Anti-plasmodial activity of some zulu medicinal plants and of some triterpenes isolated from them

Article abstract of DOI:10.3390/molecules181012313

Mimusops caffra E. Mey. ex A.DC and Mimusops obtusifolia Lam (both members of the Sapotaceae family), and Hypoxis colchicifolia Bak (family Hypoxidaceae) are used by traditional healers in Zululand to manage malaria. Anti-plasmodial investigation of the crude extracts and some triterpenes isolated from the plants showed activity against a chloroquine sensitive (CQS) strain of Plasmodium falciparum (D10). Among the crude extracts the leaves of M. caffra exhibited the highest activity, with an IC₅₀ of 2.14 μg/mL. The pentacyclic tritepenoid ursolic acid (1), isolated from the leaves of M. caffra was the most active compound (IC₅₀ 6.8 μg/mL) as compared to taraxerol (2) and sawamilletin (3) isolated from the stem bark of M. obtusifolia (IC₅₀ > 100). Chemical modification of the ursolic acid (1) to 3β-acetylursolic acid (4) greatly enhanced its anti-plasmodial activity. Compound 4 reduced parasitaemia against Plasmodium berghei by 94.01% in in vivo studies in mice. The cytotoxicity of 3β-acetylursolic acid (IC ₅₀) to two human cell lines (HEK293 and HepG2) was 366.00 μg/mL and 566.09 μg/mL, respectively. The results validate the use of these plants in folk medicine.

Full text of DOI:10.3390/molecules181012313

Molecules 2013, 18, 12313-12323; doi:10.3390/molecules181012313
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Anti-Plasmodial Activity of Some Zulu Medicinal Plants and of
Some Triterpenes Isolated from Them
Mthokozisi B. C. Simelane 1,*, Addmore Shonhai 1, Francis O. Shode 2, Peter Smith 3,
Moganavelli Singh 4 and Andy R. Opoku 1
1
Department of Biochemistry & Microbiology, University of Zululand, Private Bag X1001,
KwaDlangezwa 3886, South Africa; E-Mails: adshon@gmail.com (A.S.);
OpokuA@unizulu.ac.za (A.R.O.)
2
Department of Chemistry, University of Zululand, Private Bag X1001, KwaDlangezwa 3886,
South Africa; E-Mail: francisshode@yahoo.com
3
Division of Pharmacology, University of Cape Town, Private Bag X3, Rondebosch 7701,
South Africa; E-Mail: Peter.Smith@uct.ac.za
4
Discipline of Biochemistry, University of KwaZulu-Natal, Private Bag X54001, Durban 4000,
South Africa; E-Mail: Singhm1@ukzn.ac.za
* Author to whom correspondence should be addressed; E-Mail: simelanembc@gmail.com
Tel.: +27-35-902-6099; Fax: +27-35-902-6568.
Received: 6 August 2013; in revised form: 16 September 2013 / Accepted: 24 September 2013 /
Published: 8 October 2013
Abstract: Mimusops caffra E. Mey. ex A.DC and Mimusops obtusifolia Lam (both members
of the Sapotaceae family), and Hypoxis colchicifolia Bak (family Hypoxidaceae) are used
by traditional healers in Zululand to manage malaria. Anti-plasmodial investigation of the
crude extracts and some triterpenes isolated from the plants showed activity against a
chloroquine sensitive (CQS) strain of Plasmodium falciparum (D10). Among the crude
extracts the leaves of M. caffra exhibited the highest activity, with an IC50 of 2.14 μg/mL.
The pentacyclic tritepenoid ursolic acid (1), isolated from the leaves of M. caffra was the
most active compound (IC50 6.8 μg/mL) as compared to taraxerol (2) and sawamilletin (3)
isolated from the stem bark of M. obtusifolia (IC50 > 100). Chemical modification of the
ursolic acid (1) to 3β-acetylursolic acid (4) greatly enhanced its anti-plasmodial activity.
Compound 4 reduced parasitaemia against Plasmodium berghei by 94.01% in in vivo
studies in mice. The cytotoxicity of 3β-acetylursolic acid (IC50) to two human cell lines
(HEK293 and HepG2) was 366.00 μg/mL and 566.09 μg/mL, respectively. The results
validate the use of these plants in folk medicine.

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Keywords: Plasmodium falciparum; Mimusops caffra; Mimusops obtusifolia;
Hypoxis colchicifolia; ursolic acid
1. Introduction
Malaria is one of the major health problems in tropical Africa, South-east Asia, Central South
America and Oceania. Despite the various efforts by governmental and non-governmental organizations
aimed at eradicating the disease, malaria is said to kill a child every 30 s [1]. Malaria cases have been
reported in other areas of the World that were previously considered eradicated of malaria [2].
In Africa, herbal medicines are an important part of the culture and traditions of its people [3].
Traditional healers use different concoctions prepared from medicinal plants to treat malaria. Given
the remarkable anti-malarial properties of Cinchona bark that have been known for more than
300 years, resulting in the discovery of quinine [4] and the more recent development of artemisinin
derivatives [5], the potential of plant species to provide effective drugs for the treatment of malaria
cannot be overemphasized. Furthermore, the drug resistance of the malaria parasite to chloroquine
and sulfadoxine–pyrimethamine, and also the toxicity of the currently available drugs have stimulated
the search for alternative medicines which are naturally derived. In addition, modern health care to
the rural people is still a far-reaching goal, due to economic constraints [6] and many vulnerable
groups depend on plant-based traditional healing. The anti-malarial activity of many plants has
been reported [7–10]. An ethonobotanical survey revealed the extensive utilization of M. caffra,
M. obtusifolia and H. colchicifolia for the management of malaria in Zulu traditional medicine.
M. caffra is a small to medium-sized tree that grows up to 15 m high [11]. Its natural habitat is
the dune forest from the high tide mark in KwaZulu-Natal and the Eastern Cape Provinces of South
Africa and the Mozambique [11]. Medicinal uses of M. caffra include healing properties against sores
and wounds [12].
M. obtusifolia has a small to medium-sized tree. Bark is grey to blackish, very rough and fissured in
older specimens. Leaves are relatively broader and more rounded, 3.5–10 cm long. It is found in
Southern Africa [13]. There is little information in the literature on its pharmacological activities.
H. colchicifolia is a slow-growing plant that often reaches up to 600 mm in height, with erect
leaves; it is widespread in southern Africa [14]. It has a large underground tuber that allows it to
survive the regular grass fires common to the grassland where it is found. These tubers are used by
traditional healers to treat impotency and barrenness. Infusions are also taken as love charm emetics
and are administered for hysterical fits [15]. Pharmacological activities of H. colchicifolia include
anti-HIV and anti-diabetic properties [16,17]. This study was undertaken to investigate the
anti-malarial activity of these plants.
2. Results and Discussion
Traditional medicines are a potential rich source of new drugs against malaria and other infectious
diseases. The literature abounds with descriptions of the bioactivity of many antimalarial plants [7–10].
The observed anti-plasmodial activity of the crude extracts of M. caffra, M. obtusifolia, and

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H. colchicifolia is presented in Table 1. While the Mimusops species exhibited anti-plasmodial
activity, it is apparent that our results do not support the traditional use of the bulb of H. colchicifolia
in treating malaria. It is however worth mentioning that we observed antipyretic properties (data not
included) of the extract of H. colchicifolia. Fever is the early symptom of malaria, and it is thus likely
that the plant is used traditionally to treat the symptoms rather than the disease.
Table 1. Anti-plasmodial activity against Plasmodium falcipurum (CQS) D10 strain
(in vitro) and Plasmodium berghei (in vivo).
a Cytotoxicity (μg/mL)
a IC50
(µg/mL)
2.14
b Average %
b Average %
Sample
parasitemia
NT
suppression
NT
HEK293
HepG2
M. caffra (leaves)
M. obtusifolia (bark)
H. colchicifolia (bulb)
Ursolic acid
32.5
NT
NT
NA
NT
NT
6.8
NT
NT
Ursolic acid acetate
3-oxo-ursolic acid
Taraxerol
1.9
0.07
NT
94.01
NT
366.00
566.09
7.3
>100
>100
5.1 *
14.1 *
NT
NT
Sawamilletin
NT
NT
Artesunate
NT
NT
Chloroquine
0.07
83.43
a
b
* = ng/mL, IC50 = Inhibitory concentration, Average percentage parasitemia/suppression (in vivo),
NT = not tested, NA = not active.
Several triterpenes have been reported to possess both in vitro and in vivo anti-plasmodial
activity [18–20]. Of the three triterpenes that were isolated from M. caffra and M. obtusifolia (ursolic
acid, taraxerol and sawamilletin), only ursolic acid showed any appreciable anti-plasmodial activity
(IC50 6.8 μg/mL) at the concentration tested. Ursolic acid has been previously reported to possess
anti-plasmodial activity [21], and its presence in M. caffra could have contributed to the observed
bioactivity of the plant. The lower activity of the extracted ursolic acid (compared to the crude
M. caffra extract) could indicate a synergistic effect with other compounds, decomposition during
fractionation, or removal of a protective matrix. Chemical modification of ursolic acid to its acetate
derivative however resulted in a 72% increase in the in vitro anti-malarial activity (IC50 1.9 μg/mL).
Chemical modification of drugs has been known to improve the potency of the drugs [22,23]; it is
noteworthy that the usoric acid acetate exhibits a 94.01% suppression of parasitemia in infected mice
(in vivo).
In cytotoxic evaluations a compound is only considered significantly active with an IC50 of less
than 30 μg/mL [24]. The cytotoxicity of ursolic acid acetate to HEK293 and HepG2 cell lines is
presented in Table 1. It is apparent that, when dilution in the bloodstream is taken into account, ursolic
acid acetate should be regarded as non-toxic.

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3. Experimental
3.1. Plant Collection
Fresh plant materials of M. obtusifolia and H. colchicifolia were collected from the Manguzi area,
KwaZulu-Natal Province, South Africa, at the flowering stage in April, 2011 and M. caffra was
collected in May 2012 from Durban, KwaZulu-Natal Province, South Africa. The plants were
identified by Mrs. N. R. Ntuli, Department of Botany, University of Zululand, KwaDlangezwa.
Voucher specimens were deposited at the University Herbarium [Simelane, MBC/02 (ZULU);
Simelane, MBC/03 (ZULU); Simelane, MBC/04 (ZULU)].
3.2. Extraction and Isolation
The air-dried leaves of M. caffra (500 g) and the bark of M. obtusifolia (1.1 kg) were extracted with
dichloromethane (DCM) and ethyl acetate (EtOAc) respectively (1:5 w/v). The resultant filtered
extracts were concentrated to dryness under reduced pressure in a rotary evaporator (40 ± 2 °C). Dried
extracts (5 g) were separately subjected to column chromatograph (20 × 500 mm) using silica gel 60
(300 g; 0.063 to 0.2 mm; 70 to 230 mesh ASTM supplied by Merck (Darmstadt, Germany). The crude
extracts were chromatographed using gradient elution of hexane-ethyl acetate in a 5% stepwise
increase at a speed of 100 mL per min and collecting 20 mL fractions. The collected fractions were
combined based on their TLC (20 × 20 F254—Merck, Whitehouse Station, NJ, USA) profile to yield
combined fractions. Visualization was achieved by UV light (254 nm) and by heating with 20% H2SO4
acid in MeOH. The single spot fractions were recrystallized in methanol and hexane to obtain 270 mg
of MBCF93 from the n-hexane/EtOAc 7:3 eluate of the M. caffra extract; 310 mg of MBCF45
and 240 mg of MBCF15 were obtained from the n-hexane/EtOAc 9:1 fractions from the bark of
M. obtusifolia. No attempt was made to isolate any compounds from H. colchicifolia because the
initial crude extracts indicated no observable anti-plasmodial activity.
3.3. Structural Elucidation
Compound MBCF93 was a white powder with a melting point (mp) of 279–283 °C, MBCF45
was also a white powder with mp 280–287 °C, MBCF15 also came out as a white powder with mp
272–285 °C. The structure of compounds were established using Nuclear Magnetic Resonance (NMR)
techniques with the application of 2D-NMR (1H-1H, 13C-13C, DEPT, COSY, HMQC, HMBC and
NOESY), infrared (IR) spectra and liquid chromatography mass spectrometry (LC-MS); molecular
weights were identified by ESI-MS (positive mode). The compounds were confirmed by spectra
comparison of their spectra (1H and 13C-NMR) with reported literature (Table 2). MBCF93 was
confirmed as ursolic acid (1), MBCF45 as taraxerol (2), and MBCF15 as sawamilletin (3) (Figure 1).

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Figure 1. Chemical structures of the isolated triterpenes.
CH3
H3C
CH3
H3C
CH3
OH
CH3
CH3
CH3
CH3
O
CH3
H
H
CH3
HO
H3C
1
HO
CH3
H3C
CH3
2
H3C
CH3
CH3
Ursolic acid
Taraxerol
H3C
CH3
OH
CH3
CH3
CH3
CH3
O
CH3
O
CH3
H
H
O
O
H3C
H3C
CH3
CH3
4
H3C
3
3-acetylursolic acid
Sawamilletin
Table 2. 1H- and 13C-NMR chemical shifts (δ, ppm) of compounds 1, 4 and 2.
Carbon
Position
UA(1)
δ 13C (ppm)
38.7
UAA(4)
δ 13C (ppm)
38.3
δ 1H (ppm)
DEPT
DEPT
1
2
3
CH2
CH2
CH
C
CH2
CH2
CH
C
23.5
79.0
39.6
24.1
80.9
37.7
3.43 (1H, brs)
4
5
6
7
8
52.7
18.3
33.0
39.1
CH
CH2
CH2
C
55.3
18.2
32.9
39.5
CH
CH2
CH2
C
9
47.6
36.7
23.7
125.8
138.0
42.0
29.4
23.3
47.9
55.3
30.6
30.4
CH
C
CH
CH
C
47.9
36.7
23.3
125.8
138.0
41.9
30.6
23.6
47.5
52.6
39.0
38.8
CH
C
CH
CH
C
10
11
12
13
14
15
16
17
18
19
20
5.50 (1H, brs)
C
C
CH2
CH2
C
CH
CH
CH
CH2
CH2
C
CH
CH
CH
2.52 (1H, d, J = 11.0 Hz)

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Table 2. Cont.
δ 1H (ppm)
Carbon
Position
UA(1) δ
13C (ppm)
27.3
UAA(4) δ
13C (ppm)
30.6
DEPT
DEPT
21
22
23
24
25
26
27
28
CH2
CH2
CH3
CH3
CH3
CH3
CH3
C
CH2
CH2
CH3
CH3
CH3
CH3
CH3
C
37.0
23.4
17.0
17.0
15.5
24.2
176.0
21.1
23.4
36.9
23.6
17.1
16.7
17.1
21.3
182.6
15.5
21.2
1.24 (3H, s)
1.02 (3H, s)
0.93 (3H, s)
1.05 (3H, s)
1.22 (3H, s)
29
30
0.97 (3H, s)
0.99 (3H, d, J = 6.1 Hz)
CH3
CH3
CH3
CH3
-COCH3
28.1
-COCH3
171.0
Carbon
Position
Taraxerol (2) δ 13C (ppm)
DEPT
δ 1H (ppm)
1
2
3
4
5
6
38.0
27.2
79.1
39.0
55.6
18.8
CH2
CH2
CH
C
CH
CH2
2.0 (1H, dt, J = 3.1, 12.6 Hz,
7
35.1
CH2
H-7a)
8
9
38.8
48.8
37.6
17.5
35.8
37.6
158.1
116.9
C
CH
C
CH2
CH2
C
10
11
12
13
14
15
C
CH
5.5 (1H, dd, J = 3.2, 8.2 Hz)
1.9 (1H, dd, J = 3.0, 14.6 Hz,
H-16a)
16
36.7
CH2
17
18
19
20
21
22
23
24
25
26
27
28
29
30
37.7
49.3
41.3
28.8
33.7
33.1
28.0
15.4
15.5
29.8
25.9
29.9
33.4
21.3
C
CH
CH2
C
CH2
CH2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
0.98 (3H, s, H-23)
0.80 (3H, s, H-24)
0.93 (3H, s, H-25)
1.09 (3H, s, H-26)
0.91 (3H, s, H-27)
0.82 (3H, s, H-28)
0.95 (3H, s, H-29)
0.90 (3H, s, H-30)

Molecules 2013, 18
12319
3.4. Acetylation of Ursolic Acid
The plant derived ursolic acid (283 mg) was treated with acetic anhydride (10 mL) and pyridine
(20 mL). The mixture was stirred at room temperature overnight after which water (10 mL) was added
and stirred for at least 30 min. The resultant solid was then filtered and washed thoroughly with dilute
HCl solution under vacuum. The white powder obtained (300 mg, mp 280–282 °C) was identified, by
its spectral properties, as 3β-acetylursolic acid (4).
3.5. Preparation of 3-Oxoursolic Acid (5)
To a solution of compound 4 (100 mg) in acetone (1.5 mL) Jones’ reagent (0.4 mL) was added
dropwise in an ice-salt bath. The reaction mixture was allowed to warm up to room temperature and
stirred for 1 h. After cooling to 0 °C, 2-propanol (5 mL) was added and the solution stirred at room
temperature for 30 min. The green precipitate was collected and washed well with acetone. The
acetone solution from the combined filtrates were concentrated and dried. By purification on a silica
gel column compound 5 was obtained as a white solid (86 mg) and identified as 3-oxo-ursolic acid
(Figure 2).
Figure 2. Chemical structure of 3-oxourosolic acid.
CH3
H3C
CH3
H
CH3
COOH
H
CH3
O
H
CH3
H3C
5
3.6. Drug Sensitivity Assay
3.6.1. In Vitro Antiplasmodial Activity
The test samples (crude extracts, isolates, and chemical derivatives) were tested in triplicate against
a chloroquine sensitive (CQS) strain of Plasmodium falciparum (D10). Continuous in vitro cultures of
asexual erythrocyte stages of P. falciparum were maintained using modified method of Trager and
Jensen [25]. Quantitative assessment of anti-plasmodial activity in vitro was determined via the
parasite lactate dehydrogenase assay using a modified method described by Makler [26]. The test
samples were prepared to a 20 mg/mL stock solution in 100% DMSO and sonicated to enhance
solubility. Stock solutions were stored at −20 °C. Further dilutions were prepared on the day of the
experiment. Chloroquine and artesunate were used as the reference drugs for experiments. A full
dose-response was performed for all active compounds to determine the concentration inhibiting 50%

Molecules 2013, 18
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of parasite growth (IC50-value). Test samples were tested at a starting concentration of 100 μg/mL,
which was then serially diluted 2-fold in complete medium to give 10 preparations of variable
concentrations; with the lowest concentration being 0.2 μg/mL. The same dilution technique was used
for all samples. CQ was tested at a starting concentration of 1,000 ng/mL. The highest concentration of
solvent to which the parasite-infected erythrocytes were exposed to had no measurable effect on the
parasite viability (data not shown). The IC50-values were obtained using a non-linear dose-response
curve fitting analysis via Graph Pad Prism v.4.0 software (Graph Pad Prism, Inc: San Diego, CA,
USA, 1994–2003).
3.6.2. In Vivo Antiplasmodial Activity
This study was carried out after the approval from the Ethical Committee on Animal Use and Care
of the University of Zululand (UZREC 171110-030 PGD 2013/26). Swiss mice (20–25 g each) of both
sexes were obtained from biomedical research unit (BRU) in the University of KwaZulu Natal,
Durban. They were kept in plastic cages, and given standard laboratory diet and water ad libitum and
maintained under laboratory conditions of temperature and 12 h light and 12 h dark cycle. The animals
were allowed to acclimatize to the laboratory at a controlled temperature of 22 °C for 15 days before
being subjected to the experiments. The animals were divided into seven groups (eight in each group)
consisting of control, chloroquine (5 mg/kg/day), and the extract treated group. The extract treated
group was sub divided into five sub groups that received 50, 100, 200, 300 and 400 mg/kg/day body
weight respectively.
3.6.2.1. Parasite Inoculation
A single donor mouse infected with Plasmodium berghei parasites was bled into sterile heparinized
culture medium and the blood was diluted with RPMI 1640 medium. The healthy experimental mice
were infected intravenously via a tail vein with 0.2 mL of the diluted blood containing 1 × 107
parasitized (Plasmodium berghei) red blood cells on day one.
3.6.2.2. Evaluation of Antimalarial Activity
The antimalarial activity tests were performed using the four-day suppressive tests described by
Peters et al. [27]. The extracts dissolved in 0.5% carboxymethyl cellulose (CMC) were administered
orally using cannula (equivalent to 0.2 mL solution per mouse) for four consecutive days. Parallel tests
with chloroquine were conducted for reference purposes at the same dose in one group and with an
equivalent volume of 0.5% CMC (0.2 mL/mouse/day) in the control group. Thin smears were obtained
from the tail vein of each mouse on day five after infection. The smears were fixed with methanol and
stained with Giemsa stain. The percent parasitemia suppression was determined by counting the
number of parasitized erythrocytes out of 500 red blood cells on random fields under the microscope.
The average percentage suppression of parasitemia was calculated using the following formula:
% suppression = A – B/C
(1)

Molecules 2013, 18
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where A = % parasitemia in untreated controls, B = % parasitemia in treated groups, and C = %
parasitemia in untreated controls. The data were analyzed using the F-test. A P-value < 0.05 was
considered significant.
3.7. MTT Cell Proliferation Assay
Human embryonic kidney (HEK293) and human hepatocellular carcinoma (HepG2) cells were all
grown to confluenecy in 25 cm2 flasks. This was then trypsinized and plated into 48 well plates at
specific seeding densities. Cells were incubated overnight at 37 °C. Medium was then removed
and fresh medium (MEM + Glutmax + antibiotics) was added. Extracts (50–350 μL/mL) were then
added in triplicate and incubated for 4 h. Thereafter medium was removed and replaced by complete
medium (MEM + Glutmax + antibiotics + 10% Fetal bovine serum). After 48 h cells were subjected to
the MTT assay [28]. Data were evaluated through regression analysis using QED statistics program
and from the linear equation the IC50 values representing the lethal concentration for 50% mortality
was calculated.
3.8. Statistical Analyses
The mean and standard error mean of three experiments were determined. Statistical analysis of the
differences between mean values obtained for experimental groups were calculated using Microsoft
Excel Program, 2010 and Graph Pad Prism v.4.0 software (Graph Pad Prism, Inc: San Diego, CA,
USA, 1994–2003) for IC50. Data were subjected to one way analysis of variance (ANOVA).
P values < 0.05 were regarded as significant and P values < 0.01 as very significant.
4. Conclusion
This study was conducted to investigate the anti-malarial activity of some indigenous plants used by
Zulu traditional healers to treat malaria. Even though the observed activity of the crude plant extracts,
the isolated triterpenes, and the chemical derivative may not be as high as those reported for the
standards (chloroquine and artesunate), the activity of the extracts were dose dependent, and with low
toxicity levels, encourages the use of M. caffra in managing malaria in traditional medicine.
Acknowledgments
The authors are grateful to National Research Fund (SA) and University of Zululand Research
committee for financial support.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds (1–5) are available from the authors.
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