Aluminum Chloride prepared in Ethanol 51 Figure 4.3: TLC plate for positive Alkaloid bands the plates were developed. Chemical and TLC methods were used to determine phytochemicals from the study plant extracts and fractions.
GENERAL INTRODUCTION
INTRODUCTION
Local traditional healers also prepare concoctions from bulbs/roots to treat various diseases (Sigidi et al., 2017). Screening for toxicity early in drug development requires high-throughput assays with low amounts of the test compound (Westerink et al., 2011).
STUDY RATIONALE
Genotoxicity is one of the major concerns in drug discovery as changes in DNA content can lead to cancer and heritable effects (Tilmant et al., 2013) and thus the potential of drugs to induce genotoxicity is significant. Previous documented studies have shown that many plants used as food ingredients or in traditional medicine have mutagenic effects in vitro (Monteiro et al., 2006; Déciga-Campos et al., 2007; Razak et al., 2007) and are toxic and carcinogenic.
- SPECIFIC OBJECTIVES
Coccinia rehmanni and Jatropha zeyheri are two medicinal plants widely used in Vhembe communities (information obtained from interviews). There is a need to isolate pure active ingredients for selected medicinal plants and to investigate their phytochemical constituents, mutagenicity and any biological effects these extracts or compounds may have.
LITERATURE REVIEW
- BACKGROUND
- USE OF MEDICINAL PLANTS IN SOUTH AFRICA
- OVERVIEW OF THE STUDY PLANTS .1 JATROPHA ZEYHERI
- COCCINIA REHMANNI
- EXTRACTION AND FRACTIONATION
- EXTRACTION OF BIOACTIVE COMPOUNDS FROM THE PLANTS There are well known procedures that are used to extract bioactive compounds from
- SEPARATION AND PURIFICATION OF COMPOUNDS
- PHYTOCHEMICAL EVALUATION
- FLAVONOID
- ALKALOIDS
- TANNINS
- STEROIDS
- SAPONINS
- TERPENOIDS
- PHENOLIC COMPOUNDS
- MUTAGENICITY AND GENOTOXICITY .1 DEFINITION OF TERMINOLOGIES
- TYPES OF MUTATION
- METHODOLOGIES USED FOR DETECTING MUTAGENICITY .1 THE AMES TEST
- THE ALKALINE COMET ASSAY
- THE MICRONUCLEUS TEST
- THE UMU-C AND VITOTOX® TESTS
- PARP UNIVERSAL COLORIMETRIC ASSAY KIT
- SUMMARY OF LITERATURE REVIEW
Extraction is the first step of separation and purification of compounds from medicinal plant materials (Sasidharan et al., 2011). Phenols, coumarins, lignins, tannins and flavonoids are the simple plant phenolic compounds (Khoddami et al., 2013).
MATERIALS & METHODS
- ETHICAL CLEARANCE
- CHEMICALS AND REAGENTS
- SELECTION AND COLLECTIONS OF MEDICINAL PLANTS Medicinal plants were selected based on their extensive use for the treatment of
- EXTRACTION PROCEDURE
- FRACTIONATION OF PLANTS EXTRACTS
- GENERAL PRELIMINARY SCREENING FOR PHYTOCHEMICAL COMPOUNDS
- PHYTOCHEMICALS ANALYSES
- BIOCHEMICAL TESTS
- PHYTOCHEMICAL ANALYSIS USING TLC
- CYTOTOXICITY AND GENOTOXICITY OF THE PLANT EXTRACTS
- SAMPLE PREPARATION
- CELL CULTURE AND MAINTENANCE
- TREATMENT OF VERO CELLS
- BIOCHEMICAL ANALYSIS OF TREATED CELLS
- DATA QUANTIFICATION
- MUTAGENICITY OF THE PLANT EXTRACTS USING THE PARP UNIVERSAL COLORIMETRIC ASSAY KIT
- REAGENT PREPARATION
- PARP INHIBITOR ASSAY PROTOCOL
- DETECTION OF ANTIMUTAGENS
- STATISTICAL ANALYSIS
Two medicinal plants Coccinia rehmannii bulbs and Jatropha zeyheri rhizoids were selected and collected from the geographical locations of Vhembe District Municipality in Limpopo Province. Extraction of the plant material was performed with methanol (99.5%) as described by Wei et al (2011) with slight modifications. For each plant sample, 1000 g of the powdered materials were macerated in 10,000 mL of methanol for 24 h and filtered through Whatman No1 filter paper (Figure 3.1).
Ten microletters of the extracts were placed on a TLC plate and developed in saturated chambers using mobile phases of different polarities viz. the formation of a persistent foam was considered an indication of the presence of saponins (Astuti et al., 2011). A brown ring in the intermediate phase or a brown color was an indication of the presence of the compounds (Yadav et al., 2011).
The mobile phase was then allowed to migrate through the adsorbent phase up to 3/4 of the plate where they were then removed and allowed to dry. One hundred microliter aliquots of diluted plant extracts in fresh medium were used for cell treatment. Toxicity was determined by observing cell viability, expressed as nuclei per site of extract-treated cells compared to the untreated control.
RESULTS AND DISCUSSION
EXTRACTION RESULTS
PRELIMINARY SCREENING FOR PHYTOCHEMICALS
- JATROPHA ZEYHERI FRACTION RESULTS
- COCCINIA REHMANNI FRACTION RESULTS
It could be observed that the first fractions showed low percentage yield compared to the last collected fractions. This may be because the less polar/nonpolar solvent system was used first and could only elucidate less polar compounds, this is similar to the observation by Raaman, (2006); Braithwaite and Smith, (2012) and Houghton and Raman, (2012), who reported that when the stationary phase is polar, polar molecules spend more time adsorbed on the stationary phase, while less polar ones will be quickly carried by non-polar ones. mobile phase. Therefore, it can be concluded that the plant contained more polar compounds than non-polar compounds.
A higher percentage of polarity (methanol) was increasingly used at this stage, this solvent can elute most polar compounds as the polarity increases. The results show that Cr2 and Cr4 had lower yield percentages, it is known that these fractions are intermediate and have lower yield percentage compared to the previous following fractions.
PHYTOCHEMICAL EVALUATIONS
- PHYTOCHEMICAL ANALYSIS BY BIOCHEMICAL TESTS
- Phytochemical analysis of J. Zeyheri
- Phytochemical analysis of C. rehmanni
- PHYTOCHEMICAL ANALYSIS USING TLC
Terpenoids, cardiac glycosides, phenolics and tannins were the most detected bioactive compounds from both the crude extract and the fractions, respectively. Steroids and alkaloids were least detected as they were absent in some of the fractions or present in low concentrations when present. This may be due to the process of fractionation where the compounds are basically grouped according to their polarity as such, they end up being concentrated in some fraction and not in others (Houghton and Raman, 2012).
It can be speculated that these compounds were not detected in the crude extract due to their low content. This also proves a greater separation potential of the CEF dissolution system in these methanol extracts. Crude extracts from J zeyheri show bands for all secondary metabolites tested, but the Rf values differ from those of the fractions.
This could be because the fractions contained fewer compounds, so that the remaining compounds could move freely on the TLC plates during development. The reported spots are well separated and show many different compounds in the samples under study. Some of the bands could be seen even without UV light, but with Tannins plates were viewed under long wave UV light to observe the red bands.
CYTOTOXICITY OF PLANT EXTRACTS AND FRACTIONS
- CYTOTOXICITY EVALUATION OF J. ZEYHERI FRACTIONS
- CYTOTOXICITY EVALUATION OF C. REHMANNI FRACTIONS
This is a minimal inhibition compared to jz3 and 4 Figure 4.6 as marked which inhibited respectively (25 and 31%), the significance (p<0.01) of these extracts is observed even at 25 µg/ml comparisons. Melphalan inhibited 94% of the cells, it is a known chemical known to be toxic to cells. Jz4 toxicity on total cells was the most toxic and highly significant and had a p-value <0.01.
Studies have demonstrated the toxicity of these compounds, although it was from different plants and extraction solvent (Tshikalange et al., 2016). Fractions CR1 and 3 (Figure 4.7) showed a lower toxicity, which was shown when the number of viable cells decreased by 49 and 24%, respectively, with a statistical significance of p<0.01. This is a minimal inhibition compared to CR4 and 5 (Figure 4.7) as the label which inhibited (66 and 78%) respectively with the statistical significance p<0.0001.
CR4 and 5 toxicity was too high, too high and highly significant and had a p-value <0.0001. The higher concentrations of phenols, cardiac glycosides and terpenoids may explain the toxic nature of these fractions. Studies have shown the toxicity of these compounds, although they originated from different plants and extraction solvents (Tshikalange et al., 2016, and that the effects of flavonoids at higher doses may outweigh their beneficial effects, and that the unborn fetus is particularly at risk , because flavonoids easily cross the placenta.
GENOTOXICITY OF PLANT EXTRACTS AND FRACTIONS
- GENOTOXICITY ASSESSMENT J. ZEYHERI CRUDE EXTRACTS AND ITS FRACTIONS
- GENOTOXICITY ASSESSMENT C. REHMANNI CRUDE EXTRACTS AND ITS FRACTIONS
The fraction shows a significant increase in toxicity with the increase in concentration the cell count drops from 2400 to below 500 cells at concentration 100 µg/ml the formation of micronuclei increased significantly from concentration 12.5 to 100 µg/ml (from blowing 5 % to 10 %) (Figure 4.11a, Figure 4.11c and Figure 4.11d). Jatropha zeyheri extract fraction 4 shows a significant increase in toxicity, the relative cell count decreased from 2400 to below 500 cells (Figure 4.12a). The formation of micronuclei increased significantly from 2 % in control well to 11 % at concentration 25 µg/ml while concentration 50 and 100 µg/ml show higher percentage of micronuclei. Figure 4.12c.
In this fraction it was found that the fraction is very toxic, the relative number of cells was < 50 % at the lowest concentration 12.5 (Figure 4.12b), therefore the genotoxicity of this fraction cannot be concluded. The formation of micronuclei was significantly increased by the lower concentration compared to control cells (Figure 4.13c). The number of cells per well decreased from 2800 in the negative control to 500 cells/well at 200 µg/ml concentration (Figure 4.14a).
A significant gradual increase was observed in the formation of multinucleated cells (Figure 4.14d) up to 12 % at concentration 200 µg/ml. Coccinia rehmanni fraction 4 shows a large decrease in number of cells, in relative cell count and number of cells (Figure 4.16b) goes below <50% even in the lowest concentration. Coccinia rehmanni fraction 5 shows a large decrease in number of cells, in relative cell count (Figure 4.17b) and number of cells (Figure 4.17a) goes below <50% even in the lowest concentration.
HARP COLORIMETRIC ASSAY
Apoptosis and necrosis are reported to be directly caused by an excess level of reactive oxide species (Al-Khayal, 2017). Poly (ADP-ribose) polymerase, or PARP, is required during the repair of damaged DNA (Gohlke et al., 2015). PARP recognizes these defects by binding to NAD+, and initiates the synthesis of poly ADP-ribose chains (Pandya et al., 2010).
Thus, a plausible mechanism for genotoxicity of both plants would be direct inhibition of PARP activity which blocks the repair of DNA damage caused by excessive ROS, this in turn would lead to cell death by apoptosis or necrosis.
CONCLUSION AND RECOMMENDATIONS
Jatropha zeyheri crude extracts (Figure 4.5) show a significant toxicity with 46.5% at higher concentration (100 µg/ml) with a statistical significance p <0.0002. Jz4 was found to be more toxic than all the fractions and crude extracts. Finally, the last objective of this study was to investigate the mutagenicity/antimutagenicity of plant extracts, colorimetric test and an advanced fluorescence microscopic method was found to be effective in this regard.
As such, consuming these plants as food or as an ailment in higher doses is not recommended. This study serves as scientific proof that extensive use of this plant in traditional medicine can lead to some irreversible damage.
Phytochemical screening and comparative analysis of antimicrobial activity of root and leaf extracts of Tinospora coridifolia, Phyllanthus niruri and Abrus precator, important medicinal plants. Isolation, characterization and cytotoxic effects of antibacterial and antioxidant compounds from selected medicinal plants (Doctoral dissertation). The effect of fermentation conditions on the phytochemical composition and antioxidant activity of Jatropha zeyheri leaves.
Phytochemical study of the leaves of Adansonia digitata (Doctoral dissertation, Sudan University of Science and Technology). Antibacterial activities of selected medicinal plants used to treat sexually transmitted infections in Blouberg area, Limpopo Province (Doctoral dissertation, University of Zululand). In vitro pharmacological activity of the crude acetone extract of Erythrina caffra: antibacterial and antifungal assessment.
Comparative in vitro cytotoxic, anti-inflammatory and anti-microbiological activities of two indigenous Venda medicinal plants. Antimicrobial, antioxidant and cytotoxicity studies of medicinal plants used in the treatment of sexually transmitted diseases. In vitro and in vivo anti-inflammatory activity of leaves of Symplocos cochinchnensis (Lour) Moore ssp laurina.