Genetic, phytochemical and bioactivity studies of Clinacanthus nutans (Burm. f.) Lindau (Acanthaceae)

Fong, S 2015, Genetic, phytochemical and bioactivity studies of Clinacanthus nutans (Burm. f.) Lindau (Acanthaceae), Doctor of Philosophy (PhD), Applied Science, RMIT University.

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Title Genetic, phytochemical and bioactivity studies of Clinacanthus nutans (Burm. f.) Lindau (Acanthaceae)
Author(s) Fong, S
Year 2015
Abstract There are approximately 300,000 higher plant species in the world, but only about 10% of these have been studied extensively for possible medical applications. Therefore, these plant species have significant potential for studies on their phytochemical constituents and bioactivities. These include Clinacanthus nutans (Burm. f.) Lindau (Acanthaceae), a popular medicinal plant native to Southeast Asia, traditionally used to treat infections and several diseases. The plant is normally propagated vegetatively by stem cuttings to meet consumer demand. C. nutans leaves are often prepared in dried form to prolong storage life and the dried leaves are commonly steeped in boiling water and then consumed as a herbal tea.

The level of phytochemicals in plants varies even amongst the members of the same species. This variation can be influenced by genetics or environment alone, or as a combination of the two factors. Besides, postharvest processing of medicinal plants, such as drying and extraction, can also affect the level of phytochemicals in plants, and hence, their bioactivities. However, there is still a lack of information on how and to what extend these factors affect the phytochemical constituents in C. nutans leaves, which may consequently influence its bioactivity. Therefore, this study aimed at addressing these knowledge gaps by investigating the genetic variation, total phytochemical content and anticancer activity of C. nutans using a multidisciplinary approach that integratesdmolecular genetics, analytical chemistry and cell biology.

The leaves of C. nutans grown at higher elevations with lower air temperatures (i.e. CT5 from Chiang Dao, Chiang Mai, Thailand) had higher total phenolic and flavonoid content as determined using Folin-Ciocalteu, aluminium chloride assay and high performance liquid chromatography (HPLC) methods, compared to those grown at lower elevations with higher air temperatures. Likewise, the crude methanol extracts of C. nutans leaves from higher elevations and lower air temperatures exhibited higher cell cytotoxicity, with the leaf extract from Chiang Dao, Chiang Mai, Thailand, showed the highest activity (24 h EC50: 0.95 mg/mL and 72 h EC50: 0.77 mg/mL). This extract also showed selective cytotoxicty against cancer cells but not non-cancerous cells. The extract induced apoptosis in the D24 melanoma cells as determined by cytofluorimetry and microscopy (confocal and transmission electron). Genetic study of C. nutans using molecular markers revealed high genetic similarity, with an average of 77.0% (RAPD) and 91.6% (microsatellite) among the vegetatively propagated C. nutans samples from Malaysia, Thailand and Vietnam, with identical genetic profiles even though they were geographically distant. These results suggested that the anticancer activity of the C. nutans leaf extract against D24 melanoma cells may be determined by its phytochemical constituents, which are influenced by environmental variables, particularly elevation and air temperature, rather than genetic factors.

The effect of postharvest treatments, including drying temperature and extraction solvent, on the phenolic and flavonoid content in C. nutans leaves was investigated using the same colourimetric and HPLC methods. An increase in the level of phytochemical constituents was observed with increasing drying temperatures, where leaves dried at 100°C and 80°C had the highest total phenolic and flavonoid content, respectively. HPLC analysis also showed the greatest amount of phytochemicals in the C. nutans leaves dried at 80°C. This event may have resulted from the formation of phenolic compounds through non-enzymatic reactions and/or a decrease in peroxidase and polyphenoloxidase enzymatic activities. This suggested that low temperature drying may not be a suitable postharvest method for drying C. nutans leaves. All methods showed that the most polar solvent (water) resulted in the highest total phytochemical content being extracted from the leaves. The crude cold aqueous C. nutans leaf extract had the greatest quantities of both phenolics and flavonoids, suggesting that cold extraction (22°C) with water is the best method to prepare the crude extract of C. nutans leaves with maximum phenolics and flavonoids. Only the effect of extraction solvent on the viability of four human cancer cell lines (melanoma: D24 and MM418C1 cells and breast carcinoma: MCF7 and BT474 cells), was examined in the current study. The D24 melanoma cell line was the most sensitive to treatment and the crude aqueous (cold and hot) leaf extracts were the most active against all cancer cell lines, whereas the methanol, ethanol and dichloromethane extracts showed little or no cytotoxicity against these cell lines. The crude cold aqueous extract was the most cytotoxic against the D24 melanoma cells with a 72 h EC50 of 1.63 mg/mL. This is in agreement with that of the colourimetric methods, suggesting that the extraction with water at 22°C is the best method to extract the bioactive compound with anticancer property from C. nutans leaves, which remains to be isolated and identified. The extract also predominantly induced apoptosis in the D24 cells according to cytofluorimteric, confocal and transmission electron microscopic analyses. This study provides important information on the optimisation of postharvest processing to prepare crude extracts that preserve the bioactive phytochemicals from dry C. nutans leaves. This may increase the potential of C. nutans as a valuable medicinal plant.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Applied Science
Keyword(s) Genetics
Clinacanthus nutans
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Created: Thu, 10 Dec 2015, 13:37:16 EST by Denise Paciocco
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