Wabo Poné Josué*, Noumedem Anangmo Christelle Nadia, Komtangi Marie Claire, Yondo Jeannette and Mpoame Mbida
Department of Animal Biology, Faculty of Science, University of Dschang, Cameroon
Received date November 08, 2012; Accepted date December 17, 2012; Published date December 19, 2012
Citation: Josué WP, Nadia NAC, Claire KM, Jeannette Y, Mbida M (2014) In Vitro Sensitivity of Plasmodium falciparum Field Isolates to Methanolic and Aqueous Extracts of Cassia alata (Fabaceae). Altern Integr Med 3:159. doi:10.4172/2327-5162.1000159
Copyright: ©2014 Josué WP, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Objective: The aim of this study was to evaluate the in vitro activity of aqueous and methanolic extracts of Cassia alata leaves on the development of Plasmodium falciparum field isolates. Method: The Trager and Jensen method with slight modifications was used. For the culture, RPMI 1640 and Albumax were used to replace human serum. The extracts as well as the reference drug (chloroquine) were diluted using RPMI medium. The P. falciparum field isolates were incubated with 8 concentrations ranging from 128 to 1 μg/ml in a 96-well microplate and incubated for 48 h in a candle jar. RPMI and 1% DMSO were used as negative controls. Result: The extraction yields of C. alata were 7.96 and 13.23% for aqueous and methanolic extracts respectively. RPMI and DMSO didn’t have any harmful effect on the growth of P. falciparum. On the other hand, in the wells treated with extracts of C. alata leaves, inhibition of P. falciparum growth was registered with increasing concentrations of extracts. The inhibitory effect of the methanolic extract was stronger and we obtained the maximum mean inhibition rate of 100 ± 0.00% and 99.87 ± 0.62% at the concentrations 128 and 64 μg/ml respectively. As for the aqueous extract, it yielded a mean inhibitory rate of 99.2 ± 0.76% at the concentration of 128 μg/ml. Given the IC50 obtained that is 0.48 ± 0.02; 0.67 ± 0.11 and 0.77 ± 0.08 μg/ml for methanolic extract, aqueous extract and chloroquine respectively. The extracts of C. alata may be classified as active. This activity may be due to the presence of terpenes and tannins in the extracts.
Antiplasmodial; Activity; Cassia alata; Plasmodium falciparum; Cameroon
Malaria is a parasitic disease caused by a protozoan of the genus Plasmodium and transmitted by Anopheles mosquito vectors. In endemic regions, more than 300 million cases of malaria occur annually . This disease is responsible for about 200 million of cases worldwide and each year it kills about 600 000 of people , of which 1 million are children of less than 5 years old. In Cameroon, malaria transmission is permanent and intense . It remains a major public health problem in Cameroon as elsewhere in sub-saharan Africa . These past 30 years, malaria parasites especially P. falciparum have rapidly developed resistance to commonly used antimalarial drugs . New, more effective and affordable anti-malarial drugs are needed . Medicinal plants play a key role in the control of malaria, especially where access to modern health services is limited. Tropical rainforest plants represent a fertile source of potential candidates for the development of new alternative anti-malarial drugs. In Cameroon, many plants are used by traditional healer to cure fever. In certain rural areas, anti-malarial traditional medicine is even preferred to pharmaceutical drugs, suggesting that herbal preparations are useful and active products . More than 200 different species of plants from Cameroon possess antiplasmodial properties; but only 26 species have been investigated . Cassia alata extract was shown to possess antifungal activity on some dermatophytes especially on Trichophyton verrucosum and Epidermophyton jloccosum . This plant has also demonstrated antibacterial activity on Vibrio cholera, Bacillus subtilis, Staphylococcus aureus, and Escherichia coli . It is in this light that the present study designed to assess, using the Trager and Jensen culture technique, the antiplasmodial efficacy of Cassia alata was tested in vitro, on P. falciparum field isolates. C. alata belongs to the Fabaceae plant family, the most exploited by tradipractitioners for the treatment of malaria .
To carry out this research, an ethical clearance was obtained from the National Ethics Committee of Cameroon, in order to ensure the consent and the confidentiality of the participants.
Fresh leaves of Cassia alata (Fabaceae) were collected from Dschang-Cameroon in November 2011. The plant was identified in the National Herbal of Cameroon where a specimen was kept under number 18572/SRF-CAM. The leaves of the plant were air dried and reduced to powder before extractions were undertaken.
Two types of extracts (aqueous and methanolic) were prepared and tested on P. falciparum field isolates.
Preparation of extracts
The methanolic extract was obtained using the procedure described by Wabo Poné et al. . Briefly, 100 g of stored powder were macerated in 1.5 l methanol 90% which removes the active ingredients of plants. The mixture was daily stirred and 72 hours later, the solution was sieved and filtered using filter paper of pore size 2.5 µm. The extract was evaporated using a rotavapor Buchi-R-124 model heated at 65°C for 8 h.
A similar procedure was carried out for the aqueous extract, except that hot (distilled) water was used as solvent. The infusion took 3 h and evaporated for 7 days in a ventilated oven at a temperature of 50°C.
Dilution of extracts
200 µg of methanolic extract was diluted in 100 µl of Dimethylsulfoxide (DMSO). A quantity of RPMI was added to obtain a total volume of 1000 µl and thus a stock solution of 200 µg/ml. A series of dilutions were made with RPMI medium to obtain concentrations of 128, 64, 32, 16, 8, 4, 2 and 1 µg/ml .
Reference drug and chemicals
The reference drug, pharmaceutical chloroquine, used in this study was bought from a local pharmacy. RPMI 1640 and Albumax were obtained from SIGMA and GIBCO respectively. Chloroquine was chosen due to its availability, and also because some authors have used it for in vitro trials. This drug was diluted with RPMI in order to obtain the same concentrations with the organic and aqueous extracts. Negative controls used for the bioassay were 1% DMSO and culture medium (RPMI 1640 +Albumax).
About 4ml of blood were collected by vein puncture from patients suffering from malaria at the District Hospital of Dschang using a manual syringe of 10ml. This blood was transferred in sterile tubes with EDTA and put in on icebox. This was quickly transported to the laboratory of Biomedical Sciences for analysis. In the laboratory the specimens were distributed in eppendorf tubes and centrifuge at 800g for 10 min . These preparations were then washed 3 times by centrifugation using RPMI 1640 supplemented with sodium bicarbonate until all white blood cells were removed. The prepared blood was diluted with a washed blood, of group O+.
Evaluation of the antiplasmodial activity
To evaluate the effects of the various extracts on P. falciparum field isolates, 21 µl of infected red blood cells with a parasitemia of about 1 to 2% were distributed in 81 wells of the 96-well microplate and mixed with a volume of 189 µl of a specified tested products at various concentrations diluted with a culture medium (RPMI 1640, supplemented with 25 Mm HEPES, 0.2% Sodium bicarbonate and glucose, 5% Albumax and filter through a STERIVEX GS Millipore of 0.22 µm) . The microplate was covered and placed in a candle jar. This container was totally closed when the candle was about to go off. The candle jar was then put in an incubator for 48 h at 37°C . At 24 h of incubation, the culture medium in each well was replaced by fresh ones containing the same concentration of products. After 48h, the supernatants were removed using Pasteur pipettes. A small drop (10-15 µl) of erythrocytes from the bottom of each well was put on a clean glass microscope slide for the preparation of thin blood films. The percentage of inhibition (PI in %) was determined using the following:
All tests were repeated three times for each treatment and control in the same conditions.
Comparisons of different inhibition rates on P. falciparum growth were made using the Chi-square test. Results were regarded as significant at P<0.05. The 50% and 90% inhibitory concentrations (IC50 and IC90) were determined from linear regression curve obtained between the inhibition rate expressed in probit and the decimal logarithm of the concentrations (µg/ml).
The yields obtained after extraction with methanol and hot water solvents from 100 g of C. alata leaves powder were 13.23 % and 7.96 % respectively. The variation of the mean inhibition rate of the growth of P. falciparum field isolates according to the different concentrations of C. alata and chloroquine is shown in Figure 1.
This figure shows that RPMI 1640 and 1% DMSO did not affect (0% inhibition rate) the development of P. falciparum. In the treated wells, the inhibition rate increases with increased concentration of the tested products. For concentrations greater than 4 µg/ml, the effect of chloroquine was less than the effect of extracts with a significant difference (p<0.05), but below these concentrations the antiplasmodial activity was similar. At concentrations of 4 to 32 µg/ml the effect of the methanolic extract was higher than that of aqueous extract (58.7 to 98.13% and 48.9 to 58.1% respectively; p<0.05). After the transformation of the inhibition rate to probits (Figure 2), a linear relationship was obtained with the logarithm of the concentrations. From the equations of the regression lines the following results were obtained: 50 % inhibition concentration (IC50) of 0.48 ± 0.02, 0.67 ± 0.11 and 0.77 ± 0.08 µg/ml for methanolic extract, aqueous extract and chloroquine respectively. Also, the IC90 determined from the same regression lines was 0.7± 0.02, 1.53 ± 0.11; and 2.22 ± 0.08 µg/ml respectively for methanolic extract, aqueous extract and chloroquine. The inhibition gradient of the development of P. falciparum was: Eme > Eai > Cqi. Figure 3 shows the inhibitory concentrations (IC50 and IC90) for the growth of P. falciparum. From this figure, we observed that, no matter the tested products the IC50s are less than the IC90s.
The extracts obtained from the C. alata leaves presented different yields. The higher yield (13.23%) was obtained with methanolic extract. This finding is similar to that reported by Muganga et al.  with Fuerstia Africana. In fact, these authors obtained a yield of 13.3% for methanolic extract and 5.3% for aqueous extract of this plant. These differences observed in the various studies may be due in one hand, to the nature of the solvent and in the other hand to the method used .
From the normal growth observed in the negative control wells, the variation of parasitemia shown in the treated wells was due to the effect of tested products. The methanolic and aqueous extracts of C. alata were active against P. falciparum with IC50 values of 0.48 µg/ml and 0.67 µg/ml respectively. Even though water is used as the solvent in the traditional preparation, the methanolic extract demonstrated a slightly higher antiplasmodial activity than the aqueous extract at all concentrations. Similar observations were reported by Sotheara et al.  and Douki et al.  comparing the methanolic extracts of Brucea javanica and Staudtia gabonensis to aqueous extracts. This suggests that, more active compounds were extracted with this solvent. The antiplasmodial activities of the extracts against field isolates parasites were dose-dependent. The reported antiplasmodial activity of C. alata may be attributed to terpènes and tannins compounds present in the extracts [19-24]. In general, the IC50s obtained in this work are closer to those reported by other researchers. The IC50=0.48 µg/ml obtained with methanolic extracts of C. alata was similar to the ones reported (0.48 µg/ml) by Kayembe et al.  when testing the antiplasmodial effects of the same plant. However, IC50 (0.25 µg/ml) obtained by Kayembe et al.  with Cassia occidentalis extracts is significantly less than the one obtained in the present study. On the contrary, higher IC50s were reported by Sotheara et al.  with the methanolic extracts of Brucea javanica, Eurycoma longifolia, Phyllanthus urinaria, Stephania rotunda, Azadirachta indica, Flueggea virosa, Vernonia cinerea, Fagraea fragrans, Bixa orellana, Spondias pinnata, Anneslea fragrans, Cananga latifolia and Andrographis paniculata with IC50 of (1,7 ; 1,8 ; 2,5 ; 9 ; 16;31,5 ; 32,1 ; 33,4 ; 33,5 ; 41,2 ; 41,5 ; 48,7 ; 50 µg/ml respectively). By comparing either the IC50 of the methanolic extract or aqueous extract to that of chloroquine, we noticed that the IC50 of plant extracts were less than that of the reference drug (0.77 µg/ml). This IC50 was similar to the one obtained (IC50=0.8 µg/ml) by El Tahir et al.  when testing the effect of chloroquine on a P. falciparum chloroquinoresistant strain. However, they determined an IC50 of 0.23 µg/ml during evaluation of the activity of the same product on a P. falciparum chloroquinosensitive strain. We can therefore consider that P. falciparum field isolates used in this study was a chloroquinoresistant strain. Taking into consideration the threshold of in-vitro antiplasmodial activity proposed by Rosoanaivo et al. , the effect of the plant extract tested in this study can be considered active. The IC50 obtained with both extracts falls in the range 0.1 to 1 µg/ml.
RPMI and 1% DMSO allowed the normal growth of P. falciparum field isolates. The two tested extracts inhibited the growth of this P. falciparum strain with the means IC50 of 0.48 ± 0.02 and 0.67 ± 0.11 µg/m for methanolic and aqueous extracts respectively. Methanolic extract was the most potent on the development of P. falciparum field isolates with a maximum mean inhibition rate of 100% at 128 µg/ml concentration. Even though the IC50 of chloroquine was 0.77 ± 0.08 µg/ml, its effect remains less than the ones obtained with the tested extracts. This could be due to the resistance developed by the parasite.
We declare that we have no conflict of interest.
The authors wish to express sincere gratitude to the Laboratory of Applied Biology and Ecology (LABEA) and the Laboratory of Biomedical Sciences of the University of Dschang. We also thank the traditional healers of Dschang-Cameroon and M. FOPA François for the information on the plant specimen and for helping in the collection of P. falciparum field isolates used in this study respectively.