- Open Access
Isolation, characterization, and biological evaluation of a potent anti-malarial drimane sesquiterpene from Warburgia salutaris stem bark
© The Author(s) 2018
- Received: 16 April 2018
- Accepted: 1 August 2018
- Published: 15 August 2018
Malaria continues to be a global burden as the efficacy of most commercial anti-malarial drugs has been compromised by the evolution of parasite resistance. With the urgent need created for the development of alternative and more efficient anti-malarial drugs, this study focused on the evaluation of anti-malarial agents of the Warburgia salutaris stem bark.
The stem bark was extracted with dichloromethane followed by silica gel column chromatography that led to the isolation of iso-mukaadial acetate, a drimanoid sesquiterpene. This compound was characterized by spectroscopic analysis (1H NMR, 13C NMR, IR and MS), and its structure was confirmed by X-ray crystallography. In vitro anti-plasmodial activity was investigated using a chloroquine sensitive NF54 Plasmodium falciparum strain. Cytotoxicity was measured using the MTT assay on HEK239 and HEPG2 cell lines. Chloroquine-sensitive Plasmodium berghei was used to infect Sprague–Dawley rats for in vivo studies. The W. salutaris crude extract and iso-mukaadial acetate were administered orally at 0.5; 1.5, 2.5 and 5 mg/kg, chloroquine was used as the reference drug. Determination of percentage parasitaemia, haematological parameters, and rat body weights was done throughout the experimental study period.
The crude extract and iso-mukaadial acetate showed very good activity on the inhibition of parasite growth (IC50 0.01 ± 0.30 µg/ml) and (IC50 0.44 ± 0.10 µg/ml), respectively, with iso-mukaadial acetate having cytotoxicity activity of 36.7 ± 0.8 and 119.2 ± 8.8 (µg/ml) on HEK293 and HEPG2 cells, respectively. The crude extract and iso-mukaadial acetate reduced percentage parasitaemia in a dose-dependent manner in comparison to the control. There were no significant differences in the haematological parameters in all the experimental groups in comparison to control group. This study proves that W. salutaris contains components (including iso-mukaadial acetate) that exhibit anti-malarial activity. This study scientifically validates the use of this plant in folk medicine.
This study proves that Warburgia salutaris contains components (including iso-mukaadial acetate) that exhibit anti-malarial activity and scientifically validates the use of this plant in folk medicine.
- Plasmodium falciparum
- Anti-malarial agents
- Warburgia salutaris
- Iso-mukaadial acetate
- Plasmodium berghei
- Sprague–Dawley rats
Approximately 80% of rural African population is mainly dependent on traditional medicine to treat diseases , including malaria . Historically, most anti-malarial drugs have been derived from plants . UNESCO encourages research into local medicinal plants with the hope of finding anti-malarial drugs that are more efficient and could combat the artemisinin resistant malaria parasite . Currently artemisinin-based combination therapy is recommended for treatment of falciparum malaria and its effectiveness against other plasmodial species . In 1996 among the top 20 prescription dispensed, six were derived from natural products and the clinical use of drugs, such as artemisinin and taxol, further proved that plants are sources of novel drugs entities .
Warburgia salutaris (pepper-bark tree) is an evergreen medium-sized tree that can grow up to 10 metres in height. It is found in the southern African region, where it is used in traditional medicine. Most traditional concoctions are prepared from the inner bark of this tree . Tablet forms of W. salutaris are available in the market for the treatment of bronchitis, chest infections and ulcers ; the leaf tablet form is a natural antibiotic, effective against oesophageal and oral thrush [8, 9]. Traditional use of the roots, bark and leaves is common in the treatment of venereal diseases, influenza, abdominal pain, ulcers, respiratory complaints and malaria . There has been no scientific proof of this plant’s anti-malarial activity to date.
Some drimane sesquiterpenes have been isolated from this plant; these include: warburganal , found to possess, molluscicidal , antifungal and antibacterial [7, 13] activity; polygodial , with antifungal and antibacterial  activity; salutarisolide [15–17], muzigadial , possess antimicrobial and antifungal [7, 19]; ugandensidial, isopolygodial and mukaadial ; 11a-hydroxycinnamosmolide with anti-mycobacterial activity . The primary aim for this study is to scientifically validate the use of W. salutaris for treating malaria in Zulu traditional medicine. An attempt was made to identify the active constituent in the plant extract. Animal studies were also carried out to determine the plant’s targets in parasite progression.
Collection and preparation of plant material
Warburgia salutaris stem bark was collected from the Botanical gardens of the University of KwaZulu-Natal, Pietermaritzburg Campus, KwaZulu-Natal, South Africa (29°37′30′′S, 30°24′14′′E). The plant was identified by the horticulturist and a voucher specimen was prepared (voucher number: NU0043932) and deposited at the University of KwaZulu-Natal, Pietermaritzburg herbarium. The bark was sun-dried for 5 days and crushed into a fine powder.
Extraction and isolation
The fine stem bark powder (1 kg) was subjected to maceration with dichloromethane (1:5 w/v) for 3 days with periodic shaking. The maceration extract was filtered through Whatman (No. 1) filter paper and concentrated under reduced pressure at 45 °C. Silica gel column chromatography (60 × 1000 mm; Merck silica gel, 60: 0.063–0.200 mm) was used to isolate the compound from the DCM extract. The column was eluted with hexane: ethyl acetate gradient and the ratio 8:2 yielded 35 fractions. The fractions were grouped according to the thin layer chromatography (silica gel 60 aluminium sheets, F 254—Merck, Whitehouse Station, New Jersey, USA) profiles, into seven combined fractions (Cfrs). The spots were fixed on the TLC plates with a 20% H2SO4 in methanol mixture and viewed under UV light (254 nm). The combined fractions were then dried under a fume-hood overnight; Cfrs 2–4 produced a white amorphous powder (NN-01). NN-01 was recrystallized from methanol to give white needles (mpt. 95–98 °C); [α]D = − 11(0.1M in CHCl3); HREI-MS: MF = C17H24O5, [M+Na]+ ion at m/z 331.1619 (calcd: 331.1521).
1H- and 13C-NMR chemical shifts (δ, ppm) of cinnamodial (1)  and iso-mukaadial acetate (2)
δc (ppm) (multiplicity)
δc (ppm) (multiplicity)
Iso-mukaadial acetate (2)
1.77–1.39 (6H, m)
1.85–1.30 (6H, m)
2.05 (1H, d, J = 4.6 Hz)
2.08 (1H, d, J = 4.8 Hz)
5.87 (1H, t, J = 4.6 Hz)
5.91 (1H, t, J = 4.8 Hz)
7.04 (1H, d, J = 4.6 Hz)
7.01 (1H, d, J = 4.8 Hz)
4.01 (1H, br s)
4.07 (1H, br s)
9.75 (1H, s)
9.76 (1H, s)
9.47 (1H, s)
9.48 (1H, s)
1.02 (3H, s)
1.03 (3H, s)
1.16 (3H, s)
1.17 (3H, s)
1.34 (3H, s)
1.34 (3H, s)
2.14 (3H, s)
2.17 (3H, s)
The HepG2 (liver hepatocellular carcinoma) cells were obtained from the Highveld Biologicals (Pty) Ltd, Kelvin, SA, while the HEK293 (Human embryonic kidney) cells were provided by the University of the Witwatersrand, Medical school, Antiviral Gene Therapy Unit, SA. The modified method of Mosman  was used. The MTT assay is used to measure the cells metabolic activity, by reducing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into formazan salt using the enzyme succinate-tetrazolium reductase within the cell. The cells were developed to semi-confluency, in flasks for tissue culture (25 cm2), in Eagle’s minimal essential medium containing, 100 U/ml penicillin; 100 μg/ml streptomycin as well as 10% fetal bovine serum. Cells were then seeded at a density of 21 × 103 cells in each well, with medium (100 µl) in a 96 well plate. A 24 h incubation period (37 °C; 5% CO2) followed, after which fresh medium (100 µl) was added upon the removal of the old medium. Varying concentrations (50 µg/ml, 100 µg/ml and 150 µg/ml) of the compound were added in triplicates onto the cells then further incubated for 48 h at 37 °C. Cells without the experimental compound were used as the positive controls (100% viability).
The used medium was removed, after 48 h of incubation and, 100 µl new medium and 5 mg/ml MTT in PBS added, and this went into incubation (37 °C) for another 4 h. MTT and medium were then discarded, and 200 µl of DMSO was added per well, for dissolving the formazan. Absorbance of resulting mixture was then read with a Mindray 96A microplate reader (Vacutec, Hamburg, Germany) using a wavelength of 570 nm (detection λ) and that of 630 nm as the reference wavelength of nonspecific signals. The percentage viability of the cell was linked to absorbance and worked out in reference to the positive control using the equation: [(OD570 Treated)/(Control)]× 100. All experimentations were done in triplicates.
In vitro anti-plasmodial activity
The compound was tested against a NF54 Plasmodium falciparum strain (chloroquine sensitive); the tests were done in triplicate. Continuous in vitro cultures of asexual erythrocytes stage of the parasite were maintained following the modified method of Trager and Jensen . The viability of the parasite was evaluated by looking at the parasite lactate dehydrogenase activity, which was conducted following a modified method described by Makler et al. . Test sample was made to a stock solution of 20 mg/ml in DMSO (100%). The stock solution was preserved at − 20 °C. Dilutions of the stock were made on the day of experimentation. Artesunate and chloroquine were the drugs of reference in the experiment.
Full dose–response was conducted on the test compound to find concentrations at which 50% of parasitic growth is inhibited (IC50 value). Primary compound concentration for the trial was at 100 μg/ml, thereafter successively diluted to concentration twice as much in medium to produce ten measures with varying concentrations; minimum concentration being 0.2 μg/ml. Chloroquine initial concentration was at 1000 ng/ml. Solvent concentrations in which the infected red blood cells were subjected to, had no significant consequence on the viability of parasite. A non-linear dose–response curve fitting analysis was used to calculate the IC50 values, with the version4.0 Graph Pad Prism software (Graph Pad Prism, Inc: San Diego, CA, USA, 1994–2003).
In vivo antiplasmodial activity
Male Sprague–Dawley rats were used, 16–18 weeks old, from the University of KwaZulu-Natal Biomedical research unit. The housing and feeding conditions were maintained as per recommended standards.
Chloroquine sensitive Plasmodium berghei was obtained from the University of Cape Town, Clinical Pharmacology Division, South Africa. The parasite (105 P. berghei) was introduced to the animals via an intraperitoneal injection.
Male Sprague–Dawley rats (n = 48), were randomly grouped into non-P. berghei infected (n = 12) and P. berghei infected (n = 36) groups. Malaria induction was done via intra-peritoneal injection of 105 P. berghei strain ANKA parasitized RBCs. Following malaria induction, treatment was commenced after a 7 days pre-treatment period. The non-P. berghei infected experimental animals were further sub divided into two separate groups (n = 6/group), receiving oral dosing of 1.5 mg/kg crude extract and 1.5 mg/kg Iso-mukaadial acetate each. The P. berghei infected experimental animals were further sub divided into six separate experimental groups (n = 6/group): absolute control/not treated—vehicle group; positive control group-CQ 60 mg/kg (day 8–11); crude extract x5#—1.5 mg/kg (day 8–12); M. acetatex5#—1.5 mg/kg (day 8–12); Crude extract x1*—1.5 mg/kg (day 8); M. acetatex1*—1.5 mg/kg (day 8). The experimental animals were then monitored throughout the duration of the experimental period (21 days). *x1—treated for 1 day; #x5—treated for 5 days.
Determination of level of parasitaemia
Percentage parasitaemia was determined by thin blood smears prepared from the tail blood of each rat, fixed with methanol and stained with 20% Giemsa every day from day 3 to day 21. The animals were sacrificed on day 21 and determination of survival and haematology profiles were done.
Determination of body weight
Animal body weights were monitored to determine whether iso-mukaadial acetate and W. salutaris crude extract prevents weight loss that is commonly observed with increasing parasitaemia. Weights were taken at 3-day intervals until day 21. The rat grimace scale , from the National Centre for the Replacement Refinement and Reduction of Animals in Research (NC3Rs), was used to assess whether the animals were suffering during the experiment. Orbital tightening; nose and cheek flattening; ear change; and whisker change was used as parameters for the grimace scale.
In vitro data was analysed using one-way analysis of variance (ANOVA), the haematology data was analysed using group statistics, followed by Bonferroni post hoc test and parasitaemia and weight data was analysed using two-way analysis of variance. The mean ± SEM was calculated. The 2010 Microsoft Excel Program and version. 4.0 Graph Pad Prism software (Graph Pad Prism, Inc: San Diego, CA, USA, 1994–2003) were used for the statistical analysis of differences amongst the mean values calculated for the different trial groups, for IC50. P values ≤ 0.05 were taken as significant.
In vitro antiplasmodial and cytotoxic activity
In vitro anti-plasmodial and cytotoxic activity of Iso-mukaadial acetate (NN-01)
0.01 ± 0.30
21.30 ± 01
55.21 ± 11
0.44 ± 0.10
36.7 ± 08
119.2 ± 88
4.9 ± 0.07 ng/ml
232.45 ± 21
350.04 ± 90
< 2 ng/ml
In drug discovery, a compound is considered a success if it has an IC50 ≤ 1 µg/ml and a selectivity index that is at least ten times more active on the parasite than it is on human cell-lines . Iso-mukaadial acetate shows good in vitro anti-malarial activity. There has been no report of any other isolated anti-malarial compound from W. salutaris in literature. Katiyar et al. suggests that natural sources have a potential of delivering natural compounds as base for synthetic analogues in drug discovery . It is noteworthy to state that the high antiplasmodial activity of iso-mukaadial acetate makes it eligible for structural modification studies to reduce cytotoxicity while retaining or improving antiplasmodial activity.
In vivo antiplasmodial results
Effects of Warburgia salutaris and iso-mukaadial acetate on haematology profile
The reduce red blood cell count and haematocrit in the study is consistent with previous malaria studies of severe malarial anaemia. A study showed that red blood cell destruction and decrease in red blood cell production in response to parasite invasion may lead to reduced haematocrit and haemoglobin levels in the blood of infected animals . However, mechanisms by which haematology parameters were affected are not described in this study. Further plasma and serum studies may provide mechanisms by which observed malaria-induced haematology changes occurred. The study groups are represented as, UMA-Uninfected iso-mukaadial acetate treated; UCE-Uninfected crude extract treated; IC-infected without treatment; ICQ-infected chloroquine treated; ICE1-infected crude extract 1-day treatment; ICE2-infected crude extract 5-day treatment; IMA1-infected iso-mukaadial acetate 1-day treatment; IMA2-infected iso-mukaadial acetate 5-day treatment. Column statistics was carried out for all haematology data.
Effects of Warburgia salutaris and iso-mukaadial acetate on animal weight
The aim of this study was to scientifically validate the use of W. salutaris in Zulu traditional medicine. Results obtained show that W. salutaris possess anti-malarial activity, the active compound found to be iso-mukaadial acetate, which shows appreciable inhibition on parasite growth. The in vivo antiplasmodial studies with chloroquine-sensitive P. berghei show that W. salutaris has very good anti-malarial activity and protective effects on the survival of malaria-infected rodents. Iso-mukaadial acetate could serve as a lead compound for the development of a relatively potent drug in the treatment of malaria. However, more studies are required to determine the compounds mechanism of action on other in vitro models and on in vivo models as well. The results obtained support the use of W. salutaris in traditional medicine, to treat malaria.
MBCS conceived and designed the experiments; ZNN and PM performed the experiments; FOS, SM and ARO analysed the data. All authors read and approved the final manuscript.
We thank Peter Smith (University of Cape Town) for assisting with In vitro antiplasmodial activity.
The authors declare that they have no competing interests.
Availability of data and materials
The authors confirm that the samples of the compounds are available from the authors.
Consent for publication
Ethical approval and consent to participate
Ethical clearance for the use of animals in the study was obtained from the University of KwaZulu-Natal Ethics committee (Reference: AREC/053/016).
We thank the University of KwaZulu-Natal Research committee and the National Research Foundation (UID: 99326) for their financial support.
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- Mahomoodally M. Traditional medicines in Africa: an appraisal of ten potent African medicinal plants. Evid Based Complement Alternat Med. 2013;2013:617459.View ArticlePubMedPubMed CentralGoogle Scholar
- Ahmad HA. Review on Anogeissus leiocarpus a potent African traditional drug. Int J Res Pharm Chem. 2014;4:496–500.Google Scholar
- Oliveira A, Dolabela M, Braga F, Jácome R, Varotti F, Póvoa M. Plant-derived antimalarial agents: new leads and efficient phythomedicines. Part I. Alkaloids. An Acad Bras Ciênc. 2009;81(Suppl 4):715–40.View ArticlePubMedGoogle Scholar
- UNESCO. Promotion of ethnobotany and the sustainable use of plant resources in Africa. Paris: Unesco; 1998. p. 60.Google Scholar
- Anvikar AR, Kuepfer I, Mishra V, Bruce J, Arya T, Mishra DR, et al. Efficacy of two artemisinin-based combinations for the treatment of malaria in pregnancy in India: a randomized controlled trial. Malar J. 2018;17:246.View ArticlePubMedPubMed CentralGoogle Scholar
- Philipson JD. Phytochemistry and medicinal plants. Phytochem. 2001;56:237–43.View ArticleGoogle Scholar
- Mahmoud II, Kinghorn AD, Cordell GA, Farnsworth NR. Potential anti-cancer agents. XVI. Isolation of bicyclofarnesane sesquiterpenoids from Capsicodendron dinisii. J Nat Prod. 1980;43(Suppl 3):365–71.View ArticlePubMedGoogle Scholar
- Jansen B, de Groot A. The occurrence and biological activity of drimane sesquiterpenoids. Nat Prod Rep. 1991;8(Suppl 3):309–18.View ArticlePubMedGoogle Scholar
- Botha J, Witkowski E, Shackleton C. The impact of commercial harvesting on Warburgia salutaris (‘pepper-bark tree’) in Mpumalanga, South Africa. Biodiversity Conservation. 2004;13(Suppl 9):1675–98.View ArticleGoogle Scholar
- Van Wyk BE. Medicinal plants of South Africa. Pretoria: Briza Publications Pretoria; 1997.Google Scholar
- Khamkar AD, Motghare VM, Deshpande R. Ethnopharmacology—a novel approach for drug discovery. Indian J Pharm Pharmacol. 2015;2(Suppl 4):222–5.View ArticleGoogle Scholar
- Appleton CC, Drewes SE, Mashimbye MJ, Cunningham AB. Observations on the molluscicidal properties of (−)-warburganal on the South African Bulinus africanus (Planorbidae). J Med Appl Malacology. 1992;4:37–40.Google Scholar
- Nakanishi K, Kubo I. Studies on warburganal, muzigadial and related compounds. Israel J Chem. 1997;16:28–31.View ArticleGoogle Scholar
- Taniguchi M, Adachi T, Haraguchi H, Oi S, Kubo I. Physiological activity of warburganal and its reactivity with sulfhydryl groups. J Biochem. 1983;94:149–54.View ArticlePubMedGoogle Scholar
- Mashimbye MJ. Chemical constituents of plants native to Venda. Ph.D. thesis. University of Natal, Department of Chemistry; 1993.Google Scholar
- Mashimbye MJ, Maumela MC, Drewes SE. A drimane sesquiterpinoid lactone from Warburgia salutaris. Phytochemistry. 1999;51:435–8.View ArticleGoogle Scholar
- Frum Y, Viljoen AM. In vitro 5-lipoxygenase and anti-oxidant activities of South African medicinal plants commonly used topically for skin diseases. Skin Pharmacol Physiol. 2006;19:329–35.View ArticlePubMedGoogle Scholar
- Frum Y, Viljoen AM, Drewes SE. In vitro 5-lipoxygenase and anti-oxidant activities of Warburgia salutaris and drimane sesquiterpenoids. South African J Botany. 2005;71:447–9.View ArticleGoogle Scholar
- El-Feraly FS, McPhail AT, Onan KD. X-ray crystal structure of canellal, a novel antimicrobial sesquiterpene from Canella winterana. Chem Comm. 1978;1:75–6.View ArticleGoogle Scholar
- Rabe T, van Staden J. Isolation of an antibacterial sesquiterpenoid from Warburgia salutaris. J Ethnopharmacol. 2000;73:171–4.View ArticlePubMedGoogle Scholar
- Mashimbye MJ, Maumela MC, Drewes SE. Novel and bioactive metabolites of Warburgia salutaris indigenous to the Northern Province, South Africa. Nigerian J Nat Prod Med. 1999;3:28–30.Google Scholar
- Mosman T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–65.View ArticleGoogle Scholar
- Trager W, Jensen J. Human malaria parasites in continuous culture. Science. 1976;193(Suppl 4254):673–5.View ArticlePubMedGoogle Scholar
- Makler MT, Ries JM, Williams JA, Bancroft JE, Piper RC, Gibbins BL, et al. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drugs sensitivity. Am J Trop Med Hyg. 1993;48:739–41.View ArticlePubMedGoogle Scholar
- Sotocinal SG, Sorge RE, Zaloum A, Tuttle AH, Marton LJ. The rat grimace scale: a partially automated method for quantifying pain in the laboratory rat via facial expressions. Mol Pain. 2011;7:55.PubMedPubMed CentralGoogle Scholar
- Katiyar C, Kanjilal S, Gupta A, Katiyar S. Drug discovery from plant sources: an integrated approach. Int Q J Res Ayurveda. 2012;33(Suppl 1):10.View ArticleGoogle Scholar
- Fidock DA, Rosenthal PJ, Croft SL, Brun R, Nwaka S. Antimalarial drug discovery: efficacy models for compound screening. Nat Rev Drug Discov. 2004;3(Suppl 6):509.View ArticlePubMedGoogle Scholar
- Pink R, Hudson A, Mouriès M, Bendig M. Opportunities and challenges in antiparasitic drug discovery. Nat Rev Drug Discov. 2005;4(Suppl 9):727–40.View ArticlePubMedGoogle Scholar
- Adugna M, Feyera T, Taddese W, Admasu P. In vivo antimalarial activity of crude extract of aerial part of Artemisia abyssinica against Plasmodium berghei in mice. Glob J Pharmacol. 2014;8(Suppl 3):460–8.Google Scholar
- Evans KJ, Hansen DS, van Rooijen N, Buckingham LA, Schofield L. Severe malarial anemia of low parasite burden in rodent models results from accelerated clearance of uninfected erythrocytes. Blood. 2006;107(Suppl 3):1192–9.PubMedPubMed CentralGoogle Scholar