# A new phenyl alkyl ester and a new combretin triterpene derivative from Combretum fragrans F. Hoffm (Combretaceae) and antiproliferative activity

Isaac Silvère Gade, Corinne Chadéneau, Richard Tagne Simo, Emmanuel Talla, Alex de Theodore Atchade, Paule Seité, Brigitte Vannier, Sophie Laurent, Celine Henoumont, Armel Nwabo Kamdje, et al.

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## ► To cite this version:

Isaac Silvère Gade, Corinne Chadéneau, Richard Tagne Simo, Emmanuel Talla, Alex de Theodore Atchade, et al. A new phenyl alkyl ester and a new combretin triterpene derivative from Combretum fragrans F. Hoffm (Combretaceae) and antiproliferative activity. Open Chemistry, 2020, 18 (1), pp.1523-1531. 10.1515/chem-2020-0167. hal-03196380

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Research Article

Isaac Silvère Gade*, Corinne Chadeneau, Richard Tagne Simo, Emmanuel Talla, Alex De Theodore Atchade, Paule Seité, Brigitte Vannier, Sophie Laurent, Celine Henoumont, Armel H. Nwabo Kamdje, Jean-Marc Muller

A new phenyl alkyl ester and a new combretin triterpene
derivative from *Combretum fragrans* F. Hoffm (Combretaceae)
and antiproliferative activity

https://doi.org/10.1515/chem-2020-0167
received June 26, 2020; accepted August 20, 2020

**Abstract:** This work concerns the isolation and structure elucidation of compounds obtained from the extract of leaves and stem bark of *Combretum fragrans* F. Hoffm. Both extracts and some isolated compounds were tested for antiproliferative activity on glioblastoma (U87MG and C6 cells) and prostate (PC-3 cells) cancer cell lines using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) assay. The dichloromethane/ methanol (1:1) extract of the leaves led to the isolation of two new compounds such as fragransinate (1) and combretin C (2), alongside five known compounds such as combretin A (3), belamcanidin (4), cirsilineol (5), velutin (6), and a mixture of β-sitosterol-3-O-β-D-glucopyranoside (10a) and stigmasterol-3-O-β-D-glucopyranoside (10b), whereas the methanol extract of the stem bark led to the isolation of three known compounds betulinic acid (7), bellericagenin B (8), and a mixture of β-sitosterol

(9a) and stigmasterol (9b). The structure of compounds was elucidated by nuclear magnetic resonance and mass spectrometry data. The methanol extract of the stem bark showed a powerful antiproliferative activity on all tested cells, as well as the extract of leaves which also showed important cytotoxicity effect. Compound (3) showed good antiproliferative activity particularly on U87MG and PC-3 cells, whereas compound (5) exhibits moderated activity. Compounds (2) and (8) were not active on all tested cells.

**Keywords:** *Combretum fragrans* F. Hoffm, fragransinate, combretin C, antiproliferative activity

**1 Introduction**

The genus *Combretum* is the largest and most widespread
genus of Combretaceae. It is widely distributed in tropical
and subtropical regions (Africa, India, and China) and
comprises about 200 and 250 species that include trees,
shrubs, shrublets, or woody climbers [1]. The genus *Com-
bretum* is reported to have various biological activities
such as antioxidant, anti-inflammatory, antimalarial,
antibacterial, and cytotoxicity against tumor cells [1,2].
Phytochemical studies of *Combretum* genus showed
many secondary metabolites such as flavonoids, triter-
penes, lignans, phenanthrenes, and stilbenoids. [1,2].
*Combretum fragrans* F. Hoffm (syn: *Combretum adenogo-
nium* Steud. ex A. Rich) is a medicinal plant belonging to
the *Combretum* genus. It is a shrub or a small tree that
grows up to 10–12 m high. The plant is found in decid-
uous woodland, wooded grassland associated with sea-
sonally waterlogged clay soils, and on shallow, stony
soils [3]. *C. fragrans* F. Hoffm is used in African folk med-
icine for the treatment of various types of disease, such as
coughs, syphilis, leprosy, septic wounds, fungal infection
of the scalp, diarrhea, hypertension, incurable wound,
malaria, snake bite, gonorrhea, pain, and inflammation
[4–6]. In the northern part of Cameroon, the plant is used
to treat jaundice, diabetic wound, ulcers, and cancers.

* Corresponding author: Isaac Silvère Gade, Department of Chemistry, Faculty of Science, University of Ngaoundere, P.O. Box 454, Ngaoundéré, Cameroon; Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon, e-mail: isaac_gade@yahoo.fr, tel: +237-69-709-7902

Corinne Chadeneau, Paule Seité, Brigitte Vannier, Jean-Marc

Muller: Department of Cell Biology, UFR Sciences Fondamentales et Appliquées, Team “Récepteurs, Régulations, Cellules Tumorales” (2RCT)-EA 3842 CAPTuR, Pôle Biologie Santé-Bât. B36/B37, University of Poitiers, 1 rue Georges Bonnet-TSA 51106, 86073, Poitiers Cedex 9, France

Richard Tagne Simo, Armel H. Nwabo Kamdje: Department of Biomedical Science, Faculty of Science, University of Ngaoundere, P.O. Box 454, Ngaoundéré, Cameroon

Emmanuel Talla: Department of Chemistry, Faculty of Science,
University of Ngaoundere, P.O. Box 454, Ngaoundéré, Cameroon

Alex De Theodore Atchade: Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon

Sophie Laurent, Celine Henoumont: Department of General, Organic Chemistry and Biomedical, Laboratory of NMR and Molecular Imaging, University of MONS, Mons, Belgium

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Pharmacological investigation of *C. fragrans* F. Hoffm
extracts have been established to have antibacterial, anti-
fungal, and antiproliferative properties [7,8], whereas
phytochemical analysis of leaves, stem bark, and roots
has revealed the presence of flavonoids, triterpenes, sterols,
saponins, and tannins [9,10]. Despite the well-documented
benefits of *C. fragrans* F. Hoffm, only few phytochemical
studies have been performed on it.

The search of novel bioactive compounds against
cancer is still on, because many cancer patients develop
resistance to the existing anticancer agents during treat-
ment and because of the undesired side effects produced
by these anticancer agents. Plants offer a large possibility
to find novel anticancer agents with new mechanism of
action and weak toxicological effect. It is in this sense
that we investigated *C. fragrans* F. Hoffm. The present
work describes the isolation of compounds from extract
of leaves and stem bark of *C. fragrans* F. Hoffm and eva-
luation of antiproliferative activity of both extracts and
some isolated compounds on glioblastoma (GBM) and
prostate cancer cell lines.

**2 Materials and methods**

**2.1 General experimental procedure**

The melting point of the new compounds was recorded in
an open capillary using Electrothermal 9100 and is uncor-
rected. IR spectra were recorded on FT-IR spectrometer
(Perkin-Elmer Spectrum 100). The proton and ¹³carbon
nuclear magnetic resonance (NMR) data were recorded
on spectrometer Bruker Avance AV-500 and 600, and tetra-
methylsilane (TMS) was used as standard. Chemical
shifts are given in ppm (δ) and coupling constant (J) in
Hz. CDCl₃, MeOD, and DMSO-d₆ were used as NMR sol-
vents. Electrospray ionization-mass spectrometry (ESI-MS)
spectra were registered on a QTOF Spectrometer (Bruker,
Germany) equipped with a ZQ F1 source. The spectrometer
was operated in positive mode (scan: 150–1,500; centroid
CV = 20). The high resolution mass spectrum was regis-
tered on LC-MS-QTOF Spectrometer (Bruker, Germany)
equipped with ESI source operating in positive mode.
Column chromatography (CC) was performed on silica
gel 60 (70–230 mesh, Merck), and thin layer chromato-
graphy was performed on silica gel precoated plates
F-254 Merk (20 × 20 cm). Spots were visualized under
UV light (254 and 365 nm), sprayed with 5% of phospho-
molybdic acid prepared in ethanol, then heated. XTT (2,3-

bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-
carboxyanilide inner salt) reagent from Promega was
used to evaluate antiproliferative activity.

**2.2 Plant material**

Leaves and stem bark of *C. fragrans* F. Hoffm were col-
lected during the month of May 2017 at Padarmé located
in Bibémi subdivision, in the north region of Cameroon.
The sample was identified at the National Herbarium of
Cameroon, where a voucher specimen is deposited under
the number 39,753/HNC.

**2.3 Extraction and isolation**

The powders of 1 kg of leaves and 1 kg of stem bark of
*C. fragrans* F. Hoffm were extracted separately by macera-
tion at room temperature with 5 L of dichloromethane/
methanol (CH₂Cl₂/MeOH) (1:1) and methanol (MeOH)
respectively for 48 h with intermittent stirring. After fil-
tration on Whatman filter paper, the resulting solutions
were concentrated using a rotary evaporator. The proce-
dure was repeated thrice to optimize extraction. We
obtained 55 g of crude extract from the leaves and 40 g
from the stem bark. Then, a quantity of 30 g of each crude
extract was taken differently and each subjected to CC
separation on silica gel with the gradient eluting system
hexane-ethyl acetate (100:0 → 0:100), followed by ethyl
acetate-methanol (100:0 → 0:100). The fractions of
100 mL each were collected and concentrated on rotary
evaporator. From the extract of leaves, two new com-
pounds, fragransinate (1, 5.1 mg) and combretin C (2,
215 mg), alongside four known compounds combretin
A (3, 46 mg), belamcanidin (4, 12.3 mg), cirsilineol (5,
64 mg), and velutin (6, 60 mg), and a mixture of β-sitos-
terol-3-O-β-D-glupyranoside and stigmasterol-3-O-β-D-
glucopyranoside (10a,b, 9 mg) were isolated. From the
methanol crude extract of the stem bark, three known
compounds: betulinic acid (7, 4.3 mg), bellericagenin B
(8, 13 mg), and a mixture of β-sitosterol and stigmasterol
(9a, b, 15.6 mg) were isolated (Figure 1).

**2.3.1 Compound 1: fragransinate (3'-(3",4"-dihydroxy-5-methoxyphenyl)propyl heptadecanoate)**

Whitish solid; C₂₇H₄₆O₅; m.p. = 66.5–67.5; IR: νmax =
3,400 cm⁻¹ (–OH), 1,735 cm⁻¹ (C=O), 1,450–1,600 cm⁻¹
<figure><img src="image_5.png" /><figcaption>**Figure 1:** Chemical structures of compounds isolated from *Combretum fragrans* F. Hoffm.</figcaption></figure>

(aromatic ring), 1,100 and 1,150 cm⁻¹ (C–O); (+) ESI-MS *m/z* 496 [M + 2Na]⁺; ¹H and ¹³C NMR data, see Table 1.

HRESI-MS *m/z* 643.4149 [M + Na]⁺ (calculated for C₃₆H₆₀O₈, 643.4288); ¹H and ¹³C NMR data, see Table 2.

### 2.3.2 Compounds 2: combretin C (3-(β-D-xylopyranosyl)-15α-hydroxycycloartan-28-oic acid)

White powder; C₃₆H₆₀O₈; m.p. = 239.1–240.1; IR: νmax = 3,400 cm⁻¹ (–OH), 1,700 cm⁻¹ (C=O) and 1,000–1,025 cm⁻¹ (C–O); High resolution ESI mass spectrum (HRESI-MS) (+)

## 2.4 Biological evaluation

### 2.4.1 Cell culture and cell conditions

The U87 human GBM cells line and C6 rat GBM cells line were maintained in Dulbecco's modified Eagle's medium
<table><caption>**Table 1:** ¹H and ¹³C NMR data for compounds 1 (CDCl₃, δ in ppm, 600 and 151 MHz)</caption><thead><tr><th rowspan="2">No.</th><th colspan="2">1</th></tr><tr><th>δ<sub>H</sub> (Mult., J(Hz))</th><th>δ<sub>C</sub></th></tr></thead><tbody><tr><td>1</td><td>—</td><td>174.0</td></tr><tr><td>2</td><td>2.33 (t, j = 7.5; 2H)</td><td>34.4</td></tr><tr><td>3</td><td>1.64 (dd, j = 7.5, 14.2; 2H)</td><td>25.0</td></tr><tr><td>4–16</td><td>1.73–1.22 (m)</td><td>29.2–29.7</td></tr><tr><td>17</td><td>0.90 (t, j = 6.9; 3H)</td><td>14.1</td></tr><tr><td>1'</td><td>4.10 (t, j = 6.5; 2H)</td><td>63.5</td></tr><tr><td>2'</td><td>1.93 (dd, j = 7.6, 6.5; 2H)</td><td>32.1</td></tr><tr><td>3'</td><td>2.60 (t, j = 7.6 Hz; 2H)</td><td>30.3</td></tr><tr><td>1"</td><td>—</td><td>133.1</td></tr><tr><td>2"</td><td>6.30 (s, 1H)</td><td>103.2</td></tr><tr><td>3"</td><td>—</td><td>146.7</td></tr><tr><td>4"</td><td>—</td><td>130.5</td></tr><tr><td>5"</td><td>—</td><td>143.7</td></tr><tr><td>6"</td><td>6.45 (s, 1H)</td><td>108.3</td></tr><tr><td>MeO–</td><td>3.89 (s, 3H)</td><td>56.11</td></tr></tbody></table>

(DMEM, 1 g/L glucose) with GlutaMAX™ (Gibco) and sodium pyruvate (Invitrogen), supplemented with 10% of fetal calf serum and 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco) (antibiotics mixture). The PC-3 human prostate cancer cells line were maintained in the same condition except the use of DMEM medium with 4.5 g/L glucose. Cell cultures were carried out in a biological safety cabinet (laminar flow hood). An inverted microscope (OCO-2-KERN) was used for cell counting. All cells line were grown in a humidified incubator (95% air and 5% CO₂) at 37°C.

### 2.4.2 Antiproliferative assay

The antiproliferative activity of extracts and some isolated compounds was evaluated using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) test [11]. The assay is based on the reduction of XTT reagent (Promega) into an orange formazan dye by the metabolically active living cells. Thus, the U87 and C6 GBM cells, as well as the PC-3 prostate cancer cells were plated in 96-well plates respectively at a density of 5 × 10³, 3 × 10³, and 4 × 10³ cells per well in 100 µL of the corresponding medium. Then, cells were allowed to attach overnight in growth medium. After 24 h of incubation, the medium was removed and replaced with fresh medium containing different concentrations (3.15–150 µg/mL) of extracts or compounds, and control cells were treated with DMSO (0.5%) used to dissolve samples. After 72 h of treatment, 50 µL of XTT labeling reagent mixture solution was added in each

<table><caption>**Table 2:** ¹H and ¹³C NMR data for compounds 2 (DMSO-d₆, δ in ppm, 500 and 126 MHz)</caption><thead><tr><th rowspan="2">No.</th><th colspan="2">2</th></tr><tr><th>δ<sub>H</sub> (Mult., J(Hz))</th><th>δ<sub>C</sub></th></tr></thead><tbody><tr><td>1</td><td>1.24<sup>a</sup>, 1.08<sup>a</sup></td><td>32.3</td></tr><tr><td>2</td><td>1.81<sup>a</sup>, 1.52<sup>a</sup></td><td>28.5</td></tr><tr><td>3</td><td>3.96 (dd, j = 11.0, 4.7; 1H)</td><td>82.4</td></tr><tr><td>4</td><td>—</td><td>58.8</td></tr><tr><td>5</td><td>1.92<sup>a</sup></td><td>44.7</td></tr><tr><td>6</td><td>1.58<sup>a</sup>, 1.15<sup>a</sup></td><td>24.6</td></tr><tr><td>7</td><td>—</td><td>27.6</td></tr><tr><td>8</td><td>—</td><td>46.9</td></tr><tr><td>9</td><td>—</td><td>19.3</td></tr><tr><td>10</td><td>—</td><td>26.0</td></tr><tr><td>11</td><td>—</td><td>27.1</td></tr><tr><td>12</td><td>—</td><td>34.7</td></tr><tr><td>13</td><td>—</td><td>48.3</td></tr><tr><td>14</td><td>—</td><td>44.6</td></tr><tr><td>15</td><td>3.24 (m, 1H)</td><td>71.2</td></tr><tr><td>16</td><td>1.85<sup>a</sup></td><td>43.9</td></tr><tr><td>17</td><td>1.57<sup>a</sup></td><td>51.5</td></tr><tr><td>18</td><td>1.01 (s, 3H)</td><td>18.9</td></tr><tr><td>19</td><td>0.35 (d, j = 4.0; H-19b), 0.54 (d, j = 4.0; H-19a)</td><td>28.8</td></tr><tr><td>20</td><td>1.32<sup>a</sup></td><td>38.9</td></tr><tr><td>21</td><td>0.81 (d, 3H)</td><td>18.6</td></tr><tr><td>22</td><td>1.71<sup>a</sup>, 1.24<sup>a</sup></td><td>34.9</td></tr><tr><td>23</td><td>—</td><td>30.8</td></tr><tr><td>24</td><td>—</td><td>35.1</td></tr><tr><td>25</td><td>2.20</td><td>36.2</td></tr><tr><td>26</td><td>0.86 (brd, 3H)</td><td>25.7</td></tr><tr><td>27</td><td>0.86 (brd, 3H)</td><td>25.0</td></tr><tr><td>28</td><td>—</td><td>177.9</td></tr><tr><td>29</td><td>1.03 (s, 3H)</td><td>10.0</td></tr><tr><td>30</td><td>0.9 S (s, 3H)</td><td>14.3</td></tr><tr><td>31</td><td>1.00 (brd, 3H)</td><td>18.6</td></tr><tr><td colspan="3"><strong>Xylose</strong></td></tr><tr><td>1</td><td>4.14 (d, j = 7.5; 1H)</td><td>104.0</td></tr><tr><td>2</td><td>2.89 (brdd, 1H)</td><td>73.4</td></tr><tr><td>3</td><td>3.02 (brdd, 1H)</td><td>76.3</td></tr><tr><td>4</td><td>3.23 (brdd, 1H)</td><td>69.4</td></tr><tr><td>5</td><td>3.65 (brdd, Ha-5'); 2.97 (brdd, Hb-5')</td><td>65.5</td></tr></tbody></table>

well and the plate was incubated for 4 h. After incubation with the XTT reagent, the formazan formed is soluble in the medium and is directly proportional to the living cells. Then, cell viability was assessed by the measurement of the absorbance at 490 nm using a spectrophotometric microplate reader.

Each test was performed in triplicate. The IC₅₀ (50% inhibitory concentration, that is, the concentration of extract or compound that inhibits 50% of the proliferation of cancer cells) value was calculated automatically from the sigmoidal dose-response nonlinear regression curve using GraphPad prism software.

<footnote><sup>a</sup>Signals overlapped.</footnote>
**Ethical approval:** The conducted research is not related to either human or animal use.

# 3 Results and discussion

## 3.1 Structural elucidation of compounds

Chromatographic separation on silica gel of the CH₂Cl₂/ MeOH (1:1) crude extract of leaves of C. fragrans F. Hoffm led to two new compounds (1 and 2) and five known compounds (3, 4, 5, 6, and 10), whereas the methanol crude extract from the stem bark yielded three known compounds (7, 8, and 9). The structures of the compounds were elucidated by spectral analysis using ESI-MS and 1D, 2D NMR data.

Compound 1 was isolated as a whitish powder and melted between 66.5 and 67.5°C. Its molecular formula, C₂₇H₄₆O₅, implying five degrees of unsaturation, was deduced from its ESI-MS spectrum, which showed a pseudomolecular ion peak [M + 2Na]⁺ at m/z 496 in positive mode. Its IR spectrum showed broad absorption bands at 3,400 cm⁻¹, indicating the presence of hydroxyl group at 1,735 cm⁻¹ characteristic of a carbonyl of an ester moiety, and of aromatic ring between 1,450 and 1,600 cm⁻¹, and C–O bonds (1,100 and 1,150 cm⁻¹). The ¹H NMR analysis revealed in up-field region the presence of a terminal methyl signal at δ<sub>H</sub> 0.90 (3H, t, *j* = 6.9 Hz), with aliphatic methylene (–CH<sub>2</sub>–) signals ranging from δ<sub>H</sub> 1.37 to 1.22 ppm, corresponding to an aliphatic chain. The spectrum also displayed resonances of α and β protons adjacent to the ester function of an aliphatic chain, respectively, at δ<sub>H</sub> 2.33 (2H, t, *j* = 7.5 Hz) [12] and 1.64 (2H, dd, *j* = 7.5; 14.2 Hz), as well as the signal of an oxymethylene proton appearing at 4.10 (2H, t, *j* = 6.5 Hz). On the same spectrum, the signals of aromatics protons appeared as singlets at δ<sub>H</sub> 6.45 (1H, s) and 6.31 ppm (1H, s) suggesting the presence of phenyl ring, one methoxy proton signal at δ<sub>H</sub> 3.89 (3H, s), and a broad signal of free hydroxyl protons at δ<sub>H</sub> 5.30 ppm as singlet. All the above information suggests an alkylphenyl skeleton. The ¹³C NMR broad-band showed 16 carbon signals, and together with HSQC and DEPT spectra, these signals were unambiguously assigned as: a carbonyl of ester at δ<sub>C</sub> 174.0 (C-1), six tertiary and quaternary aromatic signals at δ<sub>C</sub> 103.2, 108.5, 130.5, 133.1, 143.7, and 146.7, one oxymethylene carbon at δ<sub>C</sub> 63.5, one methoxy carbon at δ<sub>C</sub> 56.1, and one primary carbon at δ<sub>C</sub> 14.1 corresponding to the terminal methyl of aliphatic chain. The

signals of carbon atoms of the aliphatic chain at α and β positions to the carbonyl of the ester function appear at δ<sub>C</sub> 34.4 and 25.0 respectively, and methylene carbon signals ranging from δ<sub>C</sub> 29.2 to 29.7 correspond to a long aliphatic chain. In addition, two methylene carbon signals at δ<sub>C</sub> 32.1 and 30.3 were attributed to the two methylenes between the phenyl group and the ester function. The two signals at δ<sub>C</sub> 143.7 and 146.7 indicate that two of the oxygenated quaternary carbons are in *ortho* position. The ¹H–¹H COSY experiment shows correlations between the oxymethylene proton at δ<sub>H</sub> 4.10 and the methylene proton at δ<sub>H</sub> 1.93, which in turn correlates with another methylene proton at δ<sub>H</sub> 2.59. These correlations suggest a propane chain substituted on position C-1' and C-3' enabling the assignment of chemical shifts at 4.10 to H-1', 1.93 to H-2', and 2.59 to H-3'. The spectrum also shows correlations between the α and β protons to a carbonyl (ester) respectively at 2.31 (H-2) and 1.63 (H-3). The proton in β correlates in turn with the proton at δ<sub>H</sub> 1.33 (H-4). The heteronuclear multiple bond correlation (HMBC) experiment shows correlations between the proton at 4.10 (H-1') and carbons at δ<sub>C</sub> 32.0 (C-3'), 30.5 (C-2') and a carbonyl signal at 173.9 (C-1). The spectrum also displays correlation between the proton at δ<sub>H</sub> 1.93 (H-2') and carbons at δ<sub>C</sub> 31.6 (C-3'), 63.3 (C-1') as well as with an aromatic carbon signal at 133.2 (C-1''). Cross-peak correlations were also observed between the proton at δ<sub>H</sub> 2.59 (H-3') and aromatic carbons at 108.3 (C-6''), 103.1 (C-2''), and 133.2 (C-1'') as well as carbons at δ<sub>C</sub> 30.2 (C-2'), 63.4 (C-1'). Proton H-3 is the only proton that has several correlations with carbons from phenyl ring, indicating that C-3 is directly attached to the phenyl ring on C-1'' position. This result suggests that the aliphatic chain, which is a fatty ester, is located on C-1' position of the propyl chain. In addition, the HMBC spectrum shows correlations of an aromatic proton at δ<sub>H</sub> 6.45 (C-2'') with carbons at δ<sub>C</sub> 31.9 (C-3'), 108.6 (C-6''), 130.2 (C-4''), and 143 (C-3''). The aromatic proton at δ<sub>H</sub> 6.30 (H-6'') correlates with different carbons at δ<sub>C</sub> 31.9 (C-3'), 103.2 (C-2''), 130.3 (C-4''), and 146.7 (C-5''). Proton of the methoxy group (δ<sub>H</sub> 3.89) correlates with the carbon at δ<sub>C</sub> 146.7, indicating that the methoxy group is attached on C-5'' position of the aromatic ring. Important HMBC correlations were also observed between the proton at δ<sub>H</sub> 2.31 (H-2) from the long chain and carbons at δ<sub>C</sub> 29.02 (C-4), 25.05 (C-3), and the carbonyl function at δ<sub>C</sub> 173.98 (C-1). The length of the aliphatic chain was determined in conjunction with its ¹H NMR spectrum and different fragmentation patterns observed on its mass spectrum. Characteristic fragments appeared at m/z 311, 253, and 181 (Figure S1). All these spectral data and some information
reported in literature enabled us to characterize com-
pound **1** as 3'-(3",4"-dihydroxy-5-methoxyphenyl)propyl
heptadecanoate to which the trivial name fragransinate
has been given. To the best of our knowledge, this com-
pound is isolated for the first time from natural sources.

Compound **2** was isolated as a white powder and
melted between 239.1 and 240.1°C. Its HRESI-MS mass
spectrum in positive mode shows the pseudomolecular
ion peak at *m/z* 643.4149 [M + Na]⁺ (calculated for
C₃₆H₆₀O₈, 643.4288) corresponding to the molecular for-
mula C₃₆H₆₀O₈ accounting seven degrees of unsatura-
tion. The IR spectrum exhibits absorption bands *ν*max
at 3,400 cm⁻¹, 1,700 cm⁻¹, and 1,000–1,025 cm⁻¹, which
were respectively attributable to a hydroxyl group, a car-
bonyl group, and C–O bonds. The analysis of its ¹H
NMR spectrum in conjunction with HSQC spectrum
shows in upfield region the signals of two protons as
doublet at *δ*H 0.35 (1H, d, *j* = 4.0 Hz) and *δ*H 0.54 (1H, d,
*j* = 4.0 Hz) corresponding respectively to proton H-19b
and H-19a of a cyclopropane from a cycloartane-type tri-
terpene [13]. The spectrum also revealed the presence of
six methyl groups at *δ*H 0.80, 0.86, 0.92, 1.00, 1.01, and
1.03, and two oxymethine proton signals appearing at *δ*H
3.24 (1H, m) and 3.96 (1H, dd, *j* = 11.0; 4.7 Hz). The spec-
trum also displays signals of osidic proton indicating the
presence of a sugar moiety. Signals of oxymethine pro-
tons of a sugar moiety appeared at *δ*H 3.67 (1H, dd, *j* =
11.3; 5.3 Hz), 3.24 (1H, m), 3.02; 2.97 (1H, m), 2.89 (1H, m)
as well as the anomeric proton at 4.14 (1H, d, *j* = 7.5 Hz).
The larger coupling constant (*j* = 7.5 Hz) of the anomeric
proton suggests the β-configuration of the sugar moiety.
The ¹H NMR spectrum of compound **2** was very similar to
that of Combretin B [9], except the missing signal of an
olefinic proton in **2**. Combretin B has been previously
isolated from the same plant. The sugar moiety was iden-
tified as xylose. The ¹³C NMR spectrum confirmed the
absence of olefinic function compared with that of Com-
bretin B. The spectrum shows signals of 36 carbons
including three methyl group signals as singlet at *δ*C
18.9 (C-18), 10.0 (C-29), and 14.3 (C-30); four methyl
group signals as doublet at *δ*C 18.6 (C-21), 25.7 (C-26),
25.0 (C-27), and 18.6 (C-31); two oxymethine signals at
*δ*C 82.4 (C-3) and 71.2 (C-15); one carbonyl function at
*δ*C 177.5 (C-28). The signals of sugar moiety appear at *δ*C
73.4 (C-2'), 76.3 (C-3'), 69.4 (C-4'), and 65.5 (C-5') together
with the anomeric carbon at *δ*C 104.0 attributable to
C-1'. The HMBC experiment shows important correlation
between the anomeric proton at *δ*H 4.14 and the carbon
at *δ*C 82.43 (C-3), indicating that the sugar moiety is
attached to the aglycone moiety at the C-3 position. Other
correlations were also observed between the proton at *δ*H

1.03 (H-29) and carbons at δ<sub>C</sub> 177.59 (C-28), 82.43 (C-3), and 53.54 (C-4), indicating that the carbonyl group is located on C-28 position. According to the great similarity observed between compound **2** and Combretin B, the structure of compound **2** has been proposed as 3-(β-D-xylopyranosyl)-15α-hydroxycycloartan-28-oic acid for which the trivial name Combretin C has been given. The compound is isolated for the first time as a new derivative combretin triterpene.

The known compounds were identified from their
NMR spectroscopic data and comparisons made with
those reported in the literature: combretin A (3) [9];
belamcanidin (4), cirsilineol (5), velutin (6) [14]; betu-
linic acid (7) [15], bellericagenin B (8) [16], a mixture
of β-sitosterol (9a) and stigmasterol (9b) [17]; and a
mixture of β-sitosterol-3-O-β-D-glucopyranoside (10a)
and stigmasterol-3-O-β-D-glucopyranoside (10b) [18].
Compounds 7, 8, 10a, and 10b are reported for the first
time from this plant species.

**3.2 Antiproliferative activity**

The antiproliferative activity of extracts and compounds
**2**, **3**, **5**, and **8** of *C. fragrans* F. Hoffm on U87 human GBM
cells, C6 rat GBM cells, and PC-3 prostate cancer was
evaluated by XTT test. From the results presented in
Table 3, it is deduced that the MeOH crude extract of
the stem bark of *C. fragrans* F. Hoffm was found to be
more active on all tested cells than the CH₂Cl₂/MeOH
(1:1) crude extract from the leaves. The MeOH extract
powerfully inhibits the proliferation of U87, C6, and PC-
3 cells after 72 h treatment compared to untreated control
cells. The extract was most active on PC-3 prostate cancer
cells (IC₅₀ = 11.24 µg/mL) than C6 (IC₅₀ = 12.17 µg/mL) and
U87 (IC₅₀ = 20.13 µg/mL) cancer cells. On the contrary,
the CH₂Cl₂/MeOH extract from the leaves also showed
important inhibition of C6 cells proliferation (IC₅₀ =
14.19 µg/mL) and moderate cytotoxicity effect on U87
(IC₅₀ = 31 µg/mL) and PC-3 (36.26 µg/mL) cells. The lit-
erature mentions that many species from the combretum
genus, similar to the specie studied here, are known to
possess antiproliferative activity [8].

Among the tested compounds, combretin A (3) was
the most active followed by compound cirsilineol (5),
whereas combretin C (2) and bellericagenin B (8) were
not active. Compound 3 exhibited very good inhibition on
PC-3 cells proliferation (IC50 = 22.57 µM), as well as on
U87 (IC50 = 29.30 µM) and C6 cells (58.93 µM) which is
moderated. Compound 5 also showed important
<table><caption>**Table 3:** Inhibitory concentration values of extracts and some compounds that kill or inhibit 50% of cancer cells (IC<sub>50</sub> in µg/mL for extracts, and µM for compounds)</caption><thead><tr><th>Extracts and compounds</th><th>U87 MG</th><th>C6 IC<sub>50</sub> values</th><th>PC-3</th></tr></thead><tbody><tr><td>CH<sub>2</sub>Cl<sub>2</sub>/MeOH extract</td><td>31 µg/mL</td><td>14.19 µg/mL</td><td>36.26 µg/mL</td></tr><tr><td>MeOH extract</td><td>20.13 µg/mL</td><td>12.17 µg/mL</td><td>11.24 µg/mL</td></tr><tr><td>2</td><td>>100 µM</td><td>>100 µM</td><td>>100 µM</td></tr><tr><td>3</td><td>29.30 µM</td><td>58.93 µM</td><td>22.57 µM</td></tr><tr><td>5</td><td>65.46 µM</td><td>31.61 µM</td><td>30.46 µM</td></tr><tr><td>8</td><td>>100 µM</td><td>>100 µM</td><td>>100 µM</td></tr></tbody></table>

inhibition on PC-3 (IC<sub>50</sub> = 30.46 µM) and C6 (IC<sub>50</sub> = 31.61 µM) cells' proliferation and weak effect on U87 cells growth (IC<sub>50</sub> = 65.46 µM). Compounds **3** and **5** isolated from the leaves could be responsible for the antiproliferative activity obtained with the related extract.

Combretin A (**3**) and C (**2**) are cycloartane-type triterpenes. The second is different from the first just by the presence in its structure of a sugar moiety on carbon C-3 and by the absence of a double bond on C-24 and C-31 carbons. However, combretin A showed good activity on all tested cells, whereas combretin C was inactive. The loss of the activity in combretin C could be attributable to the presence in its structure of the sugar moiety and the lack of the double bonds which negatively influences its activity. Previous study concerning structure-activity relationship showed that sugar moiety in gensenoside decreases its anticancer potential, whereas its aglycone presents good anticancer activity [19]. This study could explain our obtained results. Cycloartane triterpene types are also known for their anticancer activity [20,21], and this corroborates with the result obtained with combretin A. This is the first time that Combretin A is evaluated for its antiproliferative activity and it could be a good candidate for developing an anticancer agent in a pharmaceutical areas.

Compound (**5**) or cirsilineol is a flavonoid from flavone type. Its activity was moderated on C6 and PC-3 cells but weak on U87MG cells. Its antiproliferative activity has already been documented for the first time on a large panel of cancer cell lines [22], and the results are similar with those obtained here with PC-3 cells. Flavonoids are known to possess anticancer activity, and this activity correlates positively with the presence of methoxyl group, carbonyl on carbon C-4, and the double bond on carbon C-2 and C-3 of flavonoids [23].

Compound (**8**) or bellericagenin B is an oleanane-12-ene triterpene type polyhydroxylated and showed no

activity on all tested cancer cells. It is known that hydroxyl group correlates negatively with the anticancer activity in flavonoids [23] and other compounds [24]. Thus, the number of hydroxyl groups in compound **8** could explain its inactivity. In addition, compound **8** could be an oleanolic acid derivative and is different from this later with four hydroxyl groups. However, it is known that oleanolic acid, which possesses one hydroxyl group, has very good anticancer activity [25,26].

Betulinic acid (**7**) and stigmasterol (**9b**) were isolated from the bark and are known for their anticancer activity on many types of cancers [27,28]. Thus, the higher antiproliferative activity observed with the MeOH crude extract of the stem bark could be attributable in part to the presence of compounds **7** and **9b**. The results of the antiproliferative activity of both extracts and some compounds of *C. fragrans* support with scientific evidences the traditional use of this plant in the treatment of cancer.

## 4 Conclusion

The present work describes the isolation and antiproliferative activity of extracts and some isolated compounds from *C. fragrans* F. Hoffm on GBM and prostate cancer cells. Column chromatographic separation on silica gel of the CH<sub>2</sub>Cl<sub>2</sub>/MeOH crude extract from the leaves yielded two new compounds (**1** and **2**) and five known compounds (**3**, **4**, **5**, **6**, and **10**), whereas the MeOH crude extract from the stem bark led to three known compounds (**7**, **8**, and **9**). Compounds **7**, **8**, and **10** were detected for the first time from this species. The results of the antiproliferative activity against U87, C6, and PC-3 cancer cells showed that the methanol crude extract from the stem bark exhibits powerful antiproliferative effect on all tested cells than the CH<sub>2</sub>Cl<sub>2</sub>/MeOH extract from leaves. As well, compound **3** was found to be the most active tested compound with highest effect on PC-3 cancer cells. The antiproliferative activity of extracts and compounds from *C. fragrans* F. Hoffm against U87, C6, and PC-3 cancer cells is reported here for the first time. In the future, we plan to isolate compound **1** with the aim of obtaining more compounds and testing its antiproliferative activity. This work can also serve as a guide for further isolation of compounds from other parts of *C. fragrans* F. Hoffm and evaluation of their anticancer properties as well as their toxicity.

**Acknowledgments:** The authors thank the "Agence Universitaire de la Francophonie" for the financial
support of this study. The authors are also grateful to Annie-Claire Balandre from the University of Poitiers for her helpful technical assistance. The authors also thank the team of the Laboratory of NMR and molecular imaging, University of Mons (Belgium) and the Laboratory of NMR, University of Poitiers (France) for NMR analysis.

**Funding information:** This work was funded by "Agence Universitaire de la Francophonie" (AUF) to support research team in central Africa and great lakes: BAGL-2015-36.

**Authors' contribution:** I. S. G. - writing, conceptualization, formal analysis, and methodology; C. C., R. S. T., A. D. T. A., P. S., and B. V. - conceptualization, formal analysis, methodology, and manuscript revision; S. L. and C. H. - conducted the NMR and ESI-MS experiments; A. H. N. K. - project promoter; E. T. and J.-M. M. - supervised the research.

**Competing interests:** The authors declare that they have no competing interests that could influence the work reported in this paper.

**Data availability statement:** All data generated or analyzed during this study are included in this published article and its supplementary information files.

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