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Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Heterophynone and methyl ester of Colic acid, two
new compounds with antimicrobial activity from
Cola heterophylla (Sterculiaceae)
Martin Mbané Noah, Maurice Tagatsing Fotsing, Alex de Théodore Atchadé,
Pierre Mkounga, Fabrice Fekam Boyom, Emmanuel Talla, Celine Henoumont
& Laurent Sophie
To cite this article: Martin Mbané Noah, Maurice Tagatsing Fotsing, Alex de Théodore
Atchadé, Pierre Mkounga, Fabrice Fekam Boyom, Emmanuel Talla, Celine Henoumont &
Laurent Sophie (2020): Heterophynone and methyl ester of Colic acid, two new compounds with
antimicrobial activity from Cola�heterophylla (Sterculiaceae), Natural Product Research, DOI:
10.1080/14786419.2020.1777412
To link to this article: https://doi.org/10.1080/14786419.2020.1777412
View supplementary material Published online: 12 Jun 2020.
Submit your article to this journal Article views: 2
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Heterophynone and methyl ester of Colic acid, two new
compounds with antimicrobial activity from Cola
heterophylla (Sterculiaceae)
Martin Mban
e Noah
a
, Maurice Tagatsing Fotsing
a
, Alex de Th
eodore Atchad
e
a
,
Pierre Mkounga
a
, Fabrice Fekam Boyom
b
, Emmanuel Talla
c
, Celine Henoumont
d
and Laurent Sophie
d
a
Departement of Organic Chemistry, Faculty of Science, University of Yaound
e I, Yaound
e,
Cameroon;
b
Department of Biochemistry, Faculty of Science, University of Yaound
e I, Yaound
e,
Cameroon;
c
Department of Materials Engineering, School of Chemical Chemical Engineering and
Mineral Industries, University of Ngaound
er
e, Ngaound
er
e, Cameroon;
d
Laboratory of NMR and
Molecular Imaging, Department of General, Organic Chemistry and Biochemistry, Univerisy of Mons,
Mons, Belgium
ABSTRACT
The ethyl acetate fraction, the stem bark and the residual methanolic
extracts from the leaves of Cola heterophylla (Sterculiaceae) led to the
isolation of two new compounds: Heterophynone (1) and methyl ester
of Colic acid (6), along with four known triterpenes: betulinic acid (2),
oleanolic acid (3), ursolic acid (4) and chletric acid (5). Structures of
compounds were established by different spectroscopic methods that
included 1D and 2D NMR experiment. The antimicrobial activity of iso-
lated compounds was evaluated against two yeasts, Candida Albicans
NR 29456 and Candida Krusei 1415; and five Gram-positive bacterial,
Salmonella enteric Serovar Muenchem,Salmonella enteric Serovar
Thyphimurium, Staphylococcus aureus NR 46003, Staphylococcus aureus
NR46374 and Pseudomonas aeruginosa HM 601). Among tested
compounds, Heterophynone was found to be the most active with sig-
nificant antimicrobial activity against Salmonella enteric Serovar
Thyphimurium (MIC ¼7.82 lg/mL and MBC ¼62.5 lg/mL) and good
activity against Candida Albicans NR 29456 (MIC ¼62.5 lg/mL).
ARTICLE HISTORY
Received 24 February 2020
Accepted 23 May 2020
KEYWORDS
Cola heterophylla;
heterophynone; methyl
ester of Colic acid;
antimicrobial
CONTACT Martin Mban
e Noah mbanenoa@yahoo.fr; Alex de Th
eodore Atchad
ealexiode@yahoo.fr
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1777412.
ß2020 Informa UK Limited, trading as Taylor & Francis Group
NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1777412
1. Introduction
Infectious diseases are a public health problem. They are the cause of 3 to 4 million
cases of death worldwide, especially in developing countries (Delmas et al. 2003). This
is due to the omnipresence of bacteria that can cause various types of diseases to
human, including urinary tract infections (Sowjanya et al. 2017), nosocomial blood
infections (Mayra et al. 2015), wound infections (Irina et al. 2018), skin infections (Rau
et al. 2002) and intestinal infections (Delahaye et al. 2009). The management of these
infections has often been done through the use of topical antibiotics, which are
obtained through synthesis. They are particularly used as excellent agents against bac-
teria Gram (þ) and Gram () (Chanawanno et al. 2010; Rodica et al. 2014).
Unfortunately, the resistance of these bacteria to the said antibiotics is one of the
major problems of anti-infective medicines in the world of the pharmaceutical industry
(Laila et al. 2015). The situation remains very worrying because of the emergence of
multi-resistant strains due to bacteria genome mutations. In this context, the search
for new alternative principles is of primary interest. The use of natural products, espe-
cially those of plant origin, opens a path in search of new antimicrobial medicines and
therapeutic agents. This study aims to expand knowledge of a botanical family,
Sterculiaceae, specifically Cola genus. Cola has about 125 species that are mainly
found in tropical and subtropical regions, particularly in Africa (Chevalier and Perrot
1911; Pellegrini 1951). Traditional uses of Cola species are diverse and have sometimes
converged on different cultures, even in the absence of any contact between them.
Among these uses, Cola accuminata and Cola nitida are used as anti-bacteria, against
sexual anaesthesia that may originate from bacteria (Yinyang et al. 2014). C. hetero-
phylla is a tree in African tropical regions of 25–30 metres of height and about 1.5 m
of diameter with pale green flowers inside and yellowish flowers outside (Brenan
1954). According to the information gathered from traditional practitioners of the cen-
tral region of Cameroon, C. heterophylla is used by women to protect against microbial
infections. As far as we know, no phytochemical study has been carried out on this
plant species. The present work focuses on the isolation of Heterophynone (1), betu-
lenic acid (2), oleanolic acid (3), ursolic acid (4), chletric acid (5), and methyl ester of
Colic acid (6) and evaluates their antimicrobial activities (Figure 1).
2. Results and discussion
The powder of steam bark and leaves of C. heterophylla were extracted with methanol.
The crude methanolic dried extracts were partitioned with hexane and methanol to
hexane fraction and residual methanolic extracts after respective evaporation with a
rotavapor. The dry residual extracts containing methanol were solubilised with ethyl
acetate to give a fraction of ethyl acetate and methanol residual. Column chromatog-
raphy of the ethyl acetate fraction of steam barks and the methanol residue extract of
the leaves led to the isolation of two new compounds Heterophynone (1), and methyl
ester of Colic acid (6), and four known: betulinic acid (2) (Kumar et al. 2015,Table S6),
oleanolic acid (3) (Martins et al. 2013,Table S5), ursolic acid (4) (Martins et al. 2013,
Table S5) and chleritic acid (5) (Tao et al. 2012,Table S6).
2 M. M. NOAH ET AL.
Compound 1 was isolated as a purple solid (m.p. 108.1–110.1 C). Its IR (Figure S1)
spectrum revealed the characteristic absorption bands of the hydroxyl group
(3394.41–3278.00 cm
1
), ketone (1585.95–1569.28 cm
1
), aliphatic chain
(2921.26–2871.13 cm
1
), and methylene groups (1535.3 cm
1
)moieties.(þ)HR-ESIMS
(Figure S2) mass spectrum showed a pseudo-molecular ion [M þ4H þNa]
þ
at 481.2214,
corresponding to m/z 454 in accordance with C
27
H
34
O
6
with eleven degrees of unsatur-
ation. The UV (Figure S3)(MeOH,kmax) spectrum exhibited absorption maxima at
280 nm and 306 nm characteristic of band II and band I. Subsequently, the structure was
completely elucidated by 1D and 2D NMR spectroscopy. The
13
CNMR(Figure S4)
spectrum revealed at d
C
170.3 and 139.4, signals characteristic of carbons C-3 and C-4 of
flavonol skeleton (Fernanda et al. 2019). The
1
HNMR(Figure S5) spectrum analysis shows
four aromatic protons that appear as doublets, d
H
6.66 (d, 1H; 1,8 Hz, H-6) and 6.72 (d, 1H;
1,8 Hz, H-8); 6.35 (d, 1H; 2,4 Hz H-30) and 6.37 (d, 1H; 2,4 Hz, H-50)andatd10.30 (s) and
9.0 (s), a feature of aromatic hydroxyls at C-2 and C-3. These correlations are confirmed
by the COSY (Figure S6,Table S1) spectrum between the protons H-100/H-200 and H-400/H-
500 corresponding to the aliphatic chain at C-5. The similar correlations were observed at
Figure 1. Chemical structures of compounds (1–6) isolated from Cola heterophylla.
NATURAL PRODUCT RESEARCH 3
C-60which is linked to the second aliphatic chain. The
13
C NMR spectrum in mod DEPT
135 (Figure S7) spectrum reveals three major family of carbons: four aromatic methine at
d
C
114.3 and, 103.5, 99.3 and 107.5 corresponding to C-6 and C-8 of A ring, and C-30and
C-50of B ring; eight methylene peaks between d
C
22.3 and 39.9; and two methyles at d
56.4 and 55.6 belonging to those of methoxyl groups; and d
C
14.9 and 14.1 for aliphatic
chains. Both methoxy are confirmed by the proton spectrum at d
H
3.77 (3H, s, H-A, 7) and
3.75 (3H, s, H-B, 50) respectively. However, the analysis of the
1
Hand
13
C spectra showed
two substitutions of aliphatic at C-5 and C-60andconfirmedthepresenceofaglyconefla-
vonol at d
C
139.4. The analysis of the HMQC (Figure S8,Table S1) spectrum shows correla-
tions between C-6 (114.3) and H-6 (6.66), C-8 (103.5) and H-8 (6,72), C-30(99.3) and H-30
(6.35), C-50(107.5) and H-50(6.37, d, 2.4 Hz, H-50), C-100 (33.0) and H-100 (2.54, t, 2H, 7.8 and
15.6 Hz), C-1000 (39.9) and H-1000 (2.67, t, 2H; 7.8 and 15.6 Hz), a range of 31.8–22.3
(1.59–1.22, m), 14.9 (0.85, d, 3H; 7.2 Hz) and 14.1 (0.85, d, 3H, 7.2 Hz). Peaks in its HMBC
(Figure S9,Table S1) spectrum correlations between H-100 (2.54, t, 2H, 7.8 and 15.6 Hz)
with C-4a (122.7), C-5 (141.4) and C-6 (114.4); and 2.67 (t, 2H; 7.8 and 15.6) with C-40
(107.5) and C-60(144.7) are observed and allowed us to locate the aliphatic chain at C-5
(141.4) and C-60(144.7) of the flavonol. The EI/ESI (Figure S10) spectrum, with pseudo-
molecular ion corresponding to [M-5H]
þ
m/z 449, enabled the observation of fragmenta-
tions, including losses of CO, CO
2
and fission spectra of the C-ring. This fission is based on
the cleavage Retro Diels-Alder (RDA), while resulting fragments fall within the substitution
of cycles A and B (Fabre et al. 2001; Tsimagiannis et al. 2007). Thus, the compound under-
went subsequent losses of [M-10H-CO
2
] at m/z 400 and [M-5H-CO] at m/z 420. The frag-
mentation of the compound gave m/z 193 and m/z 256 (5) which correspond
successively to ring B and ring A-C. Also, we had a fragmentation of the residual com-
poundthathasledto[M-10H-CO
2
-OCH
3
] m/z 369 and [M-10H-CO
2
-OCH
3
-C
5
H
11
þH] m/z
299 (Figure S11). The analysis of all these combined data enabled us to determine the
structure as 20,3-dihydroxy-7,40-dimethoxy-5,60-dipentyl flavonol which is a new derivative
of flavonol to which trivial name Heterophynone (Figure S12) has been given.
Compound 6 was isolated as white powder. Its IR (Figure S13) spectrum revealed the
characteristic absorption bands of the hydroxyl group (3320.36 cm
1
), ester (1686.78 cm
1
),
aliphatic chain (2925.61 cm
1
), and methylene groups (1031.14–1376.70 cm
1
) moieties. The
spectrum of ESI
þ
(Figure S14) weight in positive mode showed a pseudo-molecular ion
[M þ5H]
þ
at m/z 553, corresponding to the formula C
32
H
52
O
7;
m/z 548, and shows 7 units
of unsaturation. The
13
C NMR spectrum (Figure S15)ofcompound6revealed30signalsof
carbon, which were deduced from DEPT 135 spectrum (Figure S16) by 5 methyl groups, 8
methylene groups, 4 methine groups, 6 quaternary carbons, 2 oxymethylene, two methine
groups, each attached to an OH, a carbonyl group of an ester and 2 olefinic carbons. A
detailed analysis of the
1
H NMR spectrum (Figure S17) showed the presence of a triterpene
skeleton of ursolic acid. Tertiary methyl groups appeared at d
H
0.74, 0.81, 0.88, 0.90 and
1.23 (3H, H-26, H-30, H-29, H-25 and H-27 respectively), while oxymethine protons were
observed at d
H
3.62 (dt, 11.5 and 5 Hz) and 3.00 (d, 5 Hz), corresponding to H-2 and H-3.
Two protons of primary alcohol were also observed in the proton spectrum d
H
3.89/4.02
(2H, d,6.5Hz,H-23a,H-23b)andtwoanotherprotonsatd
H
3.33 corresponding to oxy-
methylene group (H-24). Equally, the
1
H NMR spectrum reveals the presence of protons at
d
H
3.60 and 3.80 indicating two methoxy groups. In the HMBC spectrum (Figure S18),
4 M. M. NOAH ET AL.
alcoholic methine carbon at d
C
76.8 showed interaction
3
J
CH
with H-5 (0.68). The positions
of the hydroxyl groups in C-23 and C-24 were established from the HMQC (Figure S19)
data at d
H
3.89/4.02, 3.33 respectively. The HMQC spectrum (Figure S20) enabled us to con-
firm the presence of two methoxy groups at d
H
3.60 and 3.80, corresponding to 28-OMe
(51.7) and 24-OMe (56.0). The position of the double bond at C-12 was confirmed from the
chemical displacements at d
C
124.6 and C-13 (138.4), a feature of fragment D
12
of ursane
triterpene (Lee et al. 2011). The distribution of chemical displacements in
1
HNMRand
13
C
NMR spectra of compound 6werebasedontheHMQCandHMBCspectra.Thevaluesof
the coupling constant of these carbinolic methine hydrogens H-2 (J¼11.5 and 5 Hz) and H-
3(J¼5 Hz) enable us to determine the geometry of the molecule. Thus, the coupling
constant (J
2, 3
) of 5 Hz is typical of a periplanar (axial-axial) relationship between H-2
(a-orientation) and H-3 (a-orientation). Furthermore,
13
C NMR data showed agreement with
reported data of Euscaphic acid (Tanaka et al. 2003;Kyeongetal.2014). Therefore, the
structure of compound 6was established 2a,3a,19a, 23-tetrahydroxy-24-methoxymethyl
ester of urs-12-en-28-oic acid a new derivative, to which trivial name methyl ester of Colic
acid (Figure S21,Table S2) has been given.
The antimicrobial activity was evaluated by determining the minimal inhibitory con-
centration (MIC) and minimal bactericidal/fungicidal concentrations (MBC/MFC) of
Heterophynone (1), betulinic acid (2), oleanolic acid (3), ursolic acid (4), chletric acid
(5), and methyl ester of Colic acid (6) against multi-resistant bacteria according to the
method of Clinical Laboratory Standard Institute (Clinical Laboratory Standard Institute
(CLSI)) 2006) M7-A9 microdilution assay (Tables S3 and S4). The fungicidal and bacteri-
cidal tests were done with Gram-positive bacteria and yeast. The results displayed dis-
tinct activities of compounds against the bacterial strains tested. A gradient of MIC
was determined as 250<MIC 500 lg/mL, 62.5 <MIC 250 lg/mL and MIC
62.5 lg/mL. Based on these criteria, Heterophynone (1) was categorised as very highly
active at MIC of 7.82 lg/mL against Salmonella enterica Ser typhimirium and 62.5 lg/
mL against the bacterial strain CA NR 29456. Oleanolic acid (3) and ursolic acid (4)
reveal good inhibitory at 62.5 lg/mL against the bacterial strain SE Ser typhimirium.
Chletric acid (5) reveals a high inhibition against strain CA NR 29456 at 31.25 lg/mL.
Betulinic acid (2) has a high inhibition against strain CA NR 29456 at 31.25 lg/mL and
a moderate inhibitory against strains SE Ser Muenchen and SE Ser typhimirium at
250 lg/mL. Finally, the methyl ester of Colic acid (6) is moderately inhibitory at
250 lg/mL against the SA NR 46003 and CA NR 29456 bacterial strains. On the same
note, the antimicrobial activity was evaluated with the goal of confirming the death of
bacteria. Hence, Heterophynone displayed a very important bactericidal activity as the
MBC was 62.5 lg/mL against SE Ser typhimirium strain. The bactericidal activity was
very significant for the oleanolic acid (3) and ursolic acid (4) with an MBC at 125 lg/
mL. A moderate activity with an MBF at 250 lg/mL was observed for betulinic acid,
chlectric acid and Heterophynin against the CA NR 29456 bacterial strain. It is worth
noting that the activities of the compounds evaluated were inferior to those obtained
with the control antibiotics (Ciprofloxacin & Amphotericin B). Finally, the compounds
tested displayed significant antimicrobial activity against the positive gram bacteria
and yeast used. However, Heterophynone (1) was very active against the bacteria SE
Ser typhimirium with a MIC at 7.82 lg/mL and an MBC at 62.5 lg/mL. This very good
NATURAL PRODUCT RESEARCH 5
inhibition was due to the presence of aliphatic chains which render the compound
more lipophilic and favours its insertion within its cell. This structural ability of
Heterophynone increases its concentration and has a deadly effect on the energetic
metabolism within the microorganism (Cushnie and Lamb 2005; Ireneusz et al. 2019).
This antagonistic effect justifies the very significant activity of Heterophynone (1)asa
potential microbial agent. The results of antimicrobial controls account for the use of
species of Cola genus in traditional medicine, and point out the first investigation on
antimicrobial tests on C. heterophylla.
3. Experimental section
3.1. General experimental procedures
Melting points were measured using Electrothermal 9100 and were uncorrected.
Optical rotation was determined on a Jasco DIP-370 automatic polarimeter in MeOH
at 25 C. Electrospray Ionisation mass spectra were obtained with a QTOF
Spectrometer (Bruker, Germany) equipped with a ZQ F1 source. The spectrometer was
operated in positive mode (scan: 150–1500, centroid CV ¼20) with automatic gain
control and High-Resolution Mass Spectra were obtained with an LC-MS-QTOF
Spectrometer (Bruker, Germany) equipped with HESI source. The spectrometer was
operated in positive mode (mass range: 100–1500, with a scan rate of 1.00 Hz) with
automatic gain control to provide high-accuracy mass measurements within 0.40 ppm
deviation using Na Formate as calibrant. The NMR data were recorded with a Bruker
spectrometer and JOEL Solutions for Innovation spectrometer DELTA2_NMR with tetra-
methylsilane (TMS) as standard,
1
H NMR (500 MHz) and
13
C NMR (125 MHz).
1
H,
13
C
NMR COSY, HMBC (DMSO-d
6
) 400, 500 or 600 MHz, with the residual solvent peaks as
internal references. Column Chromatography (CC) was performed with silica gel 60
(40–63 lm and 70–230 mesh particle size) as adsorbent and thin-layer chromatography
(TLC) on pre-coated silica gel on aluminium sheets F-254 Merck (20 20 cm).
Detection of the spots was achieved with a UV lamp at 254 nm at 366 nm and by
spraying with 10% H
2
SO
4
followed by heating.
3.2. Plant material
The leaves and the stem bark of C.heterophylla were collected from Nyamanga II
located in the Centre Region of Cameroon on February 2017. The plant was localised
at 1.010 m of altitude with a ‘QK72’UTM position and joint Operation Graphic of
NA32-04. The voucher specimens were identified and authenticated by Mr NANA a
botanist of the Cameroon National Herbarium (Yaounde, Cameroon) by comparison to
a known specimen under voucher number No 4277/HNC. The plant was localised at
the geographical coordinates of 32003700 North, 111202500 East and 1.010 m of altitude
with ‘QK72’UTM position and a Joint Operation Graphic of NA32604.
6 M. M. NOAH ET AL.
3.3. Extraction and isolation
The leaves and stem bark collected were air-dried at room temperature (26–27 C) dur-
ing eight days and powdered. This powder of C. heterophylla were macerated in pure
methanol during 48 hours at room temperature 1.5 Kg leaves powdered in 10 L metha-
nol and 2 Kg stem bark powdered in 15 L methanol. Removal of the solvent from the
dark green residue extract took placed under reduced pressure. The methanol extract
(278 g) and (92 g), were partitioned the first time between methanol and hexane. Dry
methanol phase were re-extracted with ethyl acetate to give ethyl acetate fraction and
methanol residual extract. The methanol residual extract (15 g) from the leaves was
treated by chromatography column with silica gel, and eluted with gradient hexane,
dichloromethane and methanol system. At CH
2
Cl
2
/MeOH (95/5) 36 mg (2.4%) of com-
pound 3was obtained, 13 mg (0.87%) of compound 4at CH
2
Cl
2
/MeOH (90/10), 10 mg
(0.67%) of compound 5at CH
2
Cl
2
/MeOH (80/20), and 14 mg (0.93%) of compound 6at
CH
2
Cl
2
/MeOH (70/30). The ethyl acetate fraction from stem bark (8 g) was submitted
to the column chromatography with silica gel, and eluted with gradient hexane, ethyl
acetate and methanol system. At Hex/EA (85/15) and Hex/EA (70/30) two amorphous
powder were obtained (compounds 2; 8 mg (0.4%) and 1; 11 mg (0.55%)).
3.3.1. Heterophynone (1)
Purple amorphous powder HR-ESIMS [M þ4H þNa]
þ
at 481.2214 (calcd for C
27
H
34
O
6
,
454). UV (MeOH, kmax) 280 nm, band II and 306 nm, band I. NMR
13
H(d, 600 MHz):
6.66 (d, 1.8 Hz, H-6), 6.72 (d, 1.8 Hz, H-8), 6.35 (d, 2.4 Hz, H-30), 6.37 (d, 2.4 Hz, H-50), 3.77
(s, OCH
3
-7), 3.74 (s, OCH
3
-40), 2.54 (t, 7.8 and 15.6 Hz, H-100 ), 1.55 (m, H-200), 1.29 (m,H-
300/H-400), 0.85 (dd, 5.4 and 15.6 Hz, H-500), 2.67 (t, 7.8 and 15.6 Hz, H-1000), 1.59 to 1.22
(m, H-2000 to H-4000) and 0.84 (dd, 5.4 and 13.8 Hz, H-5000); NMR
13
C(d, 150 MHz): 167.2
(C-2), 139.4 (C-3), 169.1 (C-4), 141.5 (C-5), 114.3 (C-6), 151.7 (C-7), 103.4 (C-8), 123.2 (C-
4a), 156.9 (C-4b), 113.1 (C-10), 158.8 (C-20), 99.3 (C-30), 162.3 (C-40), 107.5 (C-50), 144.4
(C-60), 56.4 (OCH
3
-7), 55.6 (OCH
3
-40), 33.0 (C-100), 31.4 (C-200), 30.4 (C-300), 22.3 (C-400),
14.9 (C-500), 39.9 (C-1000), 31.8-22.3 (C-2000 to C-4000) and 14.1 (C-5000).
3.3.2. Methyl ester of Colic acid (6)
White amorphous powder, [a]25
D
5
121.8 (MeOH, c0.2), ESI
þ
m/z 553 ([M þ5H]
þ
,
(calcd. for C
32
H
52
O
7
, 548)) . NMR
13
H(d, 500 MHz): 3.62 (td, 11.5 and 5 Hz, H-2a), 3.00
(d, 5 Hz, H-3a), 0.68 (m, H-5), 1.29/1.46 (m, H-6), 1.25/1.43 (H-7), 1.45 (m, H-9), 1.51/1.92
(mH-11), 5.13 (m, H-12), 0.98/1.79 (m, H-15), 1.44 (m, H-16, 2.36 (s, H-18), 1.3 (m,H-
20), 1.54/1.57 (m, H-21), 0.90/1.52 (m, H-22), 3.89/4.02 (d, 6.5 Hz, H-23), 3.33 (m, H-24),
0.85 (s, H-25), 0.74 (s, H-26), 1.23 (s, H-27), 0.88 (s, H-29), 0.81 (d, 6.4 Hz, H-30), 3.80 (24-
OCH
3
) and 3.60 (28-OCH
3
). NMR
13
C(d, 125 MHz): 68.9 (C-2), 77.0 (C-3), 46.8 (C-4),
54.8 (C-5), 18.0 (C-6), 32.7 (C-7), 39.0 (C-8), 47.0 (C-9), 36.5 (C-10), 23.6 (C-11), 124.7 (C-
12), 138.4 (C-13), 41.7 (C-14), 30.2 (C-15), 27.5 (C-16), 46.7 (C-17), 53.6 (C-18), 71.4 (C-
19), 38.5 (C-20), 27.0 (C-21), 38.2 (C-22), 65.6 (C-23), 62.6 (C-24), 15.2 (C-25), 16.0 (C-26),
23.2 (C-27), 178.2 (C-28), 28.3 (C-29), 17.3 (C-30), 56.0 (24-OCH
3
), 51.7 (28-OCH
3
).
NATURAL PRODUCT RESEARCH 7
3.4. Yeast and bacterial strains
The microorganism using for the screening were fungal strains such as Candida
Albicans NR 29456, Candida Krusei 1415 and bacterial strains such as Salmonella enteric
Serovar Muenchem,Salmonella enteric Serovar Thyphimurium, Staphylococcus aureus NR
46003, Staphylococcus aureus NR46374 and Pseudomonas aeruginosa HM 601. Isolates
were obtained from Yaound
e Centre Hospital, Cameroon and the reference strains
from BEI resources and the American Type Culture Collection. Bacteria and fungi were
cultivated in Petri dishes containing Muller Hinton Agar (MHA) and Sabouraud
Dextrose Agar (SDA) respectively followed by an incubation period of 24 hours at
37 C. Each microorganism was sub-cultured in a new Agar plate and incubated in the
same above-mentioned conditions prior to each experiment.
3.5. Preparation of compounds
Compounds were weighted and dissolved in pure 100% dimethyl sulfoxide (DMSO) for
the final concentration of 2 mg/mL. Positive controls, Ciprofloxacin and Amphotericin
B were prepared at 1 mg/mL. The stock solutions were filter-sterilized with a 0.20 mm
syringe filter and stored at 20 C until use.
3.6. Antimicrobial assays
The antimicrobial activity of each product was done as previously described by the
Clinical and Laboratory Standards Institute (Clinical Laboratory Standard Institute
(CLSI)) 2006) using the broth microdilution method in 96-wells microplates. Briefly,
50 lL of the culture media (Mueller Hinton Broth (MHB) or Sabouraud Dextrose Agar
(SDA) depending on the test organisms) were introduced in plates and 50 lL of test
compounds were added only in wells belonging to the first time. After a gentle hom-
ogenisation, serial two-fold dilutions of test products were made by transferring 50 lL
of the mixture from the first well to the second up to the last. Fifty microliters of the
microbial inoculum standardised at 510
6
CFU/mL and 510
3
CFU/mL for bacterial and
fungal (with 0.5 McFarland standard) were respectively introduced in wells. Wells con-
taining the culture medium only served as sterility control, those containing the micro-
organisms and the culture medium were the Negative control corresponding to one
hundred percent growth (100% growth). Positive controls Ciprofloxacin and
Amphotericin B were distinctively screened for their antibacterial and antifungal activ-
ities in same conditions. All the experiments were performed in triplicate and plates
were incubated for 24 hours at 37 C. Ten microliters of resazurin (1 mg/mL in PBS)
were then added in duplicate wells and concentration in which no visible growth
(blue coloured wells) were observed in wells after the incubation. The bactericidal and
fungicidal property of active compounds were determined by transferring 50 lL ali-
quots of the clear wells (without resazurin) in plates containing 50 lL of freshly pre-
pared broth medium (MHB or SDB depending on the test organisms) followed by and
additional incubation period of 24 hours. The minimum bactericidal concentration
(MBC) and the minimum fungicidal concentration (MFC) were obtained as described
above with resazurin. Cut-off points for significant activity based on minimum
8 M. M. NOAH ET AL.
inhibitory concentration (MIC) of compound were as follow: very good (MIC <
62.5 mg/ml), good (62.5 <MIC 125 mg/ml), moderate (250 <MIC 500 mg/ml) or
weak (MIC >500 mg/ml). These intervals also correspond to those bactericidal and fun-
gicidal activities.
4. Conclusion
Natural products are attractive sources for developing antimicrobial agents, because
they can provide diverse structural chemistry and biological activities. Therefore, a
detailed phytochemical investigation by column chromatography of the stem bark
ethyl acetate fraction and the residual methanol fraction of leaves of Cola heterophylla
resulted in the isolation of two new compounds together with four known com-
pounds. The antimicrobial activity revealed that Heterophynone, a new flavone, has a
very significant antimicrobial activity against SE Ser typhimirium (MIC ¼7.82 lg/mL
and MBC ¼62.5 lg/mL) and good activity against CA NR 29456 (MIC ¼62.5 lg/mL).
The other compounds isolated showed such a significant antimicrobial activity: betu-
linic acid and chletric acid showed very good activity against CA NR 29456 (MIC ¼
31.25 lg/mL), when oleanolic acid and ursolic acid showed good activity against SE
Ser typhimirium (MIC¼62.5 lg/mL and MBC ¼125 lg/mL).
Acknowledgements
The authors thank the Department of Organic Chemistry at the University of Yaounde 1 for
helpful assistance. We gratefully acknowledge the Laboratory for phytobiochemistry and medi-
cinal plants study, Department of biochemistry of the University of Yaound
e 1, for undertaking
the biological activities; and the Laboratory of NMR and Molecular Imaging, University of Mons,
Belgium, for the spectral analysis of compounds.
Disclosure statement
No potential conflict of interest was reported by the authors.
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