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Malstat method
To determine the activity of extracts against P. Jalciparum in a preliminary in vitro assay, a slightly modified version of parasite lactate dehydrogenase assay was used (Makler et al.1993). The experiment was done in triplicate at 1 % parasitemia and 5% hematocrit and plates prepared as described in 2.2.2.2.3. After 48h, a duplicate 96-well plate was prepared by adding 100 ).11 Malstat reagent [133 ml Triton X-100, 1.33 g lactate, 0.44 g TRIS buffer and 44 mg 3-acetylpyrimidine adenine dinucleotide (APAD) made up to 200 ml] to each well together with 25 i-ll developing dye solution [160 mg nitroblue tetrazolium (NBT) and 8 mg phenazine ethosulphate (PES) to 100 ml Millipore water] and 10 ).11 from the incubated plate. The duplicate plate was incubated for 20 min in the dark and read with an ELISA plate reader at 620 nm.
Flow cytometric analysis
To determine the activity of extracts against P. jalciparum in an accurate in vitro assay, the flow cytometric method of (Schulze et al. 1997) was used. Samples of cultures with extracts or compounds, as well as controls were stained using thiazole orange. The flow cytometer was programmed to have three electronic gates, each of which counts the erythrocytes of a different fluorescence intensity. All uninfected erythrocytes were counted in gate 1, which covers the region near zero fluorescence intensity. Gate 2 counts ring-infected erythrocytes, which have a fluorescence intensity lower than that of the later-phase parasites and gate 3 counted trophozoite- and schizont-phase infected erythrocytes, which showed the highest fluorescence intensity. The percentage of parasites present in the ring-phase or later phases could then be determined.
Flow cytometric analysis of fixed parasite cultures
Parasites in the 96-well plates were pre-fixed by adding fixing solution in a 1: I ratio after which they were incubated at 4°C for at least 18 hours. The fixing solution consisted of 10% formaldehyde and 4% D-glucose fonnulated in a Tris-saline buffer (10 mM Tris, ‘ ISO mM sodium chloride and 10 mM sodium azide). The final pH was adjusted to 7.3 using sodium hydroxide. The adjustment of the pH was important in preventing lysis of the erythrocytes.
» After incubation at 4°C for 18 hours or longer, 50 fll of fixed parasite culture was added to I ml phosphate buffer saline (PBS) containing 0.25 flg thiazole orange, in plastic tubes (Coming). This amount of thiazole orange was used to ensure that there was sufficient DNA intercalating dye available, even at higher parasitemias. The parasite-PBS-thiazole orange solution was mixed carefully, by inverting the tube 2-3 times, and incubated at ambient temperature, in the dark, for I hour. The samples were then placed on ice to stop further staining of the parasite DNA prior to flow cytometric analysis. A volume of 200 fll of prepared parasite sample was analysed by the flow cytometer and a total of 1~O 000 erythrocytes were counted in each sample (Prozesky et al. 2001).
Isolation and identification of compounds
A 10 cm diameter glass column (30 cm length, 5 cm diameter) was filled with 500 g of silica gel 60 (Merck) (column A). The extract sample was dissolved in a minimal amount of solvent and mixed with silica gel. This mixture was air dried and applied to the column.
The column was developed with a solvent gradient of hexane: ethyl acetate in a 100:0 to 0: 1 00 ratio (50 ml fractions collected). Fractions containing the same compounds as detennined by TLC on silicagel 60 (Merck), were combined and each of the pooled fractions concentrated to dryness under reduced pressure. Fractions were spotted on a TLC plate and then developed. TLC plates were viewed under UV light (254 and 366 run) after development and also dipped in vanillin reagent (15 g vanillin, 500 ml ethanol and 10 ml concentrated 98% sulfuric acid) and heated to detect compounds not absorbing UV light.
Each pooled fraction was tested for antiplasmodial activity and most active fractions further purified. Figure 2.1 shows the isolation procedure. Fraction A6 crystallized and crystals were collected for identification. Triterpene I was identified by mass spectroscopy (MS) after comparison, to MS-data from a database and to a standard on TLC.
Combined chlorophyll fractions (fraction A9) collected from silica gel column A had good antiplasmodial activity. For easier isolation of the active compounds from fraction A9, the chlorophyll was removed with activated charcoal. Granular charcoal was powdered with an lKA dry mill and added to the fraction in ethyl acetate and shaken. Enough charcoal was added to remove all the green colour from the fraction. The fraction was then filtered and the charcoal repeatedly rinsed with ethyl acetate to wash the non-aromatic compounds from the charcoal. The collected liquid was dried under reduced pressure and used for further isolation. A 2.5 cm diameter glass column (30 cm long) was filled with 100g of silica gel (Column B). The extract sample was dissolved in a minimal amount of solvent and mixed with silica gel. This mixture was air dried and applied to the column. A solvent gradient of chloroform: methanol was applied to the column in 99: 1 to 98:2 ratios (5 ml fractions collected). Fractions containing the same compounds as determined by TLC, were combined and each of the pooled fractions concentrated to dryness under reduced pressure. Fractions were spotted on a TLC plate and then developed. TLC plates were viewed under UV light (254 and 366 run) after development and also dipped lil vanillin reagent and heated to detect compounds not absorbing UV light. Fraction B3 from silica column B crystallized after solvent evaporation and a compound (diterpene 1) was isolated.
1 Introduction
1.1 Secondary compounds of plants
1.2 Malaria
1.3 Croton steenkampianus
1.4 Objectives
1.5 Scope of thesis
1.6 Hypothesis
1.7 References
2 Bio-guided fractionation of extract and antiplasmodial activity of isolated compounds
2.1 Introduction
2.2 Materials and Methods
2.3 Results & Discussion
2.4 References
3 Cytotoxicity of extract and compounds
3.1 Introduction
3.2 Materials & Methods
3.3 Results & Discussion
3.4 References
4 Chloroquine reversal/synergistic effects of isolated compounds
4.1 Introduction
4.2 Methods
4.3 Results & Discussion
4.4 References
5 Mode of action of crotrene A, a new diterpene isolated from C. steenkampianus
5.1 Introduction
5.2 Methods
5.3 Results & ·Discussion
5.4 References
6 General discussion and conclusions
6.1 Introduction
6.2 Bio-guided fractionation of extract and antiplasmodial activity of isolated compounds
6.3 Cytotoxicity of extract and compounds
6.4 Chloroquine reversal/synergistic effects of isolated compounds
6.5 Mode of action of crotrene A, a new diterpene isolated from C. steenkalnpianus
7 Summary
8 Acknowledgments
Appendix 1
