymonin 8 (Broucke et al. 1982), a-terpinol-8-O-b-D-glu-
coside 9 (Yamahara et al. 1985). It should be noted that all the isolated compounds 1-
9 were reported for the first time from this genus, and among them, the two indoles
2 and 3 are described herein for the first time in Lamiaceae family.
2.2. Structural elucidation of the new compound
Compound 1 was isolated as a brown wax. Its HR-ESIþMS showed a pseudo-molecular
ion peak at m/z 508.4301 [M þ Na]þ (calcd. for C24H23NO10Na, 508.4293), consistent
with the molecular formula C24H23NO10, indicating the presence of 14 degrees of

NATURAL PRODUCT RESEARCH
3
2
1
3
R1
4: OH
5: OH
6: OH
R2
H
R3
R4
H
H
H
H
R5
OH
R6
OCH3
OCH3
OCH3
OH
OH
H
OCH3
H
OCH3 OCH3
OCH3
7: OH OH OCH3
OH
OH
8: OH OH OCH3 OCH3 OCH3
9
Figure 1. Structures of isolated compounds 1–9.
1
unsaturation. The H NMR spectrum of compound 1 showed five downfield signals of
an ABCD aromatic spin system at dH 8.02 (d, J 7.5 Hz, H-4), 7.18-7.22 (m, H-5), 7.20-7.24
(m, H-6) and 7.49 (d, J 7.7 Hz, H-7), and of a vinylic proton at dH 8.16 (s, H-2). These
main characteristic features suggested the presence of an indole alkaloid skeleton
(Levy et al. 2000). The absence of a coupling constant for H-2 proved the existence of
a substituent at C-3 of the indole ring. Indeed, the HMBC spectrum confirmed this
3
assumption with a J cross-peak between H-2 and the quaternary carbon at dC 162.8,
2
assigned to the carbonyl ester C-8, and a J correlation of this proton (H-2) with the
signal of C-3. The 13C NMR spectrum of 1 exhibited 24 carbon resonances classified
into eight quaternary carbons (dC 105.6, 125.4, 125.7, 136.4, 145.5, 148.4, 162.8 and
166.5), fifteen methine (dC 69.4, 72.5, 74.6, 76.3, 93.6, 112.4, 113.7, 114.9, 115.7, 120.5,
121.4, 121.5, 122.5, 133.5 and 145.4) and one methylene (dC 63.4) carbons. The carbon
resonances at dC 105.6, 112.4, 120.5, 121.5, 122.5, 125.4, 1334.4 and 136.4 were attrib-
uted to the indole skeleton using HMBC and HSQC correlations. The downfield signals
at dC 63.4, 69.4, 72.5, 74.6, 76.3 and 93.6 were assigned to a glucopyranosyl moiety
based in the comparison with data reported in the literature (Hagemeier et al. 2001).
1
In addition, the H NMR spectrum supported the presence of the glucosyl moiety with
the anomeric proton resonating at dH 5.63 (d, J 7.2 Hz, H-1’). The analysis of the COSY
and HSQC spectra permitted the full assignment of the glucose proton signals.
Furthermore, acid hydrolysis of 1 in refluxing MeOH and HCl (1N) solution provided
(þ)-D-glucose which was confirmed by TLC comparison with an authentic sample. The
cross-peak of the anomeric proton H-1’ and carbonyl carbon C-8 (dC 162.8) allow us to
establish the linkage point between the sugar moiety and the substituted indole skel-
eton (Figure S10, supplementary material). This linkage was confirmed by the chemical
shift value of the glucosyl carbon C-1’ at dC 93.6, which would appear at higher than

4
H. KHERKHACHE ET AL.
100 ppm in 3-O-glucosylated derivatives (Meng et al. 2017). From these results, the
partial structure of compound 1 was assigned as an indole-3-carboxylic acid-b-D-glu-
copyranosyl ester derivative (Hagemeier et al. 2001). However, the remaining part of
the molecule it was identified as a C9H7O3 moiety according to the ESI-MS spectrum.
The ABX spin system of a caffeoyl moiety appear at dH 7.05 (s, H-2"), 6.75 (d, J 7.9 Hz,
H-5") and 7.01 (d, J 7.9 Hz, H-6"), while their vinylic protons appear as two doublets at
3
dH 7.46 (H-7") and 6.28 (H-8"), with a high J 15.9 Hz characteristic of a trans configur-
ation. The remaining proton and carbon assignments were accomplished by HSQC
and HMBC spectra. The 13C NMR spectrum of the glucose moiety also showed a
downfield shift of C-6’ signal (D þ 0.9) relatively to the chemical shift values of an
unlinked carbon C-6’ (Hagemeier et al. 2001, Meng et al. 2017). This shift is expected
for a glucose C-6’ acylation (Hosokawa et al. 1997). This chemical bond was also con-
3
firmed by a J HMBC cross-peak of H-6’ signals (dH 4.16, dd, J 11.7, 6.0 Hz and 4.41, d,
J 11.7 Hz) with the caffeoyl carbonyl ester C-9" signal (dC 166.5) (Figure S10, supple-
mentary material). These findings showed that caffeic acid was linked to C-6’ of gluco-
syl moiety. All the above-mentioned NMR spectral data established the new structure
of compound 1 as indole-3-carboxylic acid-(6’-O-caffeoyl)-b-D-glucoside.
2.3. Spectral data
Indole-3-carboxylic acid-(6’-O-caffeoyl)-b-D-glucoside: Brown wax; HR-ESIþ-MS: m/z
1
508.4301 [M þ Na]þ (calcd. for C24H23NO10Na, 508.4293) for formula C24H23NO10. H
NMR (300 MHz, DMSO-d6) dH (ppm): 12.06 (s, NH), 8.16 (s, H-2), 8.02 (d, J 7.5 Hz, H-4),
7.49 (d, J 7.7 Hz, H-7), 7.46 (d, J 15.9 Hz, H-7"), 7.20-7.24 (m, H-6), 7.18-7.22 (m, H-5),
7.05 (s, H-2"), 7.01 (d, J 7.9 Hz, H-6"), 6.75 (d, J 7.9 Hz, H-5"), 6.28 (d, J 15.9 Hz, H-8"),
5.63 (d, J 7.2 Hz, H-1’), 4.41 (d, J 11.7 Hz, H-6), 4.16 (dd, J 11.7 Hz, 6.0 Hz, H-6), 3.58-3.64
(m, H-5’), 3.36-3.42 (m, H-2’ and H-3’), 3.29 (t, J 8.1 Hz, H-4’). 13C NMR (75 MHz, DMSO-
d6) dC (ppm): 162.8 (C-8), 166.5 (C-9"), 148.4 (C-4"), 145.5 (C-3"), 145.4 (C-7"), 136.4 (C-
7a), 133.5 (C-2), 125.7 (C-1"), 125,4 (C-3a), 122.5 (C-6), 121.5 (C-5), 121.4 (C-6"), 120.5 (C-
4), 115.7 (C-5"), 114.9 (C-2"), 113.7 (C-8"), 112.4 (C-7), 105.6 (C-3), 93.6 (C-1’), 76.3 (C-3’),
74.6 (C-5’), 72.5 (C-2’), 69.4 (C-4’), 63.4 (C-6’).
2.4. Evaluation of antioxidant activity
2.4.1. DPPH radical scavenging method
The antioxidant activity of the different extracts of S. satureioides was measured in
terms of hydrogen donating or radical scavenging ability using the stable DPPH radical
(Von Gadow et al. 1997). Quercetin was used as positive control. The antioxidant
power of the different extracts (Table S1, supplementary material) decreased in the
order of EtOAC (IC50 20.81 3.52 mg/mL) > CHCl3 (IC50 23.93 4.15 mg/mL) > n-BuOH
(IC50 93.23 6.43 mg/mL) > light petroleum (IC50 537.02 1.03 mg/mL). The EtOAc and
CHCl3 extracts showed the highest radical scavenging activity while the activity of
light petroleum extract was much lower. It should be noted that the substrate polarity
of extracts obtained from plants does not affect DPPH scavenging activity (Yamaguchi
et al. 1998; Pekkarinen et al. 1999; Koleva et al. 2002). However, the lower

NATURAL PRODUCT RESEARCH
5
effectiveness of the CHCl3 extract in comparison with EtOAc extract may be explained
by the weak concentration of active components in CHCl3 extract, having capacity to
donate hydrogen atoms or electrons and to capture free radicals (Kulisic et al. 2004).
Moreover, this study revealed that the major constituents of EtOAc extract are flavo-
noids known especially for their antioxidant activity.
2.4.2. β-Carotene bleaching method
This method is based on the loss of b-carotene orange colour due to its reaction with
radicals formed by linoleic acid oxidation in an emulsion system. The b-carotene
bleaching rate can be slow down in the presence of antioxidants (Pratt 1980; Mallet
et al. 1994). Butylated hydroxyanisole (BHA) was used as standard. The IC50 values of
the different extracts (Table S1, supplementary material) clearly indicated that chloro-
form extract possessed the highest antioxidant potential (IC50 40.32 5.32 mg/mL). The
weakest radical scavenging activity was exhibited by the light petroleum extract with
an IC50 908.35 0.90 mg/mL. The fact that the CHCl3 extract being more effective as
antioxidant than the semi-polar EtOAc extract (IC50 57.12 1.55 mg/mL), in comparison
with the results obtained for the DPPH assay, suggests that the antioxidant activity of
the CHCl3 extract is probably due to the presence of lipophilic active compounds
(Kulisic et al. 2004).
2.5. Evaluation of antibacterial activity
The results obtained from disc diffusion method (Bauer et al. 1996) indicated that
Staphylococcus aureus and Escherichia coli are the most sensitive microorganisms.
Gentamicin was used as positive control. As can be seen in Figure S1 (supplementary
material), n-BuOH and EtOAc extracts were found to have a good activity against
Staphylococcus aureus, Escherichia coli, Bacillus cereus and Enterobacter aerogenes,
where the n-BuOH extract exhibited
a strong antibacterial activity against
Staphylococcus aureus. In general, the weaker activity was observed for the CHCl3 and
light petroleum extracts, while this latter indicated particularly no activity against
Streptococcus pneumonia and Bacillus cereus. The results of the antibacterial activity of
the different extracts obtained from S. satureoides showed differences in inhibiting
microorganisms which could be explained by the nature and amount of active com-
pounds in the extracts.
3. Conclusion
The phytochemical study of the EtOAc extract of S. satureoides afforded indole-3-car-
boxylic acid-(6’-O-caffeoyl)-b-D-glucoside 1, a new indole derivative, together with
eight know compounds 2-9 consisting of two indoles, five methylated flavone agly-
cones and one monoterpene glucoside, detected for the first time in Saccocalyx genus.
The two isolated indoles 2 and 3 were identified for the first time from the family
Lamiaceae. The antioxidant activity evaluation using two different methods indicated
that S. satureioides extracts (EtOAc and CHCl3) possess remarkable antioxidant

6
H. KHERKHACHE ET AL.
properties. The results of the antibacterial activity suggest that polar n-BuOH and
semi-polar EtOAc extracts are the most effective in inhibiting of most tested
microorganisms.
The results of this phytochemical and biological research support the folkloric use
of this medicinal plant (Biondi et al. 2006) and indicate that it could be used as poten-
tial resource of natural antioxidants. On the other hand, the polar and semi-polar
extracts of S. satureioides showed a strong antibacterial activity, which seems to sug-
gest their use as effective herbal protecting against a wide spectrum of patho-
genic bacteria.
Disclosure statement
No conflict of interest reported by authors.
Funding
The authors wish to express thanks to the DGRSDT of the Algerian Minister of Higher Education
and Scientific Research for providing a research grant, and to the Department of Chemistry and
QOPNA (FCTUID/QUI/00062/2013, Portuguese NMR Network), University of Aveiro, Portugal for
providing research facilities and technical support.
ORCID
Artur M. S. Silva
Hamada Haba
http://orcid.org/0000-0003-2861-8286
http://orcid.org/0000-0002-7979-5007
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