Isolation, characterization and in vitro anti- salmonellal activity of compounds from stem bark extract of Canarium schweinfurthii

Abstract

Background
Bacteria belonging to the Salmonella genus are major concern for health, as they are widely reported in many cases of food poisoning. The use of antibiotics remains a main stream control strategy for avian salmonellosis as well as typhoid and paratyphoid fevers in humans. Due to the growing awareness about drug resistance and toxicities, the use of antibiotics is being discouraged in many countries whilst advocating potent benign alternatives such as phyto-based medicine. The objective of this work was to isolate, characterise the bioactive compounds of Canarium schweinfurthii ; and evaluate their anti-salmonellal activity.

Methods
The hydro-ethanolic extract of Canarium schweinfurthii was fractionated and tested for their anti-salmonellal activity. The most active fractions (i.e. chloroform and ethyl acetate partition fractions) were then explored for their phytochemical constituents. Fractionation on normal phase silica gel column chromatography and size exclusion chromatography on Sephadex LH-20 led to the isolation of four compounds (maniladiol, scopoletin, ethyl gallate and gallic acid) reported for the first time in Canarium schweinfurthii .

Results
Result indicated that scopoletin and gallic acid had greater activity than the crude extracts and partition fractions. Among the isolated compounds, scopoletin showed the highest inhibitory activity with a MIC of 16 μg/ml against Salmonella Typhimurium and Salmonella Enteritidis.

Conclusions
The overall results of this study indicates that the hydro-ethanolic extract as well as some of isolated compounds have interesting anti-salmonellal activities that could be further explored for the development of potent therapy for salmonellosis. Furthermore, the study adds credence to the folkloric applications of the plant.

Full text

R E S E A R C H A R T I C L E Open Access
Isolation, characterization and in vitro anti-
salmonellal activity of compounds from
stem bark extract of Canarium
schweinfurthii
Jean Baptiste SOKOUDJOU
1,2
, Olubunmi ATOLANI
2,3
, Guy Sedar Singor NJATENG
1
, Afsar KHAN
2
,
Cyrille Ngoufack TAGOUSOP
4
, André Nehemie BITOMBO
2,5
, Norbert KODJIO
1
and Donatien GATSING
1*
Abstract
Background: Bacteria belonging to the Salmonella genus are major concern for health, as they are widely reported
in many cases of food poisoning. The use of antibiotics remains a main stream control strategy for avian
salmonellosis as well as typhoid and paratyphoid fevers in humans. Due to the growing awareness about drug
resistance and toxicities, the use of antibiotics is being discouraged in many countries whilst advocating potent
benign alternatives such as phyto-based medicine. The objective of this work was to isolate, characterise the
bioactive compounds of Canarium schweinfurthii; and evaluate their anti-salmonellal activity.
Methods: The hydro-ethanolic extract of Canarium schweinfurthii was fractionated and tested for their anti-
salmonellal activity. The most active fractions (i.e. chloroform and ethyl acetate partition fractions) were then
explored for their phytochemical constituents. Fractionation on normal phase silica gel column chromatography
and size exclusion chromatography on Sephadex LH-20 led to the isolation of four compounds (maniladiol,
scopoletin, ethyl gallate and gallic acid) reported for the first time in Canarium schweinfurthii.
Results: Result indicated that scopoletin and gallic acid had greater activity than the crude extracts and partition
fractions. Among the isolated compounds, scopoletin showed the highest inhibitory activity with a MIC of 16 μg/ml
against Salmonella Typhimurium and Salmonella Enteritidis.
Conclusions: The overall results of this study indicates that the hydro-ethanolic extract as well as some of isolated
compounds have interesting anti-salmonellal activities that could be further explored for the development of
potent therapy for salmonellosis. Furthermore, the study adds credence to the folkloric applications of the plant.
Keywords: Ethnomedicine, Salmonellosis, Canarium schweinfurthii, Natural substances
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* Correspondence: gatsingd@yahoo.com
1
Research Unit of Microbiology and Antimicrobial substances, Faculty of
Science, University of Dschang, P.O. Box 67, Dschang, Cameroon
Full list of author information is available at the end of the article
BMC Complementary
Medicine and Therapie
s
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Background
Salmonella is a major source of food-borne illness in
humans and a major cause of morbidity, mortality and
economic loss both in the poultry and human health
sectors. The disease caused by bacteria belonging to
Salmonella genus is often called salmonellosis. This
pathology remains one of the limiting factors in the de-
velopment of poultry farming especially in developing
countries of Asia and Africa [1] because it causes huge
direct and indirect losses [2]. The genus Salmonella is
very diverse and today it is composed of more than 2500
serotypes, many of which cause enteric diseases in
humans and animals. Many serotypes of Salmonella can
infect chickens and some serotypes are well adapted
although, Salmonella Gallinarum and Salmonella Pull-
orum cannot be transmitted to human. However, some
serotypes can infect both poultry and human and among
these serotypes Salmonella Enteritidis and Salmonella
Typhimurium are more prevalent in chickens and not-
able in human disease outbreaks. These serotypes are
most commonly implicated in the human Salmonella in-
fections [3,4]. The poultry is considered one of the main
sources of Salmonella human infection usually through
poorly cooked foods [5–9] and foodstuffs of avian origin
[10]. Salmonella infection represents a considerable bur-
den in both developing and developed countries. Ubiqui-
tous non-typhoidal Salmonella (NTS) which includes
Salmonella Enteritidis and Salmonella Typhimurium an-
nually cause more than 93.8 million illnesses and 155,
000 deaths each year [11]. Salmonella Enteritidis and
Salmonella Typhimurium, both NTS are the most
frequently occurring serotypes from poultry causing
infection in human [3]. Similarly, each year worldwide,
typhoidal serotypes among which Salmonella Typhi and
Salmonella Paratyphi, cause approximately 22 million
cases of typhoid and 216,500 deaths [12].
Resistance of Salmonella to commonly used anti-
microbial agents is increasing both in the veterinary and
public health sectors and has emerged as a global health
challenge. Several Salmonella serotypes are multidrug
resistant, and there is evidence of the spread of these
strains from animals to humans. Antimicrobial resist-
ance in NTS is considered one of the major public
health threats related with food-animal production, as
well as the poultry production chain and poultry meat,
which is an additional concern in the management of
salmonellosis [13]. Many authors [14–17] have reported
that several strains of Salmonella isolated from chicken
have shown resistance to many antibiotics commonly
used in human medicine and some of these strains have
been found in humans [14]. Moreover, antibiotic resi-
dues in poultry products intended for consumption may
lead to hypersensitivity or poisoning in consumers. Due
to the growing awareness of resistance issues, the use of
antibiotics is strongly discouraged in many countries
whilst encouraging the use of plants as a better alterna-
tive due to their diverse nature of bioactive principles
[18–20]. The large majority of salmonellosis in humans
is carried by foodstuffs; mainly those of avian origin [10,
20,21], therefore controlling avian salmonellosis by
using plant could significantly reduce the prevalence of
human gastroenteritis [20]. Several studies have focused
on medicinal plants as new control strategies for human
salmonellosis [22,23] or avian salmonellosis [24–28].
But, to our knowledge, no phytomedicine has yet been
formulated to control avian salmonellosis. Canarium
schweinfurthii Engl. (Burseraceae), is a tree with a cylin-
drical bole, native to tropical West Africa and grows to
about 50 m high [29]. This plant is mainly found in
equatorial forest regions from Cameroon, Central Afri-
can Republic, Gabon to Congo [30] and is used in folk
medicine for the treatment of various diseases including
malaria, diarrhea and Typhoid fever [31,32]. Previous
studies of Sokoudjou et al. [20,28] showed that the
hydroethanolic extracts of Canarium schweinfurthii were
active both in vitro and in vivo against several serotypes
of Salmonella. The objective of this work was to isolate,
characterise the bioactive compounds of Canarium
schweinfurthii; and evaluate their anti-salmonellal
activity.
Methods
General experiment
Reagents which include ammonium cerium sulphate,
were of analytical grade. Solvents were distilled before
being used (St Louis, MO, USA). Thin Layer Chroma-
tography (TLC) was performed on pre-coated silica gel
with thickness 0.20 mm 60 F
254
plates (MerckKGaA,
Germany) and viewed under the UV light (254 and 365
nm). NMR analyses which included
1
H NMR,
13
CNMR,
DEPT 90, DEPT 135, 2D NMR (COSY, HSQC), NOESY
and ROESY were performed using deuterated solvents
(Acétone-d
6,
CD
3
OD and/or CDCl
3
) on 400 MHz NMR
(Ascend™400, Bruker) with TMS as internal reference.
ESI-MS spectra of the compounds were recorded on a
Bruker-Ion Trap MS (MicroTOF-Q mass spectrometer,
Bruker) using the positive mode.
Plant collection, identification and extraction
Canarium schweinfurthii stem bark was harvested in
West region of Cameroon and identified at the National
Herbarium at Yaoundé-Cameroon, where a voucher spe-
cimen was deposited under the reference Number
16929/SRF/Cam. The air-dried plant material (3 Kg) was
powdered and macerated at room temperature with 12 L
of ethanol-water system (50/50, v/v). After 48 h, the mix-
ture was filtrated using Whatman №1 filter paper. The
filtrate was evaporated using a Rotary evaporator (Büchi
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R200) at reduced pressure to afford the crude extract
(265 g, 8.8%).
We needed no permission to collect the sample since
Canarium schweinfurthii is not a protected species in
Cameroon.
Fractionation and isolation of bioactive compounds of
Canarium schweinfurthii
The profiling of the hydro-ethanolic extract of Canar-
ium schweinfurthii on TLC plates with several solvent
systems showed no promising separation. In order to
facilitate isolation, 260 g of extract was dissolved in
distilled water (700 mL) and successively extracted with
hexane (500 mL × 2), chloroform (500 mL × 2), ethyl
acetate (500 mL × 2) and n-butanol (500 mL × 2) yielding
respectively 5.56 g, 25.97 g, 25.92 g and 90.89 g of
fractions after evaporation to dryness. These partition
fractions were explored for their antibacterial activity
and only the most active fractions were selected for the
isolation of bioactive principles. Figure 1below shows
the protocol for isolating the bioactive principles of
Canarium schweinfurthii.
Part of Chloroform fraction (23 g) was subjected to sil-
ica gel column chromatography using n-hexane-EtOAc
(85:15 →00:100) and MeOH, gradient elution. 40 sub-
fractions of 100 mL each were collected and combined
on the basis of their TLC profiles to give 5 fractions: A
(1–3), B (4–12), C (13–22), D (23–25) and E (25–40).
Sub-fraction A (4.5 g) was purified on silica gel column
chromatography eluted with n-hexane-EtOAc (95:5 →
80:20) to give compound 1(42 mg). The purification of
sub-fraction D (4 g) on silica gel column chromatog-
raphy using n-hexane-EtOAc (70:30 →20:80) afforded
compound 2(57 mg) which was recrystallized in EtOAc-
MeOH (20:80).
Part of EtOAc fraction (23 g) was also subjected to sil-
ica gel column chromatography eluted with a gradient of
n-hexane-EtOAc (70:30 →00:100) and chloroform-
MeOH (92:5 →75:25) to afford 60 sub-fractions of 20
mL which were combined to four sub-fractions: F (1–4),
G(5–15) H (16–24), I (25–60) on the basis of their TLC
profile. Sub-fraction G (3.5 g) was purified on silica gel
column chromatography using n-hexane-EtOAc (50:
50 →00:100) to yield compound 3(21 mg) while purifi-
cation of sub-fraction H (2.6 g) on sephadex LH-20 col-
umn eluted with chloroform-methanol (50:50) afforded
compound 4(60 mg). The structures of the isolated
compounds were elucidated by combining various tech-
niques comprising 1D Nuclear Magnetic Resonance
(NMR):
1
HNMR,
13
C-NMR, DEPT 90, DEPT 135 and
2D NMR (COSY, HSQC), NOESY and ROESY as well
as Mass Spectrometry analysis (TOF-ESI-MS). The data
of the established structures were compared with those
existing in literature.
Anti-salmonellal assay
Chemicals for anti-salmonellal assay
Ciprofloxacin (BDH Chemicals, England) and oxytetra-
cyclin (BDH Chemicals, England) were used as reference
antibiotics. P-iodonitrotetrazolium chloride (Sigma-Al-
drich, Germany) was used as microbial growth indicator.
Fig. 1 Flow chart for the isolation of compounds from the hydroethanolic extract of Canarium schweinfurthii
SOKOUDJOU et al. BMC Complementary Medicine and Therapies (2020) 20:316 Page 3 of 10
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Test bacteria and culture media
Three clinical isolates (Salmonella Typhi, Salmonella
Enteritidis and Salmonella Typhimurium from Pasteur
Center, Yaoundé-Cameroon) and one bacterial strain
(Salmonella Typhi ATCC6539 from American Type
Culture Collection) were used for antimicrobial evalu-
ation. The culture media used were Salmonella-Shigella
Agar (SSA from HiMedia Laboratories, India) and Muel-
ler Hinton Broth (MHB from HiMedia Laboratories,
India).
Determination of minimal inhibitory concentrations (MICs)
and minimal bactericidal concentrations (MBCs)
The MIC values of the fractions obtained from parti-
tion and compounds from Canarium schweinfurthii
were determined in 96-wells microplates using rapid
INT colorimetric assay [33,34]. Briefly, each sample
was dissolved in 5% Dimethyl-sulfoxide (DMSO)/
MHB. The obtained solution was then added to
100 μL of MHB, and followed by two-fold serial dilu-
tion. Then 100 μL of inoculum (1.5 × 10
6
CFU/mL)
prepared in MHB were added to each well except the
negative control wells. The plates were covered with a
sterile plate sealer and incubated at 37 °C for 18 h.
The wells containing either MHB or MHB and
100 μL of inoculum served as control. After the incu-
bation, 40 μL of INT (0.2 mg/mL) was added to each
well and plates were re-incubated at 37 °C for 30 min,
and the MIC of each sample was recorded. MIC was
defined as the lowest concentration of the sample that
prevented change in colour and exhibited complete
inhibition of microbial growth. The MBC was deter-
mined by adding 50 μL aliquots of the preparations,
which did not show any growth after incubation dur-
ing MIC assays, to 150 μL of MHB. These prepara-
tions were then incubated at 37 °C for 48 h. The
MBC was recorded as the lowest concentration of test
samplewhichdidnotproduceacolourchangeafter
addition of INT as previously described. The tests
were performed in triplicates.
Results
The yield and physical appearance of each partition frac-
tion of Canarium schweinfurthii extract are as shown
below (Table 1).
Characterization of isolated compounds
The four compounds isolated and characterized from
the stem bark extract of Canarium schweinfurthii are as
depicted in Fig. 2.
Compound 1: Maniladiol (42 mg) white solid, soluble
in methanol, with molecular weight 442 calculated for
C
30
H
50
O
2
(ESI-MS: m/z 465.1 [M + Na]).
Compound 2: Scopoletin (57 mg) yellowish crystals,
soluble in acetone, with molecular weight 192 calculated
for C
10
H
8
O
4
(ESI-MS: m/z 214.9 [M + Na]).
Compound 3: Ethyl gallate (21 mg) white solid, soluble
in methanol, with molecular weight 198 calculated for
C
9
H
10
O
5
(ESI-MS: m/z 221.0 [M + Na]).
Compound 4: Gallic acid (60 mg) white solid, soluble
in methanol, with molecular weight 170 calculated for
C
7
H
6
O
5
(ESI-MS: m/z 193.1 [M + Na]).
The
1
H-NMR and
13
C-NMR data of isolated com-
pounds are presented in the Tables 2,3,4and 5.
Anti-salmonellal activity of partition fractions and isolated
compounds from stem barks extract of Canarium
schweinfurthii
Table 6shows the inhibition parameters (MIC, MBC,
MBC/MIC ratio) of the crude extract, partition fractions
and isolated compounds of Canarium schweinfurthii
against pathogenic Salmonella. The isolated compounds
have variable activity (16 ≤MIC≤1024 μg/mL) on the
tested Salmonella serotypes. It appears that the activity
of isolated compounds is greater than those of the crude
extract and partitions. Among the partition fractions,
chloroform and ethyl acetate fractions showed the best
anti-salmonellal activity while among the isolated com-
pounds, scopoletin showed the highest inhibitory activity
with a MIC of 16 μg/mL against Salmonella Typhimur-
ium and Salmonella Enteritidis. MIC values of other
compounds and extract ranged between 128 and
1024 μg/mL, while hexane and residual fractions are the
less active substances with MICs of 512 or 1024 μg/mL.
Discussion
The antimicrobial effects of some plants and their extracts
are well known today [39,40]; the diversity of plant species
is a valuable source for the search for new classes of antibi-
otics. These plants may proffer valuable alternative to ad-
dress certain human and veterinary health challenges. It is
in this perspective that the hydro-ethanolic extract of
Canarium schweinfurthii has been explored for its anti-
salmonellal activity and its bioactive compounds. Several
plants are traditionally used against human salmonellosis
[41–46] and avian salmonellosis [24–26,47]. Plants with
Table 1 Yield and physical appearance of each partition
fraction of Canarium schweinfurthii stem barks extracts
Partitioned fractions Yields
(%)
Physical characteristics
Color Physical appearance
Hexane fraction 2 Green Oily
Chloroform fraction 10 Dark brown Oily
Ethylacetate fraction 10 Brown Solid
n-butanol fraction 34 Blackish Cristalline powder
Residual fraction 38 Blackish Sticky semi-solid (Syrup)
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high anti-salmonellal potential that show promise for the
control of avian salmonellosis include Aloe secundiflora
[47], Thymus vulgaris [48], Curcuma longa and Scutellaria
baicalensis [25]andErica mannii [27]. Plant extracts as
well as traditionally improved drugs are one of the promis-
ing ways to combat human salmonellosis [23,47,49]. Sev-
eral authors [22,23,28,50–53] have shown that plant
extracts depending on their concentrations are active both
in vitro and in vivo against several Salmonella serotypes.
Most of these extracts treat salmonellosis in the same
range of time as conventional medicines. These findings
corroborate our results which showed that the hydroetha-
nolic extract of Canarium schweinfurthii is active against
Salmonella serotypes with MIC range from 64 to 128 μg/
ml, moreover this extract have previously demonstrated an
in vivo anti-salmonellal activity [20], curing avian salmon-
ellosis on day 9 and with the doses 19 and 75 mg/kg bw of
the extract. In addition to the therapeutic efficacy of the
hydroethanolic extract of Canarium schweinfurthii,the
antibacterial activity of its partitions was evaluated. Among
the partitions, chloroform and ethyl acetate fractions
showed the best anti-salmonellal activity. It also appears
that the activity of isolated compounds is greater than
those of the crude extract and partitions. This could be
due to the low concentration of these compounds in the
plant extract or to the antagonism effect of other com-
pounds present in the same extract. The anti-salmonellal
activity of plants is linked to the diversity and complexity
of their secondary metabolites. The in vitro anti-
salmonellal effect of hydroethanolic extract of Canarium
schweinfurthii found in this study and its therapeutic effi-
cacy [20] can be linked to a combined action of its second-
ary metabolites. Indeed, at the molecular level, compounds
such as gallic acid and scopoletin found in plants belonging
to Canarium genus [54] could act synergistically and could
be partly responsible for the anti-infectious activity of
Canarium schweinfurthii. In order to verify this possibility
and to have a clear idea on the active principles of this
plant, the fractionation of its stem bark extract was
performed.
Gallic acid, ethyl gallate, scopoletin and maniladiol
were isolated from the Canarium schweinfurthii stem
bark extract, these compounds were reported for the
first time in this medicinal plant species and belong to
the classes of polyphenols, triperpenes and coumarins.
From the previous reports [54], only gallic acid and sco-
poletin have been isolated from other plants belonging
to the same genus as Canarium schweinfurthii and these
compounds were reported to have antibacterial and anti-
oxidant properties. The isolated compounds have vari-
able activities (16 ≤MIC≤1024 μg/mL) against the tested
Salmonella serotypes. Among the pure isolated com-
pounds, scopoletin showed the highest inhibitory activity
with a MIC of 16 μg/mL against Salmonella Typhimur-
ium and Salmonella Enteritidis. The activity of most of
the isolated compounds was less than those of
Fig. 2 Chemical structures of isolated compounds from Canarium schweinfurthii stem barks extract
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oxyphylline B (10 μg/mL) isolated from Zizyphus oxy-
phylla Edgew against Salmonella Typhi [55] and lespe-
din (12.25 μg/ml) isolated from Brillanta isialamium
against Salmonella Typhi [56]. However the anti-
salmonellal activity of gallic acid and scopoletin against
Salmonella Typhi (32 μg/mL) was better than those of
Bafoudiosbulbins A and Bafoudiosbulbins B isolated
from Dioscorea bulbifera L. var. sativa [57]. These results
corroborate the finding of Lunga et al. [44] who showed
that the anti-salmonellal activity of isolated compounds
from Paullinia pinnata Linn ranged from 0.781 to
100 μg/mL. According to the Kuete’s classification scale
[39], the antibacterial activity of a compound is signifi-
cant when the MIC< 10 μg/mL; moderate when 10 <
Table 2
1
H-NMR and
13
C-NMR of compound 1
Compound 1 Maniladiol, Quijano et al. [35]
Positions δc
(CD
3
OD+ CDCl
3
, 100 MHz)
δ
H
(mult; J)
(CD
3
OD+ CDCl
3
, 400 MHz)
δc
(CD
3
Cl, 125 MHz)
δ
H
(mult; J)
(CD
3
Cl, 500 MHz)
1 32.9 1.40 (1H;m)
1.12 (1H; m)
38.5 1.64 (1H; m)
0.98 (1H; m)
2 24.5 1.99 (1H; m)
1.52 (1H; m)
27.1 1.62 (1H; m)
1.58 (1H; m)
3 75.4 3.35 (1H;dd; 11.9; 4.8) 78.9 3.22 (1H; dd; 11.5; 4.5)
4 37.1 –38.7 –
5 48.8 1.30 (1H;m) 55.1 0.74 (1H; dd; 11.5; 1.5)
6 18.0 1.45 (1H; m)
1.44 (1H; m)
18.3 1.58 (1H; t; 3.6)
1.41 (1H; dd; 15.5; 12.0)
7 32.4 1.62 (1H; m)
1.38 (1H; m)
32.6 1.54 (1H; t; 3.5)
1.33 (1H; t; 3.6)
8 39.9 –39.8 –
9 46.5 1.06 (1H; m) 46.8 1.51 (1H; dd; 11.0; 6.5)
10 36.6 –37.3 –
11 23.4 1.91 (2H; m) 23.5 1.92 (1H; ddd; 18.5; 11.0; 3.5)
1.86 (1H; ddd; 18.5; 7.0; 4.0)
12 122.3 5.26 (1H; t; 3.4) 122.3 5.25 (1H; t; 3.5)
13 143.7 –143.5 –
14 43.5 –43.7 –
15 34.9 1.71 (1H; m)
1.26 (1H; m)
35.5 1.67 (1H; d; 13.0)
1.31 (1H; dd; 13.0; 5.0)
16 65.0 4.16 (1H; dd; 11.5; 4.9) 66.0 4.20 (1H; dd; 11.5; 5.0)
17 37.0 –36.8 –
18 49.2 2.16 (1H; dd; 11.5; 4.9) 49.0 2.15 (1H; dd; 14.0; 4.5)
19 46.5 1.71 (1H; m)
1.06 (1H; m)
46.5 1.68 (1H; t; 14.0)
1.06 (1H; ddd; 13.5; 4.5; 2.5)
20 30.4 –30.9 –
21 34.0 1.41 (1H; m)
1.13 (1H; m)
34.1 1.36 (1H; t; 3.7)
1.15 (1H; t; 3.6)
22 30.5 1.91 (1H; m)
1.88 (1H; m)
30.5 1.83(1H; t; 3.4)
1.20(1H; t; 3.5)
23 27.8 0.95 (3H; s) 28.0 1.00 (3H; s)
24 21.7 0.86 (3H; s) 15.6 0.79 (3H; s)
25 14.8 0.99 (3H; s) 15.5 0.94 (3H; s)
26 16.24 1.03 (3H; s) 16.8 0.99 (3H; s)
27 26.4 1.27 (3H; s) 27.1 1.22(3H; s)
28 21.4 0.80 (3H; s) 21.4 0.80 (3H; s)
29 32.6 0.90 (3H; s) 33.2 0.89 (3H; s)
30 23.2 0.92 (3H; s) 23.9 0.90 (3H; s)
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MIC≤100 μg/mL and low when MIC> 100 μg/ml. With
regard to this scale, the anti-salmonellal activities of the
isolated compound from Canarium schweinfurthii are
moderate (10 < MIC≤100 μg/mL). Scopoletin and gallic
acid are significantly active against Salmonella Typhi,
Salmonella Typhi ATCC6539 and Salmonella Typhi-
murium. These results corroborate those of Okoli et al.
[58] who showed that 3β-hydroxylolean-12,18-diene iso-
lated from Canarium schweinfurthii was active on Sal-
monella with a MIC of 12.5 μg/ml against Salmonella
Typhi. It has been shown that in addition to its immu-
nomodulatory effect [59], scopoletin reduces the intra-
cellular survival of Salmonella Typhi within U937
human macrophage cell line [60]. Gallic acid has in
addition to its in vitro and in vivo antibacterial effect
against Salmonella Typhimurium [61,62], an antioxi-
dant activity. These compounds related properties cor-
roborate the findings of Sokoudjou et al. [20] who
reported that the ability of the extract of Canarium
schweinfurthii to cure salmonellosis in broilers could be
explained by its ability to directly kill Salmonella and/or
boost the immune system of the host. The dosage of the
compounds isolated from this plant can be used to
normalize the extract during the phytomedicine evalu-
ation and preparation.
Conclusion
Gallic acid, ethyl gallate, scopoletin and maniladiol were
isolated from the Canarium schweinfurthii stem bark ex-
tract. These compounds were reported for the first time
in this plant species. The four isolated compounds
showed in vitro anti-salmonellal activity against Salmon-
ella serotypes and particularly scopoletin was the most
active and highly selective against both non-typhoidal
Salmonella and typhoidal Salmonella with MIC of 16 or
32 μg/mL. The anti-salmonellal activity of the com-
pounds isolated from Canarium schweinfurthii justifies
the use of this plant in traditional medicine and con-
firms the anti-salmonellal effect of the hydroethanolic
extract thus adding credence to its use in the treatment
Table 3
1
H-NMR and
13
C-NMR of compound 2
Compound 2 Scopoletin, Mogana et al. [36]
Positions δ
C
(acétone-d
6
, 100 MHz)
δ
H
(mult; J)
(acétone-d
6
, 400 MHz)
δc
(CD
3
Cl, 100 MHz)
δ
H
(mult; J)
(CD
3
Cl, 400 MHz)
1––––
2 160.4 –161.6 –
3 112.5 6.20 (1H; d; 9.5) 111.6 6.30 (1H; d; 9.5)
4 143.6 7.86 (1H; d; 9.5) 143.3 7.63 (1H; d; 9.5)
5 102.8 6.81 (1H; s) 103.2 6.87 (1H; s)
6 144.9 –144.6 –
7 150.8 –150.2 –
8 108.9 7.20 (1H; s) 107.4 6.95 (1H; s)
9 150.0 –149.7 –
10 112.1 –113.5 –
6-OCH
3
55.9 3.92 (3H; s) 56.4 3.98 (3H; s)
7-OH –8.78 (1H; s)––
Table 4
1
H-NMR and
13
C-NMR of compound 3
Compound 3 Ethyl gallate, Ooshiro et al. [37]
Positions δc
(CD
3
OD, 100 MHz)
δ
H
(mult; J)
(CD
3
OD, 400 MHz)
δc
(CD
3
OD, 150 MHz)
δ
H
(mult; J)
(CD
3
OD, 600 MHz)
1 168.8 –168.5 –
2 121.7 –121.7 –
3/7 110.0 7.07 (2H; s) 110.0 7.04 (2H; s)
4/6 146.2 –146.4 –
5 139.6 –139.7 –
1’61.6 4.28 (2H; q; 7.1) 61.6 4.28 (2H; q; 7.3)
2’14.7 1.35 (3H; t; 7.1) 14.6 1.33 (3H; t; 7.3)
SOKOUDJOU et al. BMC Complementary Medicine and Therapies (2020) 20:316 Page 7 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Table 5
1
H-NMR and
13
C-NMR of compound 4
Compound 4 Gallic acid, Chanwitheesuk et al. [38]
Positions δc
(CD
3
OD, 100 MHz)
δ
H
(mult; J)
(CD
3
OD, 400 MHz)
δc
(acétone-d
6
, 100 MHz)
δ
H
(mult; J)
(acétone-d
6
, 400 MHz)
1 168.8 –167.3 –
2 120.7 –120.8 –
3/7 108.0 7.08 (2H; s) 109.1 7.15 (2H; s)
4/6 145.0 –144.9
5 138.1 –137.7
Table 6 Inhibition parameters (MIC, MBC) of partition fractions and isolated compounds from Canarium schweinfurthii against
different test microorganisms
Tested samples Studied
parameters
(μg/mL)
Strain/isolates
ST STs STM SE
HEE 50/50 MIC 256 128 64 128
MBC 512 512 256 512
MBC/MIC 2 4 4 4
Hexane partition MIC 1024 1024 512 > 1024
MBC > 1024 > 1024 > 1024 > 1024
MBC/MIC ––––
Chloroform partition MIC 512 1024 256 1024
MBC 1024 > 1024 > 1024 > 1024
MBC/MIC 2 –––
Ethyle acetate partition MIC 256 256 128 32
MBC > 1024 1024 > 1024 128
MBC/MIC –4–4
n-butanol partition MIC > 1024 1024 512 > 1024
MBC > 1024 > 1024 > 1024 > 1024
MBC/MIC ––––
Residual partition MIC > 1024 > 1024 > 1024 1024
MBC > 1024 512 256 > 1024
MBC/MIC ––––
Compound 1
Maniladiol
MIC 512 512 32 64
MBC > 1024 > 1024 128 256
MBC/MIC ––44
Compound 2
Scopoletin
MIC 32 32 16 16
MBC 64 128 32 64
MBC/MIC 2 4 2 4
Compound 3
Ethyl gallate
MIC 128 1024 64 1024
MBC > 1024 > 1024 > 1024 > 1024
MBC/MIC ––––
Compound 4
Gallic acid
MIC 32 32 64 128
MBC 32 32 128 256
MBC/MIC 1 1 2 2
Oxytetracycline MIC 8842
MBC 32 64 32 16
MBC/MIC 4 8 8 8
Ciprofloxacine MIC 0,5 1 4 4
MBC 2288
MBC/MIC 4 2 2 2
ST Salmonella Typhi, STs Salmonella Typhi ATCC6539, STM Salmonella Typhimurium, SE Salmonella Enteritidis, MIC Minimum inhibitory concentration, MBC Minimum
bactericidal concentration.
SOKOUDJOU et al. BMC Complementary Medicine and Therapies (2020) 20:316 Page 8 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

of avian salmonellosis. Further studies will be necessary
to verify the in vivo activity of these compounds and to
elucidate their mechanisms of action.
Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s12906-020-03100-5.
Additional file 1.
Acknowledgements
Authors thank the researchers of the Natural Products Chemistry Laboratory
of CUI, Abbottabad Campus, Pakistan for their useful suggestions.
Authors’contributions
All the authors contributed to carry out this study. JBS was the principal
investigator, OA and GSSN contributed to evaluate the anti-salmonellal activ-
ities. CNT, ANB and NK contributed to the fractionation purification and
structural elucidation of isolated compounds. NK revised the manuscript, AK
and DG co-supervised the work. All authors read and approved the final
manuscript.
Funding
This research work was supported in part by The Academy of Sciences for
the Developing World (TWAS) in collaboration with COMSATS University
Islamabad (CUI) under grant FR number 3240299471 (TWAS-CIIT
Postgraduate fellowship). The obtained fund was used for compound
isolation and characterization.
Availability of data and materials
They are available as Supporting information.
Ethics approval and consent to participate
Not applicable in this section.
Consent for publication
All authors read and approved the final manuscript.
Competing interests
Authors have declared that no competing interests exist.
Author details
1
Research Unit of Microbiology and Antimicrobial substances, Faculty of
Science, University of Dschang, P.O. Box 67, Dschang, Cameroon.
2
Natural
Products Chemistry Laboratory, Department of Chemistry, COMSATS
University Islamabad, Abbottabad Campus-22060, Islamabad, Pakistan.
3
Department of Chemistry, Faculty of Physical Sciences, University of Ilorin,
P.M.B, Ilorin 1515, Nigeria.
4
Department of Basic Scientific Studies, University
Institute of Technology, University of Ngaoundere, P.O.Box 455, Ngaoundere,
Cameroon.
5
Department of Organic Chemistry, Faculty of Science, University
of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon.
Received: 20 December 2019 Accepted: 29 September 2020
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