Biomedicine & Pharmacotherapy 144 (2021) 112264
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Hydrolyzable tannins (ellagitannins), flavonoids, pentacyclic triterpenes
and their glycosides in antimycobacterial extracts of the
ethnopharmacologically selected Sudanese medicinal plant Combretum
hartmannianum Schweinf
Enass Y.A. Salih a, d, *, 1, Riitta Julkunen-Tiitto b, Olavi Luukkanen c, Mustafa K.M. Fahmi e,
Pia Fyhrquist a
a
Faculty of Pharmacy, Division of Pharmaceutical Biosciences, Viikki Biocenter, University of Helsinki, P.O. Box 56, FIN-00014, Finland
Faculty of Science and Forestry, Department of Environmental and Biological Sciences, University of Eastern Finland, 80101 Joensuu, Finland
c
Viikki Tropical Resources Institute (VITRI), Viikki Campus, University of Helsinki, FIN-00014, Finland
d
Department of Forest Products and Industries, Shambat Campus, University of Khartoum, SUD-13314, Sudan
e
Department of Forest Management, Shambat Campus, University of Khartoum, SUD-13314, Sudan
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Combretum hartmannianum
Traditional medicine
Tuberculosis
Ellagitannins
Flavonoids
Terpenoids
In Sudanese traditional medicine, decoctions, macerations, and tonics of the stem and root of Combretum hartmannianum are used for the treatment of persistent cough, a symptom that could be related to tuberculosis (TB).
To verify these traditional uses, extracts from the stem wood, stem bark, and roots of C. hartmannianum were
screened for their growth inhibitory effects against Mycobacterium smegmatis ATCC 14468. Methanol Soxhlet and
ethyl acetate extracts of the root gave the strongest effects (MIC 312.5 and 625 µg/ml, respectively). HPLC-UV/
DAD and UHPLC/QTOF-MS analysis of the ethyl acetate extract of the root led to the detection of 54 compounds,
of which most were polyphenols and many characterized for the first time in C. hartmannianum. Among the major
compounds were terflavin B and its two isomers, castalagin, corilagin, tellimagrandin I and its derivative, (S)flavogallonic acid dilactone, punicalagin, and methyl-ellagic acid xylopyranoside. In addition, di-, tri- and tetragalloyl glucose, combregenin, terminolic acid, cordifoliside D, luteolin, and quercetin-3-O-galactoside-7-Orhamnoside-(2→1)-O-β-D-arabinopyranoside were characterized. Luteolin gave better growth inhibition against
M. smegmatis (MIC 250 µg/ml) than corilagin, ellagic acid, and gallic acid (MIC 500–1000 µg/ml). Our study
justifies the use of C. hartmannianum in Sudanese folk medicine against prolonged cough that could be related to
TB infection. This study demonstrates that C. hartmannianum should be explored further for new anti-TB drug
scaffolds and antibiotic adjuvants.
1. Introduction
Tuberculosis (TB) is a bacterial infectious disease caused mainly by
Mycobacterium tuberculosis and spreads by coughing and/or sneezing
[1]. TB was discovered in 1882 by the German scientist Robert Koch [2,
3]. Although TB mainly infects the lungs, also the bones and the central
nervous system can be affected [4]. Recently osteoarticular TB, a
chronic inflammatory disease affecting the bones and joints, has been
reported to become a serious problem in China [5]. Moreover, in Africa
Buruli ulcer, caused by Mycobacterium ulcerans, is still a significant
mycobacterial disease in rural remote regions, leading to amputations if
untreated [6]. In 2020, the WHO estimated that more than one-third of
the global population is infected with TB [7]. However, TB deaths occur
mainly in developing countries, and especially in sub-Saharan Africa
[8–10]. The high burden of TB in some countries in Africa is due to
poverty and consequently shared and crowded living conditions, which
facilitate the spread of TB. Moreover, malnutrition worsens the TB
burden in some regions of Africa. Besides, latent M. tuberculosis is known
to become activated in patients with severe immunosuppression due to
HIV [11]. In addition, in South Africa, TB patients have been observed to
* Corresponding author at: Faculty of Pharmacy, Division of Pharmaceutical Biosciences, Viikki Biocenter, University of Helsinki, P.O. Box 56, FIN-00014, Finland.
E-mail addresses: enass.salih@helsinki.fi, eyabdelkareem@uofk.edu (E.Y.A. Salih).
1
Permanent address (E. Salih): Department of Forest Products and Industries, Shambat Campus, SUD-13314, University of Khartoum, Sudan.
https://doi.org/10.1016/j.biopha.2021.112264
Received 9 June 2021; Received in revised form 14 September 2021; Accepted 27 September 2021
Available online 5 October 2021
0753-3322/© 2021 The Authors.
Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
delay their treatments as they cannot afford to travel to the treatment
centers daily for observation by health workers to take their TB drugs
[12]. Also, a low antibiotic treatment compliance and even treatment
interruption due to the complicated and long treatment regimens
contribute to increase the pool of more resistant TB strains (multi-drug
resistant, MDR-TB and extremely-drug resistant, XDR-TB). These TB
strains are resistant to one or more of the available conventional TB
drugs, such as rifampicin, isoniazid, thioacetazone, streptomycin,
ethambutol, or pyrazinamide [13–16].
Due to the relatively high cost and limited availability to synthetic
antibacterial drugs, communities in Africa have largely relied on traditional healers and plant-based drugs to treat infections, including
tuberculosis [17,18]. Higher plants, and especially those used in traditional medicine for infections, are rich in a variety of defense compounds
against microbial intrusion and could contain valuable new anti-TB drug
lead compounds [19,20]. Despite this, there are still no modern antibacterial drugs developed from plant compounds. African medicinal
plants could be a valuable source for new antimicrobial drug scaffolds
and/or antibiotic adjuvants [18,19,21].
Combretum hartmannianum (Schweinf) (in Sudanese vernacular language: Al-Habeel )ﺍﻝﻩﺏﻱﻝoccurs in the horn of Africa where it grows
mainly in Eritrea, Ethiopia, South Sudan, and Sudan Fig. 1F [22,23]. In
addition, it is common in the whole Sahel region and West Africa [24,
25]. C. hartmannianum is a shrub or small tree and grows mainly in
wooded grassland, high-rainfall savannas, and savannah woodland
habitats on well-drained alluvial soils [25]. The fruits need fire to open
and release their seeds and fire is also needed for new sprouting from the
stem, and thus regular bushfires are needed for the regeneration of this
species [26,27]. The crown is very dense and broader when compared to
other species of Combretum (Fig. 1A), and the leaves are glabrous with
characteristic, extremely extended leaf tips (Fig. 1D). In Sudanese
traditional medicine C. hartmannianum is used habitually against a wide
range of diseases including asthma, malaria, fever, jaundice, fungal and
bacterial infections, including tuberculosis [26,28–34]). Accordingly,
some in vitro studies confirm that C. hartmannianum extracts are antibacterial [30,33,35–37]. However, only three papers to date are available on the phytochemistry of C. hartmannianum [38–40].
Based on our ethnopharmacological survey in Sudan,
C. hartmannianum is used for the treatment of persistent and prolonged
coughing, that could be symptoms of tuberculosis (Table 1). However,
only two previous investigations [13,32] reported on the antimycobacterial effects of C. hartmannianum. According to the other study
[32], dichloromethane, ethyl acetate, and ethanol extracts of the stem
bark were growth inhibitory against M. aurum A+ with MIC values of
0.78, 3.12, and 0.19 mg/ml, respectively. Moreover, a leaf extract of
C. hartmannianum inhibited the growth of M. tuberculosis. However, the
root and stem wood parts were not studied for their antimycobacterial
potential, although we found that decoctions, macerations, and ethanolic tonics made from these plant parts are used for the alleviation of
prolonged coughing. Therefore, in this present screening we have chosen to study the in vitro antimycobacterial activity of the root, stem bark,
and stem wood extracts of various polarities, including traditional medicinal preparations, against a fast-growing model bacterium for
tuberculosis, Mycobacterium smegmatis. To date, the chemical constituents of the root of C. hartmannianum have not been studied. Thus,
HPLC-DAD and UHPLC/QTOF-MS were used to decipher the molecular
structures of constituents in a root ethyl acetate extract, with special
emphasis on polyphenols.
2. Material and methods
2.1. Ethnobotanical survey and collection of plant material
To study plants of the genus Combretum that are used for infectious
diseases in Sudan, ethnobotanical surveys were performed in seven localities in the Blue Nile and Kordofan regions, in South Eastern and
South Western Sudan in 2006 and 2014. The surveys were performed by
the first author in collaboration with Associate Professor Haytham H.
Geebril from the Faculty of Forestry, Department of Silviculture, University of Khartoum. A questionnaire was used for the interviews and
focus was put on questions relating to symptoms that could be a sign of
bacterial infection and TB, such as persistent coughing, fever, fatigue,
and chest pain (Appendix). Among the seven Combretum spp. that were
growing naturally in the study area, C. hartmannianum was the most
commonly used species for the treatment of TB and its symptoms, and it
was used in the same way in all of the villages we visited (Table 1).
Fig. 1. (A), C. hartmannianum occurs in savannah woodland and wooded grassland in Sudan; (B) rough and finely fissured bark; (C) samara fruits; (D) leaves with
extremely long tips that are almost as long as the leaf blade; (E), flowers borne in axillary spikes. Photos by: E. Y. A. Salih and H. H. Gibreel 2006. (F) Occurrence of
C. hartmannianum in Africa; (G), Study site in the Blue Nile region in Sudan is marked in red color on the map.
Source: African Plant Database.
2
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
Table 1
Ethnobotanical uses, occurrence, phenology, and phytochemistry of Combretum hartmannianum. Results from our ethnobotanical survey in Blue Nile State, Sudan, and
the phytochemical analysis reported in this present paper are marked with an asterisk *.
The species name,
synonyms and
Sudanese vernacular
name
Botanical data
Gum exudate and its
traditional/commercial
uses and chemical
constituents *
Chemical constituents
Traditional uses
Combretum aculeatum
Syn. Combretum
ovale R.Br. ex G.
Don; Combretum
alternifolium Spreng.
Sudanese
vernacular name:
Shuheit
4–5 m in height *[41].
Flowering/fruiting time:
March to June/July to
October *[41]
Gum was observed from
the stem *
No reports on the chemical
constituents of the gum
Palmitoleic acid, Ethyl linoleate, α-Linolenic acid, Stearic acid,
Omega-6 (Linoleic acid), Behenic acid, Dotriacontanol,
Squalene, Lycoxanthin from the aerial parts[42].
Combretun ghasalense
Engl. & Diels.
Syn. Combretum
ternifolium Engl. &
Diels.
Combretum fragrans
F.Hoffm.
Combretum
adenogonium Steud.
ex A. Rich.
Sudanese
vernacular name:
Habil Um-ismaeel
Combretum collinum
Fresen.
Syn. Combretum
bongense Engl. &
Diels.
Sudanese
vernacular name:
Habil el-gouruz
12–15 m in height *[44].
Flowering/fruiting time:
November to February
/February to March *[45]
Tapping produces a long
line of gum *
Gum contains arginine,
aspartic acid, glutamic
acid, glycine, histidine,
hydroxyproline,
isoleucine, leucine, lysine,
methionine,
phenylalanine, proline,
serine, threonine,
tyrosine, valine[46]
Arjunolic acid, Velutin (3′ ,7-dimethoxy-4′ ,5dihydroxyflavone), Asiatic acid, Belamcanidin (5-hydroxy6,7,3′ ,4′ -tetramethoxyflavone), Cirsilineol (5,4′ -dihydroxy6,7,3′ -trimethoxyflavone), Combretin B, Cirsimaritin (5,4′ dihydroxy-6,7-dimethoxyflavone), 3β-Acetoxy-20,24-epoxy11,25-hydroxy-dammarane, Combretin A[44].
10 m in height *[48].
Flowering/fruiting time:
January to February
/January to March *[41].
Myricetin-3-O-rhamnoside, Myricetin-3-O-glucoside,
Tetracosanoic acid, Squalene, Campestrol, Palmitic acid[49].
Combretum glutinosum
Perr. Ex DC.
Syn. Combretum
glutinosum var.
relictum (Hutch &
Dalziel) Aubrév.
Sudanese
vernacular name:
Habil el gebel
12 m in height *[50].
Flowering/fruiting time:
December to March /
October to December *[51].
The exudate is an edible
gum *
Gum contains sarginine,
aspartic acid, glutamic
acid, glycine, histidine,
hydroxyproline,
isoleucine, leucine, lysine,
methionine,
phenylalanine, proline,
serine, threonine,
tyrosine, valine[46]
Gum exudate observed *
Gum contains arginine,
aspartic acid, glutamic
acid, glycine, histidine,
hydroxyproline,
isoleucine, leucine, lysine,
methionine,
phenylalanine, proline,
serine, threonine,
tyrosine, valine[46].
Leaves decoctions as
laxatives and for
venereal disease; stem
against skin infections,
including leprosy; root
decoction against flu *
[42]. Decoction of the
aerial parts against TB
symptoms *[43].
Bark for treating
stomach diseases; smoke
from the wood as
perfume * .
Leaves and stem cold
and hot water extracts
are used against
gastrointestinal
infectious diseases *
Stem bark used as a tea
against malaria *
Root decoction is used
against cough *[47].
The gum is used against
toothache; root and
leaves decoctions are
used against diarrhea
and cough*
Root and stem water
extracts are used against
intestinal pain * .
Leaves infusions are
used against cough[49].
Combretum
hartmannianum
Schweinf.
Syn. Poivrea
hartmanniana
Schweinf.
Sudanese
vernacular name:
Al-Habil, Subagh
Shrub-tree grows up to 25 m
in height * . Characteristic
leaf morphology with long,
pointed leaf tips that differs
from all other species of
Combretum. Bark rough and
finely fissured. Crown
rounded *[23].
Occurrence: Horn of Africa
(Erithrea, Ethiopia, Sudan,
Somalia) and Sahel region to
Senegal. High rainfall
savanna woodland and
wooded grasslands on
well-drained alluvial soils
[23,25].
Flowering/fruiting time:
Flowering in January to
February and fruiting from
April to May *
Water-soluble and edible
gum exudate, called
kaakol* .
The gum contains the
sugars galactose,
arabinose, mannose,
xylose, rhamnose,
glucuronic acid, 4-Omethylglucuronic acid,
and galacturonic acid[58]
and the amino acids
arginine, aspartic acid,
glutamic acid, glycine,
histidine, hydroxyproline,
isoleucine, leucine, lysine,
methionine,
phenylalanine, proline,
serine, threonine,
tyrosine, valine[46].
Apigenin, isorhamnetin derivatives, phenantherenes,
kaempferol and its derivatives, quercetin and its derivatives,
chrysoeriol and its dervatives[39].
Flavogallonic acid dilactone and terchebulin[40].
Ursolic acid, pomolic acid, corosolic acid, arjunic acid,
arjunglucoside I, trachelosperoside E-1, combreglucoside,
chebuloside II, and 2α,3β,6β,19α-tetrahydroxyoleanolic acid
28-O-β-D-glucopyranoside[38].
In this present study, we have tentatively characterized the
following compounds from a root ethyl acetate extract of
C. hartmannianum: Terflavin B and its isomer, Castalagin,
Corilagin, S- flavogallonic acid dilactone, Tellimagrandin I and
its derivatives, Punicalagin, Terchebulin, Combregenin,
Isorhamnetin, Cordifoliside D, Luteolin, Ellagic acid, and its
derivatives,
Quercetin-3-O-galactoside-7-O-rhamanoside-(2–1)-O-Darabinopyranoside, Terminolic acid, mono-, di-, tri-, and
tetra-galloyl glucose * .
Combreglutinin, 2,3-(S)-hexahydroxydiphenoyl-D-glucose
[53], punicalagin, punicalin[52,54].
Root, stem, and leaves
decoction for
hypertension, as
laxatives, for stomach
pain, hepatitis,
respiratory symptoms,
and fever *[55,56].
Leaves extracts are used
to cure cough[57].
The traditional
medicinal preparation
called “Dokhan” is made
from burning the root
and stem and a smoke
bath is used to cure
sexually transmitted
infectious diseases * .
Leaves decoction and
maceration are used to
cure ascites (that can be
caused by TB), stem
bark, leaves, or fruits are
mixed with gum against
jaundice, for skin
infections and wounds;
root, leaf, and bark
water extracts are used
as a mouth wash and
against inflamed gums;
water extracts and
decoctions, as well as,
ethanolic tonics of stem
bark, and root, are used
to slow down a
persistent cough (TB),
and against eye
infections *[39,59].
(continued on next page)
3
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
Table 1 (continued )
The species name,
synonyms and
Sudanese vernacular
name
Combretum nigricans
Guill. & Perr.
Syn. Combretum
elliotii Engl. & Diels;
Combretum
lecananthum Engl. &
Diels.
Sudanese
vernacular name:
Habil
Combretum molle R.
Br. Ex G. Don.
Syn: Combretum
deserti Engl.;
Combretum
reticulatum Fresen.;
Combretum
tetragonum M.A.
Lawson; Combretum
pisoniiflorum
(Klotzsch) Engl.
Sudanese
vernacular name:
Habil Khrisha
Botanical data
Gum exudate and its
traditional/commercial
uses and chemical
constituents *
Chemical constituents
12 m in height *[63].
Flowering/fruiting time:
February to March /April to
July *[64].
The bark exudates an
edible gum* , that
contains rhamnose and
uronic acid[65].
Arjunglucoside I, arjungenin, combreglucoside, mollic acid,
combregenin, arcapitin A, 11α-acetoxy-20,24-epoxy-25hydroxy-dammar-3-one, 20,24-epoxy-11α,25-dihydroxydammar-3-one, 20,24-epoxy-12β-25-dihydroxy-dammar-3-one
[53].
15 m in height *[48].
Flowering/fruiting time:
February to May / May to
August *[45].
Exudates a gum * that
contains arginine, aspartic
acid, glutamic acid,
glycine, histidine,
hydroxyproline,
isoleucine, leucine, lysine,
methionine,
phenylalanine, proline,
serine, threonine,
tyrosine, and valine[46].
Punicalagin, punicalin[54,66], combretene A and B,
combregenin, arjungenin, arjunglucoside I, combreglucoside,
sericoside, mollic acid and its glycosides; mollic acid glucoside,
mollic acid arabinoside, mollic acid xyloside[53].
Traditional uses
Water extracts of the
barks and infusion of the
leaves for jaundice[60].
Paste and ointments
from crushed and
powdered leaves against
skin infections, and
leprosy *[55].
Stem bark used as a tea
against Malaria * .
“Dokhan”, a smoke bath
from the burning roots
and stem is used to cure
sexually transmitted
infectious diseases. The
smoke of bark and wood
against arthritis
rheumatism and to treat
dryness of skin in Sudan
*[28].
For bacterial diseases *
[61,62].
Leaf decoctions for
jaundice, external
infections on the skin,
malaria, and febrile
diseases[39].
Leaves decoction is used
for intestinal disorders,
against fever, acne,
jaundice, respiratory
symptoms, and
rheumatism * .
Paste and ointments
from leaves and stem
wood are used against
skin infections * .
The burnt leaves
produce an aromatic
smoke that is used to
induce abortion and
enhance constipation,
and as an antianthelmintic especially
against hookworms;
leaves decoction against
cough *[66].
Paste and ointments
from leaves are used
against skin infections
and leprosy * .
African plant database and The Plant List were used as sources for the scientific name synonyms.
http://www.theplantlist.org/tpl1.1/record/kew-2732601 http://www.villege.ch/musinfo/bd/cjb/africa/details.php?langue=an&id=189625
Therefore, C. hartmannianum was selected for further analysis on its
antimycobacterial extracts and compounds, and bulk collections were
done for this species. Root and stem bark material were collected from
four individuals of C. hartmannianum growing in savannah woodland
and wooded grassland habitats in the Blue Nile and Kordofan regions.
Specimens of C. hartmannianum were botanically identified by the first
author and the taxonomists, Dr. Haytham H. Gebreel and Dr. El-Sheikh
Abd Alla El-Sheikh. Voucher specimens were authenticated and deposited at the Faculty of Forestry, University of Khartoum, Sudan. The
collected plant parts were dried in the shade, grinded, and kept in paper
bags for further use. Plant material from different plant individuals was
kept separate.
2.2. Extraction techniques
2.2.1. Hot (Soxhlet) and cold methanol extractions
A hot extraction method was employed by using a Soxhlet apparatus
(Sigma-Aldrich, Germany). Fifty grams of dry and powdered plant
samples were loaded into a Soxhlet thimble in the glass chamber of a
Soxhlet apparatus. The extraction solvent, 100% methanol, was poured
into a round-bottom distillation flask that was connected to the Soxhlet
apparatus. The solvent was brought to a boil and the extraction process
was continued for 6 h whereafter the resulting extracts were cooled
down and transferred to a rotary evaporator (Heidolph VV2000). After
rotary evaporation, the extracts were further dried to complete dryness
in a freeze drier for 2–3 days (lyophilizer, HETO LyoPro 3000,
Denmark). The resulting dry extracts were dissolved in methanol for
antimycobacterial testing (50 mg/ml stock solutions).
4
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
Cold methanol extraction was made to compare the composition of
the extracts with the hot methanol extracts since hot extraction could
lead to the loss of some compounds due to molecular degradation. For
the cold methanol extractions, 20 g of various parts of C. hartmannianum
were extracted at room temperature for 24 h with 500 ml methanol and
using a magnetic stirrer (RCT, digital). The extracts were filtered with
Whatman filter paper (150 mm, Germany), whereafter they were dried
using a rotary evaporator and a water bath at + 40 ◦ C. These extracts
were then further dried for three days using a lyophilizer (HETO LyoPro
3000, Denmark).
Pasteur pipette (P 4518–5X, Accupipette ™ Pipets/ DADE and DURAN®,
ring Caps, Germany). The mobile phase consisted of methanol, water,
and orthophosphoric acid (50:50:1, v/v/v). Fluorescent and quenching
spots were observed at the wavelengths of 366 and 254 nm, respectively, using a Camaq Reprostar UV-detector (3 TLC Visualizer). Retardation factors (Rf values) of the fractions and pure compounds were
calculated according to the formula 2:
Rf value = Distance moved by the fractions or compounds / Distance moved by
the solvent
(2)
For the analysis of the antioxidant effects of extracts and compounds
of C. hartmannianum, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent
(C18H12N5O6, Sigma-Aldrich, China), was prepared in methanol to a
concentration of 0.2% (w/v). The developed thin layer plates containing
the extracts, fractions, and pure compounds were sprayed with the
DPPH reagent to qualitatively investigate the oxidative reactions of the
separated spots in visible light. The compounds and fractions with
antioxidant properties resulted in a change in the color of the stable free
radical, DPPH, from dark purple (color of the DPPH in the form of a
radical) to colorless. This color change is a result of the weak hydrogen
bonds in the antioxidant compounds present in the extracts that react
with the strong, stable DPPH radical to form the colorless DPPH-H
complex. Retardation factors of standard compounds (ellagic acid,
gallic acid, corilagin, and luteolin, (Sigma-Aldrich) were compared with
the retardation factors of the spots present in the fractions and extracts,
to localize these compounds in the extracts.
2.2.2. Sequential extraction and solvent partition
Sequential extraction, starting with lipophilic solvents and gradually
extracting with more polar solvents, was used according to the method
described in Salih et al. [67,68]. A hundred grams dry powder of the
stem bark, stem wood, and root of C. hartmannianum was extracted
overnight, first with 1000 ml n-hexane, secondly with 1000 ml of
dichloromethane or chloroform (for the stem wood and bark) and
acetone (for the root part), and finally with 80% methanol. All extractions were performed at room temperature. A magnetic stirrer was used
to facilitate the extraction. Solvent partition was performed using the
80% methanol extract that was combined with an equal volume of ethyl
acetate in a separation funnel and mixed carefully (1000 ml, a Nalgene®
FEP). This sequential extraction and solvent partition led to the separation of n-hexane, chloroform, dichloromethane, acetone, aqueous
(80% methanol), and ethyl acetate fractions. A rotary evaporator was
used to dry the fractions before they were freeze-dried for three days in a
lyophilizer (HETO LyoPro 3000, Denmark). The dried fractions were
dissolved in methanol (stock solutions of 50 mg/ml) for antimycobacterial testing.
2.3.2. HPLC-UV/DAD analysis
Phytochemical screening of the extracts and fractions of
C. hartmannianum was performed using an Agilent Chemstation HPLCUV/DAD system (Waters Corp., Milford, USA) and using the method
described as the HPLC method II in Fyhrquist et al. [73]. A
reversed-phase column, Hypersil Rp C-18 (length 10 mm; ID 2 mm) was
used for phytochemical separation. A diode-array UV detector (a 991
PDA), an automatic sample controller, and a pump (Waters 600 E) were
connected in the HPLC system. The mobile phase eluents comprised of
solvent A (aqueous solvent consisting of 1.5% tetrahydrofuran and
0.25% orthophosphoric acid) and solvent B (100% methanol). The
gradient elution was programmed to a flow rate of 2 ml/min and with a
sample injection volume of 10 µl (2 mg/ml in 50% MeOH). The UV
detector wavelengths were adjusted at 220, 270, 280, 320, 360, and
380 nm, to record the UVλ absorption maxima spectra for the identified
peaks. The retention times and UVλ absorption maxima spectra of the
detected compounds were compared with the compounds in the natural
compounds library available in our computer system Agilent Chemstation library as well as with information obtained from SciFinder,
PubChem, and other sources of literature [74,75].
2.2.3. Water extracts
Twenty grams of powdered plant materials were used for the hot
(decoctions) and cold water (macerations) extractions. For the decoctions, the samples were first brought to a boil for 5 min and then
cooled down whereafter extraction was continued at room temperature
overnight and using a magnetic stirrer. No heating was used for the
maceration method and the extraction was done overnight at room
temperature with a magnetic stirrer facilitating extraction. After
extraction, the water extracts were decanted into Eppendorf centrifuge
tubes (50 ml v/v, Germany) and centrifuged in an Eppendorf centrifuge
(AG, 5810 R, Germany) at 3000 rpm for 15 min. The supernatants of the
centrifuged extracts were frozen at −20 ◦ C for 24 h and dried using a
lyophilizer (HETO LyoPro 3000, Denmark). The dried extracts were
dissolved in methanol to a concentration of 50 mg/ml for antimycobacterial testing [69].
The percentage extraction yields of the extracts and fractions were
calculated according to the formula 1 [70]:
2.3.3. UHPLC/QTOF-MS method
Ultra-high performance liquid chromatography coupled to quadrupole time of flight mass spectrometry (UHPLC/QTOF-MS) in negative
mode was used to identify exact molecular masses [M] between 100 and
2000 m/z. A method described by Taulavuori et al. [76] and
Julkunen-Tiitto and Sorsa [77], was employed to identify the various
compounds, with emphasis on polyphenols, in a root ethyl acetate
extract of C. hartmannianum. Shortly, the Agilent Technologies
UPLC-DAD (Model 1200 Agilent Technologies)-JETSTREAM/QTOF-MS
(Model 6340 Agilent Technologies) and a reverse-phase column C18
(2.1 ×60 mm, 1.7 µm, Agilent technologies) were set into a
UHPLC/Q-TOF-MS system. Eluent (A) was aqueous 1.5% tetrahydrofuran and 0.25% acetic acid, and eluent (B) was 100% methanol.
Gradient elution was used as follows: from 0 to 1.5 min, B 0%, from 1.5
to 3 min, 0–15% B, from 3 to 6 min, 10–30% B, from 6 to 12 min,
30–50% B, from 12 to 20 min, 50–100%, and from 20 to 22 min,
100–0% B.
Percentage extraction yield (%) = weight of the dry extract/weight of dry plant
material before extraction × 100
(1)
2.3. Antioxidant analysis and phytochemical profiling
2.3.1. Thin-layer chromatography and qualitative antioxidant analysis
A reversed-phase thin layer chromatography method combining TLC
separation with DPPH-antioxidant analysis was used according to Wang
et al. [71] and Sethiya et al. [72] to detect the antioxidant compounds in
C. hartmannianum root methanolic Soxhlet extracts. A TLC was prepared
with the plates sized at 10 × 20 cm (RP-18 F 254 S TLC-plates, Darmstadt, Germany). Ten microliters (10 µl) of corilagin, gallic acid, luteolin, and ellagic acid (1 mg/ml in methanol, Sigma-Aldrich, Germany)
and 20 µl of the extracts and fractions (50 mg/ml in methanol) were
applied manually 1.5 cm from the bottom of the TLC plate using a glass
5
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
The mass accuracy (PPM) was calculated according to formula 3:
Sigma-Aldrich, China), quercetin (Merk Art. 7546, Darmstadt, Germany), corilagin (Sigma-Aldrich, Darmstadt, Germany) and apigenin
(Extrasynthese Genay, France) were two-fold diluted in Dubos broth at
concentrations ranging from 0.030 to 1000 μg/ml. Preparation for the
tests started by transferring a few colonies of M. smegmatis into 25 ml of
Dubos broth and incubating this suspension for three days in a shaking
incubator (Stuart® SI500, UK) at + 37 ◦ C, 200 rpm. The inoculum for
the test was prepared by pipetting 2 ml from this suspension into a
sterile test tube and mixing carefully. 1 ml from this suspension was
pipetted into a disposable UV cuvette (BrandTech®, USA), to measure
the turbidity at 625 nm using a UV-Visible Spectrophotometer (Pharmacia LKB-Biochrom 4060). The suspension that was left in the test tube
was diluted following the guidelines certified by the Clinical and Laboratory Standards Institute [79] to reach an absorbance of 0.1 at 625 nm
(consisting of approximately 1 × 108 CFU/ml). This suspension was
diluted further with Dubos broth so that the inoculum used for the
microdilution assay contained 5 × 105 CFU/ml. A hundred microliters
from this prepared inoculum was added to each well of a 96-well
microplate, except the sample control wells (Nunc, Nunclone,
Denmark). In addition, 100 µl of C. hartmannianum extracts, pure compounds or rifampicin were added in the wells, so that the final volume in
the wells was 200 µl and thus consisting of 2.5 × 105 colony-forming
units (CFU)/ml. The wells containing extracts, or antibiotics and bacteria were called test wells (GT). Moreover, the microplate wells consisted of growth control (GC) wells that contained 100 µl of the bacterial
suspension (containing 5 × 105 CFU/ml) and 100 µl of Dubos broth so
that the final volume of 200 µl contained an inoculum of 2.5 × 105
CFU/ml. In addition, the sample control wells (SC), made individually
for each two-fold dilutions of the extracts and rifampicin, contained a
volume of 100 µl of the extracts, pure compounds or antibiotics, and
100 µl of Dubos broth without bacterium. Sample controls were used to
subtract eventual absorption resulting from the color pigments or precipitates in the extracts and the antibiotic. Methanol and hexane were
used as solvent controls at a maximum concentration of 5%, and at this
concentration, no growth inhibition was detected. The 96-well microplates were incubated in a shaking incubator (Stuart® SI500, UK) at
+ 37 ◦ C at 100 rpm for four days. Subsequently, the turbidity of the
wells was measured at 620 nm using a spectrophotometer (Victor 1420,
Wallac, Finland). The percentage growth was calculated as the mean
percentage of growth of triplicates in relation to the growth of the
growth control (the growth control of freely growing bacteria was
defined as growing 100%). Therefore, the percentage growth inhibition
was obtained by subtracting the percentage growth value of the test
sample from the growth control (100% growth). Calculations for percentage growth and % growth inhibition are explained by the formulas
(5) and (6) below. All assays were done in triplicate, and the experiments were repeated three times. The lowest concentration that
inhibited ≥ 90% of the mycobacterial growth (resulting in no visible
growth) was defined as the minimum inhibitory concentration (MIC).
Applied formulas for the percentage of growth and growth inhibition
calculations:
ppm (parts per million mass error) mass accuracy = (M measured –M calculated) ×
(3)
106/M calculated
Where M measured is the measured mass in QTOF-MS and M calculated is
the exact calculated mass according to the molecular formula of the
compound. Since QTOF-MS was used in negative mode, the monoisotopic mass of the hydrogen atom (1.0078) was subtracted from the
calculated masses.
2.4. Antimycobacterial assays
2.4.1. Agar diffusion assay
Primary antimycobacterial testing was performed for all extracts
using an agar diffusion method according to Fyhrquist et al. [73]. Before
the test, Mycobacterium smegmatis ATCC 14468 was grown on
Löwenstein–Jensen agar (Becton–Dickinson & Company, USA) at
+ 37 ◦ C for five days. For the test, Petri dishes (Ø 14 cm, Bibby Sterilin,
UK), were used. A bottom layer that consisted of 25 ml of base agar
(Antibiotic medium Number 2, Difco, UK) and a top layer of 25 ml
Middlebrook 7H10 agar (Difco, UK), supplemented with oleic albumin
dextrose catalase (OADC, Difco supplement) were added to the Petri
dishes. The Petri dishes were inoculated with 200 µl of M. smegmatis
suspension, containing approximately 1.0 × 108 CFU/ml. Six filter
paper disks (Ø = 12.7 mm, Schleicher & Schuell) loaded with 200 µl of
the tested extracts (50 mg/ml) and the positive control rifampicin
(10 mg/ml, Sigma-Aldrich) were placed equidistantly on the inoculated
Petri dishes. Methanol and hexane were used as negative controls.
Before incubation, the Petri dishes were kept in the cold room (+4 ◦ C)
for two hours where after the dishes were incubated for five days at
+ 37 ◦ C. The tests were performed in triplicates, with each replicate
sample on a separate Petri dish. The results were calculated as the mean
of the diameters of the inhibition zones (IZD in mm) of three replicates
± standard error of the mean (SEM), with the replicates placed on
separate Petri dishes. In addition, also the activity indexes of the extracts
were calculated in relationto rifampicin according to the formula 4 [70,
73]:
AI (Activity index) = Inhibition zone of the plant extract / Inhibition zone of
rifampicin
(4)
The agar diffusion method was also employed to obtain the
approximate minimum inhibitory concentration (MIC) values for some
extracts, such as methanol extracts of the wood and bark and acetone,
cold and hot water extracts of the root. The mentioned extracts form
excess precipitation in broth, making it impossible to use the turbidimetric microplate assay for measuring MIC. For these experiments,
200 µl of two-fold dilutions of the extracts (39–5000 µg/ml) and
rifampicin (0.030–1000 µg/ml) were pipetted onto sterile filter papers.
The approximate MIC was estimated as the smallest concentration still
resulting in a visible inhibition zone (IZ ≤1 mm) around the filter paper
[69,78]). Moreover, the inhibition zone diameter (IZD) of the two-fold
dilutions were plotted against the concentrations of the extracts and
compounds to investigate the linearity (dose-dependency) of the growth
inhibition.
Percentage (%) bacterial growth = [(x‾ GT A620 – x‾ SC A620) / x‾ GC A620) ×
100]
(5)
Percentage (%) inhibition of growth = 100 (% growth of the growth control) –
[(x‾ GT A620 – x‾ SC A620) / x‾ GC A620) × 100]
(6)
2.4.2. Turbidimetric microplate assay
C. hartmannianum extracts and rifampicin were tested for their
minimum inhibitory concentration (MIC) and percentage growth inhibition against M. smegmatis ATCC 14468 using a turbidimetric microplate method as described by Salih et al. [70] and Fyhrquist et al. [73].
C. hartmannianum extracts were two-fold diluted from 39 to 5000 µg/ml
and rifampicin from 0.030 to 1000 µg/ml in Dubos broth (Difco). Besides, standard compounds, that had been detected in extracts of
C. hartmannianum, such as luteolin (Sigma-Aldrich, Darmstadt, Germany), ellagic acid (Sigma-Aldrich, England), gallic acid (G-7384,
x‾ = the average of the triplicates of the examined wells; GT A620
= turbidity of the test well (plant extracts, compounds, or antibiotics
incubated with the bacterium); SC A620 = turbidity of the sample control
wells (controls containing the plant extracts, compounds, or rifampicin
only with no bacteria); GC A620 = turbidity of the growth control.
2.4.3. Calculation of total activity
The total antimycobacterial activities were calculated to assess the
potential of the various solvents to extract antimycobacterial
6
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
compounds. The total activity indicates the maximum volume of solvent
to which 1 g of an extract could be diluted into and still possess antimicrobial activity [80]. The total activity was calculated using formula
(7) below as outlined by Eloff [80]):
(7) Total activity (ml/g) = extraction yield of extract (mg/1000 mg)
/ MIC of the extract in mg/ml.
Table 2
In vitro antimycobacterial activity of extracts and fractions of Combretum hartmannianum against M. smegmatis ATCC 14468. Results were obtained with an
agar diffusion method and are presented as the diameters of inhibition zones
(IZD) in mm. Activity indexes (AI) were calculated in relation to rifampicin.
2.5. Statistical analysis of data
Data resulting from the antimycobacterial assays (diameters of inhibition zones, IZD, and percentage inhibition of growth) were
expressed as the mean of replicates (n = 3) ± standard error of the mean
(SEM), obtained from three independent experiments. For the extraction
yields, the yield was expressed as the mean of two separate extractions
± SEM.
3. Results
3.1. Ethnopharmacological uses of Combretum spp., with special
emphasis on C. hartmannianum in the villages of the Blue Nile state, Sudan
According to our results, Combretum aculeatum, C. ghazalense, C.
collinum, C. glutinosum, C. nigricans, C. hartmannianum and C. molle occur
commonly in the Kordofan and Blue Nile state areas in Sudan and are
used for the treatment of various infectious diseases, acne and infected
wounds (Table 1). However, of these species, C. hartmannianum was the
most frequently used among the traditional healers for the treatment of
symptoms related to TB, including persistent cough. Traditional healers
in all villages in Al-Azaza, A-Rosiers, A-Damazein, Baw, and Al-Ubied
localities that we visited, use C. hartmannianum as a medicinal plant in
several traditional applications to treat infectious diseases, and among
them respiratory infections (Table 1). Water extracts (macerations and
decoctions) and tonics in ethanol from the leaves, roots, and stems of
C. hartmannianum are used to treat a persistent cough that could be due
to TB. Moreover, macerations are used for skin ulcer infection, toothache, wound inflammations, and infections in general. In addition,
tonics, pastes, and ointments of C. hartmannianum leaves, fruits, stem
wood, and roots are frequently used to cure various types of acne, skin
infections, and leprosy caused by Mycobacterium leprae and
M. lepromatosis. The tonics are prepared by cooking the plant for
30–40 min in water or as overnight macerations in ethanol, prepared
traditionally from the date palm or sorghum. For the pastes, dried plant
powder is boiled in animal lipid called "Karkar" for 2–3 h and then mixed
with clove and a little volume of water, whereafter the preparation is
cooled down before use. The pastes are used to cover infected areas
(wounds, fungal infections) of the skin. Ointments are prepared in the
same way as the pastes, by boiling the plant material in "Karkar" for
2–3 h, cooled-down and filtrated whereafter the preparation solidifies.
After solidification, the ointments are used as creams on the skin.
Extracts and
C. hartmannianum
fractions
IZD, Average
SEM
AI
R MeSox* **
R acetone
R hex
R EtoAc
R aqu
R Dic
R H2O٭٭
R Me٭٭
R HH2O* **
W MeSox
W hex
W H2O٭
W CHCl3
W HH2O
W aqu
W EtoAc
W Me٭٭
B EtOAc
B aqu
B MeSox
B hex
B CHCl3
B H2O٭٭
B Me٭٭
B HH2O
Rifampicin
31.50
25.67
19.73
24.67
20.50
13.00
21.00
20.00
22.00
20.17
19.00
18.00
14.67
18.17
19.67
20.67
24.33
23.00
19.00
24.83
15.00
16.00
20.00
24.67
20.33
47.5
0.29
0.33
0.15
0.17
0.29
0.00
0.00
0.00
0.17
0.33
0.00
0.00
0.17
0.17
0.17
0.17
0.17
0.00
0.00
0.17
0.00
0.00
0.17
0.17
0.17
0.29
0.66
0.54
0.42
0.52
0.43
0.27
0.44
0.42
0.46
0.42
0.40
0.38
0.31
0.38
0.41
0.44
0.51
0.48
0.40
0.52
0.32
0.34
0.42
0.52
0.43
1.00
W, stem wood; B, stem bark; R, root; L, leaves; Rb, root bark; Me* , cold
methanol extracts; EtOAc, ethyl acetate extracts; hex, hexane extracts; Dic,
dichloromethane extracts; CHCl3, chloroform extracts; aqu, aqueous extracts;
HH2O, hot water extracts (decoctions); MeSox, methanol Soxhlet extracts;
H2O* , Cold water extracts (macerations); NA, not active. NT, not tested; Filter
paper disks (∅ 12.7 mm) were saturated with 200 µl extracts/fractions (50 mg/
ml) and rifampicin (10 mg/ml); AI, Activity index in relation to rifampicin;
Diameter of inhibition zones (IZD) recorded in mm as mean of triplicates
(n = 3) ± SEM of three experiments. Most promising results (IZD ≥20 mm)
were indicated by bold text; the underlined extract was analyzed phytochemically with HPLC-DAD and UHPLC/QTOF-MS; * ** analyzed preliminary with
HPLC-DAD.
with a methanol Soxhlet and an ethyl acetate extract of the root with IZD
of 31.50 and 24.67 mm, respectively, and MIC values of 312.5 and
625 µg/ml, respectively. Moreover, the growth inhibition of the methanol Soxhlet extract of the root was dose-dependent (Fig. 3), and displayed the highest total activity of 660 ml/g, resulting from a high
extraction yield of 20.6% and the MIC of 312 µg/ml (Fig. 2 and Table 3).
Also stem bark extracts, such as a cold methanol and a methanol Soxhlet
extract, inhibited the growth of M. smegmatis with large inhibition zone
diameter (IZD) of 24.33 and 24.83 mm, respectively. Acetone was found
to be the best solvent by far to extract compounds from the root,
resulting in an extraction yield of 35% (Fig. 2). Moreover, the acetone
extract of the roots gave an IZD of 25.67 mm, and a MIC value of
2500 µg/ml. Therefore the total activity of the acetone extract was
140 ml/g (350/2.5 = 140). Applications used in traditional medicine, i.
e. the decoctions and macerations of the root and stem bark, showed
some growth inhibition (IZD 20–22 mm), but the MIC value for the
decoction of the root was high (5000 µg/ml) and thus resulting in low
total activity of 19.6 ml/g.
In addition, some compounds that we detected in C. hartmannianum
roots were evaluated for their antimycobacterial effects (Table 3). Of
these compounds, luteolin showed the strongest effects (MIC 250 µg/
ml), while gallic acid and ellagic acid showed MIC values of 500 µg/ml
and corilagin a MIC value of 1000 µg/ml. Moreover, apigenin and
quercetin, of which apigenin and quercetin glycosides were previously
3.2. Antimycobacterial effects, extraction yields, and total activity
A total of thirty-one extracts of various polarities from the stem
wood, stem bark, and root of Combretum hartmannianum, were subjected
to antimycobacterial screening (Tables 2 and 3). Alongside, also the
total activities, which are dependent on the extraction yields (Fig. 2) and
MIC values are shown in Table 3. The results revealed that hydrophilic
extracts of C. hartmannianum were mostly more potent growth inhibitors
than the lipophilic extracts, although this result could be due to the agar
diffusion method that was used for the primary screening (Table 2).
Moreover, MIC values were not estimated for the lipophilic extracts due
to the small volumes of samples available. When compared to the more
polar extracts, the hexane, dichloromethane, and chloroform extracts of
C. hartmannianum showed low to moderate growth inhibitory activity
against M. smegmatis with inhibition zone diameters (IZD) ranging from
13 to 19.7 mm (Table 2). The strongest growth inhibition was obtained
7
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
C. hartmannianum, all of them giving growth inhibitory effects against
M. smegmatis in our analysis (Tables 2 and 3). The results of the antioxidant analysis are shown in Fig. 4. Accordingly, those spots (compounds or fractions) on the TLC plate that show antioxidant activity
exhibit a light yellow to white color due to a color change of the DPPH
reagent from a dark purple (violet) to yellow, as a result of the reaction
and neutralization of the DPPH radical with the hydrogen atoms and
other functional groups found in the antioxidant molecules of the extracts [81]. As can be seen in Fig. 4 (1C, 2C, and 3C) the phytochemical
profiles of all three extracts look very similar. Strong antioxidant activity
could be found among compounds within a wide range of polarities in all
the studied extracts, and especially among the ellagic acid derivatives,
the ellagitannins, and gallic acid, with luteolin being less active as
judged by DPPH color change intensity (Fig. 4, 1 D and F). The results
show that the root decoction is rich in antioxidant ellagitannins that
could be important for its use to treat infections in traditional medicine.
Table 3
Minimum inhibitory concentrations (MIC) in µg/ml of extracts and fractions of
C. hartmannianum and pure compounds against Mycobacterium smegmatis ATCC
14468. The total activity was calculated as the relation between MIC and
extraction yield.
C. hartmannianum
extracts
MIC
Extraction
yield in mg
from 1 g
plant
material
(Percentage
yield)
Total activity
(ml/g)
W EtOAc
W Me*
B MeSox
B EtOAc
B Me٭
R MeSOx
R acetone
R aqu
R EtOAc
R HH2O
Rifampicin
5000 (IC 97)* *
2500
2500
5000 (IC 90)* *
5000
312.5 (IC 95)* *
2500
5000
625 (IC 99)* *
5000
39.06 (3.90, IC
98)* *
45 (4.5%)
28 (2.8%)
189 (18.9%)
97 (9.7%)
21 (2.1%)
206 (20.6%)
351 (35.1%)
200 (20.0%)
115 (11.5%)
98 (9.8%)
9
11.2
75.6
19.4
4.2
660
140
40
184
19.6
Pure compounds
Corilagin
Gallic acid
Luteolin
Quercetin
Apigenin
Ellagic acid
3.4. HPLC-DAD and UHPLC/QTOF-MS results on the phytochemistry of
a root extract
An ethyl acetate extract of the root of C. hartmannianum was chosen
for phytochemical analysis due to the fairly good antimycobacterial
activity shown by this extract. Another criterium for the use of this
extract for our phytochemical analysis was that, when compared to the
methanol extract of the root, this extract had a cleaner profile, with the
ellagitannins (ETs) better separated from each other. Moreover, according to our preliminary HPLC-DAD results, most of the ETs present in
the methanol extract also occurred in the ethyl acetate extract. HPLCDAD and UHPLC/QTOF-MS analysis led to the identification of 54
compounds of which most were phenolic compounds, including hydrolyzable tannins and flavonoids (Fig. 5, Fig. 6, and Table 4).
1000 (IC 94)* *
500 (IC 98)* *
250 (IC 91)* *
250 (IC 94)* *
250 (IC 97)* *
500 (IC 98)* *
W, stem wood; B, stem bark; R, root; L, leaves; Me* , cold methanol extracts;
EtOAc, ethyl acetate extracts; HH2O, hot water extracts (decoctions); MeSox,
methanolic Soxhlet extracts; H2O* , Cold water extracts (macerations); IC, the
inhibitory concentration that indicates the percentage growth inhibition at the
MIC concentration; * *, MIC measured with microdilution method and other
MIC results obtained with an agar diffusion method. Corilagin, gallic acid, and
luteolin were characterized from an ethyl acetate extract of the root of C. hartmannianum in this study.
3.4.1. Ellagitannins, ellagic acid and gallic acid derivatives
Ten ellagitannins (ETs) with molecular weights ranging between 470
and 1084 Da were tentatively identified in an ethyl acetate extract of the
root of C. hartmannianum based on their retention times and UVλ absorption maxima in HPLC-DAD (Figs. 5 and 7) and their [M-H]- ions
resulting from UHPLC/QTOF-MS (Table 4, Figs. 5 and 7). Six other
compounds were also identified as ellagitannins based on their UVλ
absorption maxima spectra in HPLC-DAD, with three distinct absorption
maxima (Fig. 7), although the molecular masses could not be determined for these compounds (Table 4). Among the tentatively identified
ellagitannins were corilagin (7), terflavin B (6), and its two isomers (4
and C), castalagin (5), tellimagrandin I (9), a tellimagrandin I derivative
detected in the leaves of C. hartmannianum [39], showed a MIC value of
250 µg/ml.
3.3. Antioxidant effects
Since the antioxidant capacity of a plant extract could be an
important second therapeutic ability in addition to its antimycobacterial
effect, we assessed the qualitative antioxidant capacity for a methanol
Soxhlet extract, a decoction, and an ethyl acetate extract of the root of
Fig. 2. Percentage extraction yields resulting from (A), Sequential extraction and solvent partition extraction and (B), single solvent extraction from the roots, stem
bark, and stem wood of Combretum hartmannianum. Red bars = root; blue bars = stem bark; green bars = stem wood. Ethyl acetate (EtOAc); aqueous (aqu, obtained
from 80% MeOH); acetone (acet); hot water extracts (HH2O); hexane extracts (hex); dichloromethane extracts (Dic); cold water extracts (H2O*); methanolic Soxhlet
extract (MeSox); and cold methanol extracts (Me*); (R), root; (W), stem wood and (B), stem bark.
8
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
Fig. 3. Concentration-dependent growth inhibition of a methanolic Soxhlet extract of Combretum hartmannianum root against Mycobacterium smegmatis.
Fig. 4. Thin layer chromatograms (RP 18-TLC) of a
(1) methanolic Soxhlet extract, an (2) ethyl acetate
extract, and a (3) decoction of the root of
C. hartmannianum. The same extract is photographed
at (A) 254 nm; (B) 366 nm; and (C) after treatment
with the DPPH reagent. Rf = retardation factors for
the standard compounds. The red braces in 1, 2, and
3 show the ellagitannin-zone (ETs). The dotted line
shows the positions of the standard compounds in
the extracts. D and F contain eluted standard
compounds.
(12), α-punicalagin (11), β-punicalagin or terchebulin (13) and (S)flavogallonic acid dilactone (8) (Fig. 5). Besides, gallic acid (1a), an
ellagic acid derivative (14), methyl-3,4,5-trihydroxy benzoate (1b),
methyl ellagic acid xyloside (18), and epigallocatechin gallate (1 C)
were characterized from the root of C. hartmannianum.
good agreement with compounds found in our natural compounds library (Agilent Chem Station software), as well as with triterpenes in
SciFinder and PubChem databases, and with the literature [90–92].
Among these terpenes, the tetrahydroxylated pentacyclic triterpene,
terminolic acid (S) (Syn. 2α,3β,4α,6β)− 2,3,6,23-tetrahydroxyolean-12-en-28-oic acid) was tentatively identified. Terminolic acid is
also known as the aglycone of chebuloside II (Fig. 6 G) [93,94]. In
addition, the saponin combregenin (J) and the norditerpene furan
glycoside, cordifoliside D (Q) were tentatively identified in the root of
C. hartmannianum.
3.4.2. Flavonoids
Twelve peaks in the C. hartmannianum root ethyl acetate extract
HPLC-DAD-chromatograms could be assigned to the group of flavonoids
based on their retention times and UVλ absorption spectra, showing two
main UV–visible absorption maxima bands; one at 240–285 nm for the B
ring and another at 300–400 nm for the A & C rings [82–89]. Moreover,
three flavonoids could be tentatively characterized as luteolin (H), isorhamnetin
(L),
and
quercetin-3-O-galactoside-7-O-rhamnoside-(2→1)-O-β-D-arabinopyranoside (F) (Table 4, Fig. 5).
4. Discussion
4.1. Results from this present study on the antimycobacterial effects of
extracts of C. hartmannianum in relation to previous studies of African
species of Combretum
3.4.3. Triterpenes
Our UHPLC/QTOF-MS analysis of the ethyl acetate extract of the
C. hartmannianum roots revealed for the first time the presence of three
terpenes in this plant (Table 4 and Figs. 5 and 6). Their UV–visible
spectroscopic data and retention times (Table 4, Figs. 5 and 7) were in
Various traditional medicinal preparations, such as decoctions,
macerations, fumigations, tonics, ointments, and pastes of Combretum
hartmannianum are used in Sudanese, West African, and Sahelian
traditional medicine for the treatment of infectious diseases and
9
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
Fig. 5. HPLC-DAD chromatogram at 270 nm of an ethyl acetate extract of the root of Combretum hartmannianum. Compound (1a), Gallic acid; (2), unknown
ellagitannin; (3), unknown ellagitannin; (C), Terflavin B isomer I; (4), Terflavin B isomer II; (5), Castalagin; (6), Terflavin B; (7), Corilagin; (8), S-flavogallonic acid
dilactone; (9), Tellimagrandin I; (1b), Methyl 3,4,5-trihydroxybenzoate; (10), Di-galloyl glucose; (11), α-Punicalagin; (12), Tellimagrandin I derivative; (13),
β-Punicalagin or Terchebulin; (14), Ellagic acid derivative; (15), Tri-galloyl glucose; (16), Tetra-galloyl glucose; (17), ellagic acid derivative; (18), Methyl ellagic
acid xyloside; (19), Unknown ellagitannin; (20), gallotannin; (1 C), Epigallocatechin gallate; (F), Quercetin 3-O-galactoside-7-O-rhamnoside – (2→1)-O-D-arabinopyranoside; (H), Luteolin; (J), Combregenin; (L), Isorhamnetin; (Q), Cordifoliside D; (S), Terminolic acid; (A, B, D, E, G, I, K, M, N, O, P, and R), are unknown
compounds including unknown flavonoids. Their UV absorption maxima are shown in Fig. 7, Table 4.
Fig. 6. Ellagitannins, pentacyclic triterpenes, and a diterpene were tentatively characterized in C. hartmannianum roots in this study. (A), Terchebulin; (B),
α-Punicalagin R = H, R1 = OH and β-Punicalagin R = OH and R1 = H; (C), Castalagin, a c-glycosidic ellagitannin with an open chain glucose core; (D), Tellimagrandin I (1-Desgalloyleugeniin); (E), Terflavin B and its triphenyl (flavogallonyl) ester group in red color; (F), Corilagin; (G), Terminolic acid (Chebuloside II);
(H), Combregenin; (I), Cordifoliside D. HHDP; hexahydroxydiphenic acid; NHTP, nonahydroxytriphenic acid in blue color.
infections on the skin, including symptoms related to tuberculosis, as
could be observed both in this present study and by other authors [61,
62,95]. In this present study, we found that in seven villages in Sudan,
C. hartmannianum was the most commonly used species of the Combretum spp. included in our study for the treatment of symptoms related
to TB. Despite the common use of decoctions, tonics and macerations of
C. hartmannianum for bacterial infections and cough [28,61,62], there
are only two studies to date that have confirmed that C. hartmannianum
possesses antimycobacterial potential; Eldeen and Van Staden [32]
showed that ethanol extracts of the leaves of C. hartmannianum gave
good growth inhibitory effects against Mycobacterium aurum A+ with a
MIC value of 190 µg/ml and Ahmed [13] reported that leaf and seed
extracts of C. hartmannianum inhibit the growth of clinical strains of
M. tuberculosis. These results are in agreement with our findings that
10
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
Table 4
HPLC-DAD and UHPLC/ QTOF- MS data of polyphenolic compounds and derivatives in the root of C. hartmannianum.
Combretum hartmannianum
Root ethyl acetate extract
Molecular
formula
Rt HPLCDAD
Gallic acid (1a)
Unknown (A)
Unknown (B)
Gallotannin
Gallotannin
Ellagitannin (2)
Ellagitannin (3)
Terflavin B isomer I (C)
Castalagin (5)
Terflavin B isomer II (4)
Terflavin B (6)
Corilagin (7)
(S)-Flavogallonic acid dilactone (8)
Flavonoid (D)
Tellimagrandin I (9)
Methyl gallate (Methyl 3,4,5-trihydroxybenzoate) (1b)
Gallotannin
Di-galloyl-β-D-glucose (10) together with
α-Punicalagin (11)
Tellimagrandin I derivative (12)
Gallotannin
β-Punicalagin or Terchebulin (13)
Gallotannin
Ellagic acid derivative (14)
Tri-О-galloyol-β-D-glucose (15)
Ellagitannin
1,2,3,6-Tetragalloylglucose (16)
Ellagic acid derivative (17)
Methyl ellagic acid xyloside (18)
Ellagic acid derivative
Flavonoid (E)
Epigallocatechin gallate (1 C)
Gallotannin
Quercetin 3-O-galactoside-7-O-rhamnoside –
(2→1)-O-D-arabinopyranoside (F)
Flavonoid (G)
C7H6O5
1.74
4.8
5.3
6.02
6.81
8.30
8.88
8.90
10.87
10.1
11.30
12.10
12.85
13.4
13.6
14.8
Luteolin (H)
Flavonoid (I)
Combregenin (2α,3β,6β,19α,23pentahydroxyolean-12-en-28-oic acid) (J)
Ellagitannin (19)
Unknown (K)
Isorhamnetin (L)
Flavonoid (M)
Gallotannin (20)
Unknown (N)
Unknown flavonoid (O)
Unknown (P)
Cordifoliside D (Q)
Unknown flavonoid (R)
Terminolic acid (S)
C34H24O22
C41H26O26
C34H24O22
C34H24O22
C27H22O18
C21H10O13
C34H26O22
C8H8O5
C20H20O14
C48H28O30
C34H26O22
C48H28O30
C27H24O18
C34H28O22
C20H16O12
C22H18O11
C32H38O20
14.9
15.1
15.6
15.9
16.5
17.6
17.7
18.6
18.9
19.8
19.9
20.9
21.9
22.0
22.1
24.3
24.3
25.4
Rt UHPLCDAD (min)
[M-H]-
Exact
calculated
mass (M+)
PPM
value
UV λ absorbtion
max.
216, 272
214, 225
214,225
216, 272
220, 282
214, 260, 380
218, 260, 380
218, 260, 380
215, 259, 377
218, 258, 380
216, 258, 378
216, 260, 382
214, 256, 380
216, 256, 280
216,256,380
216, 280
1.426
169.0147
170.0213
7.05
2.975
3.125
3.541
3.641
3.824
783.0709
933.0640
783.0699
783.0701
633.0725
469.0059
784.0750
934.0702
784.0750
784.0750
634.0798
470.0247
4.71
1.71
3.44
3.44
0.78
– 4.47
3.908
4.157
785.0860
183.0301
786.0906
184.0369
4.07
5.43
4.123
4.424
4.657
483.0801
1083.0574
785.0869
484.0846
1084.0654
786.0906
6.81
– 0.18
5.21
5.473
1083.0600
1084.0654
2.21
5.539
635.0899
636.0954
3.61
6.472
8.316
8.504
787.1030
788.1062
5.83
447.0659
448.0636
-4.44
9.940
457.0768
458.0843
0.65
10.502
741.4083
742.5644
-4.01
28.9
C15H10O6
30.2
12.933
285.0782
286.2070
-5.12
C30H48O7
30.8
31.9
13.050
519.3359
520.6374
-5.75
C16H12O7
C26H34O12
C30H48O6
33.0
33.9
34.9
36.2
36.9
37.1
38.9
39.9
40.6
41.1
43.2
14.982
315.1000
316.2306
-4.91
15.847
537.1568
538.4872
-4.90
16.599
503.3402
504.6384
-5.94
218, 286
216, 278
216, 256,
210, 258,
218, 274
216, 256,
210, 278
210, 254,
216, 278
218, 258,
224, 276
254, 362
254, 368
210, 254,
216, 262,
210, 276
216, 298
218, 258,
340
220, 270,
370
214, 264,
314
216, 280,
218, 260,
216, 256,
210, 230,
218, 274,
218, 278,
216, 298
230, 264,
216, 280,
210, 290
218, 276,
218, 280,
220, 266,
Peak area
(%)
from HPLCDAD
1.1663
1.2229
1.2172
0.1446
0.0448
0.5014
1.6658
6.3051
2.5805
3.4495
7.0732
0.2506
0.2506
0.0372
284,
0.0426
0.9788
14.2385
0.1969
0.4149
0.9735
0.0622
1.5896
1.5234
0.3178
0.3025
2.3194
9.7547
0.5374
1.2071
0.1298
1.1498
1.6271
300,
1.6434
280,
2.9317
314
282
1.1143
3.5554
362
280
300
302
0.9248
0.7443
4.0821
0.9841
1.1489
1.5567
0.9987
1.2079
4.0442
0.3638
1.0830
380
364
278
380
360
366
280
372
314
300
302
370
The exact calculated mass was obtained from the molecular formula and is the sum of the monoisotopic masses and numbers of the atoms in the molecule; Compounds
were detected at 270 nm; UVλ absorption maxima were obtained from HPLC-DAD. Peak area % was obtained from an HPLC-DAD chromatogram at 270 nm.
methanol and ethyl acetate extracts of the root of C. hartmannianum
possess fairly good antimycobacterial effects against M. smegmatis (MIC
312 and 625 µg/ml, respectively) (Table 3). However, although results
from our ethnopharmacological survey of the traditional medicinal uses
of C. hartmannianum in Sudan indicate that decoctions and macerations
are used for the treatment of persistent cough that could be related to
TB, we found that these preparations were not as effective as the
methanol and ethyl acetate extracts against M. smegmatis (MIC
5000 µg/ml for the decoction) (Tables 1 and 3). Also, the most
outstanding growth inhibitory effects in our study were shown by a
methanol extract (MIC 312 µg/ml), which could imply that ethanol
extracts (that would contain approximately the same composition of
compounds as the methanol extract, both qualitatively and quantitatively) of the root could have better potential than decoctions and
macerations as a traditional medicinal preparation for the treatment of
TB. This view is also supported by the study of Eldeen and Van Staden
[32], who found that ethanol extracts of C. hartmannianum were effective inhibitors of the growth of Mycobacterium aurum A+ . Therefore, the
results of this present investigation and the results of Eldeen and Van
Staden [32] could justify the use of ethanolic tonics of C. hartmannianum
for the treatment of persistent cough (Table 1).
Our results on the antimycobacterial effects of extracts of
C. hartmannianum are in agreement with those of many authors who
report that several African species of Combretum possess
11
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
Fig. 7. UVλ-visible absorption maxima spectra of ellagitannins and flavonoids in an ethyl acetate root extract of Combretum hartimannianum. (2), Unknown ellagitannin; (3), Unknown ellagitannin; (4), Terflavin B isomer II; (5), Castalagin; (8), S-flavogallonic acid dilactone; (10), Di-galloyl glucose; (11), α-punicalagin; (12),
Tellimagrandin I derivative; (D, E, G, I, M, N, O, P and R), unknown flavonoids; (C), Terflavin B isomer I; (F), Quercetin 3-O-galactoside-7-O-rhamnoside – (2→1)-OD-arabinopyranoside; (H), Luteolin; (J), Combregenin; (L), Isorhamnetin; (S), Terminolic acid (Chebuloside II); (Q), Cordifoliside.
antimycobacterial potential. For example, Eldeen and Van Staden [32]
reported that ethyl acetate and ethanol extracts of the leaves, root, and
bark of C. kraussii showed growth inhibitory effects against Mycobacterium aurum A+ , with MIC values ranging from 196 to 1500 µg/ml. In
addition, Asres et al., [66] reported that acetone extracts of the stem
bark of C. molle were mildly growth inhibitory against M. tuberculosis
(MIC ˃ 1000 µg/ml) and Lall and Meyer [96] showed that an acetone
extract of the bark of C. molle gave a MIC value of 500 µg/ml against
M. tuberculosis H37Rv. Moreover, Fyhrquist et al. [97] found that stem
bark and root extracts of Combretum padoides, C. zeyheri, and C. psidioides
had good antimycobacterial potential against M. smegmatis, and a
methanol extract of the stem bark of C. psidioides gave the lowest MIC of
625 µg/ml. Acetone extracts of the leaves of C. hereroense and an ethanol
extract of the leaves of C. imberbe, gave good antimycobacterial effects
(MIC 125 μg/ml and 470 μg/ml, respectively) against M. smegmatis [98,
99]. In addition, alkaloid enriched extracts from leaf extracts of
C. zeyheri, C. molle, and C. platypetalum were antimycobacterial against
M. smegmatis [100]. Interestingly, Queiroz et al., [101] demonstrated
that water decoctions of the leaves, flowers, and stem of C. aculeatum,
which are used for tuberculosis in Senegalese traditional medicine, gave
good antimycobacterial effects in a host-pathogen assay using Mycobacterium marinum, and Acanthamoeba castellanii. This would imply that
decoctions of Combretum aculeatum, and possibly also other Combretum
spp., and amongst them C. hartmannianum, could contain compounds
useful for the treatment of the more drug-resistant, dormant TB found in
infected people without symptoms.
different polarities with antioxidant activities, such as ellagitannins,
ellagic acid derivatives, flavonoids, and gallic acid (Fig. 4). This would
imply that the traditional preparations, such as ethanolic tonics and
decoctions would be effective for the treatment of infections also via
their antioxidant effects. To the best of our knowledge, there are no
previous reports on the antioxidant effects of C. hartmannianum root
extracts. However, Taha et al., [25], Mariod et al., [102], and Hassan
et al., [103], demonstrated that methanol leaf and stem bark extracts of
C. hartmannianum possess good antioxidant effects. Moreover, Hassan
et al., [103] attributed the strong DPPH scavenging activity of the leaves
of C. hartmannianum mostly to the high flavonoids content of this
extract. However, we found that in the root, mainly ellagitannins,
including corilagin and punicalagin and ellagic acid derivatives give
good antioxidant effects (Fig. 4 C). In agreement with our results, corilagin and punicalagin were found to possess strong antioxidant effects
[104,105]. We found that punicalagin was quantitatively the main ET in
the root of C. hartmannianum (Table 4, Figs. 5 and 6), and could thus
contribute to the antimycobacterial effects of the root extracts by stimulating the immune cells of the host via its antioxidant effects. Moreover, terchebulin and ellagic acid are good DPPH radical scavengers
[106]. In our study another ET in the root of C. hartmannianum, which
has the same MW as punicalagin, but a longer retention time in
HPLC-DAD could be terchebulin, and could thus contribute to the
antioxidant effect of the root extract. Interestingly, Seeram et al., [105]
found that punicalagin alone was not as antioxidative as pomegranate
juice, and therefore they implied that the ellagitannins in the juice
potentiate the antioxidant effects of each other. Therefore, similarly, the
combination of ETs in the root extracts of C. hartmannianum could be
more effective than individual ellagitannins (and other polyphenols)
from this extract. This warrants quantitative analysis of the antioxidant
capacity of root extracts of C. hartmannianum in relation to that of single
4.2. Antioxidant effects of C. hartmannianum
We found that a methanol, an ethyl acetate, and a hot water extract
of the root of C. hartmannianum contain a range of compounds of
12
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
ETs separated from this extract.
traditional medicinal preparations, such as decoctions and macerations
of Combretum spp. are rich in ETs [97]. To date, there are six African
species of Combretum that have been studied for their ETs [40,52,66,97,
101,122]. The ETs might have a greater impact than has previously been
understood on the medical value of traditional preparations of Combretum spp. [101].
In our study, ten ellagitannins were tentatively characterized from
the root of C. hartmannianum for the first time. The characteristic UV
absorption with three maxima at 214–220, 254–256, and 370–380 nm
revealed that these compounds contain gallic and ellagic acid units
(Table 4, Figs. 5, 6, and 7; [123,124]. An ellagitannin at tR 15.6 min in
HPLC-DAD was the quantitatively most abundant ellagitannin and is
tentatively suggested to be the α-anomer of punicalagin (Table 4, Figs. 5,
6 and 7). Moreover, another ellagitannin at HPLC-DAD tR 17.6 min,
with the same molecular mass as punicalagin (1084.0654), is suggested
to be either the β-anomer of punicalagin or terchebulin, since both
ellagitannins have longer retention times than α-punicalagin, and terchebulin has a mass identical to punicalagin [101,125]. Terchebulin
differs from α-punicalagin in containing terchebulic acid, a gallic acid
tetramer [126], whereas α-punicalagin contains hexahydroxydiphenic
acid [127], (Fig. 6 A and B). Separations of the two anomers of punicalagin by HPLC-DAD have been described by many authors and the
differences in their retention times is based on slight differences in the
positions of the OH-group and the H-atom linked to the anomeric C1
atom (Fig. 6 B), affecting both the polarity and the UVλmax absorption
spectra (Table 4) of these anomers [43,101,128–131]. Punicalagin has
been found in some other African Combretum spp., such as C. psidioides
and C. zeyheri [97], C. molle [66,122], C. glutinosum [52], and in
C. aculeatum [43,101]. Terchebulin was earlier characterized from the
stem bark of C. hartmannianum [40]. Moreover, punicalagin and terchebulin were reported from many Terminalia spp. [67,69,75,105,125].
Regarding the potential of ETs as inhibitors of mycobacterial growth,
only a couple of studies have been made: For example, punicalagin from
the stem bark of C. molle gave mild in vitro growth inhibitory activity
against M. tuberculosis typus humanus ATCC 27294 with a MIC
> 600 µg/ml [66]. In addition, Queiroz et al., [101], found that the two
isomers of punicalagin, α- and β-punicalagin, isolated from a water
decoction of Combretum aculeatum, effectively inhibited the growth of
Mycobacterium marinum in Acanthamoeba castellanii host cells. In this
same investigation, Queiroz et al., [101], also included the punicalagin
metabolites, the urolithins, which were not as effective growth inhibitors as punicalagin. However, they concluded that the high consumption of tannins that would be achieved by taking the traditional
decoction would lead to plasma levels of the urolithins that would be
relevant for their antimycobacterial effects. Therefore, intake of decoctions and ethanolic tonics of C. hartmannianum root, preparations
that are used to treat persistent cough in Sudanese traditional medicine,
could likewise lead to the same in vivo antimycobacterial effect, as we
have found that these preparations are rich in punicalagin.
Punicalagin and terchebulin were the principle components in an
aqueous ethanolic extract of the root of Terminalia macroptera and were
suggested to be responsible for the anti-Helicobacter pylori effects of this
extract [132]. In addition, terchebulin from Terminalia laxiflora showed
good growth inhibitory effects against Propionibacterium acnes [106].
Recently, punicalagin was shown to give moderate growth inhibitory
effects against Staphylococcus aureus (MIC 600 µg/ml) and to inhibit the
growth of Escherichia coli as one of the more effective ETs among 23
tested ellagitannins [133]. Besides, punicalagin A (α-) and B (β-)
inhibited the growth of Streptococcus spp. [30,130] and MRSA
[134–136].
In our study, we could tentatively confirm the presence of the
monomeric ellagitannin corilagin ([M-H]- 633.0725) in small quantities
in the root of C. hartmannianum (Table 4, Figs. 5 and 6). Previously,
corilagin was characterized in one other Combretum spp., C. psidioides
[97], in many Terminalia species [125,132,137,138], and Lumnitzera
spp. (also a genus within the Combretaceae family) [139]. Corilagin
4.3. Phytoconstituents characterized in this study in a root extract of
C. hartmannianum and their possible contributions to its antimycobacterial
activity
Limited data is available on the phytochemistry of C. hartmannianum,
and to the best of our knowledge, no research has been performed on the
phytoconstituents of the roots of this species.
4.3.1. Flavonoids
Among the 12 flavonoids that were identified in this present study in
the root of C. hartmannianum, luteolin (H), isorhamnetin (L), and a
quercetin glycoside; quercetin-3-O-galactoside-7-O-rhamnoside-(2→1)O-β-D-arabinopyranoside (F) could be tentatively identified. Luteolin
and quercetin-3-O-galactoside-7-O-rhamnoside-(2→1)-O-β-D-arabinopyranoside were not described earlier in C. hartmannianum. Luteolin and
isorhamnetin have been found in other Combretum spp. such as the African species, C. dolichopetalum, and C. aculeatum, and the Asian and
South-American species, C. latifolium, and C. lanceolatum [42,107–109].
Earlier, Ali et al., [39] studied the polyphenol composition in a leaf
extract of C. hartmannianum, resulting in the characterization of 16
flavonoids, of which many were either glycosides or methylated,
methoxylated and hydroxylated flavonoids, including methoxylated and
glucosylated isorhamnetin derivatives, glycosylated quercetin derivatives, kaempferol ([M+H]+ 287) and its glycosylated and
methoxylated derivatives, chrysoeriol ([M+H]+ 301) and its glycosylated derivative, apigenin ([M+H]+ 271), and hydroxylated and
methoxylated flavone derivatives. The methoxylated and hydroxylated
flavonoids have been reported to be common within the genus Combretum [107].
The flavonoids that we have characterized in the ethyl acetate
extract of the root of C. hartmannianum could contribute to the antimycobacterial effect of this extract presented in our study (Table 3,
Fig. 4), since flavonoids in general are considered to form part of the
plant defense system against pathogenic bacteria [110,111]. However,
we found that luteolin and apigenin gave rather high MIC values of
250 µg/ml against M. smegmatis (Table 3). Accordingly also Lechner
et al., [112] found that flavonoids gave weak to mild growth inhibitory
effects against mycobacteria, with quercetin, isorhamnetin, and luteolin
inhibiting the growth of M. smegmatis mc2 155 with MIC values ranging
from 128 to 256 µg/ml.
Flavonoids could act as antimycobacterial agents via their ability to
act as protein chelators and thus affect the function of many important
bacterial enzymes and proteins including the aminoglycoside-modifying
enzymes (AME:s), efflux pumps, enzymes involved in the respiratory
chain as well as enzymes involved in membrane and cell wall synthesis
(Fatty acid synthase complex I and II, FASI and II) [113,114]. Moreover,
flavonoids could also exert their antimicrobial effects by complexing
with the bacterial cell wall, and by disrupting the microbial cell membrane [113–115]. For example, more lipophilic flavonoids, such as
apigenin and quercetin are known to disrupt bacterial membranes [116,
117]. Interestingly, some flavonoids, for example, quercetin, are known
to act as inhibitors of the mycobacterial proteasome, a viable and relatively newly discovered drug target for new anti-TB drugs [118]. The
proteasome is involved in the maintenance of the non-replicating form
of mycobacteria, for which there are no available effective conventional
drugs [118,119]. Moreover, the structure-activity relationship (SAR)
studies have revealed that for the flavonoids the presence of a C-ring, as
well as, the number, and positions of free 5-, and 7- hydroxyl groups, and
the position, and number of sugar residues are important for their
antimycobacterial activity and bioactivity in general [120,121].
4.3.2. Ellagitannins
Ellagitannins (ETs) are generally not well studied within the genus
Combretum compared to the genus Terminalia [53,75], although
13
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
consists of one hexahydroxydiphenoyl group (HHDP) connected with a
di-ester bond to 3-O and 6-O of D-glucose (Fig. 6 F). According to our
result, corilagin gave a weak growth inhibitory effect against
M. smegmatis (MIC 1000 µg/ml) as compared to the crude methanol or
ethyl acetate extracts showing MIC-values of 312 and 625 µg/ml,
respectively (Table 3). Therefore, corilagin and the other ETs in the
C. hartmannianum extracts are suggested to act synergistically with each
other and other compounds to enhance the antimycobacterial effect of
the extract. To the best of our knowledge, corilagin has not been
investigated for its effects against mycobacteria previously. Thus, more
testing of this and other ETs in C. hartmannianum, alone and in combinations, should be performed against M. tuberculosis, with dormant
phenotypes included. Other authors have reported that corilagin possesses mostly weak antibacterial effects. For example, Puljula et al.,
[133], found that many other ETs, such as castalagin and tellimagrandin
I, of which both are present in the root of C. hartmannianum according to
our result, were much more antibacterial than corilagin. However, in
another study, corilagin gave moderate growth inhibitory effects against
S. aureus, E. coli, and C. albicans (MIC 31.25–62.5 µg/ml) and the effect
was stronger than for erythromycin against E. coli [140]. Moreover,
corilagin was found to affect the membrane permeability of E. coli and
C. albicans in a dose-dependent manner, but did not affect S. aureus
[140]. Interestingly, corilagin had potentiating effects on conventional
antibiotics, and reduced the MIC of various β-lactams by 100- to
2000-fold against MRSA, and worked synergistically together with
oxacillin to produce a bactericidal effect [141]. This warrants further
research in the antimycobacterial potency of corilagin and its combinations effects with rifampicin and other antibiotics used for the treatment of TB. Corilagin could have a good potential as a new antibacterial
(and anti-TB) agent since it is a small enough molecule to be bioavailable
and was found to occur in rat and mice blood plasma after oral intake
and showed no toxicity in acute toxicity tests [142].
Our results showed that the small molecular hydrolyzable tanninrelated tri-galloyl compound, (S)-flavogallonic acid dilactone ([M-H]469.0059) was present in the root of C. hartmannianum in high concentrations (Table 4; Fig. 5). (S)-flavogallonic acid dilactone was reported to occur also in the stem bark of C. hartmannianum [40]. To the
best of our knowledge, no other species of Combretum has been found to
contain (S)-flavogallonic acid dilactone. Moreover, a tannin-related to
(S)-flavogallonic acid dilactone, that possesses an open ring instead of
the lactone ring, (S)-flavogallonic acid (MW 458.32), was found in
Terminalia laxiflora and showed good growth inhibitory effects against
Propionibacterium acnes [106]. In addition, (S)-flavogallonic acid, isolated from the root of T. sericea, showed broad-spectrum antibacterial
activity with MIC values ranging from 110 to 750 µg/ml [143].
We found that the root of C. hartmannianum contains castalagin. To
the best of our knowledge castalagin has not been reported to occur in
the genus Combretum before. However, castalagin was found to be one of
the major ETs in the stem bark of Anogeissus leiocarpa, also a species
belonging to the Combretaceae family [123]. Castalagin ([M-H]- ion at
m/z 933.0640) is a rare C-glycosidic ET having an open-chain glucose
core with the bond configuration of (C-O-C) and shows an S-S stereochemical arrangement of the triphenyl moiety (Fig. 6 C) [144–146]. In a
recent study, Araú jo et al., [147] reported that castalagin gave bactericidal effects against methicillin-resistant S. aureus, and affected the
assembly of peptidoglycan units in the bacterial cell wall. In agreement
with this study, also Puljula et al., [133] found that castalagin effectively
inhibits the growth of S. aureus, and was among the most effective ETs in
this respect compared to 22 other ETs. Moreover, castalagin was found
to suppress the growth of E. coli with a MIC value of 705 µg/ml, as well
as to inhibit the growth of Pseudomonas aeruginosa, Clostridia, and
Staphylococcus aureus [148,149].
We found that the monomeric ET, tellimagrandin I ([M-H]785.0860) and its derivative were present in the root extract of
C. hartmannianum. Tellimagrandin I is a carbon-carbon bond ET (C-C)
and has one hexahydroxydiphenyl group and two galloyl groups
connected with the glucose core molecule (Fig. 6 D) [144–146]. Tellimagrandin I was found to potentiate the activity of β-lactams against
MRSA [150]. Besides, in a large screening of the antimicrobial activities
of 23 ETs, tellimagrandin I was among the more antibacterial ETs
against S. aureus and E. coli [133]. Tellimagrandin I was earlier characterized in Syzygium aromaticum (Myrtaceae) [151]) and many Terminalia spp. [138]), but has not been reported in a Combretum species
before.
In this present study, we found that the root of C. hartmannianum
contains terflavin B and two isomers. Terflavin B was not reported
before in C. hartmannianum. Terflavin B, which is known to be the
biosynthetic precursor of punicalagin, has a unique chemical structure,
where the triphenyl (flavogallonoyl) ester group is attached to a glucopyranose part (Fig. 6 E) [152,153]. Terflavin B was found in several
Terminalia spp. [138].
4.3.3. Ellagic acid derivatives
We found that the roots of C. hartmannianum contain several ellagic
acid derivatives (Table 4, Fig. 5), of which methyl ellagic acid-xyloside
was present in a high concentration, and could be important for the
antimycobacterial activity we have observed for this extract. Besides, we
studied the antimycobacterial effect of pure ellagic acid (EA), which had
a weak inhibitory effect on the growth of M. smegmatis with a MIC value
of 500 µg/ml. However, di-methyl ellagic acid-xyloside and 4-galloyl-dimethyl ellagic acid-xyloside from Terminalia superba were found to be
potent growth inhibitors of M. smegmatis and M. tuberculosis, with MIC
values of 4.88 µg/ml [154]. Thus, it seems that methylation and
glycosylation are important for the antimycobacterial activity of EA
derivatives, whereas EA itself is not very active.
4.3.4. Terpenoids
We have tentatively identified two oleanolic acid-based pentacyclic
triterpenes in the root of C. hartmannianum, namely combregenin (tR
HPLC-DAD 31.9 min) and terminolic acid (tR HPLC-DAD 43.2 min)
(Table 4, Fig. 6 G). In agreement with many authors, we found that an
increase in the polarity of the terpenoids resulted in shortened retention
times in reversed-phase HPLC-DAD [155]. Consequently, we found that
combregenin, which contains six hydroxyl-groups, was eluted earlier in
the HPLC-DAD system than terminolic acid, containing five
hydroxyl-groups (Figs. 5 and 6). Terminolic acid is the aglycone of
chebuloside II [94]. Terminolic acid and combregenin were identified in
the leaves of Combretum zeyheri, the root of C. racemosum, stem bark of
C. nigricans, and C. molle, the wood of Terminalia brassii, T. complanata, T.
ivorensis, and T. impediens, and bark of T. macroptera [52,74,91,92,
156–160]. To the best of our knowledge, there are no previous reports
on the occurrence of combregenin and terminolic acid in the root of
C. hartmannianum. Morgan et al., [161], found eight pentacyclic triterpenes in the stem bark extract of C. hartmannianum, including ursolic
acid, pomolic acid, corosolic acid, arjunic acid, arjunglycoside 1, trachelosperoside E-1, and combreglucoside.
Terminolic acid, isolated from C. racemosum root, was antibacterial
against E. faecalis, E. coli, and S. aureus, with MIC values ranging from 64
to 128 μg/ml [157]. Moreover, six triterpenes, including terminolic acid
from a Centella asiatica extract were patented due to their antimycobacterial effect against M. avium, the causative agent of paratuberculosis in cattle [161]. In addition, ursolic acid that was also found
in Combretum racemosum [157], inhibited the growth of M. tuberculosis
H37Ra with a minimum inhibitory concentration of 20 μg/ml [162].
Imberbic acid, isolated from Combretum imberbe, was found to be a
powerful inhibitor of the growth of M. fortuitum (MIC 1.56 μg/ml)
[163]. Given this, it is possible that the triterpenes, terminolic acid and
combregenin, which we have found in the root extract of
C. hartmannianum, could contribute to its antimycobacterial effects.
In addition to the triterpenes, we tentatively identified the norditerpene glycoside, cordifoliside D in the root of C. hartmannianum
(Fig. 5). Although terminolic acid and cordifoliside D have the same
14
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
number of hydroxyl groups (Fig. 6), cordifoliside D had a shorter
retention time in HPLC-DAD compared to terminolic acid due to its
glucose sugar residue. Moreover, the shorter retention time could also be
due to the presence of a methoxy group in cordifoliside D, which decreases its polarity compared to terminolic acid [155]. To the best of our
knowledge, cordifoliside D has not been identified before from the genus
of Combretum. Cordifoliside D was originally found in Tinospora cordifolia (Menispermaceae) [164,165]. Moreover, an extract from Tinospora
cordifolia was found to inhibit the growth of M. tuberculosis at 1:50000
fold dilutions [166]. Interestingly, the combination of powders obtained
from Terminalia chebula, T. bellerica, and Tinospora cordifolia has a long
history in Asian and African traditional medicine for the treatment of
cough [167–169]. The presence of a glycosidic part and the beta
(β)-configurations of the hydroxyl and methyl groups in cordifoliside D
(Fig. 6 I), as well as the third carbon atom (C-6) with a γ-hydroxyl
substitution that makes it sensitive to steric effects [170], might be
important molecular features to impair the function of tyrosine kinase,
an important virulence modulator in the mycobacterial cell [171–173].
Conflict of interest statement
5. Conclusion
Appendix A. Supporting information
In this present study, extracts of various polarities of the stem bark,
stem wood, and root of C. hartmannianum showed growth inhibitory
activity against Mycobacterium smegmatis. In general, the polar extracts
were more active, and this could be due to the high diversity of polyphenols in these extracts. The study can justify the use of
C. hartmannianum in Sudanese traditional medicine to cure persistent
cough, a symptom that could be related to TB infection. However, the
traditional medicinal preparations, decoctions, and macerations, were
not as active as a methanol extract of the root that gave the best growth
inhibitory effect of the tested extracts. Therefore, this study indicates
that for the traditional medicinal use to treat persistent cough (that
could be related to TB), ethanolic tonics would have a superior effect
over water extracts and decoctions.
This study confirmed that C. hartmannianum contains a large number
of polyphenolic compounds, with ETs as prominent constituents. Some
of the ETs could play a role in the antimycobacterial effects of the
C. hartmannianum extracts in which they are suggested to enhance the
effects of each other and other compounds. In addition, several flavonoids, gallic acid, and ellagic acid derivatives, including methyl ellagic
acid-xyloside, and terpenoids were present in the root of
C. hartmannianum. A large number of potentially active antimycobacterial compounds in the root of C. hartmannianum warrant for
the further isolation of the compounds and testing against various
mycobacterial strains, including clinical strains of M. tuberculosis.
Moreover, studies on the impact of extracts and compounds from
C. hartmannianum on mycobacterial biofilms could be relevant.
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.biopha.2021.112264.
The authors declare no conflict of interest.
Acknowledgment
The first author is grateful for the initial funding of this research
provided by the Ministry of Higher Education and the University of
Khartoum, Sudan. The financial support provided by the Ekhaga Foundation, Stockholm, Sweden (2017-7) and the Swedish Cultural Foundation in Finland (Grant number 159102, 2020) is greatly
acknowledged by the first and the last author. Sincere thanks go to Dr.
Haytham Hashim Gibreel and Dr. El-Sheikh Abd Alla El-Sheikh for
assisting the first author in plant identification during the fieldwork, as
well as to Bashir Abaker, Hateel Al Qasim and Balla Alfadel for their
support during the fieldwork. Finally, many thanks to Helsinki University Library (HuLib) in Finland, for funding the article open access
processing charge (APC).
References
[1] World Health Organization (WHO). Tuberculosis and Air Travel: Guidelines for
Prevention and Control, third ed., WHO/HTM/TB/2008.399,, Geneva,
Switzerland, 2008.
[2] E. Cambau, M. Drancourt, Steps towards the discovery of Mycobacterium
tuberculosis by Robert Koch, 1882, Clin. Microbiol Infect. 20 (3) (2014) 196–201.
[3] T. Kaura, P. Sharmaa, G.K. Guptac, F. Ntie-Kangd, D. Kumara, Treatment of
Tuberculosis by natural drugs: a review, Plant Arch. 19 (2) (2019) 2168–2176.
[4] I. Smith, Mycobacterium tuberculosis pathogenesis and molecular determinants of
virulence, Clin. Microbiol. Rev. 16 (3) (2003) 463–496.
[5] W. Liu, J. Zhou, F. Niu, F. Pu, Z. Wang, M. Huang, X. Zhao, L. Yang, P. Tao, P. Xia,
J. Feng, Mycobacterium tuberculosis infection increases the number of osteoclasts
and inhibits osteoclast apoptosis by regulating TNF-α-mediated osteoclast
autophagy, Exp. Ther. Med. 20 (3) (2020) 1889–1898.
[6] P.V. Tsouh Fokou, A.K. Nyarko, R. Appiah-Opong, L.R. Tchokouaha Yamthe,
M. Ofosuhene, F.F. Boyom, Update on medicinal plants with potency on
Mycobacterium ulcerans, Biomed. Res Int. 2015 (2015), 917086, https://doi.org/
10.1155/2015/917086.
[7] World Health Organization (WHO) , 2020. Tuberculosis. https://www.who.int/
news-room/fact-sheets/detail/tuberculosis.
[8] C. Dye, K. Floyd, Tuberculosis, in: D.T. Jamison, J.G. Breman, A.R. Measham,
G. Alleyne, M. Claeson, D.B. Evans, P. Jha, A. Mills, P. Musgrove (Eds.), Disease
Control Priorities in Developing Countries, Oxford University Press, Washington,
DC, 2006, pp. 289–309. ISBN-10: 0-8213-6179-1.
[9] D.M. Castañeda-Hernández, A.J. Rodriguez-Morales, Epidemiological burden of
tuberculosis in developing countries, in: Alfonso J. Rodriguez-Morales (Ed.),
Current Topics in Public Health, IntechOpen, 2013, https://doi.org/10.5772/
53363 (https://www.intechopen.com/books/current-topics-in-public-health/
epidemiological-burden-of-tuberculosis-in-developing-countries).
[10] H. Mohajan, Tuberculosis is a fatal disease among some developing countries of
the world, Am. J. Infect. Dis. Microbiol. 3 (1) (2015) 18–31. https://mpra.ub.
uni-muenchen.de/82851/1/MPRA_paper_82851.pdf.
[11] P. Habibi, H. Daniell, C.R. Soccol, M.F. Grossi-de-Sa, The potential of plant
systems to break the HIV-TB link, Plant Biotechnol. J. 17 (10) (2019) 1868–1891.
[12] S. Russell, The economic burden of illness for households in developing countries:
a review of studies focusing on malaria, tuberculosis, and human
immunodeficiency virus/acquired immunodeficiency syndrome, Am. J. Trop.
Med. Hyg. 71 (2_suppl) (2004) 147–155.
[13] Ahmed E.O. Ibnouf, Susceptibility of Mycobacterium tuberculosis isolates to six
local medicinal plants. Thesis Submitted to the Faculty of Pharmacy, University of
Khartoum, Sudan, 2005. https://khartoumspace.uofk.edu/handle/12345
6789/8207.
[14] Centers for Disease Control and Prevention (CDC), Estimates of future global
tuberculosis, MMWR Morb. Mortal. Wkly. Rep. 42 (1993) 49. https://pubmed.
ncbi.nlm.nih.gov/8246861/.
[15] P. Gopal, T. Dick, The new tuberculosis drug Perchlozone® shows cross-resistance
with thiacetazone, Int. J. Antimicrob. Agents 45 (4) (2015) 430–433.
[16] B. Katende, T.M. Esterhuizen, A. Dippenaar, R.M. Warren, Rifampicin resistant
tuberculosis in lesotho: diagnosis, treatment initiation and outcomes, Sci. Rep. 10
(1) (2020) 1–8, https://doi.org/10.1038/s41598-020-58690-4.
[17] World Health Organization (WHO) . 2003. Calls On African Governments to
Formally Recognize Traditional Medicine. Johannesburg, South Africa. htt
Funding
This research was funded by the Ekhaga Foundation, project grant
2017-7 (Stockholm, Sweden), the Swedish Cultural Foundation in
Finland (2020, Grant number 159102), and the Ministry of Higher Education and University of Khartoum, Sudan at the earlier stage of this
research work. Open access was funded by Helsinki University Library
(HuLib), Finland.
Authors’ contributions
E.S. conducted ethnobotanical fieldwork in Sudan in 2006 and 2014.
E.S. and P.F. designed the research and wrote the manuscript. E.S.
conducted the experiments. E.S., P.F., and R.T. contributed to phytochemical analysis and analytical tools. O.L and M.F. contributed to the
writing process. All authors read and approved the manuscript.
15
E.Y.A. Salih et al.
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
Biomedicine & Pharmacotherapy 144 (2021) 112264
ps://www.afro.who.int/news/who-calls-african-governments-formally-reco
gnize-traditional-medicine.
S.B. Obakiro, A. Kiprop, I. Kowino, E. Kigondu, M.P. Odero, T. Omara,
L. Bunalema, Ethnobotany, ethnopharmacology, and phytochemistry of
traditional medicinal plants used in the management of symptoms of tuberculosis
in East Africa: a systematic review, Trop. Med. Health 48 (1) (2020) 1–21.
M.J. Cheesman, A. Ilanko, B. Blonk, I.E. Cock, Developing new antimicrobial
therapies: are synergistic combinations of plant extracts/compounds with
conventional antibiotics the solution? Pharmacogn. Rev. 11 (22) (2017) 57–72,
https://doi.org/10.4103/phrev.phrev_21_17.
H. Chandra, P. Bishnoi, A. Yadav, B. Patni, A.P. Mishra, A.R. Nautiyal,
Antimicrobial resistance and the alternative resources with special emphasis on
plant-based antimicrobials—a review, Plants 6 (2) (2017) 16.
M.F. Mahomoodally, Traditional medicines in Africa: an appraisal of ten potent
African medicinal plants, Evid. Based Complement. Altern. Med. eCAM 2013
(2013), 617459, https://doi.org/10.1155/2013/617459.
K.S. Walter, H.J. Gillett (Eds.), 1997 IUCN Red List of Threatened Plants, IUCN,
1998.
H.M. Elamin, Trees and Shrubs of the Sudan, Ithaea Press, Exeter, 1990.
H.J. Von Maydell, Trees and Shrubs of the Sahel, Their Characteristics and Uses,
GTZ, Germany, 1986. ISBN: 3880853185.
E. Taha, A. Mariod, S. Abouelhawa, M. El-Geddawy, M. Sorour, B. Matthäus,
Antioxidant activity of extracts from six different Sudanese plant materials, Eur.
J. Lipid Sci. Tech. 112 (11) (2010) 1263–1269.
E.Y.A. Salih, 2019. Ethnobotany, phytochemistry and antimicrobial activity of
Combretum, Terminalia and Anogeissus species (Combretaceae) growing
naturally in Sudan. Tropical Forestry Reports-URN:ISSN:0786–8170, 2019.
Doctoral thesis submitted to University of Helsink, Finland, 193 pages.
http://urn.fi/URN:ISBN:978-951-51-5421-7.
Dayamba SD. , 2010. Fire, Plant-derived Smoke and Grazing Effects on
Regeneration, Productivity and Diversity of the Sudanian Savanna- woodland
Ecosystem. Doctoral Thesis. Acta Universitatis agriculturae Sueciae, p. 73. ISSN
1652–6880: ISBN 978–91-576–7510-1. Print house: SLU Service/Repro, Alnarp
2010. https://pub.epsilon.slu.se/2365/1/Dayamba_S_D_101011.pdf.
G.B. El Ghazali, M.S. El Tohami, A.B. El Egami, W.S. Abdalla, M.G. Mohammed,
Medicinal plants of the Sudan. Part IV. Medicinal Plants of Northern Kordofan,
Omdurman Islamic University Press, Khartoum, Sudan, 1997.
H. Ali, G.M. König, S.A. Khalid, A.D. Wright, R. Kaminsky, Evaluation of selected
Sudanese medicinal plants for their in vitro activity against hemoflagellates,
selected bacteria, HIV-1-RT and tyrosine kinase inhibitory, and for cytotoxicity,
J. Ethnopharmacol. 83 (3) (2002) 219–228.
A.A. Elegami, E.I. El-Nima, M.E. Tohami, A.K. Muddathir, Antimicrobial activity
of some species of the family Combretaceae, Phytother. Res. Int. J. Devoted
Pharmacol. Toxicol. Eval. Nat. Prod. Deriv. 16 (6) (2002) 555–561, https://doi.
org/10.1002/ptr.995.
A.Z. Al Magboul, A.K. Bashir, A.M. Salih, A. Farouk, S.A. Khalid, Antimicrobial
activity of certain Sudanese plants used in flokloric medicine: screening for
antibacterial activity (V), Fitoterapia 59 (1) (1988) 57–62.
I.M.S. Eldeen, J. Van Staden, Antimycobacterial activity of some trees used in
South African traditional medicine, S. Afr. J. Bot. 73 (2) (2008) 248–251.
A.A. Mariod, N. Fadle, A.A. Hasan, Antimicrobial screening of wood extracts of
Combretum hartmannianum, Acacia seyal and Terminalia brownii, Eur. J. Mol. Biol.
Biochem 1 (2) (2014) 77–80, e - ISSN - 2348–2206: Print ISSN - 2348–2192.
Idris, M. , 2014. Chemical Characterization and Biological Activity of Flavonoids
in Some Medicinal Plants. Doctoral thesis submitted to Sudan University of
Science and Technology, Graduate College. Khartoum, Sudan. 89 pages.
I.M.S. Eldeen, J. Van Staden, In vitro pharmacological investigation of extracts
from some trees used in Sudanese traditional medicine, S. Afr. J. Bot. 73 (3)
(2007) 435–440.
B.O. Burham, 2016. Antimicrobial Activity of Petroleum Ether, Ethyl acetatate
and Methanolic Fraction of Five Sudanese Medicinal Plant. Int. J. Sci. Res. 5(4).
Paper ID: NOV162454.
A.M. Muddathir, T. Mitsunaga, Evaluation of anti-acne activity of selected
Sudanese medicinal plants, J. Wood Sci. 59 (1) (2013) 73–79.
A.M. Morgan, A.E. Mohamed, C. Saophea, S.U. Park, Y.H. Kim, Pentacyclic
triterpenes from the stem bark of Combretum hartmannianum Schweinf, Biochem
Syst. Ecol. 77 (2018) 48–50.
H.A. Ali, A.A. Hamza, A.E. Abass, O.E. Ahmed, LC/PDA/ESI-MS/MS polyphenols
profiling in the in vitro active leaves extracts of Combratum hartmannianum
against human pathogens with special emphasis to Madurella mycetomatis,
J. Chem. Biol. Phys. Sci. 7 (3) (2017) 757, https://doi.org/10.24214/jcbps.
B.7.3.75774.
E.A.M. Mohieldin, A.M. Muddathir, T. Mitsunaga, Inhibitory activities of selected
Sudanese medicinal plants on Porphyromonas gingivalis and matrix
metalloproteinase-9 and isolation of bioactive compounds from Combretum
hartmannianum (Schweinf) bark, Evid. Based Complement. Altern. Med 17 (1)
(2017) 1–11, https://doi.org/10.1186/s12906-017-1735-y.
C. Orwa , A. Mutua , R. Kindt, R. Jamnadass, S. Anthony , 2009. Agrofores tree
Database: a tree reference and selection guide version 4.0. http://apps.
worldagroforestry.org/treedb2/AFTPDFS/Combretum_aculeatum.PDF.
K.M. Hamad, M.M. Sabry, S.H. Elgayed, A.R. El Shabrawy, A.M. El-Fishawy, G.A.
A. Jaleel, Anti-inflammatory and phytochemical evaluation of Combretum
aculeatum Vent growing in Sudan, J. Ethnopharmacol. 242 (2019), 112052.
E.A. Diop, J. Jacquat, N. Drouin, E.F. Queiroz, J.L. Wolfender, T. Diop,
J. Schappler, S. Rudaz, Quantitative CE analysis of punicalagin in Combretum
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
16
aculeatum extracts traditionally used in Senegal for the treatment of tuberculosis,
Electrophoresis 40 (21) (2019) 2820–2827.
A. Dawé, M. Mbiantcha, F. Yakai, A. Jabeen, M.S. Ali, M. Lateef, B.T. Ngadjui,
Flavonoids and triterpenes from Combretum fragrans with anti-inflammatory,
antioxidant and antidiabetic potential, Z. Naturforsch. C J. Biosci. 73 (5–6)
(2018) 211–219.
H.H. Gibreel, A Taxonomic Study on Trees and Shrubs of El Nour Natural Forest
Reserve, University of Khartoum, Blue Nile, Sudan, 2008, p. 185 (M. Sc. thesis),
https://core.ac.uk/download/pdf/71672105.pdf.
D.M.W. Anderson, J.F. Howlett, C.G.A. McNab, Amino acid composition of gum
exudates from some african Combretum, Terminalia and Anogeissus species,
Phytochemistry 26 (3) (1987) 837–839.
S.M. Maregesi, O.D. Ngassapa, L. Pieters, A.J. Vlietinck, Ethnopharmacological
Survey of the Bunda district, Tanzania: plants Used to Treat Infectious Diseases,
in: J Ethnopharmacol, 113, 2007, pp. 457–470.
H.M. , Burkill, 1995. The useful plants of west tropical Africa, Vols. 1–3. The
useful plants of west tropical Africa, Vols. 3, Families J-L. ed. 2. Royal Botanic
Gardens, Kew. Richmond TW9 3AB United Kingdom.
P. Marquardt, R. Seide, C. Vissiennon, A. Schubert, C. Birkemeyer, V. Ahyi,
K. Fester, Phytochemical characterization and in vitro anti-inflammatory,
antioxidant and antimicrobial activity of Combretum collinum Fresen leaves
extracts from Benin, Molecules 25 (2) (2020) 288.
G.G. Alowanou, A.P. Olounlade, E.V.B. Azando, V.F.G.N. Dedehou, F.D. Daga,
M. Hounzangbe-adote, A review of Bridelia ferruginea, Combretum glutinosum and
Mitragina inermis plants used in zoo therapeutic remedies in West Africa:
historical origins, current uses and implications for conservation, J. Appl. Biosci.
87 (2015) 8003–8014.
Combretum glutinosum Perrot. ex DC., in: H. Vautier, M. Sanon, M. Sacandé,
L. Schmidt (Eds.), Seed Leaflet, 2007.
A. Jossang, J.L. Pousset, B. Bodo, Combreglutinin, a hydrolyzable tannin from
Combretum glutinosum, J. Nat. Prod. 57 (6) (1994) 732–737.
A. Dawe, S. Pierre, Habtemariam Phytochemical Constituents of Combretum Loefl,
(Combretaceae) Pharm. Crop 438 (2013) 59.
T. Yoshida, Y. Amakura, M. Yoshimura, Structural features and biological
properties of ellagitannins in some plant families of the order Myrtales, Int. J.
Mol. Sci. 11 (1) (2010) 79–106.
G.E.B. El Ghazali, M.S. El Tohami, A.A.B. El Egami, Medicinal plants of the Sudan.
Part III. Medicinal plants of the White Nile province. National Center for
Research, Khartoum, Sudan, Omdurman Islamic University Press, Khartoum,
Sudan, 1994.
H.H. EL-Kamali, Ethnopharmacology of medicinal plants used in North Kordofan
(Western Sudan), Ethnobot. Leafl. 13 (2009) 89–97.
N.F. Amako, J.C. Nnaji, GC/MS analysis and antimicrobial activity of Pentacyclic
Triterpenoids isolated from Combretum glutinosum Perr. Ex. DC. stem bark,
J. Chem. Soc. Niger. 41 (2016) 2.
D.M. Anderson, P.C. Bell, Analytical and structural features of the gum exudate
from Combretum hartmannianum schweinf, Carbohydr. Res. 49 (1976) 341–349.
E.Y.A. Salih, M.T. Kanninen, R. Julkunen-Tiitto, M.H. Sipi, M.O. Luukkanen, H.J.
Vuorela, P.J. Fyhrqvist, 2018a. Ellagitannins in antimycobacterial extracts of
Combretum hartmannianum, a savannah woodland tree. Conference paper: 66th
Congress and Annual Meeting of the Society for Medicinal Plant and Natural
Product Research (GA), 24.8–30.8.2018, Shanghai, China. Planta Medica -GA
2018a. 〈https://coms.events/GA2018/data/abstracts/en/abstract_0112.html〉.
A.H. Albagouri, A.A. Elegami, W.S. Koko, E.E. Osman, M.M. Dahab, In vitro
anticercarial activities of some Sudanese medicinal plants of the family
Combretaceae, J. For. Prod. Ind. 3 (2) (2014) 93–99.
M.M. Iwu, Handbook of African Medicinal Plants, CRC Press, Boca Raton, Florida,
USA, 1993.
H. Neuwinger, 1996. African Ethnobotany Poisons and Drugs Chemistry
(Germany). Chapman and Hall Gm bH, D-69469 Weinheim (Bundes Republik
Deutschland), ISBN 3–8261-0077–8.
J.C. Weber, C.S. Montes, T. Abasse, C.R. Sanquetta, D.A. Silva, S. Mayer, G.
I. Muniz, R.A. Garcia, Variation in growth, wood density and carbon
concentration in five tree and shrub species in Niger, N. For. 49 (1) (2018) 35–51.
Combretum nigricans Lepr. ex Guill. & Perr, in: M. Sacandé, M. Sanon, L. Schmidt
(Eds.), Seed Leaflet, 2007.
D.M.W. Anderson, J.R.A. Millar, W. Weiping, The gum exudate from Combretum
nigricans gum, the major source of West African ‘gum combretum’, Food Addit.
Contam. 8 (4) (1991) 423–436.
K. Asres, F. Bucar, S. Edelsbrunner, T. Kartnig, G. Höger, W. Thiel, Investigations
on antimycobacterial activity of some Ethiopian medicinal plants, Phytother. Res.
15 (4) (2001) 323–326.
E.Y.A. Salih, M. Kanninen, M. Sipi, O. Luukkanen, R. Hiltunen, H. Vuorela,
R. Julkunen-Tiitto, P. Fyhrquist, Tannins, flavonoids and stilbenes in extracts of
African savanna woodland trees Terminalia brownii , Terminalia laxiflora and
Anogeissus leiocarpus showing promising antibacterial potential, S. Afr. J. Bot. 108
(2017) 370–386.
E.Y. Salih, P. Fyhrquist, A. Abdalla, A.Y. Abdelgadir, M. Kanninen, M. Sipi,
O. Luukkanen, M.K. Fahmi, M.H. Elamin, H.A. Ali, LC-MS/MS tandem mass
spectrometry for analysis of phenolic compounds and pentacyclic triterpenes in
antifungal extracts of Terminalia brownii (Fresen), Antibiotics 6 (4) (2017) 37.
E.Y. Salih, R. Julkunen-Tiitto, A.M. Lampi, M. Kanninen, O. Luukkanen, M. Sipi,
M. Lehtonen, H. Vuorela, P. Fyhrquist, Terminalia laxiflora and Terminalia brownii
contain a broad spectrum of antimycobacterial compounds including
ellagitannins, ellagic acid derivatives, triterpenes, fatty acids and fatty alcohols,
J. Ethnopharmacol. 227 (2018) 82–96.
E.Y.A. Salih et al.
Biomedicine & Pharmacotherapy 144 (2021) 112264
[96] N. Lall, J.J.M. Meyer, In vitro inhibition of drug-resistant and drug-sensitive
strains of Mycobacterium tuberculosis by ethnobotanically selected South African
plants, J. Ethnopharmacol. 66 (3) (1999) 347–354.
[97] P. Fyhrquist, E.Y. Salih, S. Helenius, I. Laakso, R. Julkunen-Tiitto, HPLC-DAD and
UHPLC/QTOF-MS analysis of polyphenols in extracts of the African species
Combretum padoides, C. zeyheri and C. psidioides related to their antimycobacterial
activity, Antibiotics 9 (8) (2020) 459.
[98] P. Masoko, K.M. Nxumalo, Validation of antimycobacterial plants used by
traditional healers in three districts of the Limpopo province (South Africa), Evid.
Based Complement Altern. Med 2013 (2013), 586247, https://doi.org/10.1155/
2013/586247.
[99] R. Magwenzi, C. Nyakunu, S. Mukanganyama, The Effect of selected Combretum
species from Zimbabwe on the growth and drug efflux systems of Mycobacterium
aurum and Mycobacterium smegmatis, J. Microb. Biochem. Technol. 3 (2014) 003.
[100] T. Nyambuya, R. Mautsa, S. Mukanganyama, Alkaloid extracts from Combretum
zeyheri inhibit the growth of Mycobacterium smegmatis, BMC Complement Alter.
Med. 17 (1) (2017) 1–11, https://doi.org/10.1186/s12906-017-1636-0.
[101] E.F. Queiroz, L. Marcourt, S. Kicka, S. Rudaz, T. Diop, T. Soldati, J.L. Wolfender,
Antimycobacterial activity in a single-cell infection assay of ellagitannins from
Combretum aculeatum and their bioavailable metabolites, J. Ethnopharmacol. 238
(2019), 111832.
[102] A. Mariod, B. Matthäus, I.H. Hussein, Antioxidant activities of extracts from
Combretum hartmannianum and Guiera senegalensis on the oxidative stability of
sunflower oil, Emir. J. Food Agric. 18 (2006) 20–28, https://doi.org/10.9755/
ejfa.v12i1.5136.
[103] L.E.A. Hassan, M.B.K. Ahamed, A.S.A. Majid, H.M. Baharetha, N.S. Muslim, Z.
D. Nassar, A.M.A. Majid, Correlation of antiangiogenic, antioxidant and cytotoxic
activities of some Sudanese medicinal plants with phenolic and flavonoid
contents, BMC Complement Alter. Med. 14 (1) (2014) 1–14, https://doi.org/
10.1186/1472-6882-14-406.
[104] N. Wu, Y. Zu, Y. Fu, Y. Kong, J. Zhao, X. Li, J. Li, M. Wink, T. Efferth, Antioxidant
activities and xanthine oxidase inhibitory effects of extracts and main
polyphenolic compounds obtained from Geranium sibiricum L, J. Agric. Food
Chem. 58 (8) (2010) 4737–4743.
[105] N.P. Seeram, L.S. Adams, S.M. Henning, Y. Niu, Y. Zhang, M.G. Nair, D. Heber, In
vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic
acid and a total pomegranate tannin extract are enhanced in combination with
other polyphenols as found in pomegranate juice, J. Nutr. Biochem. 16 (6) (2005)
360–367.
[106] A.M. Muddathir, K. Yamauchi, T. Mitsunaga, Anti-acne activity of tannin-related
compounds isolated from Terminalia laxiflora, J. Wood Sci. 59 (5) (2013)
426–431.
[107] L.C.J. Araujo, V.C. da Silva, E.L. Dall’Oglio, J. Teixeira de Sousa, Flavonoids from
Combretum lanceolatum Pohl, Biochem. Syst. Ecol. 49 (2013) 37–38.
[108] M. Chairat, J.B. Bremner, S. Samosorn, W. Sajomsang, W. Chongkraijak,
A. Saisara, Effects of additives on the dyeing of cotton yarn with the aqueous
extract of Combretum latifolium Blume stems, Color Technol. 131 (4) (2015)
310–315.
[109] U.F. Ntite, Determination of antimicrobial potentials of ethanol extract of
Combretum dolichopentalum leaves by total dehydrogenase activity assay, Int. J.
Pharm. Phytochem. Ethnomed. (2017) 27.
[110] M.L. Falcone Ferreyra, S. Rius, P. Casati, Flavonoids: biosynthesis, biological
functions, and biotechnological applications, Front. Plant Sci. 3 (2012) 222.
[111] S. Kumar, A.K. Pandey, Chemistry and biological activities of flavonoids: an
overview, Sci. World J. 2013 (2013) 1–16.
[112] D. Lechner, S. Gibbons, F. Bucar, Modulation of isoniazid susceptibility by
flavonoids in Mycobacterium, Phytochem. Lett. 1 (2) (2008) 71–75.
[113] I. Górniak, R. Bartoszewski, J. Króliczewski, Comprehensive review of
antimicrobial activities of plant flavonoids, Phytochem Rev. 18 (1) (2019)
241–272.
[114] S. Mickymaray, F.A. Alfaiz, A. Paramasivam, Efficacy and mechanisms of
flavonoids against the emerging opportunistic nontuberculous mycobacteria,
Antibiotics 9 (8) (2020) 450.
[115] G. Gutiérrez-Venegas, J.A. Gómez-Mora, M.A. Meraz-Rodríguez, M.A. FloresSánchez, L.F. Ortiz-Miranda, Effect of flavonoids on antimicrobial activity of
microorganisms present in dental plaque, Heliyon 5 (12) (2019) 03013.
[116] F.J. Osonga, A. Akgul, R.M. Miller, G.B. Eshun, I. Yazgan, A. Akgul, O.A. Sadik,
Antimicrobial activity of a new class of phosphorylated and modified flavonoids,
ACS Omega 4 (7) (2019) 12865–12871.
[117] A. Adamczak, M. Ożarowski, T.M. Karpiński, Antibacterial activity of some
flavonoids and organic acids widely distributed in plants, JCM 9 (1) (2020) 109.
[118] Y. Zheng, X. Jiang, F. Gao, J. Song, J. Sun, L. Wang, X. Sun, Z. Lu, H. Zhang,
Identification of plant-derived natural products as potential inhibitors of the
Mycobacterium tuberculosis proteasome, BMC Complement Altern. Med. 14 (1)
(2014) 1–7, https://doi.org/10.1186/1472-6882-14-400.
[119] A. Pawar, P. Jha, M. Chopra, U. Chaudhry, D. Saluja, Screening of natural
compounds that targets glutamate racemase of Mycobacterium tuberculosis reveals
the anti-tubercular potential of flavonoids, Sci. Rep. 10 (1) (2020) 1–12.
[120] A. Suksamrarn, A. Chotipong, T. Suavansri, S. Boongird, P. Timsuksai,
S. Vimuttipong, A. Chuaynugul, Antimycobacterial activity and cytotoxicity of
flavonoids from the flowers of Chromolaena odorata, Arch. Pharm. Res. 27 (5)
(2004) 507–511.
[121] A. Seyoum, K. Asres, F.K. El-Fiky, Structure–radical scavenging activity
relationships of flavonoids, Phytochemistry 67 (18) (2006) 2058–2070.
[70] E.Y. Salih, R. Julkunen-Tiitto, O. Luukkanen, M. Sipi, M.K. Fahmi, P.J. Fyhrquist,
Potential anti-tuberculosis activity of the extracts and their active components of
Anogeissus leiocarpa (Dc.) guill. and perr. with special emphasis on polyphenols,
Antibiotics 9 (7) (2020) 364.
[71] J. Wang, Y.D. Yue, F. Tang, J. Sun, TLC screening for antioxidant activity of
extracts from fifteen bamboo species and identification of antioxidant flavone
glycosides from leaves of Bambusa. textilis McClure, Molecules 17 (10) (2012)
12297–12311.
[72] N.K. Sethiya, M.M. Raja, S.H. Mishra, Antioxidant markers based TLC-DPPH
differentiation on four commercialized botanical sources of Shankhpushpi (A
Medhya Rasayana): a preliminary assessment, J. Adv. Pharm. Technol. Res. 4 (1)
(2013) 25–30.
[73] P. Fyhrquist, I. Laakso, S.G. Marco, R. Julkunen-Tiitto, R. Hiltunen,
Antimycobacterial activity of ellagitannin and ellagic acid derivate rich crude
extracts and fractions of five selected species of Terminalia used for treatment of
infectious diseases in African traditional medicine, S. Afr. J. Bot. 90 (2014) 1–16.
[74] J. Conrad, B. Vogler, S. Reeb, I. Klaiber, S. Papajewski, G. Roos, E. Vasquez, M.
C. Setzer, W. Kraus, Isoterchebulin and 4, 6-O-isoterchebuloyl-d-glucose, novel
hydrolyzable tannins from Terminalia macroptera, J. Nat. Prod. 64 (3) (2001)
294–299.
[75] B. Pfundstein, S.K. El Desouky, W.E. Hull, R. Haubner, G. Erben, R.W. Owen,
Polyphenolic compounds in the fruits of Egyptian medicinal plants (Terminalia
bellerica, Terminalia chebula and Terminalia horrida): characterization,
quantitation and determination of antioxidant capacities, Phytochemistry 71 (10)
(2010) 1132–1148.
[76] K. Taulavuori, R. Julkunen-Tiitto, V. Hyöky, E. Taulavuori, Blue mood for
superfood, J. Nat. Prod. Commun. 8 (2013) 1–2.
[77] R. Julkunen-Tiitto, S. Sorsa, Testing the effects of drying methods on willow
flavonoids, tannins and salicylates, J. Chem. Ecol. 27 (2001) 779–789.
[78] P. Singariya, P. Kumar, K.K. Mourya, An activity guided isolation and evaluation
of various solvent extracts of the leaves of Anjan grass, Hygeia J. D. Med 4 (2)
(2012) 49–56.
[79] Clinical and Laboratory Standards Institute , 2012. Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved
Standard-Ninth Edition. 950 West Valley Road, Suite 2500, Wayne, Pennsylvania
19087, USA.
[80] J.N. Eloff, A proposal on expressing the antibacterial activity of plant extracts- a
small first step in applying scientific knowledge to rural primary health care in
South Africa, S. Afr. J. Sci. 96 (2000) 116–118.
[81] İ. Gülçin, Z. Huyut, M. Elmastaş, H.Y. Aboul-Enein, Radical scavenging and
antioxidant activity of tannic acid, Arab. J. Chem. 3 (1) (2010) 43–53.
[82] K.R. Markham, T.J. Mabry, Ultraviolet-visible and proton magnetic resonance
spectroscopy of flavonoids. In The Flavonoids, Springer, Boston, MA, 1975,
pp. 45–77.
[83] Z. Jurasekova, J.V. Garcia-Ramos, C. Domingo, S. Sanchez-Cortes, Surfaceenhanced Raman scattering of flavonoids, J. Raman Spectrosc. 37 (11) (2006)
1239–1241.
[84] R. Julkunen-Tiitto, N. Nenadis, S. Neugart, M. Robson, G. Agati, J. Vepsäläinen,
G. Zipoli, L. Nybakken, B. Winkler, M.A. Jansen, Assessing the response of plant
flavonoids to UV radiation: an overview of appropriate techniques, Phytochem.
Rev. 14 (2) (2015) 273–297.
[85] Q. Dong, Y. Jiang, Q. Wang, M. Liu, N. Wang, H. He, H. Zhou, T. Zhang, X. Luo,
2017, June. Analysis on the polyphenols, flavonoids and antioxidant activities of
broccoli. In 2017 Global Conference on Mechanics and Civil Engineering (GCMCE
2017). Atlantis Press. 〈https://www.atlantis-press.com/proceedings/gcmce-17/
25882885〉.
[86] J. Tošović, S. Marković, Reproduction and interpretation of the UV–vis spectra of
some flavonoids, Chem. Zvesti 71 (3) (2017) 543–552.
[87] T.Y. Wang, Q. Li, K.S. Bi, Bioactive flavonoids in medicinal plants: structure,
activity and biological fate, Asian J. Pharm. Sci. 13 (1) (2018) 12–23.
[88] R.T. Ramos, I.C. Bezerra, M.R. Ferreira, L.A.L. Soares, Spectrophotometric
quantification of flavonoids in herbal material, crude extract, and fractions from
leaves of Eugenia uniflora Linn, Pharmacogn. Res. 9 (3) (2017) 253–260.
[89] X. Wang, G. Ding, B. Liu, Q. Wang, Flavonoids and antioxidant activity of rare and
endangered fern: Isoetes sinensis, Plos One 15 (5) (2020), 0232185.
[90] B.T. Schaneberg, J.R. Mikell, E. Bedir, I.A. Khan, V. Nachname, An improved
HPLC method for quantitative determination of six triterpenes in Centella asiatica
extracts and commercial products, Die Pharm. Int. J. Pharm. Sci. 58 (6) (2003)
381–384.
[91] B.K. Ponou, L. Barboni, R.B. Teponno, M. Mbiantcha, T.B. Nguelefack, H.J. Park,
K.T. Lee, L.A. Tapondjou, Polyhydroxyoleanane-type triterpenoids from
Combretum molle and their anti-inflammatory activity, Phytochem Lett. 1 (4)
(2008) 183–187.
[92] W.M. Oluyemi, B.B. Samuel, H. Kaehlig, M. Zehl, S. Parapini, S. D’Alessandro,
D. Taramelli, L. Krenn, Antiplasmodial activity of triterpenes isolated from the
methanolic leaf extract of Combretum racemosum P. Beauv, J. Ethnopharmacol.
247 (2020), 112203.
[93] A.P. Kundu, S.B. Mahato, Triterpenoids and their glycosides from Terminalia
chebula, Phytochemistry 32 (4) (1993) 999–1002.
[94] F.R. Garcez, W.S. Garcez, A.L. Santana, M.M. Alves, M.D.F.C. Matos, A.D.
M. Scaliante, Bioactive flavonoids and triterpenes from Terminalia fagifolia
(Combretaceae), J. Braz. Chem. Soc. 17 (7) (2006) 1223–1228.
[95] Ghazali, G.E.B. El, Medicinal plants of Sudan, Part IV. Medicinal Plants of the
White Nile Province. Khartoum, University Press, Khartoum, Sudan, 1998.
17
Biomedicine & Pharmacotherapy 144 (2021) 112264
E.Y.A. Salih et al.
[122] K. Asres, F. Bucar, E. Knauder, V. Yardley, H. Kendrick, S.L. Croft, In vitro
antiprotozoal activity of extract and compounds from the stem bark of Combretum
molle, Phytother. Res. 15 (7) (2001) 613–617.
[123] M.N. Shuaibu, K. Pandey, P.A. Wuyep, T. Yanagi, K. Hirayama, A. Ichinose,
T. Tanaka, I. Kouno, Castalagin from Anogeissus leiocarpus mediates the killing of
Leishmania in vitro, Parasitol. Res. 103 (6) (2008) 1333–1338.
[124] J. Moilanen, Ellagitannins in Finnish Plant Species–Characterization, Distribution
and Oxidative Activity, Doctoral dissertation submitted to University of Turku,
Finland, 2015, p. 112. https://www.utupub.fi/bitstream/handle/10024/105203/
AnnalesAI518Moilanen.pdf?sequence=2&isAllowed=y.
[125] O. Silva, E.T. Gomes, J.L. Wolfender, A. Marston, K. Hostettmann, Application of
high performance liquid chromatography coupled with ultraviolet spectroscopy
and electrospray mass spectrometry to the characterisation of ellagitannins from
Terminalia macroptera roots, Pharm. Res 17 (11) (2000) 1396–1401.
[126] T.C. Lin, G. Nonaka, I. Nishioka, F.C. Ho, Tannins and related compounds. CII:
structures of terchebulin, an ellagitannin having a novel tetraphenylcarboxylic
acid (terchebulic acid) moiety, and biogenetically related tannins from Terminalia
chebula Retz, Chem. Pharm. Bull. 38 (1990) 3004–3008.
[127] S. Wu, L. Tian, Diverse phytochemicals and bioactivities in the ancient fruit and
modern functional food pomegranate (Punica granatum), Molecules 22 (10)
(2017) 1606.
[128] A. Mehta, , 2012. Principle of Reversed-Phase Chromatography HPLC/UPLC (with
Animation)’’. PharmaXchange.info. https://pharmaxchange.info/2012/11/thinlayer-chromatography-tlc-principle-with-animation/.
[129] L. Feng, Y. Yin, Y. Fang, X. Yang, Quantitative determination of punicalagin and
related substances in different parts of pomegranate, Food Anal. Method. 10 (11)
(2017) 3600–3606.
[130] G. Millo, A. Juntavee, A. Ratanathongkam, N. Nualkaew, J. Peerapattana,
S. Chatchiwiwattana, Antibacterial inhibitory effects of Punica granatum gel on
cariogenic bacteria: an in vitro study, Int. J. Clin. Pediatr. Dent. 10 (2) (2017)
152–157.
[131] V. Sorrenti, C.L. Randazzo, C. Caggia, G. Ballistreri, F.V. Romeo, S. Fabroni,
N. Timpanaro, M. Raffaele, L. Vanella, Beneficial effects of pomegranate peel
extract and probiotics on pre-adipocyte differentiation, Front Microbiol. 10
(2019) 660.
[132] O. Silva, S. Viegas, C. de Mello-Sampayo, M.J.P. Costa, R. Serrano, J. Cabrita, E.
T. Gomes, Anti-Helicobacter pylori activity of Terminalia macroptera root,
Fitoterapia 83 (5) (2012) 872–876.
[133] E. Puljula, G. Walton, M.J. Woodward, M. Karonen, Antimicrobial activities of
ellagitannins against Clostridiales perfringens, Escherichia coli, Lactobacillus
plantarum and Staphylococcus aureus, Molecules 25 (16) (2020) 3714.
[134] S.H. Mun, O.H. Kang, R. Kong, T. Zhou, S.A. Kim, D.W. Shin, D.Y. Kwon,
Punicalagin suppresses methicillin resistance of Staphylococcus aureus to oxacillin,
J. Pharm. Sci. 137 (4) (2018) 317–323.
[135] C. Santiago, E.L. Pang, K.H. Lim, H.S. Loh, K.N. Ting, Inhibition of penicillinbinding protein 2a (PBP2a) in methicillin resistant Staphylococcus aureus (MRSA)
by combination of ampicillin and a bioactive fraction from Duabanga grandiflora,
BMC Complement Altern. Med 15 (1) (2015) 1–7, https://doi.org/10.1186/
s12906-015-0699-z.
[136] P.X. Guo, S. Erickson, D. Anderson, A small viral RNA is required for in vitro
packaging of bacteriophage phi 29 DNA, Science 236 (4802) (1987) 690–694.
[137] S. Kinoshita, Y. Inoue, S. Nakama, T. Ichiba, Y. Aniya, Antioxidant and
hepatoprotective actions of medicinal herb, Terminalia catappa L. from Okinawa
Island and its tannin corilagin, Phytomedicine 14 (11) (2007) 755–762.
[138] Z. Chang, Q. Zhang, W. Liang, K. Zhou, P. Jian, G. She, L. Zhang, A comprehensive
review of the structure elucidation of tannins from terminalia linn, Evid. Based
Complement. Altern. Med. eCAM 2019 (2019), 8623909, https://doi.org/
10.1155/2019/8623909.
[139] T.C. Lin, F.L. Hsu, J.T. Cheng, Antihypertensive activity of corilagin and
chebulinic acid, tannins from Lumnitzera racemosa, J. Nat. Prod. 56 (4) (1993)
629–632.
[140] N. Li, M. Luo, Y.J. Fu, Y.G. Zu, W. Wang, L. Zhang, L.P. Yao, C.J. Zhao, Y. Sun,
Effect of corilagin on membrane permeability of Escherichia coli , Staphylococcus
aureus and Candida albicans, Phytother. Res. 27 (10) (2013) 1517–1523.
[141] M. Shimizu, S. Shiota, T. Mizushima, H. Ito, T. Hatano, T. Yoshida, T. Tsuchiya,
Marked potentiation of activity of β-lactams against methicillin-resistant
Staphylococcus aureus by corilagin, Antimicrob. Agents Chemother. 45 (11) (2001)
3198–3201.
[142] A. Gupta, A.K. Singh, R. Kumar, R. Ganguly, H.K. Rana, P.K. Pandey, G. Sethi,
A. Bishayee, A.K. Pandey, Corilagin in cancer: a critical evaluation of anticancer
activities and molecular mechanisms, Molecules 24 (18) (2019) 3399.
[143] C.P. Anokwuru, S. Tankeu, S. van Vuuren, A. Viljoen, I.D. Ramaite,
O. Taglialatela-Scafati, S. Combrinck, Unravelling the antibacterial activity of
Terminalia sericea root bark through a metabolomic approach, Molecules 25 (16)
(2020) 3683.
[144] S. Quideau, T. Varadinova, D. Karagiozova, M. Jourdes, P. Pardon, C. Baudry,
P. Genova, T. Diakov, R. Petrova, Main structural and stereochemical aspects of
the antiherpetic activity of nonahydroxyterphenoyl-containing C- glycosidic
ellagitannins, Chem. Biodivers. 1 (2004) 247–258.
[145] S. Quideau, M. Jourdes, D. Lefeuvre, D. Montaudon, C. Saucier, Y. Glories,
P. Pardon, P. Pourquier, The chemistry of wine polyphenolic C-glycosidic
ellagitannins targeting human topoisomerase II, Chem. Eur. J. 11 (2005)
6503–6513.
[146] G.-I. Nonaka, K. Ishimaru, M. Watanabe, I. Nishioka, T. Yamauchi, A.S.C. Wan,
Tannins and related compounds. LI. Elucidation of the stereochemistry of the
[147]
[148]
[149]
[150]
[151]
[152]
[153]
[154]
[155]
[156]
[157]
[158]
[159]
[160]
[161]
[162]
[163]
[164]
[165]
[166]
[167]
[168]
[169]
[170]
[171]
[172]
[173]
18
triphenoyl moiety in castalagin and vescalagin, and isolation of 1-O-galloyl
castalagin from Eugenia grandis, Chem. Pharm. Bull. 35 (1987) 217–220.
A.R. Araú jo, A.C. Araújo, R.L. Reis, R.A. Pires, Vescalagin and castalagin present
bactericidal activity toward methicillin-resistant bacteria, ACS Biomater. Sci. Eng.
7 (3) (2021) 1022–1030.
M.K. Reddy, S.K. Gupta, M.R. Jacob, S.I. Khan, D. Ferreira, Antioxidant,
antimalarial and antimicrobial activities of tannin-rich fractions, ellagitannins
and phenolic acids from Punica granatum L, Planta Med. 53 (05) (2007) 461–467.
S. Štumpf, G. Hostnik, M. Primožič, M. Leitgeb, J.P. Salminen, U. Bren, The effect
of growth medium strength on minimum inhibitory concentrations of tannins and
tannin extracts against E. coli, Molecules 25 (12) (2020) 2947.
S. Shiota, M. Shimizu, J.I. Sugiyama, Y. Morita, T. Mizushima, T. Tsuchiya,
Mechanisms of action of corilagin and tellimagrandin I that remarkably
potentiate the activity of β-lactams against methicillin-resistant Staphylococcus
aureus, Microbiol. Immunol. 48 (1) (2004) 67–73.
M. Toda, J. Kawabata, T. Kasai, α-Glucosidase inhibitors from clove (Syzgium
aromaticum ), Biosci. Biotechnol. Biochem. 64 (2) (2000) 294–298.
T. Tanaka, G.I. NONAKA, I. NISHIOKA, Tannins and Related Compounds. XLII.:
isolation and Characterization of Four New Hydrolyzable Tannins, Terflavins A
and B, Tergallagin and Tercatain from the Leaves of Terminalia catappa L, Chem.
Pharm. Bull. 34 (3) (1986) 1039–1049.
F.J. Mininel, C.S. Leonardo Junior, L.G. Espanha, F.A. Resende, E.A. Varanda, C.
Q.F. Leite, W. Vilegas, L.C. Dos Santos, Characterization and quantification of
compounds in the hydroalcoholic extract of the leaves from Terminalia catappa
Linn. (Combretaceae) and their mutagenic activity, Evid. Based Complement
Altern. Med 2014 (2014), 676902, https://doi.org/10.1155/2014/676902.
V. Kuete, T.K. Tabopda, B. Ngameni, F. Nana, T.E. Tshikalange, B.T. Ngadjui,
Antimycobacterial, antibacterial and antifungal activities of Terminalia superba
(Combretaceae), S. Afr. J. Bot. 76 (1) (2010) 125–131.
M. Dembek, S. Bocian, Pure water as a mobile phase in liquid chromatography
techniques, TrAC Trends Anal. Chem. 123 (2020), 115793.
D.K.B. Runyoro, S.K. Srivastava, M.P. Darokar, N.D. Olipa, C.J. Cosam, I.N.
M. Mecky, Anticandidiasis agents from a Tanzanian plant, Combretum zeyheri,
Med. Chem. Res. 22 (2013) 1258–1262.
D.P.A. Gossan, A.A. Magid, P.A. Yao-Kouassi, J. Josse, S.C. Gangloff, H. Morjani,
L. Voutquenne-Nazabadioko, Antibacterial and cytotoxic triterpenoids from the
roots of Combretum racemosum, Fitoterapia 110 (2016) 89–95.
L.A. Collins, S.G. Franzblau, Microplate alamar blue assay versus BACTEC 460
system for high-throughput screening of compounds against Mycobacterium
tuberculosis and Mycobacterium avium, Antimicrob Agents Chemother. 41 (5)
(1997) 1004–1009.
A. Jossang, M. Seuleiman, E. Maidou, B. Bodo, Pentacyclic triterpenes from
Combretum nigricans, Phytochemistry 41 (2) (1996) 591–594.
S. Roy, D. Gorai, R. Acharya, R. Roy, Combretum (combretaceae): Biological
activity and phytochemistry, Am. J. Pharm. 4 (2014) 11.
N., Magrone, JR , 2017. Novel Combination of Naturally Occurring Compounds to
Assist With Suspected Mycobacterial Infections Related to Autoimmune
Conditions. U.S. Patent Application 15/354,975.
M.A. Jyoti, T. Zerin, T.H. Kim, T.S. Hwang, W.S. Jang, K.W. Nam, H.Y. Song, In
vitro effect of ursolic acid on the inhibition of Mycobacterium tuberculosis and its
cell wall mycolic acid, Pulm. Pharm. Ther. 33 (2015) 17–24.
D.R. Katerere, A.I. Gray, R.J. Nash, R.D. Waigh, Antimicrobial activity of
pentacyclic triterpenes isolated from African Combretaceae, Phytochemistry 63
(1) (2003) 81–88.
V.D. Gangan, P. Pradhan, A.T. Sipahimalani, A. Banerji, Norditerpene furan
glycosides from Tinospora cordifolia, Phytochemistry 39 (5) (1995) 1139–1142.
S.K. Dwivedi, A. Enespa, Tinospora cordifolia with reference to biological and
microbial properties, Int. J. Curr. Microbiol Appl. Sci. 5 (6) (2016) 446–465.
R. Perumal Samy, P. Gopalakrishnakone, Therapeutic potential of plants as antimicrobials for drug discovery, Evid. Based Complement. Altern. Med. 7 (3) (2010)
283–294, https://doi.org/10.1093/ecam/nen036.
K. Spelman, Traditional and clinical use of Tinospora cordifolia , Guduchi, Aust. J.
Med. Herbal. 13 (2) (2001).
P. Nidhi, P. Swati, R. Krishnamurthy, Indian Tinospora species: natural
immunomodulators and therapeutic agents, Int. J. Pharm. Biol. Chem. Sci. 2
(2013) 1–9.
M.M. Pandey, S. Rastogi, A.K.S. Rawat, Indian traditional ayurvedic system of
medicine and nutritional supplementation, Evid. -Based Complement. Altern.
Med. 2013 (2013) 1–12, https://doi.org/10.1155/2013/376327.
P. Pradhan, V.D. Gangan, A.T. Sipahimalani, A. Banerji, Stereochemical
assignment of tertiary hydroxyl group in diterpene furan glycosides by pyridine
induced Shifts, 13C-and 2D NMR spectroscopy, Spectrosc. Lett. 30 (7) (1997)
1467–1474, https://doi.org/10.1080/00387019708006737.
K. Chow, D. Ng, R. Stokes, P. Johnson, Protein tyrosine phosphorylation in
Mycobacterium tuberculosis, FEMS Microbiol. Lett. 124 (2) (1994) 203–207.
K. Grāve, M.D. Bennett, M. Högbom, Structure of Mycobacterium tuberculosis
phosphatidylinositol phosphate synthase reveals mechanism of substrate binding
and metal catalysis, Commun. Boil. 2 (1) (2019) 1–11.
A. Niesteruk, H.R. Jonker, C. Richter, V. Linhard, S. Sreeramulu, H. Schwalbe, The
domain architecture of PtkA, the first tyrosine kinase from Mycobacterium
tuberculosis , differs from the conventional kinase architecture, J. Biol. Chem. 293
(30) (2018) 11823–11836, https://scifinder.cas.org. (accessed 12 March 2021).
https://www.tballiance.org/why-new-tb-drugs/global-pandemic. (accessed 24
August 2020).