ISOLATION AND CHARACTERIZATION OF BIOACTIVE COMPOUNDS FROM CENTAUREA SENEGALENSIS [DC]

Centaurea senegalensis DC.is a plant commonly used in Nigeria traditional medicine for treatment of stomach ache and pains. The aim is to isolate and characterize bioactive compound from the aerial part of Centaurea senegalensis. The plant was extracted using maceration method to give the crude methanol extract which was sequentially fractionated using hexane, dichloromethane and ethyl acetate respectively. The crude methanol extract and solvent fractions were subjected to phytochemical screening and found to contain alkaloids, flavonoids, terpenes, tannins, saponins and glycosides. Purification of the n-hexane (CSH) and dichloromethane (CSD) fractions using silica gel column chromatography led to the isolation of taraxasterol and eupatorin respectively. The structural elucidations of the compounds were determined using FT- IR,1D and 2D-NMR.The crude methanol extract, fractions and the isolated compounds were subjected to antimicrobial activity against selected gram-positive and gram-negative bacteria (Shigella dysenteriae, Salmonella typhi, Escherichia coli, Staphylococcus aureus, Vancomycin Resistant Enterococci, Methicillin Resistant Staphylococcus aureus) results indicated varying growth inhibitions with zones of inhibition of 16-28 mm for crude methanol extract,13-31 mm for solvent fractions and 10-15mm for isolated compounds. The minimum inhibitory concentration (MIC) of the fractions was12.5 mg/ml on the Methicillin Resistant Staphylococcus aureus and Vancomycin Resistant Enterococci. However, the isolated compounds were found to demonstrate activity on Methicillin resistance Staphylococcus aureus and Shigelladysenteriae with 25 mg/ml as the MIC and 50 mg/mlas the MBC. In conclusion, the results of this study have shown the importance of Centaurea senegalensis as a source of bioactive compounds with potential for the treatment of bacterial infections.

 

COVER PAGE  I 

TITLE PAGE   II 

DECLARATION   III 

CERTIFICATION    IV 

DEDICATION V

ACKNOWLEDGEMENT   VI 

ABSTRACT VII

TABLE OF CONTENT VIII 

LIST OF FIGURES   XI

LIST OF TABLES    XII

ABBREVIATION AND ACRONYMS   XIII

2.2.2 Sesquiterpene lactones9

2.3.3 Flavonoids 10

3.3.3.7 Test for steroids 23

3.3.7 Column chromatography separation of dichloromethane fraction (CSD) 24

 

Figure2.1: Formation of triterpenoids precursors GPP and FPP14 

Figure2.2: Biosynthesis of flavonoids 16

Figure 4.1: 1H- NMR Spectrum of CSH5 34 

Figure 4.2: DEPTSpectrum of CSH535 

Figure 4.3: COSY Spectrum of CSH536 

Figure 4.4: HMBC Spectrum of CSH537 

Figure 4.5: HSQC Spectrum of CSH5 38 

Figure 4.6: FT-IR Spectrum of CSH539 

Figure 4.7 Taraxasterol40

Figure 4.8: 1H-NMR Spectrum of CSD540 

Figure 4.9: 13C-NMR Spectrum of CSD541 

Figure 4.10: 1H-1H-COSY Spectrum of CSD542 

Figure 4.11: HMBC Spectrum of CSD543 

Figure 4.12: HSQC Spectrum of CSD544

Figure 4.13: FT-IR Spectrum of sample CSD545 

Figure 4.14: Eupatorin62

Figure 5.02D Correlation of Compound CSH560

Figure 5.1: COSY and HSQC Correlation of Compound CSD565

 

Table 2.1: Some of the Centaurea species used in traditional medicine 7

Table 4.1: Percentage Extracted and Phytochemical Screening of Centaurea senegalensis29 

Table 4.2: Column Chromatographic Separation of n-Hexane Fraction of C. senegalensis30 

Table 4.3: Repeated Column Chromatographic Separation of Column Fraction 31

Table 4.4: Column Chromatographic Separation of the sample(DCM) Fraction of C.

senegalensis32

Table 4.5: TLC Profile of isolated compounds CSH5 and CSD533

Table 4.6: Comparison of 13C-NMR and 1H-NMR of CSH5 (ppm) data with literature value 59 

Table 4.7: Comparison of13C-NMR and 1H-NMR of CSD5(ppm) data with literature value64 

Table 4.8: Antibacterial activities of Centaurea senegalensis extract and fraction 49

Table 4.9: Antibacterial activity of isolated compounds 50

Table 4.12: MBC of Extract/Fraction 53 Table 4.13: MBC of CSH5 and CSD554

 

Natural products chemistry has undergone tremendous growth owing to advances in isolation techniques, synthetic methods, physico-chemical measurements, and new concepts, as well as for the wide range of biological properties exhibited by the substances biosynthesized by living organisms (Cragg et al., 1997). Extracts from the various plant parts (leaves, stem bark and roots) of various higher plants are used in herbal medicine production (Sofowora, 1993). Plants extracts are given ordinarily or as concoctions for the treatment of various ailments. In actual sense more than 75% of the world population depends on these various forms of concoctions and herbal decoctions for the treatment of infections (Robenson and Zhang, 2011).

Plants are natural reservoir of medicinal agents and are increasingly gaining acceptance, even among the literates in urban settlements, probably due to the increasing inefficacy of many modern drugs used for the control of many infections such as typhoid fever, gonorrhoea and tuberculosis as well as increase in resistance by several bacterial to various antibiotics and the increasing cost of prescription drugs, for maintenance of personal health (Smolinski et al., 2003). Plants as a potential source of drugs are of great relevance, especially with the current global shift to obtain drugs from plant sources. Several ailments including fever, asthma, constipation, diarrhoea, oesophageal cancer and hypertension have been treated with traditional medicinal plants. Different plant parts and components have been employed in the treatment of infectious diseases (Dutta et al., 2013).

The rapid increase in the antibiotic resistance of microorganism to available synthetic drugs and their discomforting side effects necessitates the search for alternative source of antibiotics. Antimicrobial resistance is an increasingly problematic issue that leads to millions of deaths every year (WHO, 2013). Most bacteria and fungi always develop resistance to antibiotics that are used to kill them. This is a big challenge in controlling infectious diseases and hence, a need to search for new substances, especially from natural sources to solve the problem.

Many species of the genus Centaurea (Asteraceae) have been used in traditional medicine since ancient times. These plants are generally rich in flavonoids and sesquiterpene lactones, which are the main constituents responsible for biological activity (Akkal et al., 1997).

Sesquiterpene lactones (SLs) demonstrate a broad spectrum of biological activity including disrupting the cell wall of fungi and invasive bacteria, whereas others protect the plant from environmental stress that would otherwise cause oxidative damage. Many of the compounds are effective due to their bitter flavor, which has obvious implications for human consumers (Chadwick et al., 2013). Plants of Centaurea genus are used as remedy against infectious diseases in Nigeria traditional medicine. Preliminary phytochemical screening of Centaurea senegalensis extracts indicated the presence of spots on TLC. However, previous isolation and structural elucidation has been reported on flavonoids only (Aqil et al., 1998).

Antibacterial drug resistance is an increasingly problematic issue that leads to numerous deaths yearly (WHO, 2013). It is a global challenge to antibiotic chemotherapy and hence, the need to search for natural sources for more effective compounds or molecular scaffold for the synthesis and development of new antibacterial agents.

The aim of the study was to isolate and characterize bioactive compounds from Centaurea senegalensis and evaluation of antibacterial activity of the pure compounds.

 

The objectives of the study were to:

The genus Centaurea has been known to be used in traditional medicine. The need to scientifically establish the medicinal claims on the plant. This may result to discovery of more potent compounds against drug resistance pathogenic microorganisms. However, although flavonoids have been reported from the plant, there is no documented report to the best of my knowledge on the isolation, characterization and antibacterial activity of other class of compounds from Centaurea senegalensis.