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Glycohydrolase enzyme assays
Determination of activity against HIV was based on the measure of inhibition of the glycohydrolase enzymes: a-glucosidase and b-glucuronidase. Two glycohydrolase enzymes (a- glucosidase and b- glucuronidase) and the substrates p-nitrophenyl- a-Dglucopyranoside and p-nitrophenyl-b -D-glucuronide were obtained from Sigma Chemical (MO, U.S.A). The glycohydrolase assay was performed in a colorimetric 96-well microtiter plate-based assay, determining the amount of p-nitrophenol released. The method described by Collins et al. (1997) was followed. The enzymes were diluted in 50mM of an appropriate buffer (sodium acetate, pH 5.0 for b-glucuronidase and Mes-NaOH, pH 6.5 for a- glucosidase). Appropriate substrates of the respective enzymes were added to microtiter wells. The assay was calibrated relative to enzyme concentration and ~ 0.25 μg enzyme was used per assay. After the addition of the enzymes, substrate and extracts, the plates were left at room temperature for 15 min. The reaction was stopped by the addition of 50 μl of 2 mM glycine-NaOH, pH 10, and measurement of absorbance undertaken at 412 nm. The extracts were tested at concentration of 200 μg/ml and the experiment was carried out in triplicate. The positive control Doxorubicin was tested at 100 μg/ml against both the enzymes.
Reverse transcriptase (RT) assay
The effect of plant extracts on RT activity in vitro was evaluated with a non-radioactive HIV-RT colorimetric ELISA kit (Roche, Germany). The assay was carried out in triplicate. Adriamycin, an anticancer drug and also an inhibitor of viral reverse transcriptase (Goud et al., 2003) was used as a positive control. In each test well, 20 μl of diluted recombinant HIV-1 reverse transcriptase (4-6 ng), 20 μl of diluted extract, and 20 μl of reaction mixture was dispensed. The final concentration of each extract in each well was 200 μg/ml. Since this part of the experiment was not conducted at the University of Pretoria, but at Nelson Mandela Metropolitan University; due to cost implications, only one concentration was selected. Negative control wells contained 40 μl of lysis buffer and 20 μl of reaction mixture. The concentration of positive drug control (Adriamycin) was 100 μg/ml. Positive
control wells contained 20 μl diluted recombinant HIV-1 Reverse transcriptase (4-6 ng), 20 μl of lysis buffer containing 10 % DMSO, and 20 μl of reaction mixture. The wells of the microtiter plate modules were washed five times with 250 μl of washing buffer per well for 30 seconds each. The washing buffer was then carefully removed and 200 μl of anti-DIGPOD working solution was dispensed into each well. Incubation at 37oC followed once again for 1 hour after the microtiter plate modules were covered with foil. The wells were then washed in the same manner as before, the washing buffer was carefully removed from the wells, and 200 μl of ABTS substrate was dispensed into the wells. Incubation then commenced for 10-30 min at room temperature (15-25oC). The absorbencies of the samples were measured at 405 nm (reference wavelength: 492 nm). The percentage inhibitory activity of the extracts samples were then calculated, with reference to the positive control.
Description and traditional use of Lippia javanica
The are about 200 species of Lippia includes herbs, shrubs and small trees (Terblanché & Kornelius, 1996). In general, the genus appears to present consistent profiles of chemical composition, pharmacological activities. The most common use of Lippia species is for the treatment of respiratory disorders (Pascual et al., 2001). Lippia javanica (Burm.f.) Spreng (Figure 4.1) is an erect woody shrub up to two meters high, with strong aromatic leaves, which give off a lemon smell when crushed (Van Wyk & Gericke, 2000). The plant occurs in many parts of southern Africa and tropical Africa (Van Wyk & Gericke, 2000). Its infusion made from its leaves is commonly used in Africa as tea for various chest ailments, influenza, measles, rashes, malaria, stomach problems, fever, colds, cough and headaches (Smith, 1966; Watt & Breyer-Brandwijk, 1962; Hutchings, 1966 and Hutchings & van Staden, 1994). Hutchings (2003) reported the clinical use of L. javanica for the treatment of HIV in Ngwelezane Hospital, Kwazulu Natal (South Africa).
CHAPTER 1
LITERATURE REVIEW AND OBJECTIVES
1.1 Introduction
1.2 The value of plants used in ethnomedicine for drug discovery
1.3 Antiviral compounds from plants
1.4 Mozambican traditional medical practice
1.5 Hypothesis and motivation of study
1.6 Objectives of the study
1.7 Scope of this thesis
1.8 References
CHAPTER 2
ANTITUBERCULOSIS AND ANTIBACTERIAL ACTIVITY OF MEDICINAL PLANTS COLLECTED IN MOZAMBIQUE
Abstract
2.1 Introduction
2.3 Results and discussion
2.4 Conclusion
2.5 References
CHAPTER 3
ANTIVIRAL ACTIVITY OF MOZAMBICAN MEDICINAL PLANTS AGAINST HUMAN IMMUNUDEFICIENY VIRUS
Abstract
3.1 Introduction
3.2 Materials and methods
3.3 Results and discussion
3.4 Conclusion
3.5 References
CHAPTER 4
ISOLATION AND IDENTIFICATION OF COMPOUNDS FROM LIPPIA JAVANICA
Abstract
4.1 Introduction
4.2 Materials and methods
4.3 Results and discussion
4.4 Conclusion
4.5 References
CHAPTER 5
ISOLATION AND IDENTIFICATION OF COMPOUNDS FROM HOSLUNDIA OPPOSITA VAHL.
Abstract
5.1 Introduction
5.2 Materials and methods
5.3 Results and discussion
5.4 Conlusion
5.5 References
CHAPTER 6
ANTIBACTERIAL ACTIVITY OF THE COMPOUNDS ISOLATED FROM LIPPIA JAVANICA AND HOSLUNDIA OPPOSITA
Abstract
6.1 Introduction
6.2 Material and methods
6.3 Results
6.4 Conclusion
6.5 References
CHAPTER 7
ANTIMYCOBACTERAL ACTIVITY OF ISOLATED COMPOUNDS FROM LIPPIA JAVANICA AND HOSLUNDIA OPPOSITA
Abstract
7.1 Introduction
7.2 Materials and Methods
7.3 Results and Discussion
7.4 Conclusion
7.5 References
CHAPTER 8
ANTI- HIV ACTIVITY OF ISOLATED COMPOUNDS FROM LIPPIA JAVANICA AND HOSLUNDIA OPPOSITA
Abstract
8.1 Introduction
8.2 Materials and Methods
8.3 Results and discussion
8.4 Conclusion
8.5 References
CHAPTER 9
CYTOTOXICITY OF CRUDE EXTRACTS AND THE ISOLATED COMPOUNDS FROM LIPPIA JAVANICA AND HOSLUNDIA OPPOSITA
Abstract
9.1 Introduction
9.2 Materials and Methods
9.3 Results and discussion
9.4 Conclusion
9. 5 References
CHAPTER 10
GENERAL DISCUSSION AND CONCLUSION
10.1 Motivation for this study
10.2 Screening of plant species for biological activity
10.3 Isolation and identification of active compounds in plants
10.4 Cytotoxicity of selected plant extracts
10.5 Conclusion
