Characterisation, bioactivity and qsar studies of natural products from selected Namibian red marine algae

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Date
2020
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University of Namibia
Abstract
The objective of this research was to discover new drug leads from Namibian marine algae. Plocamium extracts were screened and their phytochemical contents were quantified. Both the antioxidant activity and the antimicrobial activity of the crude extracts were determined, as well as the dose-response relationship for Plocamium extracts in BALB/c mice. This was done by using acute and sub-acute toxicity parameters. In addition, the structural elucidation of the major metabolite found in the crude extract was determined and the Quantitative Structure Activity Relationships (QSARs) of the compound were determined. Methods: Frozen Plocamium leaves were soaked in dichloromethane (DCM) and methanol (MeOH) in a ratio 1:1 (v/v) for 48 hours. Concentrated Plocamium extracts were screened for phytochemical constituents. Total phenolic and flavonoid contents as well as antioxidant activity were quantified. Dried algal extracts were also reconstituted with different solvents and tested in vitro for antimicrobial activity against 12 pathogens using the Kirby Bauer disc diffusion method. Mice of known weights were infected with Escherichia coli and Pseudomonas aeruginosa by intravenous injection and sub-cutaneous methods respectively. The mice were later treated with gentamycin and ampicillin injections. Other groups of mice were treated with different concentrations of Plocamium extract over a period of five days. E. coli and P. aeruginosa loads in the faeces of the test mice were quantified daily. Plocamium extracts were purified using HPLC to fractionate the extracts. Major fractions were collected and identified by means of one and two dimensional NMR spectroscopy data and MS analysis. In terms of QSAR, the structures of the metabolites were theoretically optimized using the Merck Molecular Force Field. In addition, several physicochemical properties were computed by using the B3LYP variant of Density Functional Theory in conjunction with the 6-31G (d) basis set. Results: Plocamium cornutum and Plocamium rigidum extracted using DCM had total phenolic content of 132.85 ± 0.82 mg and 188.65 ± 0.45 mg Gallic acid equivalents per gram respectively. The IC50 values for Plocamium rigidum and Plocamium cornutum were 28.87 ± 0.82 μM and 40.11 ± 0.38 μM respectively. Ethanolic extracts of Plocamium rigidum showed a zone of inhibition of 6.35 ± 0.25 mm against Listeria monocytogenes while the standard ampicillin had no activity. From the probit plot, the LD50 was calculated to be 3556 mg/kg. A therapeutic dosage of P. rigidum of 355 mg/kg in BALB/c mice reduced E. coli load to pre-evaluation levels on the fifth day. The chemical structure of Plocamium sample I and II (Plocamium rigidum and Plocamium cornutum respectively) yielded two known compounds namely, 3,4-erythro-7-dichloromethyl-3-methyl-3,4,8-trichloro-1,5E,7E-octatriene (from sample I) and 1E,3R,4S,5E,7Z-1-bromo-3,4,8-trichloro-7-(dichloromethyl)-3-methylocta-1,5,7-triene (from sample II) respectively. CPKOvality and HLgap are physicochemical properties that best describe the variation in biological activity of the metabolites. The equation of the best fit was determined as: pIC50 = 9.91CPKovality + 0.270HL-gap-17.149 (R2 = 0.71, Adj. R2 = 0.56, R = 0.84, Std error = 0.31, and q2 = 0.55) Conclusion: DCM is a better solvent than methanol for the extraction of natural products from Plocamium species. P. rigidum showed inhibition against E. coli and L. monocytogenes in vitro. Although P. rigid inhibited the growth of E. coli, the possible development of liver lesions (in vivo) after chronic exposure is an indication of liver injury which is a sign of the chronic toxicity of P. rigidum, even at low concentrations. The probable compound responsible for this action is sample (II) above. The variables in the equation above are the parameters that best describe the variation in biological activity of the halogenated monoterpenes extracted and identified. The equation also shows that CPKovality (moderate size and shape of ligands) and a small H-L gap with high reactivity are the parameters that best optimised structure to predict improved biological activity of Plocamium metabolites.
Description
A dissertation submitted in fulfilment of the requirements for the reward of the Degree of Doctor of Philosophy in Pharmaceutical Chemistry
Keywords
Qsar studies, Algae
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