Ethnobotanical Leaflets 12: 811-19. 2008.

Preliminary Phytochemical Screening and Antimalarial Studies of Spathodea campanulatum P. Beauv Leaf Extracts

Rangasamy Dhanabalan¹*, Asirvatham Doss¹, Muthusamy Jagadeeswari¹, Rajamanickam Karthic¹, Muthusamy Palaniswamy² and Jeyaraman Angayarkanni²

Department of Microbiology¹, RVS College of Arts and Science, Coimbatore,Tamilnadu, India

Department of Microbiology^2, Karpagam Deemed University,Coimbatore,Tamilnadu, India

Department of Microbial Technology^3, Bharathiyar University,Coimbatore,Tamilnadu, India

*Corresponding author: [email protected]

Issued 30 October 2008

ABSTRACT

Spathodea campanulata P. Beauv is extensively used in Indian traditional and folklore medicines to cure various human ailments. The preliminary phytochemical screening of the leaves revealed the presence of alkaloids, flavonoids, saponins, tannins, steroids and terpenoids. The organic leaf extracts of this species was evaluated for antimalarial activity against clinically isolated Plasmodium falciparum chloroquine-resistant and sensitive strains. The in vitro antimalarial activity was performed following schizont maturation inhibition assay, (SMI). The ethanol fraction exhibited very good antiplasmodial activity against both chloroquine sensitive and resistant Plasmodium falciparum isolates followed by the butanol and chloroform extracts.

Key words: Spathodea campanulata, Antimalarial activity, Schizont maturation assay and ethanol fraction.

INTRODUCTION

Malaria is the worldwide most important parasitic disease with an incidence of almost 300 millions clinical cases and over one million deaths per year. There were an estimated 247 million malaria cases among 3.3 billion people at risk in 2006, causing nearly a mil­lion deaths, mostly of children under 5 years. 109 countries were endemic for malaria in 2008, 45 within the WHO Afri­can region (WHO, 2008). While we wait for malaria vaccine, effective chemotherapy remains the mainstay of malaria control (Winstanley, 2000). Plasmodium falciparum, the potentially lethal malaria parasite has shown itself capable of developing resistance to nearly all used antimalarial drugs and resistant strains have rapid extension (Plowe et al., 1995). The lost of effectiveness of chemotherapy constitute the greatest threat to the control of malaria. Therefore, to overcome malaria, new knowledge, products and tools are urgently needed; especially new drugs are required (Omulokoli et al., 1997; Rasoanaivo et al., 1999).

The use of traditional medicine and medicinal plants in most developing countries, as a normative basis for the maintenance of good health, has been widely observed (UNESCO, 1996). Furthermore, an increasing reliance on the use of medicinal plants in the industrialized societies has been traced to the extraction and development of several drugs, chemotherapeutics from these plants as well as from traditionally used rural herbal remedies (UNESCO, 1998). The practice of traditional medicine is widespread in China, India, Japan, Pakistan, Sri Lanka and Thailand.

Spathodea campanulata is a species belonging to the Bignoniaceae family, native from equatorial Africa. The Siddha/Tamil name of this species is Patadi and in folk it is popularly called as Ruugatuuraa. It is very commonly found and planted in the coffee estates of Munnar, South Tamilnadu and denoted by the name Malaria Maram(tree). In English the species is called as Syringe tree, Fountain tree, African Tulip tree. It is a medium-size tree (15-25 m high), characterized by red garish flowers. It is often employed in gardening in tropical and subtropical areas, including South America (Joly, 1985). Flowers and stem bark extracts have shown molluscicidal activity and also employed in diuretic, anti-inflammatory treatments. The leaves are used against kidney diseases, urethra inflammations and as an antidote against animal poisons. The stem bark preparations are employed against enemas, fungus skin diseases, herpes, stomachaches and diarrhea (Jardim et al., 2003; Mendes et al., 1986). Hypoglycemic, anti-HIV and antimalarial activities were also observed in stem bark extracts (Niyonzima et al., 1999; Makinde et al., 1988).

Several phytochemical studies were performed with different parts of S. campanulata, including stem barks, leaves, flowers and fruits (Niyonzima et al., 1999; Ngouela et al., 1990; Ngouela et al., 1988; Amusan et al., 1995; Amusan et al., 1996). The leaves have furnished spathodol, caffeic acid, other phenolic acids and flavonoids (Ngouela et al., 1991; Subramanian et al., 1973; El-Hela, 2001a; El-Hela, 2001b). Banerjee and De (2001) showed the presence of anthocyanins in flowers of S. campanulata. A qualitative fungitoxic activity of S. campanulata roots against Cladosporium herbarum CCT 0279 has been evaluated and reported (Pianaro et al., 2007). In vitro antibacterial activity of leaf extracts of this plant was evaluated (Parek.J and Chanda.S, 2007). No reports on antimalarial activity of S. campanulata leaf extracts/fractions have been reported till date. Thus, the present work is aimed to evaluate the antimalarial activity of S. campanulata leaf extracts/fractions against the clinically isolated Plasmodium falciparum chloroquine-resistant and sensitive strains.

MATERIAL AND METHODS

Plant materials

            Fresh plant leaf samples were collected from the Munnar Kundala Tea Estates, Kerala, India during March 2008. The taxonomic identity of the plant was confirmed by Botanical Survey of India, Southern Circle, Coimbatore, India and the voucher specimen of the plants was preserved in RVS College Microbiology Laboratory. Fresh plant material was washed under running tap water, air dried, homogenized to fine powder and stored in airtight bottles.

Preparation of crude extracts and fractions

            Crude extraction with 50% ethanol was done using soxhlet apparatus and cold crude extract was prepared with 80 % ethanol (Klayman, 1985). For solvent extraction, 50 g of air-dried powder was taken in 250 ml of organic solvent (Chloroform, butanol, hexane, ethanol and ethyl acetate) in a conical flask, plugged with cotton wool and then kept on a rotary shaker at 190-220 rpm for 24 h. After 24 hours the supernatant was collected and the solvent was evaporated at room temperature. The procedure was repeatedly followed for all the solvents individually. The extracts were stored at 4 ⁰C in airtight bottles.

Phytochemical Screening Test

The phytochemical screening of the plant extract was carried out by following methods used by Amarasingham et al.,(1964), Das and Bhattacharjee, (1970), Gibbs, (1974), Trease and Evans (1978), Santaram and Harborne (1984) to detect the presence or absence of certain bioactive compounds.

Antiplasmodial activity

Preparation of medium for the cultivation of malaria parasite

            Around 10.4 g of RPMI 1640 containing 25mM of triple HEPES buffer was dissolved in 960 ml of triple distilled water. To this 2g of glucose and 40µg/ml of gentamycin sulphate were added. The solution was sterilized by passing through a Millipore filter of 0.22µm porosity and stored at 4^oC as 96 ml aliquots in glass media bottle. An incomplete media (washing media) was prepared by adding 4.2 ml of freshly prepared 5% sodium bicarbonate to 96 ml of stock RPMI 1640. Simultaneously, AB ⁺ve blood serum without anticoagulant was aseptically collected placed in aliquots. The serum was inactivated in 56^oC water bath/half an hour and stored in deep freezer at -20 ^oC. A complete media was prepared by adding 10 ml of normal inactivated AB⁺ve human serum to 90 ml of incomplete media. A⁺ve blood was collected with anticoagulant and the RBCs were pelleted by centrifugation to which equal amount of complete media was added and stored for use when required.

Collection of Plasmodium falciparum positive blood samples from the field

The parasite collection was carried out in RVS Hospital Diagnostic Lab and Government Hospital, Palladam. Prior to collection peripheral blood smears of patients with no history of intake of antimalarials was checked for the presence of healthy asexual parasites. 3ml of blood was drawn from the positive patients and centrifuged at 1500 rpm/10 in a heparinised centrifuge tube. Supernatant/plasma was removed and the cells were suspended in an equal volume of cryoprotectant (28ml of glycerol to 72 ml of 4.2% sorbitol in normal saline and filter sterilized) and then distributed in cryotubes and frozen quickly in liquid nitrogen (-196 ^oC). Initiation of culture and sub-culturing was performed whenever required for tests (Connelly et al., 1996).

Revival of Cryopreserved parasites

The culture vials from liquid nitrogen was thawed quickly in water bath at 37 ^oC, the contents were centrifuged at 1500 rpm/10 min and equal volume of NaCl (3.5%) was added by removing the supernatant. The suspension was centrifuged again, supernatant removed and the pellets were washed twice with complete medium supplemented with 15% serum. After washing the cryopreserved cells the culture was setup by adding fresh washed erythrocytes.

In vitro screening of extract/ fraction against Plasmodium falciparum

            The cultures was synchronized using 5% aqueous solution of sorbitol (1 portion of pellet and 9 portion of sorbitol) and kept for 5-7min in room temperature to ensure the killing of all other stages of parasite except ring forms. The whole suspension was centrifuged   at 1500 rpm/5 min and the pellets were washed with incomplete media twice. The parasitaemia was adjusted to about 1% for the assay by diluting with fresh washed RBC’s. The plant material to be tested was dissolved in DMSO so that the concentration of DMSO never exceeded 0.1% in the experiment. The stock solution was diluted with RPMI1640 to obtain different concentrations.

The test was performed in 96 well plates using chloroquine sensitive and resistant isolates isolated from the patients. Different concentration of crude extracts and fractions were dispensed in 96 well plates in duplicate. The first well in all rows were without any drug but with DMSO and considered to be control. The synchronized parasites were inoculated in all the wells to get a final concentration of 5% haematocrit. The plates were incubated at 37^oC for 24-30 hours depending on the maturation of the schizont. After conformation of the schizont maturation, smears were prepared from the wells. The experiment is considered to be invalid, if the schizont maturation is below 10% (Draper et al., 1988). The smears were stained in Giemsa stain and numbers of schizonts were counted/200 asexual parasites.

The values were compared between control and test wells. The inhibition percentage of schizont for each concentration of extract/fraction was calculated as follows (Anon, 1979).

                                  M: number of schizonts in the test wells

From dose response curve, IC₅₀values (concentration at which inhibition of parasite growth represents 50%) were calculated from the plot of probability of chloroquine activity and logarithm of drug concentration by linear regression analysis (Sharma Poonam, 1999).

RESULTS

Screening tests of plant extracts/fractions

            Crude ethanol extracts (50% and 80%) were prepared from Spathodea campanulatum and subjected to fractionation using ethanol, chloroform, butanol, hexane and ethyl acetate. The percentage yield obtained is given in Table 1. Preliminary phytochemical analysis of the leaf extracts of Spathodea campanulatum revealed the presence of flavonoids, alkaloids, terpenoids, saponins, tannins, steroids and cardio amino glycoside Table 2. The presence of alkaloids, flavonoids, tannins and steroids, terpenoids were detected in ethanol and hexane extracts respectively. Saponins were detected in water extracts.

Antiplasmodial activity

            The crude ethanol (50% and 80%) extracts and fractions of Spathodea campanulatum were tested in vitro for its antimalarial activity using clinically isolated laboratory adapted chloroquine sensitive and resistant Plasmodium falciparum parasites. The assay followed was schizont maturation inhibition assay, (SMI).         The specific changes in morphology produced by particular extract may hint at different modes of action of the putative active principles in the extracts. The parasitaemia also decreased with increasing concentration of the extract reflecting an inhibitory activity on parasitic replication. This may be indicative of a significant potential for isolating purer compound.

            The crude extract (both 50% and 80%) of Spathodea campanulatum tested in vitro did not show profound antimalarial activity. The IC₅₀ value of crude ethanol extract (50%) against chloroquine sensitive Plasmodium falciparum isolates was 88.3±2.83µg/ml where as the IC₅₀ value against chloroquine resistant Plasmodium falciparum isolates was 108.2±8.20µg/ml. The IC₅₀ value of crude ethanol extract (80%) against chloroquine sensitive Plasmodium falciparum isolates was 68.5±10.60µg/ml and against chloroquine resistant Plasmodium falciparum isolates was 100.8±3.53µg/ml. The chloroform, butanol, hexane, ethanol and ethyl acetate fractions when tested in vitro showed IC₅₀ value of 18.7±2.23µg/ml, 12.3±1.32µg/ml, 90.0±11.12µg/ml, 7.5±1.25µg/ml and 14.2±1.20µg/ml respectively against chloroquine sensitive Plasmodium falciparum isolates and the IC₅₀ value  for chloroquine resistant Plasmodium falciparum isolates was 19.0±2.42µg/ml, 21.3±1.82µg/ml, 118.3±2.23µg/ml, 8.9±1.30µg/ml and 51.0±3.10µg/ml respectively (Table 3)

DISCUSSION AND CONCLUSION

          Crude extracts are simplest of available of medications and are still promoted by WHO policies as emerging alternative systems of medicine to reach the large population not covered by formal medical care in remote areas. In this regard the result of this preliminary study is very much encouraging. This is the first report on in vitro antimalarial effect of Spathodea campanulatum using leaf extracts (Table 3). Previous reports on this plant leaf extracts have specified only about in vitro antibacterial activity (Parek.J and Chanda.S, 2007). Stem bark extracts were used for purgative, antiviral and antimalarial works (Niyonzima et al., 1999) and roots in fungitoxic activity (Pianaro et al., 2007).  In the present work crude plant extracts did not show profound antimalarial activity, upon fractionation has yielded purer compounds with potent antimalarial activity (Wright and Phillipon, 1990). The ethanol fraction exhibited very good antiplasmodial activity against both chloroquine sensitive and resistant Plasmodium falciparum isolates followed by the butanol and chloroform extracts. Further studies can be carried out for the isolation of active principle and elucidation of chemical structure with an objective of exploring the possibility of using the component as oral/parenteral drug for treating malarial infection.

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Table 1. Preparation of crude extracts from Spathodea campanulatum.

Table 2. The phytochemical profile of the leaf extracts Spathodea campanulatum.

Table 3. In vitro antiplasmodial effect of the crude extract/fractions of Spathodea campanulatum against chloroquine resistant and sensitive Plasmodium falciparum isolates.