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Research and Reviews: Journal of Pharmacognosy and
Phytochemistry
Anti-plasmodial and Radical Scavenging Activities of Croton megalobotrys
Anthonia O Abosi1, Runner R T Majinda2,*
World Health Organization Afro Regiona TB Focal Office, Kenya
2
Department of Chemistry, Faculty of Science, University of Botswana, Botswana
1
Research Article
Received date: 21/07/2015
Accepted date: 21/12/2015
ABSTRACT
Published date: 30/12/2015
*
For Correspondence
Runner RT. Majinda. Department of Chemistry,
Faculty of Science, University of Botswana,
Botswana, Kenya.
E-mail: majindar@mopipi.ub.bw
Keywords: Croton megalobotrys; Stem
bark extract; bioassay-guided fractionation,
Antimalarial; Anti-plasmodial; DPPH radical
scavenging.
The root, stem bark and leaves of Croton megalobotrys were extracted
and tested for their anti-plasmodial activity. In in vivo screening test against
P. berghei (ANKA) infection in NMRI albino mice, the stem bark extract
produced a statistically significant suppressive effect (74.5%; p<0.05) in
early infection and a residual inhibitory effect of 86.9%. In an established
infection, a mean survival time (MST) of 16.2 days was achieved although
parasitaemia was not completely eliminated. Impressive anti-plasmodial
activity was observed in in vitro test with IC50 values of 1.74 ± .47 µg/mL and
3.78 ± 1.03 µg/mL for the hexane fraction of the stem bark extract against
D6 and W2 strains of P. falciparum respectively. The chloroform fraction of
the stem bark extract yielded a cinnamate derivative (E)-tetratriacontyl3-(4-hydroxy-3-methoxyphenyl)-2-propenate, while the fourth semi-purified
fraction of the chloroform fraction (AA-CC4) in qualitative DPPH assay
showed activity at a loading dose of 0.05 µg, thus exhibiting radical
scavenging activity comparable to that of ascorbic acid.
INTRODUCTION
Medicinal plants are used for treatment of a variety of diseases. They are sources of modern medicine and a major source
of remedy in developing countries. Even when some knowledge of traditional use of plants in Africa was lost due to lack of
documentation [1], active experimentation on medicinal plants by local population continued [2]. The emergence of new diseases
such as HIV/AIDS and resurgence and development of resistance of others such as malaria have encouraged medicinal plant
use. Scientific research is sometimes carried out to support or refute claims of medicinal value of the plants used in traditional
medicine. Some medicinal plants of Botswana have been evaluated for their antimicrobial [3,4], antimalarial [5,6] and radical
scavenging [7] activities. Most of them, collected from medicinal plant vendors, have been shown to contain useful compounds
with known biological activities [8].
In the present work the leaf, stem bark and root extracts of C. megalobotrys (Euphorbiaceae) were evaluated for antimalarial
activity in vivo against P. berghei in mice. The crude stem bark extract (AA-CCR) which produced more than 70% parasite
suppression was partitioned by liquid–liquid extraction into the n-hexane (AA-CHE), chloroform (AA-CCE), n-butanol (AA-CBE) and
residual aqueous (AA-CWE) fractions. These fractions, together with the crude extract, and six semi-purified fractions (AA-CC1 to
AA-CC6) from the chloroform fraction were evaluated in vitro against two strains of P. falciparum. The essence was to determine
the effect of the extract on chloroquine-sensitive and chloroquine-resistant strains of P. falciparum as well as the fraction likely to
contain the antimalarial activity. Radical scavenging activity of the extracts was also determined.
MATERIAL AND METHODS
Plant material
Croton megalobotrys, leaves, and roots (voucher code: A2003/3) were collected from Maun in Ngami District of Botswana in
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July 2003. The stem bark (voucher code: A2003/4) was collected from Mapoka, the North East District, Botswana in June 2003.
They were authenticated in the herbarium of Biological Sciences Department, University of Botswana.
General methodology
The 1D [1H (300 MHz), 13C (75.4 MHz), DEPT] and 2D [COSY, HMQC, HMBC) spectra were acquired on Bruker Avance DPX
300 and referenced to residual solvent signals. Low-resolution mass spectra were obtained on Finnigan MAT LCQDECA instrument.
The ultraviolet and visible (UV-VIS) spectra were taken on Shimadzu UV-2101PC UV-Vis Scanning Spectrophotometer. Infrared
(IR) spectrum was measured on a Perkins Elmer System 2000 FT-IR Spectrophotometer using KBr pellets. Melting points were
recorded using Stuart Scientific melting point apparatus. Analytical thin layer chromatograms were run on readymade 0.25 mm
thick layer of Merck silica gel 60 F254+366 coated aluminium foil. Spots on the chromatograms were detected by observing in UV
light (254 or 366 nm) and/or sprayed with vanillin-sulphuric acid spray. Preparative thin layer chromatograms were run on 0.5
mm thick layer Merck silica gel 60 HF254+366 containing CaSO4 (binder) coated on 20×20 cm glass plates. Normal chromatography
was conducted using different sizes of columns packed with Merck silica gel 60, particle size 0.0400-0.0630 mm and Sephadex
LH-20.
Extraction and isolation
The air dried and powdered leaf (15 g) and root (17.9 g) of C. megalobotrys were extracted in methanol by Soxhlet extraction
method [9] to yield upon solvent evaporation, the crude leaf (CML,1.6 g) and crude root (CMR, 1.8 g) extracts respectively. The
stem bark was scraped off the stem wood, dried in air and powdered. The dried and pulverized material (622.4 g) was extracted
in n-hexane/chloroform/methanol/water (1:14:4:1). Removal of the solvent from the extract gave a brown crude extract (AA-CCR,
54.6 g), part of which (50.0 g) was dissolved in minimal amount of water and subjected to liquid–liquid partitioning sequentially
with hexane, chloroform and butanol to yield the n-hexane (AA-CHE, 0.9 g), chloroform (AA-CCE, 4.5 g), n-butanol (AA-CBE, 11.6 g)
and the residual aqueous (AA-CWE, 10.6 g) fractions. Since the n-hexane fraction was rather small, no further fractionation work
could be done on it. The chloroform fraction was divided into two portions. The first portion (1.5 g) was adsorbed in 1.5 g of silica
gel and loaded on a silica gel column (150 g) packed and eluted with CHCl3/EtOAc (8:2) to yield (E)-tetratriacontyl-3-(4-hydroxy3-methoxyphenyl)-2-propenoate (10 mg). The second portion (3.0 g) was subjected to vacuum liquid chromatography and eluted
with 100% hexane, Hex/CHCl3 (1:1), CHCl3 (100%), CHCl3/MeOH (8:2), CHCl3/MeOH (1:1) and MeOH (100%) to give six fractions;
AA-CC-1, AA-CC-2, AA-CC-3, AA-CC-4, AA-CC-5 and AA-CC-6. These fractions were kept for the anti-plasmodial and DPPH free radical
scavenging activity tests.
In vivo anti-plasmodial activity
The dried leaf (CML,1.6 g), root (CMR, 1.8 g) and stem bark (AA-CCR, 3.0 g) crude extracts of C. megalobotrys were evaluated
for anti-plasmodial activity in NMRI white albino mice infected with 1 x 107 P. berghei (ANKA) parasitized erythrocytes. The effects
of the extracts were assessed on early infection and established infection as described in Abosi and Raseroka5. The residual
effect of the extracts was also assessed using the Repository or Prophylactic test.
Evaluation of the repository activity or prophylactic test
This method is a modification of that of Peters [10], used to assess any possible repository activity of the extract. Twentyfive NMRI mice weighing 18 ± 2 g, housed in groups of fives in plastic cages at room temperature (20°C) and kept in constant
experimental conditions were used in the experiment. They had constant supply of dog feed and water and allowed free movement.
Each group of mice was given a different plant extract for three consecutive days. A dose of 1.2 mg/kg per day of pyrimethamine,
a prophylactic drug, was given to a standard control group. Sterile distilled water was given a placebo. An inoculum size of 1 x 107
P. berghei parasitized erythrocytes obtained from a donor mouse previously infected with P. berghei parasites was then passage
into each mouse on the fourth day of treatment. Seventy-two hours later, tail blood smears were made, stained by Giemsa and
percentage parasitaemia assessed. Mean percentage suppression of parasitaemia was calculated using the formula [11]:
a= b − c x 100
b
Where:
a=Mean percentage suppression of parasitaemia
b=Mean% parasitaemia in placebo group
c=Mean% parasitaemia in extracts tested group.
In vitro anti-plasmodial activity
The anti-plasmodial activity test was based on the method of Desjardin., et al [12]. Chloroquine-resistant (W2) and chloroquinesensitive (D6) isolates of P. falciparum continuously maintained in culture by standard methods [13,14] were used in the bioassay.
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For the test, two fold dilutions of test samples were prepared over a 64-fold concentration range. 25 µL aliquots of each dilution
were added to each well of a 96-well flat bottom microculture plate. 200 µL aliquots of a 0.9% parasitized erythrocytes in culture
medium were added to all the test wells. Parasitized and non-parasitized erythrocytes and solvent controls were incorporated
in all tests. The plates were incubated at 37°C in a gas mixture of 3% O2, 6% CO2 and 91% N2 and pulsed with 25 µL of culture
medium containing 0.5 µCi of [G3H]-hypoxanthine after 24 hr incubation. It was further incubated for 18 hours and harvested on to
glass fiber filters (Packard Filtermate Harvester Unifilter-96), washed thoroughly with distilled water and radioactivity measured by
liquid scintillation. Raw data representing the parasite counts was generated and directly imported to the data analysis software
(Oracle), which gave the results as IC50 values.
Radical scavenging activity
The preliminary screening method
12 mg of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was dissolved in 50 ml of methanol (0.24 µg/ml) and placed
in a spray bottle. The crude extracts and fractions obtained from stem bark of C. megalobotrys were spotted on TLC plates and
developed appropriately. These were done in duplicates. The TLC plates were then dried using hair dryer and visualized using
UV 254 nm. One of the dried plates was sprayed with the DPPH reagent and the other with the vanillin-sulphuric acid spray. The
zone of inhibition which appeared as a yellow spot on a purple background due to the disappearance of the purple color of DPPH
indicated the radical scavenging properties. The DPPH sprayed plates were compared with the vanillin-sulphuric acid sprayed
plate to locate the probable position of compounds with radical scavenging properties.
The semi-quantitative method
The crude extracts and fractions obtained from the stem bark of C. megalobotrys were spotted on TLC plates in amounts
ranging from 0.05 µg to 100 µg, dried and sprayed with DPPH reagent. Ascorbic acid was tested in the same way and it served as
a standard. The zone of yellow colour in a purple background due to the disappearance of the purple colour of DPPH was a sign of
activity and this was compared with that of the standard. The minimum inhibitory loading dose is the minimum amount of sample
applied to the TLC that caused the inhibition.
RESULTS
Isolated compound from the chloroform fraction (AA-CCE) of the stem bark:
The chloroform fraction (AA-CCE) of C. megalobotrys was subjected to silica gel chromatography to afford white amorphous
powder. Its EI-MS spectrum showed a molecular ion peak at m/z 670 [M]+ consistent with a molecular formula C44H78O4. The IR
spectrum showed absorption bands at 3500, 1700, 1670 and 1260 cm-1 suggesting the presence of free -OH, -C=O, -C=C- and
-C-O respectively. The 1H NMR spectrum (Table 1) and 1H-1H COSY spectra showed the presence of aromatic ABX proton spin
system signals [δH 6.92 (1H, d, J=8.1), δH 7.09 (1H, dd, J=8.2, 1.8 Hz) & δH 7.05 (1H, d, J=1.8 Hz)], olefinic trans coupled proton
signals [δH 7.62 (1H, d, J=15.9 Hz) & δH 6.30 (1H, d, J=15.9 Hz)] and signals due to the protons attached to a long chain carbon
atoms [δH 4.20 (2H, t, J=6.7 Hz), δH 1.71 (2H, m), δH 1.27 (42H, m) and δH 0.89 (3H, t, J=6.9 Hz)]. The presence of the methoxy
group was evident from the sharp singlet signal at δH 3.94 (δC 56.3). The 13C NMR spectrum (Table 1) showed nine aromatic
carbons signals [δC 167.7, 148.2, 147.1, 144.9, 127.4, 123.4, 116.1, 115.0 and 109.7]. The DEPT and HMQC spectra indicated
that five of these carbons are protonated [δC 144.9, 127.4, 123.4, 116.1, 115.0, 109.7] which suggested a phenyl propanoid
skeleton. An olefinic proton resonating at δH 7.62 (δC 145.0) showed HMBC correlation with a conjugated carbonyl (δC 167.7),
olefinic carbon (δC 116.1) and aromatic carbons [C-1 (δC 127.4), C-6 (δC 123.4), C-2 (δC 109.7)] deducing the assignment of
carbons resonating at δC 116.1 & 145.0 to C-1' and C-2' respectively. The methoxy protons (δH 3.94) showed HMBC correlation
to the carbon resonating at δC 147.1 indicating its attachment on the aromatic ring. Based on the HMQC and HMBC spectral
data analysis protons at δH 4.21, 1.71 and 0.89 were assigned to C-1″ (δC 65.0), C-2″ (δC 29.1) and a terminal C-28″ (δC 14.4)
of the long chain respectively. The ESI-MS spectral fragmentation ions by McLafferty rearrangement resulted into fragments at
m/z 194 [ferulic acid] indicating a loss of 476 mass unit [-CH2=CH (CH2)31CH3]. This compound was therefore identified as (E)tetratriacontyl-3-(4-hydroxy-3-methoxyphenyl)-2-propenoate
O
1'
3'
1
5
HO
1''
2'
2''
34''
O-CH2 - CH2 - (CH2)31 - CH3
3
OCH3
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Table 1. 1H (300 MHz) and 13C (75.4 MHz) of (E)-tetratriacontyl-3-(4-hydroxy-3-methoxyphenyl)-2-propenoate in CDCl3.
δ1H
Position
1
2
3
4
5
6
1′
2′
3′
1′′
2″
3″-27″
28″
OCH3
7.05, d (1.8)
6.92, d, (8.1)
7.09, dd, (8.2, 1.8)
7.62, d (15.9)
6.30, d (15.9)
4.20, t (6.7)
1.71 m
1.27, s
0.89, t (6.3)
3.94, s
δ13C
127.4
109.7
147.1
148.2
115.0
123.4
144.9
116.1
167.7
65.0
29.1
23.0-32.3
14.4
56.3
In vivo anti-plasmodial activity
The results of the in vivo anti-plasmodial activity of the extracts are shown in Table 2. In the established infection; the
parasitaemia initiated by the standard inoculum of 1 x 107 increased gradually with time in all groups. A decrease in parasite
count was observed in the group treated with root, stem bark and the leaf extracts when compared to the placebo. The stem bark
extract demonstrated a strong anti-plasmodial activity at highest dose employed as evidenced by the mean survival time of 16.2 ±
1.3 days it produced at 500 mg/kg per day indicating a dose dependent effect. It also exhibited good mean parasite suppression
in both early infection (74.5%) and the repository state (86.9%) at the same concentration.
Table 2. Antimalarial activity of C. megalobotrys extracts against P. berghei in mice.
Plant part
Dose a
Leaf (CML)
Root (CMR)
Stem -AACCR
Stem-AACCR
Stem-AACCR
Chloroquine
Placebo
Pyrimethamine
500
500
500
250
125
5
0
1.2
Early infection
Countb
Suppressionc
23.4 ± 2.5
41.5
20.2 ± 1.6
49.5
10.2 ± 1.8
74.5
20.0 ± 3.5
23.0 ± 2.5
5.4 ± 1.4
86.5
40.0 ± 0.9
0
NT
Repository infection
Countd
Suppressione
NT
NT
NT
NT
3.0 ± 0.9
86.9
7.8 ± 2.3
66.1
22.4 ± 2.6
2.6
NT
NT
23.0 ± 2.3
0
2.2 ± 0.2
90.4
MSTf
10.2
13
16.2
11.6
11.2
30
6.8
NT
Dose of extract in mg/kg per day
Mean parasite count ± standard error in early infection
c
Mean percent suppression in early infection
d
Mean parasite count ± standard error in repository state of extract
e
Mean percent suppression in repository state
f
Mean survival times obtained in established infection
a
b
In vitro anti-plasmodial activity
The results obtained from the in-vitro anti-plasmodial activity assessment of C. megalobotrys stem bark extract against
both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum are shown in Table 3. This result showed a very
good anti-plasmodial activity for a crude extract. On partitioning of the crude extract into the n-hexane, chloroform, n-butanol
and aqueous fractions, it was possible to assess the nature of the compounds causing the anti-plasmodial activity. The n-hexane
fraction representing the least polar group of compounds produced an IC50 of 1.74 ± 0.47 μg/ml and 3.78 ± 1.03 μg/ml for D6
and W2 isolates respectively. The chloroform fraction produced an IC50 of 8.34 ± 1.66 μg/ml for D6 isolates and 10.28 ± 3.68 μg/
ml for W2 isolates. The n-butanol fraction and the aqueous extract were inactive at the highest concentration (50 μg/ml) tested.
Radical Scavenging activity of C. megalobotrys
The preliminary test showed that the AA-CC1 and AA-CC2 did not have the radical scavenging characteristics whereas all
other fractions demonstrated the radical scavenging activity (Table 4). The semi-quantitative assay showed that the activity was
dose dependent as judged by wider zones of dis colourazation observed with higher concentration. Inactive fractions did not
discolor DPPH. Ascorbic acid and fractions AA-CC4 showed activity at the lowest concentration used. AAVC4, therefore, had the
highest DPPH radical scavenging activity being able to decolorized DPPH at a loading of 0.05 µg.
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Table 3. In vitro anti-plasmodial activity of C. megalobotrys against two strains of P. falciparum.
IC50 (µg/mL)
Fractions
D6
W2
AA-CCR
3.12 ± 0.68
5.34 ± 0.78
AA-CHE
1.74 ± 0.47
3.78 ± 0.10
AA-CCE
8.34 ± 1.66
10.78 ± 0.68
AA-CWE
>50
40.28 ± 6.83
AA-CBE
>50
>50
AA-CC1
23.34 ± 0.45
23.85 ± 0.52
AA-CC2
44.24 ± 1.52
>50
AA-CC3
33.39 ± 0.91
37.38 ± 0.79
AA-CC4
7.98 ± 0.73
15.36 ± 2.63
AA-CC5
15.84 ± 1.13
20.43 ± 1.71
AA-CC6
5.64 ± 0.48
9.04 ± 0.78
Chloroquine
0.016 ± 0.003
0.029 ± 0.002
All values are the mean ± SD of four separate experiments carried out on different days.
CCR: C. megalobotrys crude extract, CHE: C. megalobotrys hexane fraction, CCE: C. megalobotrys chloroform fraction, CWE: C.
megalobotrys aqueous fraction, CBE: C. megalobotrys n-butanol fraction. AA-CC1-6=six semi-purified fractions from chloroform
fraction. Cut off point for activity of crude extract is IC50=49.9 μg/ml.
Table 4. Radical scavenging activities of C. megalobotrys stem bark crude extract and fractions.
*
Fractions
Activity
AA-CCR
AA-CHE
AA-CCE
AA-CBE
AA-CWE
AA-CC1
AA-CC2
AA-CC3
AA-CC4
AA-CC5
AA-CC6
Ascorbic acid
yes
yes
yes
yes
yes
no
no
yes
yes
yes
yes
yes
Minimum loading dose (MLD)*
(µg)
0.5
0.5
2.0
0.2
1.0
>20
>20
5.0
0.05
0.2
0.2
0.05
MLD: Minimum Loading Dose at which activity was observed.
DISCUSSION
The Croton species are distributed throughout the tropics and are generally used as a fodder for livestock. Many of them are used
in traditional medicine [15] for the treatment of a variety of ailments [16-18] including malaria [19]. Croton megalobotrys is one of the six
croton species found in Botswana [20]. The root, seed and stem bark are used in traditional medicine to treat abdominal pains, dropsy,
malaria, and to induce abortion in humans [21,22]. The stem bark and seed were well known among early pioneers in malarious areas
as a cure as well as prophylactic for fevers [23] while the stem bark is known to cause paralysis in fish [24]. The oil from the seed is a very
effective purgative, toxic to mice [25] and in combination with Ricinus communis, acts against round and tape worms [26]. Although little is
known about its biological activity, our results showed that it possesses anti-plasmodial and possible antimalarial activities both in vitro
and in vivo. C. guatemalensis [27] and C. pseudochellus [28] exhibited antimalarial activity hence supporting this finding. The crude extract
of C. megalobotrys gave an IC50 of 3.12 ± 1.68 μg/ml for D6 isolates and 5.34 ± 1.78 μg/ml for W2. This activity could be attributed
to the activity observed in the n-hexane fraction which produced IC50 of 1.74 ± 0.47 μg/ml for D6 isolates and 3.78 ± 1.03 μg/ml for
W2 isolates. The fourth sub-fraction of chloroform fraction (AA-CC4) also exhibited a good radical scavenging activity. These findings
probably suggest that the successful use of C. megalobotrys as a remedy for malaria in traditional medicine might not only be due to
its schizontocidal activity. Its ability to scavenge free radical known to be generated in malaria infection [29] could have had an effect in
maintaining low levels of parasitaemia that were symptom less.
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