South African Journal of Botany 126 (2019) 340–344

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South African Journal of Botany

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Isolation and characterisation of sesquiterpene lactones from Schkuhria

pinnata and their antibacterial and anti-inflammatory activities
R.G. Kudumela a, O. Mazimba b, P. Masoko a,⁎
a
Department of Biochemistry, Microbiology and Biotechnology, Faculty of Science and Agriculture, University of Limpopo, Turfloop Campus, Private Bag X1106, Sovenga, Limpopo 0727,
South Africa
b
Chemical and Forensic Sciences, Botswana International University of Science and Technology, Plot 10017 Palapye, Private Bag 16, Palapye, Botswana

a r t i c l e i n f o a b s t r a c t

Article history: Schkuhria pinnata (Lam.) Kuntze ex Thell is used traditionally to treat a number of diseases linked with bacterial
Received 26 January 2019 infections and inflammation such as eye infections, diarrhoea, diabetes and stomach problems. Despite these
Received in revised form 4 April 2019 uses, there is still insufficient information on the isolation and evaluation of active compounds from this plant.
Accepted 9 April 2019
Thus, the study was aimed at identification and characterisation of the antibacterial and anti-inflammatory active
Available online 8 May 2019
extracts of Schkuhria pinnata. Antibacterial activity was evaluated by the micro-broth dilution method. Bioassay-
Edited by JJ Nair guided fractionation aided the isolation of four compounds from the dichloromethane extracts. The structures of
the isolated compounds were elucidated using NMR data. The compounds were further tested for antibacterial,
cytotoxic and anti-inflammatory activity using serial dilutions. Four sesquiterpene lactones were identified,
6α-[4′,5′-dihydroxytigloyloxy]-inuviscolide 1 and, 3-desacetyl-3-isovaleroyleucannabinolide 2, eucannabinolide
3 and 6α-[4′,5′-dihydroxytigloyloxy]-isoinuviscolide 4. These compounds exhibited less antibacterial activity as
compared to the crude extracts and fractions. Anti-inflammatory and cytotoxic activities were also demon-
strated. The activities reported for the extracts support the traditional uses of this plant for treatment of infections
linked with microorganisms and inflammatory related disorders.
© 2019 The Authors. Published by Elsevier B.V. on behalf of SAAB. This is an open access article under the CC BY-NC-
ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

List of abbreviations
1. Introduction

13
C NMR Carbon 13 Nuclear Magnetic Resonance Plants have been providing medicinal agents to people for centuries
1
H NMR Proton Nuclear Magnetic Resonance and quite a number of potent modern drugs have been isolated from
CLSI Clinical and Laboratory Standards Institute
plants based on their traditional use, phytochemical and pharmacolog-
NCCL National Committee for Clinical Laboratory
LPS Lipopolysaccharide ical evaluations (Sufian et al., 2012). Medicinal plants are now explored
H2DCF-DA Dihydrodichlorofluorescein diacetate as sources of antimicrobials due to the constant increase in antimicro-
DCF Dichlorofluorescein bial resistance amongst the species of pathogenic bacteria (Ushimaru
MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium et al., 2012). On the other hand inflammation often results from several
bromide]
SL Sesquiterpene lactones
types of infections including bacterial infections. Therefore, having ther-
ROS Reactive oxygen species apies with both antibacterial and anti-inflammatory properties would
Amp ampicillin have an added advantage since current anti-inflammatory agents only
Avg average supress inflammation by relieving pain and not elimination of the caus-
I, II, III Different extraction repeats (serial exhaustive extraction)
ative bacteria (Pincus, 2005).
E.c E. coli
P.a P. aeruginosa Schkuhria is a small genus from the Asteraceae family of angio-
E.f E. faecalis sperms; it consists of about 15 species (Ganzer and Jakupovic, 1990).
S.a S. aureus. A number of compounds have been isolated from members of this
MIC Minimum Inhibitory concentration genus, these include; sesquiterpene lactones (Ganzer and Jakupovic,
LC50 50% Lethal concentration
1990; Kimani et al., 2018) such as eucannabinolides (Herz and
⁎ Corresponding author.
Govindan, 1980), heliangolides (Bohlmann and Zdero, 1981),
E-mail addresses: mazimbao@biust.ac.bw (O. Mazimba), Peter.Masoko@ul.ac.za schkuhriolides, and melampolide schkuhrioidin (de Vivar et al., 1980)
(P. Masoko). and some acyl phenylpropanoids (León et al., 2009).

https://doi.org/10.1016/j.sajb.2019.04.002
0254-6299/© 2019 The Authors. Published by Elsevier B.V. on behalf of SAAB. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).
 R.G. Kudumela et al. / South African Journal of Botany 126 (2019) 340–344
Schkuhria pinnata (Lam.) Kuntze ex Thell is commonly known as
Dwarf Marigold in English and ‘Sebabane’ in Sepedi. It has divided
leaves and branched flower heads, with yellow disc and ray florets. It
is native to South America and usually occurs in gardens and along road-
sides, but it is widespread and distributed as a weed in South Africa
(Deutschländer et al., 2009). Its traditional medicinal applications are
in the treatment of various disorders linked to infectious microorgan-
isms including dermatitis, eczema and acne (McGaw and Eloff, 2008).
The mixture of the whole plant is used for the treatment of diabetes,
hypertension, stomach problems, oedema, or gonorrhoea. The roots or
leaves are chewed and swallowed for dysmenorrhoea (Mahwasane
et al., 2013). The aerial parts are used for the treatment of eye infections,
pneumonia, diarrhoea and heart-water (Luseba et al., 2007). These
ethnopharmacological uses encouraged the investigations for the anti-
bacterial, anti-inflammatory and cytotoxic effects of compounds from
the plant.
Sesquiterpene lactones are a large class of secondary metabolites
mainly isolated from members of the Asteraceae family (Zhang et al.,
2014). The biological activities of sesquiterpene lactones include;
induction of detoxifying enzymes (Zidorn, 2008), anticancer, anti-
inflammatory and immunomodulatory effects, anti-ulcer, antimicro-
bial and antiviral activities (Ivanescu et al., 2015). Some plant
extracts also contain toxic components (Street et al., 2008). Hence,
the need for toxic evaluations. In vitro preliminary screening of bio-
logical activity of the extracts of Schkuhria pinnata showed promising
antibacterial activity. The acetone and methanol extract of Dombeya
rotundifolia and those of S. pinnata were shown to have MIC values of
320 μg/mL, as well as the dichloromethane, acetone, and methanol
extract of Elephantorrhiza elephantine against E. coli. (Kudumela and
Masoko, 2018; Kudumela et al., 2018) and anti-inflammatory activity
(Kudumela et al., 2018). This prompted the bio-guided fractionation
of its dichloromethane extract to isolate and identify these com-
pounds in an attempt to establish a correlation between the chemical
constituents and traditional uses. Thus, we report herein the isola-
tion and bioactivities of extracts and unusual sesquiterpene lactones
from Schkuhria pinnata.
2. Methods and materials
2.1. Plant collection and preparation
The whole plant excluding the roots of Schkuhria pinnata ((Lam.)
Kuntze ex Thell) (UNIN 12298) were collected in February 2016 at the
University of Limpopo, South Africa. The specimen was deposited at
the Larry Leach Herbarium (UNIN) for authentication. About 1.2 kg of
the ground plant material was extracted with 6 L of n-hexane in a
glass bottle. The bottle was vigorously shaken for a day at 200 rpm.
The supernatant was filtered, concentrated using rotary evaporator at
50 °C, and transferred into pre-weighed labelled beakers. The same pro-
cess was repeated three times. The same plant residues were extracted
as mentioned above in the following order with 6 L of dichloromethane,
ethyl acetate, acetone, and methanol.
2.2. Antibacterial activity assays
2.2.1. Microorganisms used in this study
Gram-positive bacteria (Staphylococcus aureus ATCC 29213 and En-
terococcus faecalis ATCC 29212) and Gram-negative bacteria
(Escherichia coli ATCC 28922 and Pseudomonas aeruginosa ATCC
27853), which are the strains primarily recommended for use by the
Clinical and Laboratory Standards Institute (CLSI), were selected for
the study (NCCL, 1992). The bacterial cultures were maintained on nu-
trient agar at 4 °C and inoculated in nutrient broth and incubated at 37
°C for 12 h prior to screening tests.
341
2.2.2. Quantitative antibacterial activity assay by microbroth dilution
method
Antibacterial activity was evaluated by the determination of the
minimum inhibitory concentration (MIC) for each extract on E. faecalis,
E. coli, P. aeruginosa and S. aureus following the micro-dilution method
developed by Eloff (1998).
2.3. Isolation of antibacterial compounds by bioassay-guided fractionation
The dichloromethane crude extract (102.88 g) was subjected to
open column chromatography (35 × 4 cm) on silica gel 60 (Fluka) and
eluted using solvent mixtures of increasing polarity of n-hexane/ethyl
acetate and ethyl acetate/methanol. Fifteen fractions (1–15) were
collected and analysed on TLC. The fractions of 10–30% n-hexane were
further fractionated on another column chromatography (39 × 4 cm)
which was eluted up to 100% ethyl acetate. Sub-fractions were collected
and pooled to 10 sub-fractions (F1-F10) based on TLC profiles. Sub-
fractions F1, F3 and F7-F9 were combined based on TLC profiles to
make one sub-fraction which was further purified using column chro-
matography on silica gel 60 eluted with ethyl acetate:acetone (70:30).
Sub-fractions iii–v from the pooled five sub-fractions were labelled
compound 1 to 4 and allowed to dry. These were tested for biological
activities and their spectral profiles determined by 400 MHz NMR Spec-
trometer (Bruker). The 1H and 13C NMR spectral data are in agreement
with earlier reports (Kimani et al., 2018; Pacciaroni et al., 1995; Cheng
et al., 2011).
2.4. Biological activities of compounds
2.4.1. Antibacterial activity by micro broth dilution assay
The minimum inhibitory concentration (MIC) of the compound
on E. faecalis, E. coli, P. aeruginosa and S. aureus was determined using
the micro-dilution method (Eloff, 1998). The compounds were
re-dissolved in acetone to a final concentration of 1000 μg/mL.
Two-fold serial dilutions of the compounds (250–1.95 μg/mL) were
prepared in 96-well microtitre plates. All samples were assayed in
triplicate.
2.4.2. Anti-inflammatory activity assay
The anti-inflammatory assay was carried out according to the
method described by Sekhar et al. (2015) with modifications. This
assay uses stimulants such as lipopolysaccharide (LPS) to induce oxida-
tive stress and dihydrodichlorofluorescein diacetate (H2DCF-DA) to
detect the presence of reactive oxygen species in excess. In the presence
of reactive oxygen species H2DCF-DA is oxidised to fluorescent 2,7-
dichlorofluorescein (DCF).
2.4.3. Cytotoxicity assay
The toxic effects of the compounds on African green monkey kidney
(Vero) cells was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983).
2.5. Statistical analysis
Each experiment was performed in triplicate and the results were
expressed as mean values. Linear regression analysis was used to calcu-
late LC50 values. Microsoft Excel® was used to enter and capture data.
Various graphs and tables were extracted from these data. Data were
then exported to SPSS for further analysis. The results were analysed
using one-way analysis of variance (ANOVA). P value b0.05 was consid-
ered as significant. SPSS 25.0 was employed for statistical analysis.
342 R.G. Kudumela et al. / South African Journal of Botany 126 (2019) 340–344

Table 1
MIC values (μg/mL) of the serial exhaustive extraction extracts of S. pinnata.

Microorganisms Extractants Amp (μg/mL)

n-Hexane Dichloromethane Ethyl acetate Acetone Methanol Avg

I II III I II III I II III I II III I II III

E. coli 1050 840 740 190 350 190 370 430 1670 430 430 840 530 1250 1250 700 30
P. aeruginosa 320 320 80 160 80 80 320 320 640 160 160 160 320 2500 2500 540 20
E. faecalis 80 160 160 40 160 40 80 80 160 80 320 160 640 640 1250 270 30
S. aureus 1050 940 740 640 530 430 320 370 320 1250 1250 640 1570 1300 1250 840 80
Average 630 570 430 260 280 190 270 300 700 480 540 450 770 1420 1560
Total average 540 240 420 490 1250

Key; Amp = ampicillin; Avg = average, (I – first extraction, II – second extraction, III – third extraction).

Table 2
MIC values (μg/mL) of fractions from column chromatography against four tested bacteria.

Microorganisms MIC values (μg/mL) Amp (μg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Avg

E. coli 2500 2500 320 160 160 80 40 160 320 1250 1250 640 320 640 640 730 30
P. aeruginosa 530 1250 1420 530 270 130 270 680 860 120 250 500 330 1360 1460 670 20
E. faecalis 80 130 130 90 90 20 80 80 80 290 160 90 30 120 250 110 30
S. aureus 80 160 80 40 80 20 80 40 40 2500 1250 640 320 2500 1250 610 80
Average 800 1010 500 210 150 60 120 240 330 1040 730 470 250 1160 900

Key: Avg = average; Amp = ampicillin; 1 = 100% n-hexane; 2 = 90% n-hexane: ethyl acetate; 3 = 80% n-hexane: ethyl acetate; 4 = 70% n-hexane: ethyl acetate; 5 = 50% n-hexane: ethyl
acetate; 6 = 30% n-hexane: ethyl acetate; 7 = 10% n-hexane: ethyl acetate; 8 = 100% ethyl acetate; 9 = 90% ethyl acetate: methanol; 10 = 80% ethyl acetate: methanol; 11 = 70% ethyl
acetate: methanol; 12 = 60% ethyl acetate: methanol; 13 = 50% ethyl acetate: methanol; 14 = 40% ethyl acetate: methanol; 15 = 100% methanol.

3. Results and discussion dihydroxytigloyloxy moiety, as it was previously reported to be residues

of SL from Schkuhria pinnata (Kimani et al., 2018; Pacciaroni et al.,
3.1. Antibacterial activity of extracts and fractions 1995). Thus, compound 1 was proposed to be 6α-[4′,5′-
dihydroxytigloyloxy]-inuviscolide. Compound 4 was obtained as the
Bioactivity-guided fractionation aided the isolation of the antibacte- minor mixture of the yellowish oil with 1. The data for compound 4
rial compounds from dichloromethane extracts of S. pinnata. The anti- were similar to that reported for a guaiane-type SL 6 with a C-9 olefinic
bacterial properties determined using the micro-broth dilution
method are presented in Tables 1 and 2. Ampicillin was used as a posi-
tive control and its MIC values ranged from 20 to 80 μg/mL. As suggested
by Rios and Recio (2005) only MIC values less than 100 μg/mL were con- Table 3
NMR data (1H & 13C) for compound 1, 4–6 and 13C shifts from Literature.
sidered as noteworthy. These were exhibited by n-hexane and dichloro-
methane extracts against P. aeruginosa and n-hexane, dichloromethane, No. 1 δH, multi, J in 1 δC 5a 4 δH, multi, J 4 δC 6a
ethyl acetate and acetone extracts against E. faecalis (Table 1). Fractions Hz in
Hz
6 and 7 showed higher activity against E. coli, fractions 1, 4–9, 12 and 13
showed high activity against E. faecalis, while fractions 1, 3–9 were 1 3.25, d, 6.2 48.6 40.15 CH 2.64, s 48.6 38.7 CH
2 2.39, brs 29.7 25.3 CH2 2.39, brs 29.9 22.8 CH2
active against S. aureus (Table 2). Kudumela and Masoko (2018)
2.72, brs 2.72, brs
also documented the antibacterial activities of S. pinnata against E. coli, 3 2.39, s 43.7 40.19 CH2 2.64, brs 43.9 40.5 CH2
P. aeruginosa and E. faecalis. Meanwhile, Luseba et al. (2007) reported 2.64, s 2.72, brs
on the antibacterial activity (0.31 mg/mL) of the methanol and dichloro- 4 – 83.5 81.6 C – 77.8 80.5 C
methane extracts against S. aureus and E. coli. In our previous study, we 5 2.97, brs 59.3 64.5 CH 4.40, s 64.4 56.6 CH
6 5.20, brs 76.2 75.5 CH 5.31, s 74.1 74.9 CH
observed same results when testing antibacterial activity of S. pinnata 7 3.25, d, 6.2 48.7 49.7 CH 3.25, d 6.2 48.0 51.5 CH
against E. coli which was 0.32 mg/mL. On the contrary no potent activi- 8 5.20, brs 77.8 76.9 CH 5.31, s 80.5 76.8 CH
ties were observed with methanol extracts against any of the tested bac- 9 2.29,s 52.7 40.4 CH2 5.31, s 126.5 125.3 CH
teria in this study, probably because other solvents had extracted the 2.31, s
10 – 145.9 146.0 C – 145.9 140.9 C
active plant material components. These antibacterial properties could
11 – 139.4 136.9 C – 139.2 137.6 C
therefore, justify the traditional uses of this plant for treatment of disor- 12 – 169.8 170.6 C – 169.9 170.9 C
ders linked to infectious microorganisms. 13 5.92, brs 124.6 125.8 CH2 5.92, brs 124.6 124.1 CH2
6.21, brs 6.21, brs
3.2. Identification of the active compounds 14 4.82, brs 111.4 111.7 CH2 2.04, brs 20.8 21.2 CH3
4.90, brs
15 1.82, s 22.7 23.2 CH3 1.29, s 23.1 22.2 CH3
Separation and purification of the dichloromethane extract afforded 1’ – 170.9 169.5b C – 169.8 167.6c C
four compounds. The structure of the sesquiterpene lactones (SL) 1 and 2’ – 139.2 136.6 C – 136.1 136.6 C
4 was identified by the analysis of the NMR data and comparison of the 3’ 6.84, dd, 5.2, 143.4 145.5 CH 6.62, m 139.4 140.4 CH
10.2
spectroscopic data to Inula falconeri and Inula aucheriana sesquiterpene
4’ 4.29, s 59.8 56.9 CH2 2.97, s 59.3 63.6 CH2
lactones reported in the literature (Cheng et al., 2011; Gohari et al., 5 4.40, s 61.4 59.2 CH2 4.40, s 60.1 59.6 CH2
2015).
400 MHz, Acetone-d6.
The 1H and 13C NMR signals resembled those of 6α- a
Cheng et al. (2011).
hydroxyinuviscolide 5, a guaiane SL with a five-membered lactone b
Pacciarone et al., 1995 (200 MHz, CDCl3).
ring. The identity of the residue was readily elucidated to be a 4′,5′- c
Kimani et al., 2018 (600 MHz, CDCl3).
 R.G. Kudumela et al. / South African Journal of Botany 126 (2019) 340–344 343

H2C 14 H3C
H H
OH
10

4'
1
8 O O

2'
5

1'
4
6 12
H3C15 H3C
11

5'
H O H O O OH
OH OH
RO RO A
H2C13 H2C

R R O
1 A 4 A
5 H CH3 6 H
14
9 B
1 OR
10
7
3 R R1
R1O 5 2 A B
O 11 13 3 A Ac
CH2
H3C 15 12

Fig. 1. The structure of compounds 1–6.

methine (Cheng et al., 2011). Thus, 4 was elucidated to be 6α-[4′,5′- 3.3. Antibacterial, anti-inflammatory and cytotoxic activities of the bioac-
dihydroxytigloyloxy]-isoinuviscolide, Table 3 and Fig. 1. tive compounds
The 1 H and 13 C NMR spectroscopic data of compound 2 and 3
were characteristic of a heliangolide sesquiterpene lactones bearing The minimum inhibitory concentrations of the compounds ranged
4,5-dihydroxytiglate and 2-hydroxyisovalerate moieties at C-3 from 46.8 to b250 μg/mL meanwhile those of the positive control (am-
or C-8. Comparison of the NMR data with those published picillin) ranged from 62.5 to b250 μg/mL. (Table 4). The highest activity
established the structures of compound 2 as 3-desacetyl-3- was observed for the mixture of compounds 1&4 with the lowest MIC
isovaleroyleucannabinolide and compound 3 as eucannabinolide value (46.8 μg/mL) against P. aeruginosa. However, the compounds
(Herz and Govindan, 1980; Kimani et al., 2018; Pacciaroni et al., were less active compared to the extracts and fractions suggesting
1995), Fig. 1. that these compounds were acting synergistically to exhibit activity
and this was disrupted by the repeated fractionation and separation
during isolation of the compounds.
The compounds were tested for their ability to inhibit reactive oxy-
Table 4
The LC50 on Vero cells, antibacterial MIC and selectivity index values of the isolated
gen species (ROS) production. All the compounds showed moderate
compounds. activity to inhibit ROS production in a dose dependant manner on LPS
induced RAW 264.7 macrophage cells (Fig. 2). Curcumin which shows
Compounds LC50 MIC values (μg/mL) Selectivity indexes (SI)
anti-inflammatory activity was used as a positive control. Curcumin in-
(μg/mL)
E.c P.a E.f S.a E.c P.a E.f S.a hibition of ROS production was demonstrated at the highest tested
Compound 1 & 4 b10 125 46.88 125 62.5 0.08 0.21 0.16 0.16 concentration (50 μM). Controlling ROS levels is important to protect
Compound 2 13.5 N250 250 N250 N250 0.05 0.05 0.05 0.05 against degenerative diseases since ROS play an important role in
Compound 3 14.2 N250 N250 N250 N250 0.06 0.06 0.06 0.06 inflammation (Hussain et al., 2016). Thus, the result suggest potential
Key: E.c = E. coli; P.a = P. aeruginosa; E.f = E. faecalis; S.a = S. aureus. anti-inflammatory activity. Ivanescu et al. (2015) demonstrated the

120
ROS production inhibition (%)

100

80

60 1.95 µg/mL

32.3 µg/mL
40
80 µg/mL

20

0
1&4 2 3 Curcumin Untreated

Concentration of compounds (µg/mL)

Fig. 2. The inhibition of ROS production activity in LPS induced RAW 264.7 macrophage cells by the isolated compounds.
344 R.G. Kudumela et al. / South African Journal of Botany 126 (2019) 340–344
anti-inflammatory potential of guanolides through the inhibition of
the expression of nuclear factor NF-κB (nuclear factor kappa-light-
chain-enhancer of activated B cells).
On the in-vitro cytotoxicity assay, the compounds showed cytotoxic
effects on Vero cells with LC50 values ranging from b 10 to 14.2 μg/mL
(Table 4). According to the United States National Cancer institute a
pure compound is considered toxic if its LC50 is ≤4 μg/mL after 48 and
72 h of exposure (Sufian et al., 2012). Therefore, based on the US NCI
guidelines all the isolated compounds were moderately toxic to the
monkey Vero cells, since they had LC50 values that were considerably
higher than the cut-off point. These values were even higher compared
to doxorubicin (2.29 μg/mL) (Dzoyem et al., 2014). This study recom-
mends in-vivo studies on the isolated compounds to evaluate their
toxic effects and their genotoxicity on important biomolecules. It was
important to determine if the observed activity was not due to toxicity
in order to prevent misadministration since the use of medicinal plants
in not standardised in South Africa (Madikizela et al., 2014). Therefore,
the selectivity indexes were used to relate cytotoxicity and antibacterial
activities of the isolated compounds. The efficacy of biological activity is
considered not to be due to toxicity if the selectivity index is ≥ 10
(Caamal-Fuentes et al., 2011). These values ranged from 0.05 to 0.21
(Table 4). As such the observed antibacterial activities of the isolated
compounds are likely to be due to toxicity because their selectivity
index values were very low.
4. Conclusion
This study has demonstrated the antibacterial activities of the n-hex-
ane, dichloromethane, ethyl acetate and acetone extracts of S. pinnata
against P. aeruginosa and E. faecalis as well as different fractions against
E. coli, E. faecalis and S. aureus. Bioactivity-guided fractionation has led to
the isolation and identification of sesquiterpene lactones from Schkuhria
pinnata (Lam.) Kuntze ex Thell. However, the compounds had less anti-
bacterial activity as compared to the extracts suggesting the disruption
of synergistic interactions by the isolation process. Nevertheless, the ob-
served biological activities support the traditional uses of the plant ex-
tracts for treatment of infections linked to microorganisms and
inflammatory related disorders.
Declaration of interest
The authors declare that there is no conflict of interest.
Authors' contributions
RGK, carried out the experiments and analysed the data; OM, did
structural elucidation and PM was involved in the conception and de-
sign of the study. All authors read and approved the final manuscript.
Acknowledgement
We would like to thank University of Limpopo (Grant: R800) and
National Research Foundation of South Africa for financial assistance
(Grant: SFH160713177859). Ms. D.F Mangokoana for assistance with
anti-inflammatory assay and Ms. J.G. Ramakadi of the Chemistry De-
partment for assistance with NMR spectroscopy.
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