Hindawi
BioMed Research International
Volume 2022, Article ID 7856305, 10 pages
https://doi.org/10.1155/2022/7856305
Research Article
Chemotaxonomy and Antibacterial Activity of the Extracts and
Chemical Constituents of Psychotria succulenta
Hiern. (Rubiaceae)
Darille Claudia Ngnokam Jouogo,1 Jean-De-Dieu Tamokou ,2
Rémy Bertrand Teponno ,1 Germaine Matsuete-Takongmo,2
Laurence Voutquenne-Nazabadioko ,3 Léon Azefack Tapondjou ,1 and David Ngnokam1
1
Research Unit of Applied and Environmental Chemistry, Department of Chemistry, Faculty of Science, University of Dschang, P.
O. Box 67, Dschang, Cameroon
2
Research Unit of Microbiology and Antimicrobial Substances, Department of Biochemistry, Faculty of Science,
University of Dschang, P.O. Box 67, Dschang, Cameroon
3
Groupe Isolement et Structure, Institut de Chimie Moléculaire de Reims (ICMR), CNRS UMR 7312, Bat. 18, B.P. 1039,
51687 Reims Cedex 2, France
Correspondence should be addressed to Jean-De-Dieu Tamokou; jtamokou@yahoo.fr
and Rémy Bertrand Teponno; remyteponno@gmail.com
Received 9 February 2022; Revised 26 May 2022; Accepted 3 June 2022; Published 15 June 2022
Academic Editor: Ali Imran
Copyright © 2022 Darille Claudia Ngnokam Jouogo et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
The use of natural products for medicinal purposes is becoming more and more common nowadays, as evidenced by the presence
in plants of secondary metabolites with different potentials such as antioxidant and antibacterial properties. We evaluated in this
work the antimicrobial activities of the extracts and some isolated compounds from the seeds of Psychotria succulenta Hiern.
(Rubiaceae), a Cameroonian medicinal plant traditionally used to cure microbial infections. The ethanol extract was prepared
by maceration and extracted with ethyl acetate and n-butanol. The EtOAc (m = 168 g) and n-BuOH (m = 20 g) extracts were
further fractionated by silica gel column chromatography to isolation of compounds. Their structures were elucidated by
spectroscopic analysis and by comparison with published data. The antibacterial activity of extracts and compounds was
assessed by evaluating the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against
pathogenic bacteria. Thirteen compounds including four alkaloids (veprisine (1), naucleofficine III (2), vepridimerine B (3),
and vepridimerine C (4)), three triterpenes (barbinervic acid (5), 3-O-α-L-rhamnopyranosyl quinovic acid (6), and oleanolic
acid (7)), one steroid (β-sitosterol-3-O-β-D-glucopyranoside (8)), four phenolic compounds (scopoletin (9), gallic acid (10),
quercetin-3-O-β-D-glucopyranoside (11), and kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside (12)), and
one iridoid (borreriagenin (13)) were isolated from the EtOAc and n-BuOH extracts. These compounds were identified by 1D
and 2D NMR combined analysis as well as by melting point comparison. The EtOH, EtOAc, and n-BuOH extracts exhibited
significant antibacterial activities (MIC = 32‐128 μg/mL; MBC = 64‐256 μg/mL) against Staphylococcus aureus (Gram-positive
bacterium), Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumonia (Gram-negative bacteria). Among the isolated
compounds, scopoletin (9) showed a moderate activity against Klebsiella pneumoniae with MIC and MBC values of 16 μg/mL
and 32 μg/mL, respectively. It appears that, chemotaxonomically, some of the isolated compounds have already been obtained
from the genus Psychotria but to the best of our knowledge, this is the first report on the phytochemical investigation of P.
succulenta. Although many other studies need to be achieved, our results support the use of P. succulenta in traditional
medicine to cure infectious diseases particularly those caused by the tested bacteria.
2
1. Introduction
Infectious diseases caused by bacteria, viruses, fungi, and
other parasites continue to cause enormous damage in the
world. Gram-negative or Gram-positive bacteria are able to
acquire resistance mechanisms to face environmental
aggression (natural environment, competing bacteria, host
defense, or antibiotics) either by the modification of sites
of action of anti-infective molecules or by production of degradative enzymes. It is therefore important to develop new
drugs using natural plants to fight antibiotic resistance and
to limit undesirable side effects. Psychotria succulenta Hiern.
(Rubiaceae) is a shrub of varying size between 1 and 2 m
with yellow fruits of ripeness commonly found in most tropical regions [1]. Plants of the genus Psychotria (leaves, roots,
barks, and rhizomes) are commonly used in traditional medicine for treating bronchial and gastrointestinal disorders
[2]. They are also used for curing infections of female reproductive system [3].
To the best of our knowledge, no phytochemical nor
pharmacological works have been achieved on P. succulenta.
Previous works carried out on Psychotria species have shown
that the different extracts (petroleum ether, chloroform,
ethyl acetate, dichloromethane, ethanol, and methanol
extracts), the fractions, and some isolated compounds exhibited interesting biological activities such as antibacterial,
cytotoxic, antioxidant, antimycobacterial, and antimutagenic
properties [4–7]. Plants of this genus are characterized as an
abundant source of indole, monoterpene indole, quinoline,
and isoquinoline alkaloids as well as flavonoids [8]. In the
course of our search of bioactive compounds from some
medicinal plants growing in Cameroon [9, 10], we undertook the phytochemical study of P. succulenta, leading to
the isolation and structure elucidation of thirteen compounds. Furthermore, the crude EtOH extract, the EtOAc
and n-BuOH extracts as well as some of the isolated secondary metabolites were evaluated for their antibacterial activity,
and the results are also presented.
2. Materials and Methods
2.1. Plant Material. The seeds of P. succulenta were collected
in October 2018 in Foreke-Dschang (5° 26 ′ 0″ N, 10° 4 ′ 0″
E), West Region of Cameroon, and identified at the National
Herbarium of Cameroon by comparison to the voucher
specimen deposited under the reference no. 42155/HNC.
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2.3. Isolation of Secondary Metabolites. One hundred and
sixty grams (160 g) of the EtOAc extract was subjected to silica
gel column chromatography eluted with n-hexane-EtOAc
(95 : 5 → 20 : 80), then with EtOAc-MeOH (95 : 5 → 80 : 20)
to yield five major fractions A-E. Fraction B (18 g) was purified
by column chromatography on silica gel eluted with n-hexaneEtOAc (80-20) to yield compounds 1 (9 mg), 5 (25 mg), 6
(27 mg), and 7 (20 mg). Fraction C (26 g) was subjected to a silica gel column chromatography eluted with n-hexane-EtOAc
(30-70) followed by Sephadex LH-20 (Eluted with MeOH) to
afford compound 9 (35 mg). Treatment of fraction D (40 g)
with column chromatography on silica gel by using n-hexaneEtOAc (50-50) as eluent gave compounds 2 (18 mg), 3
(28 mg), and 4 (23 mg). Fraction E (8 g) was chromatographed
on Sephadex LH-20 gel column using MeOH as eluent followed
by column chromatography on silica gel eluted by EtOAcMeOH (95-5) to give compounds 8 (33 mg), 10 (22 mg), and
11 (11 mg). An amount of the n-BuOH fraction (17 g) was first
chromatographed on Sephadex column eluted with MeOH to
yield two subfractions coded F and G. The subfraction G was
submitted to a silica gel column eluted with EtOAc-MeOHH2O (9-1-1) to give compounds 12 (21 mg) and 13 (15 mg).
2.4. General Experimental Procedures
2.4.1. Chromatographic Methods. Column chromatography
was carried out on Merck silica gel 60 (70–230 mesh) and
gel permeation on Sephadex LH-20, while TLC was carried
out on precoated silica gel 60 F254 (Merck) plates developed
with different solvents and mixture of hexane-EtOAc,
EtOAc-MeOH, MeOH-H2O, and EtOAc-MeOH-H2O.
Detection was done by using UV light (254 and 365 nm)
and by spraying with 10% H2SO4 followed by heating at
100°C.
2.4.2. NMR Analysis. The 1H and 13C-NMR spectra were
recorded on a Bruker Avance III 500 spectrometer equipped
with a cryoplatform (1H at 500 MHz and 13C at 125 MHz).
2D NMR experiments were achieved using standard Bruker
microprograms (Xwin-NMR version 2.1 software). All
chemical shifts (δ) are given in parts per million (ppm) with
the solvent signal as reference relative to TMS as internal
standard, while the coupling constants (J) are given in hertz
(Hz). Deuterated solvents such as methanol (methanol-d4),
dimethyl sulfoxide (DMSO-d6), and chloroform (CDCl3)
were used as solvents.
2.5. Antimicrobial Assay
2.2. Extraction Procedure. The seeds of P. succulenta were
dried at room temperature and then crushed in fine powder
to give 2.8 kg. Two kilograms (2 kg) of this powder was
extracted with ethanol (3 × 12 L) for 72 hours to yield
287.2 g of crude ethanol extract after evaporation of the solvent under reduced pressure. A part of this crude extract
(250 g) was suspended in distilled water (600 mL), then
extracted with EtOAc and n-BuOH, respectively. After evaporation of each solvent under reduced pressure, 168 g and
20 g of EtOAc and n-BuOH extracts were obtained,
respectively.
2.5.1. Microorganisms. The extracts and some isolated compounds were tested for their antibacterial activities against
four bacterial strains, namely, Staphylococcus aureus ATCC
25923 (Gram-positive bacterium), Pseudomonas aeruginosa
ATCC 76110, Escherichia coli ATCC 25922, and Klebsiella
pneumonia 22 (Gram-negative bacteria). These microorganisms were taken from the Research Unit of Microbiology
and Antimicrobial Substances. The different bacterial species
were maintained at +4°C and activated on BBL® nutrient
agar (NA, Conda, Madrid, Spain) for 24 h before any antibacterial testing.
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2.5.2. Determination of the Inhibition Parameters. The
determination of the minimum inhibitory concentration
(MIC) was performed using the broth microdilution
method [11]. Bacterial suspensions were prepared from
the 18-hour-old cultures. Three colonies of the bacterium
were then taken and diluted separately with sterile 0.9%
NaCl solution to give a turbidity comparable to that of
the 0.5 point on the McFarland scale corresponding to
approximately 1:5 × 108 CFU/mL. This suspension was
again diluted to 1/100 and adjusted to obtain an absorbance of 0.100 at 600 nm corresponding to a bacterial concentration of 106 CFU/mL. Microtiter plates (96
microwells) were made, and each well received 85 μL of
Mueller Hinton broth and 5 μL of inoculum. 10 μL of test
sample stock solution at a corresponding concentration
was then added to each well to reach final concentrations
ranging from 0.25 to 256 μg/mL. The positive control was
made with the appropriate liquid medium and bacterial
suspension only while the negative control was made with
10% DMSO aqueous solution in place of the inoculum.
Ciprofloxacin was used as reference antibiotic. The plates
were covered and incubated under agitation at 35°C for
24 h. Bacterial growth was determined by introducing
5 μL of a 0.2 mg/mL para-iodonitrotetrazolium solution.
Any change in colour from yellow to violet indicates bacterial growth. The minimum inhibitory concentration was
defined as the smallest concentration of the substance that
prevents this colour change. 10 μL of the contents of each
well were aseptically collected and spread separately on the
surface of Mueller Hinton agar medium for the purpose of
determining the minimum bactericidal concentrations
(MBC), which are defined as the smallest concentrations
that result in a negative subculture or only one colony.
Three replicates were performed for each test sample.
3. Results
3.1. Chemical Analysis. The EtOAc and n-BuOH extracts
from the EtOH crude extract of P. succulenta were subjected
to repeated column chromatography on silica gel and Sephadex LH-20 to yield thirteen metabolites (1-13). Their structures were elucidated by spectroscopic and spectrometric
analysis as well as by comparison with literature data (Supplementary materials/figures (available here)). The isolated
compounds were identified as veprisine (1) [12], naucleofficine III (2) [13], vepridimerine B (3) [14], vepridimerine C
(4) [14], barbinervic acid (5) [15], quinovic acid 3β-O-α-Lrhamnoside (6) [16], scopoletin (9) [17], gallic acid (10)
[18], quercetin 3-O-β-D-glucopyranoside (11) [19], kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside (12) [20], and borreriagenin (13) [21]. Compounds 7
and 8 were identified by co-TLC with authentic samples
and melting point measurement as oleanolic acid [22] and
β-sitosterol 3-O-β-D-glucopyranoside [23], respectively
(Figure 1).
Veprisin (1): yellow amorphous powder; 1H-NMR
(500 MHz, methanol-d4): δ ðppmÞ = 7:75 (1H, d, J = 9:0 Hz,
H-5), 7.09 (1H, d, J = 9:0 Hz, H-6), 6.61 (1H, d, J = 9:9 Hz,
H-4 ′ ), 5.61 (1H, d, J = 9:9 Hz, H-3 ′ ), 3.98 (3H, s, OCH3),
3
3.91 (3H, s, NCH3), 3.80 (3H, s, OCH3), and 1.51 (6H, s,
CH3). 13C-NMR (125 MHz, methanol-d4): δ ðppmÞ = 162:8
(C-1), 156.1(C-7), 155.8 (C-3), 137.0 (C-8), 133.8 (C-9),
125.6 (C-3 ′ ), 118.8 (C-5), 116.7 (C-4 ′ ), 111.5 (C-4), 108.1
(C-6), 102.9 (C-2), 78.6 (C-3 ′ ), 60.4 (OCH3), 54.6 (OCH3),
33.2 (NCH3), and 26.1 (CH3)
Naucleofficine III (2): white amorphous powder; 1HNMR (500 MHz, CDCl3) δ ðppmÞ = 7:53 (1H, d, J = 7:7 Hz,
H-9), 7.37 (1H, d, J = 7:9 Hz, H-12), 7.23 (1H, dd, J = 14:3
, 6:8 Hz, H-11), 7.17 (1H, dd, J = 13:2, 6:3 Hz, H-10), 5.54
(1H, dd, J = 14:9, 4:8 Hz, H-19), 5.28 (1H, d, J = 7:5 Hz, H17), 5.17 (1H, d, J = 7:7 Hz, H-5a), 5.04 (1H, m, H-3), 4.32
(1H, d, J = 13:1 Hz, H-21a), 4.20 (1H, d, J = 13:4 Hz, H21b), 3.04 (1H, m, H-4a), 3.02 (1H, m, H-5b), 2.77 (1H, m,
H-4b), 2.74 (1H, m, H-15), 2.58 (1H, m, H-14a), 2.56 (1H,
t, J = 6:5 Hz; H-16), 2.10 (1H, m, H-14b), and 1.62 (1H, d,
J = 11:2 Hz, H-18). 13C-NMR (125 MHz, CD Cl3): δ ðppmÞ
= 169:1 (C-22), 136.0 (C-13), 133.4 (C-20), 133.0 (C-2),
127.4 (C-8), 122.4 (C-11), 122.2 (C-19), 120.2 (C-10),
118.4 (C-9), 111.1 (C-12), 111.0 (C-7), 93.8 (C-17), 68.0
(C-21), 53.0 (C-3), 48.6 (C-16), 42.0 (C-5), 29.2 (C-15),
28.0 (C-14), 21.0 (C-6), and 12.7 (C-18)
Vepridimerine C (3): white amorphous powder; 1HNMR (500 MHz, CDCl3): δ ðppmÞ = 8:08 (1H, d, J = 9:0 Hz
, H-4), 7.71 (1H, d, J = 9:0 Hz, H-13), 6.96 (1H, d, J = 9:0
Hz, H-3), 6.84 (1H, d, J = 9:0 Hz, H-12), 3.95 (3H, s, 2OCH3), 3.90 (3H, s, 10-OCH3), 3.77 (3H, s, 1-OCH3), 3.74
(3H, s, 11-OCH3), 3.92 (3H, s, NCH3), 3.76 (3H, s, NCH3),
3.96 (1H, m, H-19a), 3.20 (1H, q, J = 3:2 Hz, H-7), 2.74
(1H, td, J = 12:7, 4:2 Hz, H-16a), 2.15 (1H, m, H-16x), 1.91
(3H, s, (CH3-6)), 1.72 (3H, s, (CH3-15)), 1.60 (1H, dd, J =
12:5, 3:4 Hz, H-6a), 1.48 (1H, m, H-16y), 1.34 (1H, m, H19b), and 1.41 (3H, s, (CH3-6)). 13C-NMR (125 MHz,
CDCl3): δ ðppmÞ = 176:5 (C-17), 163.4 (C-8), 157.7(C-4b),
155.4 (C-2), 155.0 (C-13b), 154.9 (C-10), 137.6 (C-11),
136.7 (C-1), 134.8 (C-18a), 134.1 (C-9a), 122.0 (C-4), 118.8
(C-13), 121.4 (C-4a), 112.7 (C-13a), 112.1 (C-7a), 108.4
(C-3), 107.0 (C-12), 100.1 (C-16b), 84.7 (C-6), 78.5 (C-15),
61.7 (OCH3), 61.3 (OCH3), 56.3 (OCH3), 56.2 (OCH3),
52.3 (C-6a), 39.3 (C-19), 35.8 (NCH3), 33.8 (NCH3), 31.1
(C-16), 29.2 (15-CH3), 28.6 (6-CH3), 25.7 (C-16a), 25.6 (C7), and 21.0 (6-CH3)
Vepridimerine B (4): white amorphous powder; 1HNMR (500 MHz, CDCl3): δ ðppmÞ = 7:70 (1H, d, J = 9:0 Hz
, H-13), 7.66 (1H, d, J = 9:0 Hz, H-4), 6.88 (1H, d, J = 9:0
Hz, H-12), 6.84 (1H, d, J = 9:0 Hz, H-3), 3.98 (3H, s, 1OCH3), 3.95 (3H, s, 10-OCH3), 3.84 (3H, s, 2-OCH3), 3.76
(3H, s, 11-OCH3), 3.93 (3H, s, NCH3), 3.87 (3H, s, NCH3),
3.84 (2H, m, H-19a), 3.24 (1H, q, J = 3:2 Hz, H-7), 2.62
(1H, td, J = 12:7, 4:2 Hz, H-16a), 2.17 (1H, m, H-16x), 1.92
(3H, s, 6-CH3), 1.71 (3H, s, 15-CH3), 1.59 (1H, dd, J = 12:5
, 3:4 Hz, H-6a), 1.47 (1H, m, H-16y), 1.32 (1H, m, H-19b),
and 1.39 (3H, s, (CH3-6)). 13C-NMR (125 MHz, CDCl3): δ
ðppmÞ = 164:3 (C-17), 163.3 (C-8), 155.8 (C-4b), 155.0 (C1), 154.9 (C-10), 154.6 (C-13b), 136.8 (C-2), 136.5 (C-11),
134.2 (C-18a), 134.0 (C-9a), 119.0 (C-4), 118.8 (C-13),
112.8 (C-4a), 112.7 (C-13a), 112.2 (C-7a), 107.6 (C-3),
107.0 (C-12), 105.4 (C-16b), 81.9 (C-6), 78.5 (C-15), 61.7
4
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O
O
OH
H
N
O
O
O
N
H
N
H
H
O
O
H
O
N
O
H
O
N
O
H
2
O
O
1
O
O
O
3
O
N
H
H
HO
O
12
O
18
H
O
N
O
COOH
COOH
28
1
4
O
HO
7
3
HO
5
COOH
27
O
1’
O
HO
HO
6
OH
H3CO
COOH
4
6
HO
2
O
8
O
9
RO
HO
R = Glc
7
8
R”
O
OH
OH
COOH
RO
HO
5
3
OH
10
OH
O
7
4
5
OH
11
O
4’
4
H
2
H
6
10
OR’
8
OH
O
11: R’ = Glc, R” = OH, R = H
12: R = R’ = Rha, R” = H
3
HO
13
Figure 1: Structures of compounds 1-13 isolated from P. succulenta. 1: veprisine; 2: naucleofficine III; 3: vepridimerine B; 4: vepridimerine
C; 5: barbinervic acid; 6: quinovic acid 3β-O-α-L-rhamnoside; 7: oleanolic acid; 8: β-sitosterol 3-O-β-D-glucopyranoside; 9: scopoletin; 10:
gallic acid; 11: quercetin 3-O-β-D-glucopyranoside; 12: kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside; 13:
borreriagenin.
(OCH3), 61.6 (OCH3), 56.3 (OCH3), 56.2 (OCH3), 33.8
(NCH3), 33.3 (NCH3), 52.3 (C-6a), 39.7 (C-19), 31.1 (C16), 29.3 (15-CH3), 28.5 (6-CH3), 26.3 (C-16a), 25.5 (C-7),
and 21.0 (6-CH3)
Barbinervic acid (5): brown powder; 1H-NMR
(500 MHz, methanol-d4): δ ðppmÞ = 5:30 (1H, t, J = 3:4 Hz,
H-12), 3.62 (1H, dd, J = 11:4, 4:6 Hz, H-3), 3.55 (1H, d, J
= 10:9 Hz, H-24b), 3.32 (1H, d, J = 10:9 Hz, H-24a), 2.52
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(1H, s, H-18), 1.35 (3H, s, H-27), 1.21 (3H, s, H-29), 0.99
(3H, s, H-25), 0.94 (3H, s, H-30), 0.81 (3H, s, H-26), and
0.72 (3H, s, H-23). 13C-NMR (125 MHz, methanol-d4): δ ð
ppmÞ = 180:8 (C-28), 138.7 (C-13), 128.1 (C-12), 72.6 (C3), 72.2 (C-19), 66.1 (C-24), 53.8 (C-18), 45.5 (C-17), 47.4
(C-5), 47.1 (C-9), 41.8 (C-4), 41.6 (C-20), 41.2 (C-14), 39.6
(C-8), 38.1 (C-1), 37.6 (C-22), 36.5 (C-10), 32.3 (C-7), 28.1
(C-15), 26.0 (C-2), 25.9 (C-21), 25.7 (C-29), 24.9 (C-16),
23.4 (C-27), 23.2 (C-11), 17.9 (C-6), 16.1 (C-26), 15.1 (C30), 14.8 (C-25), and 11.3 (C-23)
Quinovic acid 3β-O-α-L-rhamnopyranoside (6): brown
powder; 1H-NMR (500 MHz, methanol-d4): δ ðppmÞ = 5:61
(1H, dd, J = 5:2, 2:5 Hz, H-12), 3.07 (1H, dd, J = 11:4, 4:8
Hz, H-3), 2.28 (2H, m, H-9, H-18), 1.00 (3H, s, H-25),
0.94 (3H, s, H-24, H-30), 0.92 (3H, s, H-29), 0.91 (3H, s,
H-26), 0.81 (3H, s, H-23), and 0.78 (1H, m, H-5). L-rhamnose: 4.75 (1H, d, J = 1:6 Hz, H-1 ′ ), 3.84 (1H, dd, J = 3:2,
1:7 Hz, H-2 ′ ), 3.65 (1H, dd, J = 9:5, 3:3 Hz, H-3 ′ ), 3.35
(1H, t, J = 9:5 Hz, H-4 ′ ), 3.71 (1H, dq, J = 6:3, 9:4 Hz, H-5 ′
), and 1.20 (3H, d, J = 6:3 Hz, H-6 ′ ). 13C-NMR (125 MHz,
methanol-d4): δ ðppmÞ = 180:1 (C-28), 177.5 (C-27), 132.5
(C-13), 129.0 (C-12), 103.0 (C-1 ′ ), 88.9 (C-3), 72.6 (C-4 ′ ),
71.4 (C-3 ′ ), 71.1 (C-2 ′ ), 68.4 (C-5 ′ ), 55.8 (C-14), 55.3 (C5), 54.1 (C-18), 47.9 (C-17), 46.6 (C-9), 39.3 (C-8), 38.9
(C-19), 38.6 (C-4), 38.4 (C-1), 36.8 (C-10, C-20), 36.4 (C22), 36.2 (C-7), 29.8 (C-21), 27.3 (C-24), 25.3 (C-2, C-15),
25.1 (C-16), 22.4 (C-11), 20.1 (C-30), 18.0 (C-6), 16.6 (C26, C-29), 17.6 (C-6 ′ ).15.4 (C-23), and 15.4 (C-25)
Scopoletin (9): yellow needle; 1H-NMR (500 MHz,
methanol-d4): δ ðppmÞ = 7:80 (1H, d, J = 9:4 Hz, H-4), 7.12
(1H, s, H-5), 6.80 (1H, s, H-8), 6.21 (1H, d, J = 9:4 Hz, H3), and 3.91 (3H, s, O-CH3). 13C-NMR (125 MHz, methanol-d4): δ ðppmÞ = 162:9 (C-2), 151.4 (C-8a), 150.0 (C-7),
145.6 (C-6), 144.7 (C-4), 111.3 (C-4a), 111.2 (C-3), 108.4
(C-5), 102.7 (C-8), and 55.6 (O-CH3)
Gallic acid (10): white amorphous powder; 1H-NMR
(500 MHz, methanol-d4): δ ðppmÞ = 7:07 (2H, s, H-2/H-6).
13
C-NMR (125 MHz, methanol-d4): δ ðppmÞ = 168:9 (C = 0
), 145.3 (C-3), 138.2 (C-4), 120.5 (C-1), and 108.8 (C-2/C-6)
Quercetin 3-O-β-D-glucopyranoside (11): yellow amorphous powder; 1H-NMR (500 MHz, methanol-d4): δ ðppmÞ
= 7:72 (1H, d, J = 2:1 Hz, H-2 ′ ),7.61 (1H, dd, J = 8:4, 2:1
Hz, H-6 ′ ), 6.89 (1H, d, J = 8:4 Hz, H-5 ′ ), 6.43 (1H, d, J =
2:1 Hz, H-8), 6.23 (1H, d, J = 2:1 Hz, H-6), 5.27 (1H, d, J
= 7:6 Hz, H-1″ ), 3.72 (1H, m, H-6a″ ), 3.58 (1H, m, H-6b″
), 3.50 (1H, m, H-2a″ ), 3.44 (1H, m, H-3″ ), 3.37 (1H, m,
H-4″ ), and 3.28 (1H, m, H-5″ ). 13C-NMR (125 MHz, methanol-d4): δ ðppmÞ = 179:1 (C-4), 165.7 (C-7), 162.7 (C-5),
161.5 (C-2), 158.5 (C-9), 149.2 (C-4 ′ ), 145.6 (C-3 ′ ), 135.3
(C-3), 122.4 (C-1 ′ ), 122.8 (C-6 ′ ), 116.9 (C-2 ′ ), 116.5 (C-5 ′
), 105.3 (C-10), 103.8 (C-1″ ), 99.3 (C-6), 94.2 (C-8), 78.0
(C-5″ ), 77.6 (C-3″ ), 62.3 (C-6″ ), 75.2 (C-2″ ), and 70.7 (C4″ )
Kaempferol
3-O-α-L-rhamnopyranoside-7-O-α-Lrhamnopyranoside (12): yellow amorphous powder; 1HNMR (500 MHz, CDCl3+methanol-d4): δ ðppmÞ = 7:77
5
(1H, d, J = 8:8 Hz, H-2 ′ /H-6 ′ ), 6.94 (1H, d, J = 8:8 Hz, H3 ′ /H-5 ′ ), 6.70 (1H, d, J = 2:1 Hz, H-8), 6.45 (1H, d, J = 2:1
Hz, H-6), 5.54 (1H, d, J = 1:2 Hz, H-1″ ), 5.40 (1H, d, J =
1:5 Hz, H-1‴ ), 4.26 (1H, dd, J = 3:2, 1:6 Hz, H-2‴ ), 4.05
(1H, dd, J = 3:3, 1:7 Hz, H-2″ ), 3.86 (1H, dd, J = 9:5, 3:4
Hz, H-2″ ), 3.74 (1H, m, H-3‴ ), 3.62 (1H, dq, J = 12:3, 6:1
Hz, H-5″ ), 3.50 (1H, d, J = 9:5 Hz, H-4″ ), 3.34 (1H, m, H4‴ ), 3.33 (1H, m, H-5‴ ), 1.28 (3H, d, J = 6:2 Hz, H-6″ ),
and 0.94 (3H, d, J = 5:6 Hz, H-6‴ ). 13C-NMR (125 MHz,
CDCl3+methanol-d4): δ ðppmÞ = 178:5 (C-4), 162.0 (C-7),
161.5 (C-5), 160.1 (C-4 ′ ), 158.5 (C-2), 156.8 (C-9), 135.2
(C-3), 130.7 (C-2 ′ /C-6 ′ ), 121.1 (C-1 ′ ), 115.4 (C-3 ′ /C-5 ′ ),
106.5 (C-10), 102.0 (C-1″ ), 99.5 (C-6), 98.3 (C-1‴ ), 94.4
(C-8), 72.3 (C-4 ′″ ), 71.9 (C-4″ ), 70.8 (C-3‴ ), 70.4 (C-2″ ),
70.6 (C-4‴ ), 70.2 (C-2″ ′), 70.6 (C-5″ ), 69.8 (C-5 ′″ ), 17.1
(C-6″ ′), and 16.6 (C-6″ )
Borreriagenin (13): yellow oil; 1H-NMR (500 MHz,
methanol-d4): δ ðppmÞ = 5:85 (1H, d, J = 1:7 Hz, H-7), 5.41
(1H, d, J = 7:6 Hz, H-6), 4.20 (1H, m, H-10a), 3.92 (1H,
dd, J = 10:8, 4:7 Hz, H-3a), 3.87 (1H, dd, J = 10:9, 4:7 Hz,
H-3b), 3.77 (1H, m, H-10b), 3.59 (1H, dd, J = 11:2, 4:9 Hz,
H-1a), 3.52 (1H, dd, J = 10:8, 4.7 Hz, H-1b), 3.34 (1H, m,
H-5), 3.11 (1H, m, H-9), and 2.97 (1H, m, H-4). 13C-NMR
(125 MHz, methanol-d4): δ ðppmÞ = 179:6 (C-11), 151.9
(C-8), 123.6 (C-7), 86.7 (C-6), 63.0 (C-1), 61.4 (C-3), 53.9
(C-10), 48.6 (C-9), 44.6 (C-4), and 42.6 (C-5)
3.2. Antibacterial Activity. The results of in vitro activities
of the EtOH, n-BuOH, and EtOAc extracts as well as
some isolated compounds against pathogenic bacteria are
presented in Table 1. The n-BuOH and EtOAc extracts
showed antibacterial activity against Gram-positive and
Gram-negative bacteria (MIC = 32 – 64 μg/mL; MBC = 64
– 256 μg/mL) whereas the EtOH extract was active only
on Gram-negative bacteria (MIC = 32 – 128 μg/mL; MBC
= 64 – 256 μg/mL). The antibacterial activity of the plant
extracts can be classified as significant (MIC < 100 μg/mL
), moderate (100 < MIC ≤ 625 μg/mL), and weak
(MIC > 625 μg/mL) [24]. According to this classification,
the inhibition potential of the tested extracts could be considered as significant to moderate. The n-BuOH extract
was the most active with a lowest MIC value of 32 μg/
mL against Pseudomonas aeruginosa ATCC 76110, Staphylococcus aureus ATCC 25923, and Klebsiella pneumoniae
22 and of 64 μg/mL against Escherichia coli ATCC 25922
followed by the EtOH extract which displayed a MIC
value of 32 μg/mL on K. pneumoniae 22 and of 64 μg/
mL on P. aeruginosa ATCC 76110, S. aureus ATCC
25923, and K. pneumoniae 22. The isolated secondary
metabolites showed inhibition ranging from moderate to
weak according to the scale which states that antimicrobial
activity of pure compounds can be classified as significant
(MIC < 10 μg/mL), moderate (10 < MIC ≤ 100 μg/mL), and
weak (MIC > 100 μg/mL) [24]. Scopoletin (9) exhibited a
moderate activity with a MIC value of 16 μg/mL against
S. aureus ATCC 25923, K. pneumoniae 22, and a MIC
value of 32 μg/mL against P. aeruginosa ATCC 76110
6
BioMed Research International
Table 1: Antibacterial activity of the extracts and some isolated compounds from P. succulenta seeds.
Samples
Parameters
EtOH extract
EtOAc extract
n-BuOH extract
1
2
3
4
5
6
9
10
11
12
13
Ciprofloxacine
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
Pseudomonas aeruginosa
128/256
64/256
32/64
128/128
64/64
64/128
64/64/128
128/32/64
128/128/64/128/2/4
Bacterial species
Staphylococcus aureus
Escherichia coli
-/64/128
32/128
-/128/64/128
64/32/64
-/16/32
-/128/-/128/8/16
64/128
-/64/128
32/128
128/128
-/-/64/64
-/64/128
-/-/-/64/8/16
Klebsiella pneumoniae
32/64
64/128
32/128
128/128
32/64
128/128
-/32/64
128/16/32
64/128/32/-/8/16
MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; MIC and MBC in μg/mL; -: not active at concentration up to 256 μg/
mL for the compounds and 2048 μg/mL for the extracts.
while barbinervic acid (5) showed a moderate activity
against P. aeruginosa ATCC 76110 and K. pneumoniae
22 with a MIC value of 32 μg/mL.
4. Discussion
4.1. Chemotaxonomy. The present study reports the first
phytochemical investigation of P. succulenta which led to
the isolation and structure elucidation of thirteen compounds including four alkaloids (veprisine (1), naucleofficine III (2), vepridimerine B (3), and vepridimerine C (4)),
three triterpenes (barbinervic acid (5), 3-O-α-L-rhamnopyranosyl quinovic acid (6), and oleanolic acid (7)), one steroid
(β-sitosterol-3-O-β-D-glucopyranoside (8)), four phenolic
compounds (scopoletin (9), gallic acid (10), quercetin-3-Oβ-glucopyranoside (11), and kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside (12)), and one iridoid (borreriagenin (13)). Although all these compounds
are isolated from P. succulenta for the first time, some of
them have already been obtained from other Psychotria species. It is the case of barbinervic acid (5) previously found in
P. stachyoides [25], oleanolic acid (7) and β-sitosterol-3-Oβ-D-glucopyranoside (8) isolated from P. viridis [26], and
scopoletin (9) obtained from P. vellosiana and P. stachyoides
[25, 27]. The results obtained are in agreement with the chemotaxonomy of plants of the genus Psychotria since according to Calixto et al. (2016), they are characterized as an
abundant source of indole, monoterpene indole, quinoline,
and isoquinoline alkaloids as well as flavonoids. Furthermore, approximately 52% of the metabolites reported were
characterized as alkaloids, followed by triterpenes (12%)
and flavonoids (6%) along with constituents of other classes
[28]. The monoterpene indole alkaloid naucleofficine III (2)
isolated during our investigation has already been obtained
from the stems of Nauclea officinalis [13] also belonging to
the family Rubiaceae. Nevertheless, the isolation from P. succulenta of a metabolite belonging to this class of compounds
reported to derive biosynthetically from the coupling of
tryptophan, and the iridoid seccolaganin is not surprising
since many congeners have been isolated from other Psychotria species [28–30]. Furthermore, regarding the distribution
of the major secondary metabolites in Rubiaceae, indole
alkaloids are indicated as the main chemical markers of this
family [31].
Veprisine (1), vepridimerine B (3), and vepridimerine C
(4) are quinolone-terpene alkaloids occurring mainly in
plants of the Rutaceae family [12, 14, 32, 33]. To the best
of our knowledge, this is the first report of their isolation
from Rubiaceae. Nevertheless, the cooccurrence of indole
and quinoline alkaloids in the same plant species is well documented. These two classes of alkaloids were obtained from
Araliopsis soyauxii (Rutaceae) [33], Melodinus yunnanensis
(Apocynaceae) [34], Alstonia scholaris (Apocynaceae) [35],
and Clausena lansium (Rutaceae) [36]. This further confirmed the fact that biosynthetically, quinoline alkaloids
may be derived from ring expansion of indole alkaloids
[35]. This seems to be the first report on the isolation of
the flavonoid glycosides quercetin-3-O-β-glucopyranoside
(11) and kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-Lrhamnopyranoside (12) from a plant of the Psychotria genus
although these secondary metabolites have already been
found in the family Rubiaceae, precisely in Hedyotis diffusa
and Hedyotis verticillata, respectively [31]. The iridoid borreriagenin (13) obtained during this work has already been
isolated from some plants of the Rubiaceae family including
Borreria verticillata [21] and Morinda longifolia [37], but to
the best of our knowledge, this is the first report of its isolation from a plant of the genus Psychotria.
BioMed Research International
4.2. Antibacterial Activity. The findings of the present study
showed differences between the antibacterial activities of
extracts from P. succulenta seeds. This suggests that P. succulenta contains several active principles with different polarities
as shown by the nature of the isolated compounds. Indeed, the
antibacterial activities of medicinal plants are correlated with
the presence in their extracts of one or more bioactive secondary metabolites [38]. The n-BuOH extract was the most active
following in decreasing order by the EtOAc extract and
MeOH extract. This result reinforces the concept that P. succulenta contains also polar antibacterial compounds. These differences in antibacterial activities from different solvents had
also been observed [9, 11]. Hence, the n-BuOH extract was
expected to produce significant active principles in this
research. However, the results showed that ethyl acetate was
the better solvent compared to the n-BuOH to isolate the phytochemicals (compounds 2, 5, and 9) that are most active
toward the tested bacteria from P. succulenta. The ethyl acetate
is a semipolar solvent and could effectively extract active principles with semipolar properties such as alkaloids, sterols, terpenoids, flavonoids, and glycosides from the plant [39].
Different parts (leaves, roots, barks, and rhizomes) of
plants of the genus Psychotria are commonly used in traditional medicines for treating bronchial and gastrointestinal
disorders such as cough, bronchitis, ulcer, and stomachache
[2, 40]. They are also used to cure infections of the female
reproductive system [3]. Previous pharmacological works
carried out on other Psychotria species like P. microlabastra
(leaves, stem, and roots bark), P. gardineri (branches and
leaves), and P. nigra (branches and leaves) have shown that
methanol, dichloromethane, and hexane extracts exhibit
antibacterial activities [41]. Our results allow us not only to
validate the use of P. succulenta in traditional medicine but
also to approve the literature data.
The findings of the present study showed that the MBC
values are in general fourfold lesser than the MIC values
on the corresponding bacteria; suggesting that the extracts
and some isolated compounds from P. succulenta seeds have
a bactericidal effect on the sensitive bacteria [42].
The results of the antibacterial activity of some isolated
compound from P. succulenta seeds are in agreement with
those of the literature. Indeed, veprisine isolated from the
root wood of Teclea maniensis (Rutaceae) exhibited moderate to higher antimycobacterial activity against two mycobacterial strains, namely, Mycobacterium madagascariense
DSM 44641 and Mycobacterium indicus pranii DSM 45239
with the MIC values of 657.9 μM and 2:63 × 103 μM, respectively [12]. Oleanolic acid isolated from Miconia species displayed antibacterial effect with MIC values ranging from
30 μg/mL to 70 μg/mL [43]. A phenolic coumarin scopoletin
(7-hydroxy-6-methoxycoumarin) from Lasianthus lucidus
Blume (Rubiaceae) proved to be effective against Pseudomonas aeruginosa ATCC 27853 (AmpC β-lactamase producing
strain) and P. aeruginosa DMSC 37166 [44]. It was found
that gallic acid had antimicrobial activity against P. aeruginosa, E. coli, S. aureus, and Lysteria monocytogenes through
hydrophobicity changes, decrease of negative surface charge,
and occurrence of local rupture or pore formation in the cell
7
membranes with consequent leakage of essential intracellular constituents [45]. Finally, 10-acetyl borreriagenin isolated from the aerial parts of Hedyotis pilulifera
(Rubiaceae) showed antibacterial activity against Staphylococcus aureus, with an MIC value of 100 μg/mL [46]. To
the best of our knowledge, this is the first report on the antibacterial activities of the extracts, naucleofficine III, vepridimerine B, vepridimerine C, barbinervic acid, 3-O-α-Lrhamnopyranosyl quinovic acid, quercetin-3-O-β-D-glucopyranoside, kaempferol 3-O-α-L-rhamnopyranoside-7-O-αL-rhamnopyranoside, and borreriagenin from P. succulenta
seeds. The overall study emphasizes the potential of P. succulenta seeds as a sustainable source of broad spectrum antibacterial agents.
5. Conclusion
In conclusion, the phytochemical investigation of the seeds
of P. succulenta led to isolation and characterization of thirteen compounds, namely, veprisine (1), naucleofficine III
(2), vepridimerine B (3), vepridimerine C (4), barbinervic
acid (5), quinovic acid 3β-O-α-L-rhamnoside (6), oleanolic
acid (7), β-sitosterol-3-O-β-D-glucopyranoside (8), scopoletin (9), gallic acid (10), quercetin 3-O-β-D-glucopyranoside
(11), kaempferol 3-O-α-L-rhamnopyranoside-7-O-α-Lrhamnopyranoside (12), and borreriagenin (13). All these
secondary metabolites were isolated from this plant species
for the first time although some of them have already been
isolated from plants of the genus Psychotria. Our results
clearly showed that P. succulenta has a close chemotaxonomic relationship with other plants of the genus Psychotria.
Furthermore, the extracts and some isolated compounds
showed antibacterial activity against pathogenic bacteria,
confirming the use of P. succulenta in traditional medicine
to cure infectious diseases. Barbinervic acid (5) and scopoletin (9) were the most antibacterial principles of P.
succulenta.
Abbreviations
13
C-NMR:
H-NMR:
2D NMR:
ATCC:
CC:
CFU:
COSY:
DMSO:
EtOAc:
HMBC:
HNC:
HSQC:
INT:
IR:
MBC:
MDR:
MeOH:
MHA:
1
Carbon thirteen nuclear magnetic resonance
Proton nuclear magnetic resonance
Two-dimension nuclear magnetic resonance
American Type Culture Collection
Column chromatography
Colony forming unit
Correlation spectroscopy
Dimethylsulfoxide
Ethyl acetate
Heteronuclear multiple bond connectivities
Herbier National du Cameroun
The heteronuclear single-quantum coherence
p-Iodonitrotetrazolium
Infrared
Minimum bactericidal concentration
Multidrug-resistant
Methanol
Mueller Hinton agar
8
MHB:
MBC:
MIC:
NA:
n-BuOH:
NMR:
Rf:
TLC:
TMS:
UV:
BioMed Research International
Mueller Hinton broth
Minimum bactericidal concentration
Minimum inhibitory concentration
Nutrient agar
n-Butanol
Nuclear magnetic resonance
Retention factor
Thin layer chromatography
Tetramethylsilane
Ultraviolet.
9. Figure S23: 1H NMR spectrum of compound 10. Figure
S24: 13C NMR spectrum of compound 10. Figure S25: 1H
NMR spectrum of compound 11. Figure S26: 13C NMR
spectrum of compound 11. Figure S27: 1H NMR spectrum
of compound 12. Figure S28: 13C NMR spectrum of compound 12. Figure S29: 1H NMR spectrum of compound
13. Figure S30: 13C NMR spectrum of compound 13. Figure
S31: Microtiter plate images after INT colorimetric assay.
(Supplementary Materials)
References
Data Availability
The datasets generated and analyzed during the current
study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Authors’ Contributions
DCNJ contributed to the investigation, methodology, and
writing of the original draft. JDT designed the study, did
the biological assays, and helped in manuscript writing.
RBT supervised the extraction, isolation, and structure elucidation and contributed to manuscript preparation. GMT
contributed to the data collection and analysis. LVN performed spectroscopic analysis and structure elucidation.
JDT, RBT, LVN, LAT, and DN supervised and revised the
manuscript critically. All the authors read and approved
the final manuscript.
Acknowledgments
The authors are grateful to the Alexander von Humboldt
Foundation and the University of Dschang for the financial
support of this work.
Supplementary Materials
Figure S1: 1H NMR spectrum of compound 1. Figure S2: 13C
NMR spectrum of compound 1. Figure S3: 1H NMR spectrum of compound 2. Figure S4: 13C NMR spectrum of compound 2. Figure S5: 1H NMR spectrum of compound 3.
Figure S6: 13C NMR spectrum of compound 3. Figure S7:
1
H-1H COSY spectrum of compound 3. Figure S8: HSQC
spectrum of compound 3. Figure S9: HMBC spectrum of
compound 3. Figure S10: NOESY spectrum of compound
3. Figure S11: 1H NMR spectrum of compound 4. Figure
S12: 13C NMR spectrum of compound 4. Figure S13:
1
H-1H COSY spectrum of compound 4. Figure S14: HSQC
spectrum of compound 4. Figure S15: HMBC spectrum of
compound 4. Figure S16: NOESY spectrum of compound
4. Figure S17: 1H NMR spectrum of compound 5. Figure
S18: 13C NMR spectrum of compound 5. Figure S19: 1H
NMR spectrum of compound 6. Figure S20: 13C NMR spectrum of compound 6. Figure S21: 1H NMR spectrum of
compound 9. Figure S22: 13C NMR spectrum of compound
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