Tagousop et al.

BMC Complementary and Alternative Medicine (2018) 18:252

https://doi.org/10.1186/s12906-018-2321-7

RESEARCH ARTICLE Open Access

Antimicrobial activities of flavonoid

glycosides from Graptophyllum
grandulosum and their mechanism of
antibacterial action
Cyrille Ngoufack Tagousop1, Jean-de-Dieu Tamokou2* , Steve Endeguele Ekom2, David Ngnokam1*
and Laurence Voutquenne-Nazabadioko3

Abstract
Background: The search for new antimicrobials should take into account drug resistance phenomenon. Medicinal
plants are known as sources of potent antimicrobial compounds including flavonoids. The objective of this
investigation was to evaluate the antimicrobial activities of flavonoid glycosides from Graptophyllum grandulosum,
as well as to determine their mechanism of antibacterial action using lysis, leakage and osmotic stress assays.
Methods: The plant extracts were prepared by maceration in organic solvents. Column chromatography of the n-
butanol extract followed by purification of different fractions led to the isolation of five flavonoid glycosides. The
antimicrobial activities of extracts/compounds were evaluated using the broth microdilution method. The
bacteriolytic activity was evaluated using the time-kill kinetic method. The effect of extracts on the red blood cells
and bacterial cell membrane was determined by spectrophotometric methods.
Results: Chrysoeriol-7-O-β-D-xyloside (1), luteolin-7-O-β-D-apiofuranosyl-(1 → 2)-β-D-xylopyranoside (2), chrysoeriol-
7-O-β-D-apiofuranosyl-(1 → 2)-β-D-xylopyranoside (3), chrysoeriol-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-(4"-
hydrogeno sulfate) glucopyranoside (4) and isorhamnetin-3-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-glucopyranoside (5)
were isolated from G. grandulosum and showed different degrees of antimicrobial activities. Their antibacterial
activities against multi-drug-resistant Vibrio cholerae strains were in some cases equal to, or higher than those of
ciprofloxacin used as reference antibiotic. The antibacterial activities of flavonoid glycosides and chloramphenicol
increased under osmotic stress (5% NaCl) whereas that of vancomycin decreased under this condition. V. cholerae
suspension treated with flavonoid glycosides, showed a significant increase in the optical density at 260 nm,
suggesting that nucleic acids were lost through a damaged cytoplasmic membrane. A decrease in the optical
density of V. cholerae NB2 suspension treated with the isolated compounds was observed, indicating the lysis of
bacterial cells. The tested samples were non-toxic to normal cells highlighting their good selectivity index.
Conclusions: The results of the present study indicate that the purified flavonoids from G. glandulosum possess
antimicrobial activities. Their mode of antibacterial activity is due to cell lysis and disruption of the cytoplasmic
membrane upon membrane permeability.
Keywords: Graptophyllum glandulosum, Acanthaceae, Flavonoid glycosides, Antibacterial, Antifungal, Mode of action

* Correspondence: [email protected]; [email protected];

[email protected]; [email protected]
2
Research Unit of Microbiology and Antimicrobial Substances, Department of
Biochemistry, Faculty of Science, University of Dschang, P.O. Box 67,
Dschang, Cameroon
1
Research Unit of Environmental and Applied Chemistry, Department of Chemistry,
Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon
Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Tagousop et al. BMC Complementary and Alternative Medicine (2018) 18:252 Page 2 of 10

Background Although several ethnobotanical reports have emphasized

The development of resistance by microorganisms to the pharmacological importance of this species for condi-
existing antimicrobial agents has been known for a long tions that appear to be associated with microbial infec-
time. In several findings, the emergence of multidrug re- tions, there is very limited literature concerned with the
sistant strains of Vibrio cholerae O1 and Shigella flexneri identification of the antimicrobial compounds from this
has been reported due to different genetic factors includ- plant. However, a few reports on the in vitro antimicrobial
ing transfer of plasmids, integrons and allelic variation in activity of plants have been published [14, 15]. The object-
the specific genes [1]. In developing countries, fluoroqui- ive of this investigation was to evaluate the antimicrobial
nolones, widely used for the treatment of many bacterial activities of flavonoid glycosides from G. grandulosum, as
diseases, including cholera and shigellosis, could contrib- well as to determine their mechanism of action using lysis,
ute to the emergence of multidrug resistance among po- leakage, and osmotic stress assays.
tential enteric pathogens. Although significant progress
has been made in microbiological research and in the con- Methods
trol of many diseases caused by infectious microorganisms, General experimental procedures
recurrent epidemics due to drug-resistant microorganisms Melting point
as well as the appearance of new microbial pathogenic A Schorpp Gerätetechnik (Germany) apparatus was used
strains demand the discovery of new antibiotics. The need to take the melting points of different compounds.
for ecologically safe compounds as therapeutic agents
against drug-resistant microorganisms has driven many NMR analysis
studies toward medicinal plants. Literature shows thou- The 1D (1H and 13C-NMR) and 2D (COSY, NOESY,
sands of plant species that have been tested in vitro against HSQC and HMBC) spectra were performed in deuterated
many fungal and bacterial strains, and a good number of solvents (CD3OD) on Bruker Avance III 600 spectrometer
medicinal plant extracts and pure compounds have now at 600 MHz/150 MHz. All chemical shifts (δ) are given in
been proven to be active against fungi, Gram-positive and ppm units with reference to tetramethylsilane (TMS) as
Gram-negative bacteria [2–5]. Medicinal plants contain internal standard and the coupling constants (J) are in Hz.
therapeutic amount of secondary metabolites including
flavonoids. These are polyphenolic and C6-C3-C6 com- Spectrometric analysis
pounds in which the two C6 groups are substituted ben- The mass spectra (HR-TOFESIMS) were carried out on
zene rings, and the C3 is an aliphatic chain which contains Micromass Q-TOF micro instrument (Manchester, UK).
a pyran ring [6]. They occur as O- or C-glycosides or in Samples were introduced by direct infusion in a solution
the free state as aglycones with hydroxyl or methoxyl of MeOH at a rate of 5 μL/min.
groups [7]. The sugar moiety is an important factor deter-
mining their bioavailability. Flavonoids may be divided into Chromatographic methods
seven types: flavones, flavonols, flavonones, flavanes, isofla- Silica gel 60 Merck, 70–230 mesh and sephadex LH-20
vones, biflavones and chalcones [8]. Flavonoids are well were used to perform column chromatography while pre-
documented for their pharmacological effects, including coated silica gel 60 F254 (Merck) plates, were used to per-
antimicrobial, anticancer, antiviral, antimutagenic and anti- form thin layer chromatography. The spots were
inflammatory activities [9–11]. Biological properties of fla- visualized by an UV lamp multiband UV-254/365 nm
vonoids are linked to their ability to act as strong (ModelUVGL-58 Upland CA 91786, U.S.A) followed by
antioxidants and free radical scavengers, to chelate metals, spraying with 50% H2SO4 and heating at 100 °C for 5 min.
and to interact with enzymes, adenosine receptors and
biomembranes [7]. Plant material
Graptophyllum glandulosum Turrill (Acanthaceae) is a The aerial parts of G. grandulosum were harvested in a
shrub with 4-angled, nearly glabrous branches, normal small village called Foto situated in the Menoua Division,
green leaves, and red-purple flowers 1 in. or more long. It Western region of Cameroon) in November 2015. The
is one of several shrubs and trees of Graptophyllum that Plant was identified and authenticated by a Cameroonian
mainly grow in West and Central Africa but also in the Botanist (Mr. Fulbert Tadjouteu) at the National Herbarium
pacific regions [12]. This plant contains some important where a voucher specimen was archived (No 65631/HNC).
secondary metabolites such as polyphenols, flavonoids
and glycosides [13]. Leaves, roots and other parts of G. Extraction and isolation
glandulosum, are used in folk medicine in Cameroon to The extraction and isolation of compounds were done
treat wounds, abscesses, skin diseases, respiratory tract in- as previously described [13]. Briefly, the aerial part of G.
fections and diarrhea. Medical importance of this plant grandulosum was air-dried and powdered. The powder
attracted us to explore its antimicrobial properties. was macerated at room temperature with MeOH to

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Tagousop et al. BMC Complementary and Alternative Medicine (2018) 18:252 Page 3 of 10

afford the MeOH extract. Part of this extract (235 g) was (C-3′), 152.3 (C-4′), 116.7 (C-5′), 121.9 (C-6′), 56.6
suspended in water (300 mL) and successively parti- (C-7′) for aglycone; 100.6 (C-1′′), 78.6 (C-2′′), 77.9
tioned with EtOAc and n-BuOH to yield 37 and 13 g of (C-3′′), 70.9 (C-4′′), 66.9 (C-5′′), 110.0 (C-1′′′), 78.1
extracts, respectively. Column chromatography of the (C-2′′′), 80.7 (C-3′′′), 65.8 (C-4′′′), 75.4 (C-5′′′) for
n-BuOH extract followed by purification of different sugar moiety. 1H NMR data (CD3OD, 600 MHz) δH:
fractions led to the isolation of five compounds. 6.70 (1H, s, H-3), 6.45 (1H, d, J = 2.1 Hz, H-6), 6.77 (1H,
d, J = 2.1 Hz, H-8), 7.52 (1H, d, J = 2.1 Hz, H-2′), 6.95
Structural identification of the isolated compounds (1H, d, J = 8.4 Hz, H-5′), 7.56(1H, dd, J = 8.4 and 2.1 Hz,
The structures of isolated compounds were determined H-6′), 3.98 (3H, s, H-7′) for aglycone; 5.16 (1H, d, J =
after interpretation of their physical, spectrometric and 7.1 Hz, H-1′′), 3.68 (1H, dd, J = 9.0 and 7.1, H-2′′), 3.63
spectroscopic data summarized in this subsection. (1H, m, H-3′′), 3.62 (1H, m, H-4′′), 3.98 (1H, m, H-5′′
Chrysoeriol-7-O-β-D-xyloside (1): yellow amorphous a), 3.48 (1H, t, J = 9.6, H-5′′b), 5.46 (1H, d, J = 1.7 Hz,
powder; molecular formula C21H20O10; 13C NMR H-1′′′), 3.98 (1H, m, H-2′′′), 3.56 (2H, brs, H-4′′′),
(CD3OD, 150 MHz) δC: 165.3 (C-2), 104.3 (C-3), 184.1 4.05 (1H, d, J = 9.4, H-5′′′a), 3.84 (1H, d, J = 9.4, H-5′′′
(C-4), 161.7 (C-5), 99.6 (C-6), 163.1 (C-7), 95.8 (C-8), b) for sugar moiety.
158.5 (C-9), 107.0 (C-10), 123.1 (C-1′), 110.5 (C-2′), Chrysoeriol-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-(4′
148.2 (C-3′), 151.0 (C-4′), 116.7 (C-5′), 121.9 (C-6′), ′-hydrogenosulfate) glucopyranoside (4): yellow amorph-
55.2 (C-7′) for aglycone; 100.9 (C-1′′), 74.4 (C-2′′), 77.3 ous powder; molecular formula C28H31NaO18S. 13C NMR
(C-3′′), 70.7 (C-4′′), 66.9 (C-5′′) for sugar moiety. 1H data (CD3OD, 150 MHz) δC: 166.8 (C-2), 104.5 (C-3),
NMR data (CD3OD, 600 MHz) δH: 6.62 (1H, s, H-3), 184.1 (C-4), 163.1 (C-5), 101.1 (C-6), 164.0 (C-7), 96.2
6.38 (1H, d, J = 2.1 Hz, H-6), 6.71 (1H, d, J = 2.1 Hz, (C-8), 159.0 (C-9), 107.2 (C-10), 123.6 (C-1′), 110.8
H-8), 7.44 (1H, d, J = 2.1 Hz, H-2′), 6.84 (1H, d, J = (C-2′), 149.6 (C-3′), 152.3 (C-4′), 116.9 (C-5′), 122.0
8.4 Hz, H-5′), 7.47 (1H, dd, J = 8.4 and 2.1 Hz, H-6′), (C-6′), 56.7 (C-7′) for aglycone; 100.9 (C-1′′), 74.5 (C-2′
3.87 (3H, s, H-7′) for aglycone; 4.95 (1H, d, J = 7.1 Hz, ′), 76.8 (C-3′′), 77.5 (C-4′′), 75.3 (C-5′′), 67.1 (C-6′′),
H-1′′), 3.37 (1H, m, H-2′′), 3.36 (1H, m, H-3′′), 3.49 102.4 (C-1′′′), 71.9 (C-2′′′), 72.3 (C-3′′′), 74.2 (C-4′′′),
(1H, m, H-4′′), 3.38 (1H, m, H-5′′a), 3.87 (1H, m, H-5′′ 69.8 (C-5′′′), 17.9 (C-6′′′) for sugar moiety. 1H NMR
b) for sugar moiety. data (CD3OD, 600 MHz) δH: 6.73 (1H, s, H-3), 6.56 (1H,
Luteolin-7-O-β-D-apiofuranosyl-(1 → 2)-β-D-xylopyra- d, J = 2.1 Hz, H-6), 6.84 (1H, d, J = 2.1 Hz, H-8), 7.55 (1H,
noside (2): yellow powder; molecular formula d, J = 2.1 Hz, H-2′), 6.98 (1H, d, J = 8.4 Hz, H-5′), 7.59
C25H26O14. m.p. = 203 °C. 13C NMR data (CD3OD, (1H, dd, J = 8.4 and 2.1 Hz, H-6′), 3.99 (3H, s, H-7′) for
150 MHz) δC: 165.3 (C-2), 103.5 (C-3), 182.5 (C-4), aglycone; 5.15 (1H, d, J = 7.8 Hz, H-1′′), 3.61 (1H, dd, J =
162.9 (C-5), 99.7 (C-6), 163.1 (C-7), 94.9 (C-8), 157.4 9.1 and 7.8 Hz, H-2′′), 3.84 (1H, t, J = 9.1 Hz, H-3′′), 4.32
(C-9), 105.5 (C-10), 121.7 (C-1′), 114.2 (C-2′), 146.4 (1H, dd, J = 9.9 and 9.1 Hz, H-4′′), 3.89 (1H, m, H-5′′),
(C-3′), 150.5 (C-4′), 116.6 (C-5′), 119.7 (C-6′) for agly- 4.10 (1H, m, H-6′′a), 3.68 (1H, m, H-6′′b), 4.75 (1H, d, J
cone; 99.0 (C-1′′), 76.0 (C-2′′), 77.0 (C-3′′), 70.0 (C-4′ = 1.3 Hz, H-1′′′), 3.95 (1H, dd, J = 3.4 and 1.3, H-2′′′),
′), 66.1 (C-5′′), 109.3 (C-1′′′), 76.5 (C-2′′′), 79.7 (C-3′′ 3.72 (1H, dd, J = 9.5 and 3.4 Hz, H-3′′′), 3.32 (1H, t, J =
′), 64.5 (C-4′′′), 74.4 (C-5′′′) for sugar moiety. 1H 9.5 Hz, H-4′′′), 3.62 (1H, m, H-5′′′), 1.21 (3H, d, J =
NMR data (CD3OD, 600 MHz) δH: 6.75 (1H, s, H-3), 6.2 Hz, H-6′′′) for sugar moiety.
6.40 (1H, d, J = 2.1 Hz, H-6), 6.75 (1H, d, J = 2.1 Hz, Isorhamnetin-3-O-α-L-rhamnopyranosyl-(1 →
H-8), 7.44 (1H, d, J = 2.1 Hz, H-2′), 6.90 (1H, d, J = 6)-β-D-glucopyranoside (5): yellow amorphous powder;
8.4 Hz, H-5′), 7.47 (1H, dd, J = 8.4 and 2.1 Hz, H-6′) for molecular formula C28H32O15. 13C NMR data (CD3OD,
aglycone; 5.18 (1H, d, J = 7.1 Hz, H-1′′), 3.52 (1H, dd, J 150 MHz) δC: 159.8 (C-2), 135.7 (C-3), 179.8 (C-4),
= 9.0 and 7.1 Hz, H-2′′), 3.43 (1H, m, H-3′′), 3.41 (1H, 162.4 (C-5), 104.3 (C-6), 160.1 (C-7), 100.2 (C-8), 157.5
m, H-4′′), 3.78 (1H, dd, J = 9.7 and 3.4 Hz, H-5′′a), 3.42 (C-9), 108.6 (C-10), 123.0 (C-1′), 114.5 (C-2′), 148.4
(1H, dd, J = 9.7 and 3.4 Hz, H-5′′b), 5.34 (1H, d, J = (C-3′), 151.1 (C-4′), 116.2 (C-5′), 124.4 (C-6′), 56.7
1.3 Hz, H-1′′′), 3.75 (1H, m, H-2′′′), 3.30 (2H, d, J = (C-7′) for aglycone; 104.0 (C-1′′), 75.9 (C-2′′), 78.1
3.4 Hz, H-4′′′), 3.88 (1H, d, J = 9.3 Hz, H-5′′′a), 3.65 (C-3′′), 71.8 (C-4′′), 77.4 (C-5′′), 68.7 (C-6′′), 102.6
(1H, d, J = 9.3 Hz, H-5′′′b) for sugar moiety. (C-1′′′), 72.1 (C-2′′′), 72.3 (C-3′′′), 73.8 (C-4′′′), 69.8
Chrysoeriol-7-O-β-D-apiofuranosyl-(1 → 2)-β-D-xylo- (C-5′′′), 18.0 (C-6′′′) for sugar moiety. 1H NMR data
pyranoside (3): yellow powder; molecular formula (CD3OD, 600 MHz) δH: 6.71 (1H, d, J = 2.2 Hz, H-6),
C26H28O4; melting point = 181.8 °C. 13C NMR (CD3OD, 7.09 (1H, d, J = 2.2 Hz, H-8), 7.91 (1H, d, J = 2.2 Hz,
150 MHz) δC: 166.6 (C-2), 104.5 (C-3), 184.0 (C-4), H-2′), 6.95 (1H, d, J = 8.5 Hz, H-5′), 7.72 (1H, dd, J = 8.5
162.9 (C-5), 100.9 (C-6), 164.4 (C-7), 95.9 (C-8), 158.9 and 2.2 Hz, H-6′), 3.98 (3H, s, H-7′) for aglycone; 5.30
(C-9), 107.0 (C-10), 123.4 (C-1′), 110.4 (C-2′), 149.5 (1H, d, J = 7.6 Hz, H-1′′), 3.49 (1H, dd, J = 9.1 and

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Tagousop et al. BMC Complementary and Alternative Medicine (2018) 18:252 Page 4 of 10

7.6 Hz, H-2′′), 3.45 (1H, t, J = 9.1 Hz, H-3′′), 3.23 (1H, suspension was removed at the end of the various incuba-
t, J = 9.1 Hz, H-4′′), 4.40 (1H, m, H-5′′), 3.84 (1H, dd, J tion times and mixed with 1.95 mL of Buffer. The optical
= 12.1 and 1.9 Hz, H-6′′a), 3.42 (1H, m, H-6′′b), 4.53 densities were read in the same way.
(1H, d, J = 1.3 Hz, H-1′′′), 3.58 (1H, dd, J = 3.4 and 1.3,
H-2′′′), 3.47 (1H, dd, J = 9.5 and 3.4 Hz, H-3′′′), 3.25 Bacteriolytic assay
(1H, t, J = 9.5 Hz, H-4′′′), 3.41 (1H, m, H-5′′′), 1.10 The bacteriolytic activities of the isolated compounds were
(3H, d, J = 6.2 Hz, H-6′′′) for sugar moiety. determined using the time-kill kinetic method as previously
described [20] with slight modifications. Full growth of V.
Antimicrobial assay cholerae NB2 in MHB was diluted 100 times and incubated
Microorganisms at 37 °C to produce an OD600 of 0.8 as starting inoculum.
The microorganisms used in this study were consisted of Sample solutions were added to the starting bacterial
five bacterial strains namely Staphylococcus aureus ATCC suspension to give a final concentration of 2 × MIC and
25923, Vibrio cholerae NB2, PC2, SG24 (1) and CO6 [16]. incubated at 37 °C with shaking, then 100 μL was removed
Also included were two fungi Candida albicans ATCC from each tube at 0, 15, 30, 60, and 120 min and the optical
9002 and Cryptococcus neoformans IP95026. These bac- density measured at 600 nm. Vancomycin and tetracycline
teria and yeasts were obtained from our local stocks. were used as positive controls and the tubes without
isolated compounds served as negative controls.
Determination of minimum inhibitory concentration and
minimum microbicidal concentration Hemolytic assay
The minimum inhibitory concentration (MIC) values Whole blood (10 mL) from albino rats was collected by
were determined using the broth micro-dilution method cardiac puncture in EDTA tubes. The study was con-
as described earlier [17]. The MIC values were defined ducted according to the ethical guidelines of the Com-
as the lowest sample concentration that prevented the mittee for Control and Supervision of Experiments on
change in color indicating a complete inhibition of mi- Animals (Registration no. 173/CPCSEA, dated 28 Janu-
crobial growth. The lowest concentrations that yielded ary, 2000), Government of India, on the use of animals
no growth after the subculturing were taken as the mini- for scientific research. Erythrocytes were harvested by
mum microbicidal concentration (MMC) values [18]. centrifugation at room temperature for 10 min at 1000 x
Ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) and g and were washed three times in PBS buffer [21]. The
amphotericin B (Merck, Darmstadt, Germany) were used cytotoxicity was evaluated as previously described [21].
as positive controls for bacteria and yeast respectively.
Statistical analysis
Study on mode of action Data were analyzed by one-way analysis of variance
MIC and MBC changes under osmotic stress condition followed by Waller-Duncan post hoc test. The experi-
Osmotic stress was induced by adding 5% NaCl (w/v) to mental results were expressed as the mean ± Standard
MHB. The MHB supplemented with 5% NaCl was then ster- Deviation (SD). Differences between groups were con-
ilized and used for the determination of a new MIC and sidered significant when p < 0.05. All analyses were per-
MBC values of the samples as previously described [17]. The formed using the Statistical Package for Social Sciences
incubation time was increased from 24 to 48 h at 37 °C. (SPSS, version 12.0) software.

Effect of isolated compounds on cell membrane Results

The alteration of cell membrane of V. cholerae NB2 was Chemical analysis
evaluated by measuring the optical densities at 260 nm of The structures of five known flavonoid glycosides isolated
the bacterial suspensions in the presence and absence of from the n-BuOH fraction of leaves of G. grandulosum
compounds 1–5 using the method described by Carson et (Fig. 1) were determined using spectroscopic analysis and
al. [19]. For this purpose, the compounds were tested at NMR spectra in conjunction with 2D experiments (COSY,
their MIC using 1 mL of the bacterial suspension (ap- NOESY, HSQC and HMBC). Direct comparison with
proximately 108 CFU/mL). The mixture was then incu- published information led to the identification of
bated at 37 °C at different time intervals (0: immediately chrysoeriol-7-O-β-D-xyloside 1 [22], luteolin-7-O-β-D-a
after addition of the compound; 15; 30; 60 min), 50 μL of piofuranosyl-(1 → 2)-β-D-xylopyranoside 2 [23], chrysoe
the mixture was taken and mixed with 1.95 mL of Phos- riol-7-O-β-D-apiofuranosyl-(1 → 2)-β-D-xylopyranoside 3
phate Buffered Saline (PBS Buffer). The absorbance was [13], chrysoeriol-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-
measured on the spectrophotometer at 260 nm against (4′′-hydrogeno sulfate) glucopyranoside 4 [13] and
the blank (PBS). For the negative control, 1 mL of bacter- isorhamnetin-3-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-glu-
ial suspension was incubated at 37 °C and 50 μL of the copyranoside 5 [24].

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Tagousop et al. BMC Complementary and Alternative Medicine (2018) 18:252 Page 5 of 10

7' 7'
OCH3 OR
3' OH 3' OH
2' 2'
4'' 4' 4'
5'' O 5'' O
HO 8 HO 4'' 8
O O 5' O O
HO 9 HO 9 5'
2'' 1' 2'' 1'
3'' OH 1'' 7 2 6' 3'' O 1'' 7 2 6'
O
6 10 3 6 10 3
5 4 5''' 1''' 5 4
4''' OH
OH O OH O
3''' 2'''
1 OH OH 2R=H
3 R = CH3
6''' OH

5''' 4'''
7'
OH OCH3
O
3'''
3' OH
2'
1''' 4'
2''' OH 7'
OCH3 8
O 5'
O 9
3' 1'
6'' OH 7 2 6'
O 2' 3 1'' HO 3''
4'' 4' 2''
5'' O 6
O 8 10 OH
O O 5 4 O
S HO 9 5' O OH
Na O 2'' 1' 5''
3'' OH 1'' 7 2 6' OH O 4''
O 6''
6 10 3 O
5 4 HO 2'''
1'''
OH O 5
3''' O
4 HO
4''' 5'''

OH

Fig. 1 Chemical structures of flavonoids (1–5) isolated from n-BuOH extract of aerial parts of G. grandulosum Turill. 1:chrysoeriol-7-O-β-D-xyloside;
2:luteolin-7-O-β-D-apiofuranosyl-(1 → 2)-β-D-xylopyranoside; 3: chrysoeriol-7-O-β-D-apiofuranosyl-(1 → 2)-β-D-xylopyranoside; 4: chrysoeriol-7-O-α-
L-rhamnopyranosyl-(1 → 6)-β-D-(4"-hydrogenosulfate) glucopyranoside; 5:Isorhamnetin-3-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-glucopyranoside

Antimicrobial activity lowest MIC value of 4 μg/mL was recorded on C. neofor-

The in vitro activities of MeOH, n-BuOH and EtOAc ex- mans with compound 4 and on S. aureus with compounds
tracts as well as their isolated compounds against patho- 1, 2 and 4 whereas the lowest MMC value was obtained on
genic bacteria and fungi are presented in Table 1. The test S. aureus with compound 4. However, the highest MIC
samples demonstrated varying degrees of inhibitory activ- value for compounds (64 μg/mL) was recorded with com-
ities against the bacterial and fungal strains. Fungal strains pound 3 against V. cholerae CO6, and with compounds 2
were generally more susceptible to the effects of the com- and 5 against C. albicans, while the highest MBC value of
pounds, but less susceptible to extracts. The EtOAc and 128 μg/mL was obtained on V. cholerae CO6 with com-
n-BuOH extracts were active against C. albicans and C. pound 3 and on C. albicans with compound 5.
neoformans which were not susceptible to the MeOH ex-
tract. The MIC values obtained with the EtOAc and Antibacterial activity of flavonoid glycosides under
n-BuOH extracts were smaller than those obtained with osmotic stress condition
the MeOH extract. These observations suggest that the The MIC values of flavonoid glycosides against
fractionation of the MeOH extract enhanced its anti- Gram-negative and Gram-positive bacteria are reported in
microbial activity. The lowest MIC values were recorded Table 2. The results clearly showed that the MIC values of
on S. aureus; suggesting that this microorganism was the flavonoid glycosides obtained under osmotic stress (in the
most susceptible to all the test samples. The EtOAc ex- presence of 5% NaCl) are smaller than those obtained
tract showed the highest antimicrobial activity when com- under normal conditions (0% NaCl). This result suggests
pared with the MeOH and n-BuOH extracts. an increase in the activity of purified flavonoid glycosides
The antimicrobial activities of the isolated compounds under osmotic stress. As demonstrated under normal
from G. glandulosum were as follows: compound 4 > com- condition, compound 4 was still the most effective under
pound 1 > compound 2 > compound 5 > compound 3. The osmotic stress, followed in decreasing order by compounds

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Tagousop et al. BMC Complementary and Alternative Medicine (2018) 18:252 Page 6 of 10

Table 1 Antimicrobial activities of extracts, isolated compounds and reference antimicrobial drugs
Extracts/Compounds Inhibition V. cholerae V. cholerae V. cholerae V. cholerae S. aureus C. albicans C. neoformans
parameters SG24 (1) CO6 NB2 PC2 ATCC 25923 ATCC 9002 IP95026
MeOH extract MIC 512 512 256 512 256 > 2048 > 2048
MMC 512 512 512 1024 512 / /
MMC/MIC 1 1 2 2 2 / /
n-BuOH extract MIC 256 256 128 128 128 2048 2048
MMC 256 256 128 256 128 > 2048 > 2048
MMC/MIC 1 1 1 2 1 / /
EtOAc extract MIC 64 128 64 64 64 1024 512
MMC 128 128 64 64 64 1024 1024
MMC/MIC 2 1 1 1 1 1 2
1 MIC 16 8 8 8 4 32 8
MMC 16 8 16 8 8 64 8
MMC/MIC 1 1 2 1 2 2 1
2 MIC 16 16 8 8 4 64 16
MMC 32 16 16 8 8 64 32
MMC/MIC 2 1 2 1 2 1 2
3 MIC 32 64 32 32 8 32 16
MMC 64 128 32 64 16 32 16
MMC/MIC 2 2 1 2 2 1 1
4 MIC 8 8 8 8 4 8 4
MMC 16 8 8 8 4 8 8
MMC/MIC 2 1 1 1 1 1 2
5 MIC 32 16 16 16 8 64 32
MMC 32 16 16 16 8 128 64
MMC/MIC 1 1 1 1 1 2 2
Refa MIC 32 4 16 16 0.5 0.5 0.25
MBC 32 4 16 16 0.5 0.5 0.25
MBC/MIC 1 1 1 1 1 1 1
/: not determined; MIC Minimum Inhibitory Concentration, MMC Minimum Microbicidal Concentration; the MIC and MMC were measured in μg/mL; a:
amphotericin B for yeasts and ciprofloxacin for bacteria

1 and 2. The MIC values of chloramphenicol determined occurred during the initial period (≤ 15 min), followed
under osmotic stress condition were smaller than those de- by a slight increase with prolonged incubation period.
termined under normal conditions. However, all the MIC At the same time, the OD260 of the control without
values of vancomycin determined under osmotic stress compound was not changed. These results suggest that
were higher than those determined under normal condi- flavonoid glycosides from G. glandulosum damage the
tions. Table 1 further shows that under osmotic stress, the cytoplasmic membrane and cause loss of intracellular
antibacterial activities of compounds 1, 2 and 4 against V. components. The highest values of OD260 were recorded
chorae SG24 (1), V. chorae CO6, V. chorae NB2 and V. with compound 4 for all the V. cholerae strains, whereas
chorae PC2 were higher than that of vancomycin. the least OD260 values were noticed with compound 2,
indicating that compound 4 released the highest
Effect of flavonoid glycosides on cell membrane amounts of nucleic acids followed in decreasing order by
The effect of flavonoid glycosides of G. glandulosum was compound 1, then 2.
evaluated in terms of leakage of UV 260 absorbing ma-
terial through the bacterial cell membrane (Fig. 2). After Bacteriolytic effect of compounds 1, 2 and 4
treatment with flavonoid glycosides at MIC values of The result on the leakage of 260 nm absorbing material
compounds 1, 2 and 4, the OD260 values of filtrates of was consistent with that of bacteriolysis (Fig. 3). This re-
all test strains increased and most of the leakage sult showed a decrease in the optical density of

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Tagousop et al. BMC Complementary and Alternative Medicine (2018) 18:252 Page 7 of 10

Table 2 Antibacterial activities in terms of MIC (μg/mL) of compounds 1, 3 and 4 under osmotic stress condition against bacterial strains
Bacteria Compound 1 Compound 2 Compound 4 Chloramphenicol Vancomycin
0% NaCl 5% NaCl 0% NaCl 5% NaCl 0% NaCl 5% NaCl 0% NaCl 5% NaCl 0% NaCl 5% NaCl
V. chorae SG24 (1) 16 8 16 16 8 4 4 1 16 64
V. cholerae CO6 8 4 16 4 8 2 16 2 16 32
V. cholerae NB2 8 4 8 2 8 2 64 1 32 64
V. cholerae PC2 8 4 8 2 8 2 16 1 32 64
S. aureus 4 2 4 2 4 1 32 0.5 0,5 1

suspension treated with compounds 1, 2 and 4. After the test microbial species, the result can be considered
120 min, compounds 1, 2 and 4 induced a decline in cell as promising for the development of new antimicrobial
turbidity of 93.20, 94.36 and 95.16%, respectively in bac- drugs. The antimicrobial activities of purified flavonoids
teria suspension compared to time 0, indicating the lysis corroborated with those of early reports against bacteria
of bacterial cells. and fungi [5, 11, 26–28]. The antibacterial activity of the
samples against V. cholerae and S. flexneri are particu-
Haemolytic activity larly noteworthy since these strains were MDR clinical
The haemolytic activities of extracts and compounds 1–5 isolates which were resistant to commonly used drugs
against red blood cells (RBCs) were investigated using Tri- such as ampicillin, streptomycin, nalidixic acid, furazoli-
ton X-100 as a positive control. The positive control done and co-trimoxazole [16, 29, 30].
showed about 100% lysis, whereas the phosphate buffer Antimicrobial cutoff points have been defined in the lit-
saline (PBS) showed no lysis of RBCs. Interestingly, none erature to enable the understanding of the effectiveness of
of the tested samples showed haemolytic activities against pure compounds as follows: highly active: MIC below
RBCs at concentrations up to 256 and 2048 μg/mL for 1 μg/mL (or 2.5 μM), significantly active: 1 ≤ MIC ≤10 μg/
isolated compounds and extracts respectively (results not mL (or 2.5 ≤ MIC < 25 μM), moderately active: 10 < MIC
shown). This finding highlights the fact that the observed ≤100 μg/mL (or 25 < MIC ≤250 μM), weakly active: 100 <
biological efficacy was not due to haemolysis. MIC ≤1000 μg/mL (or 250 < MIC ≤2500 μM and not ac-
tive: MIC > 1000 μg/mL (or > 2500 μM) [25]. Based on
Discussion this, the antimicrobial activities of all the tested flavonoid
The antimicrobial activity of a plant extract is considered glycosides could be considered as significant or moderate
to be highly active if the MIC < 100 μg/mL; significantly against the specific microorganisms.
active when 100 ≤ MIC ≤512 μg/mL; moderately active The antimicrobial activities of the isolated compounds
when 512 < MIC ≤2048 μg/mL; weakly active if MIC > from G. glandulosum were in this order: compound 4 >
2048 μg/mL and not active when MIC > 10,000 μg/mL compound 1 > compound 2 > compound 5 > compound
[25]. Hence, the EtOAc extract of G. glandulosum was 3. Very little is known about the structure–function rela-
highly active (MIC < 100 μg/mL) against V. cholerae SG24 tionships of natural antimicrobials, but it seems that dif-
(1), V. cholerae NB2, V. cholerae PC2 and S. aureus; signifi- ferent substituent groups within the compounds had a
cantly active (100 ≤ MIC ≤512 μg/mL) against V. cholerae great influence on their biophysical and biological prop-
CO6 and C. neoformans; moderately active (512 < MIC erties [31]. Structural features such as the presence of an
≤2048 μg/mL) on C. albicans. The MeOH and n-BuOH aromatic ring, the sugar moiety or the numbers of hy-
extracts were significantly active against the test bacterial droxyl and methoxyl groups can significantly change
species; weakly and moderately active against the yeast membrane permeability and subsequent affinity to exter-
cells respectively. nal and internal binding sites in the bacteria, thus influ-
In this study, we also investigated if the mode of action encing the compound’s antimicrobial properties [32].
of flavonoid compounds is bactericidal or bacteriostatic. The antibacterial activities of flavonoid glycosides and
The results of the MMC values were fourfold lesser than chloramphenicol increased under osmotic stress (5%
their corresponding MIC values. This observation sug- NaCl) whereas that of vancomycin decreased under this
gests that the actions of extracts from G. glandulosum and condition. The results were supported by the observa-
their isolated flavonoid glycosides were bactericidal [11]. tion that certain bacterial strains (E. coli, S. aureus, P.
The antibacterial activities of flavonoid glycosides were aeruginosa) can survive under osmotic stress conditions
in some cases equal to, or higher than those of cipro- [33]. At low water activity, lipid composition of bacterial
floxacin used as reference antibiotic, suggesting that they cell membrane was changed [34]. This incident might
might be effective antibiotics against these pathogenic lead to occurrence of more antibacterial binding site on
bacteria. Taking into account the medical importance of cell membrane of bacteria and cause less resistance to

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Tagousop et al. BMC Complementary and Alternative Medicine (2018) 18:252 Page 8 of 10

Fig. 2 Appearance of 260-nm-absorbing material in the filtrates of V. cholerae SG24 (1), PC2, NB2 and CO6 after treatment with compounds 1, 2
and 4. Bars represent the mean ± standard deviation of the triplicate OD at each incubation time. At the same incubation time, letters a-d
indicate significant differences between samples according to one way ANOVA and Waller Duncan test; p < 0.05

Fig. 3 Bacteriolytic effect of compounds 1, 2 and 4 against V. cholerae NB2. Results represent the mean ± standard deviation of the triplicate OD
at each incubation time

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Tagousop et al. BMC Complementary and Alternative Medicine (2018) 18:252 Page 9 of 10

antibacterial substance. Therefore, the presence of the Abbreviations

13
salt triggered changes in the membrane lipid compos- C-NMR: Carbon Thirteen Nuclear Magnetic Resonance; 1H NMR: Proton
Nuclear Magnetic Resonance; 2D NMR: Two-dimension Nuclear Magnetic
ition. This is possible to increase the antibacterial activ- Resonance; ATCC: American Type Culture Collection; CC: Column
ity of flavonoid glycosides and chloramphenicol. Chromatography; COSY: Correlation Spectroscopy; DMSO: Dimethylsulfoxide;
However, the mechanisms that make bacteria more sen- EtOAc: Ethyl acetate; HMBC: Heteronuclear Multiple Bond Connectivities;
HNC: Herbier National du Cameroun; HR-EI-MS: High Resolution Electron
sitive to certain antibiotics under osmotic stress condi- Impact Mass Spectrometry; HR-TOFESIMS: High-Resolution Time of Flight
tions are still unknown. The results of vancomycin Electrospray Ionization Mass Spectrometry; HSQC: The Heteronuclear Single
activity are in agreement with those of McMahon and Quantum Coherence; IR: Infra-red; MBC: Minimum bactericidal concentration;
MDR: Multi-Drug-Resistant; MeOH: Methanol; MHA: Mueller Hinton agar;
coworkers [35] who demonstrated a decrease in the ac- MHB: Mueller Hinton broth; MIC: Minimum inhibitory concentration;
tivity of amikacin, ceftriaxone and trimethoprim against MMC: Minimum Microbicidal Concentration; NA: Nutrient agar; n-BuOH:
E. coli and S. aureus under osmotic stress conditions. n-Butanol; NMR: Nuclear Magnetic Resonance; Rf: Retention factor; TLC: Thin
Layer Chromatography; TMS: Tetramethylsilane; TOF-ESIMS: Time of Flight
Marked leakage of cytoplasmic material is considered Electrospray Ionization Mass Spectrometry; UV: Ultra-violet
indicative of gross and irreversible damaged to the cyto-
plasmic membrane. Many antibacterial compounds that Availability of data and materials
The datasets used and/or analyzed during the current study are available
act on the bacterial cytoplasmic membrane induce the from the corresponding author on reasonable request.
loss of 260 nm-absorbing materials (nucleic acids) in-
cluding chlorohexidine, hexachlorophene, phenetyl alco- Authors’ contributions
CNT and SEE contributed to the data collection and analysis. JDT designated
hol, tetracycline, polymixin, α-pinene, and lemon grass the study, did the biological assays and helped in manuscript writing and
oil [19]. The V. cholerae suspension treated with flavon- editing. JDT, DN and LVN supervised and revised the manuscript critically for
oid glycosides, showed a significant increase in the op- important intellectual content. All authors read and agreed on the final
version of the manuscript.
tical density at 260 nm, suggesting that nucleic acids
were lost through a damaged cytoplasmic membrane. Ethics approval and consent to participate
Our observations confirm that the antimicrobial activ- Not applicable.
ity of flavonoid glycosides results from their ability to Consent for publication
disrupt the permeability barrier of microbial membrane Not applicable.
structures. This mode of action is similar to that of other
Competing interests
broad-spectrum, membrane-active disinfectants and pre- The authors declare that they have no competing interests.
servatives, such as phenol derivatives, chlorohexidine
and para benzoic acid derivatives [36]. Furthermore, Publisher’s Note
Devi and Kapila [37], reported the antibacterial mechan- Springer Nature remains neutral with regard to jurisdictional claims in
ism as disruption of plasma membrane by the phyto- published maps and institutional affiliations.
chemicals in the extracts of Indian liverworts. Author details
The fact that flavonoids-induced damage to cell mem- 1
Research Unit of Environmental and Applied Chemistry, Department of Chemistry,
brane structure accompanied by the decline in the ab- Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon.
2
Research Unit of Microbiology and Antimicrobial Substances, Department of
sorbance of bacterial cell suspension treated with Biochemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang,
compounds has confirmed it as the most likely cause of Cameroon. 3Groupe Isolement et Structure, Institut de Chimie Moléculaire de Reims
cell death. Our result is supported by the observation (ICMR), CNRS UMR 7312, Bat. 18 BP.1039, 51687 Reims cedex 2, France.
that other flavonoid compounds such as epigallocatechin Received: 9 May 2018 Accepted: 6 September 2018
gallate and galangin induced 3-log reduction or more in
viable counts of S. aureus [38, 39].
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