Analysis of phenolics and other phytochemicals in selected Malaysian traditional vegetables and their activities in vitro

Mat Ali, Mohd Shukri (2008) Analysis of phenolics and other phytochemicals in selected Malaysian traditional vegetables and their activities in vitro. PhD thesis, University of Glasgow.

Abstract

A fruit and vegetable-rich diet has been associated with decreased risk of developing chronic diseases such as cardiovascular disease and cancer in humans. These protective effects have been attributed in part, to the presence of phytochemicals in fruit and vegetables, in particular flavonoids and phenolic compounds. Some plants have been used in traditional medicine for healing, ritual ceremonies and as health tonics or food supplements. Recent interest in the health-promoting properties of Malaysian traditional vegetables has been based on claims about their uses in health and medicine. However, scientific information to support these claims is largely unexplored. The overall objectives of the present study were to investigate, determine and quantify the phytochemicals, particularly phenolic compounds, in the seven samples from five species of selected Malaysian traditional vegetables (Anacardium occidentale, Centella asiatica, Colubrina asiatica, Pluchea indica and Premna cordifolia) and to evaluate their activities in vitro, including antioxidant and antibacterial activities of extracts of these plants and individual phytochemicals. In the first section of this project, discussed in Chapter 3, Malaysian traditional vegetable extracts were screened for phenolic compounds using several complimentary techniques, namely high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry and the total phenolic content determined using the Folin-Ciocalteu assay. Flavonol glycosides were predominant in most of the species, particularly A. occidentale with levels ranging from 6434 to 12420 µg/g fresh weight. Chlorogenic acids were the main components identified and quantified in C. asiatica and P. indica. The total phenolic content of the vegetables were between 100 ± 7.8 and 415 ± 20 mg/ kg gallic acid equivalent (GAE) in batch 1 but lower in batch 2 ranging from 62 ± 2.5 to 386 ± 41 mg/ kg GAE. The total phenolic content of plant extracts was positively correlated with total antioxidant capacity, determined by 2, 2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant potential (FRAP) assays.

A. occidentale exhibited the highest total phenolic content and total antioxidant activity, whereas Colubrina asiatica, which had the lowest total phenolic content, also had low antioxidant activity in vitro. Phenolic content and antioxidant activity were significantly (p<0.05) influenced by environmental factors, as in this study, plant materials in batch 1 which was harvested in rainy season, had a higher total phenolic and antioxidant content than batch 2, which was harvested in the dry season.

Based on the hypothesis that other components in addition to phenolics also contributed to the total antioxidant activities in the plants, the next objective, which was presented in Chapter 4, was to investigate the occurrence of phytochemicals such as triterpenes, carotenoids, α-tocopherol and vitamin C. The level of total triterpenes, biomarkers of C. asiatica was not significantly different between batches. The main component was madecassoside with 91 ± 4.8 µg/g fresh weight in batch 1 and 77 ± 3.4 µg/g fresh weight in batch 2. The level of carotenoids and vitamin C were low compared to previous reports. This was almost certainly due to dried samples being used in the present study, as some of the compounds would have broken down during drying process. This would have particularly affected the levels of vitamin C, which contributed only 0.9 to 5.5% to the total antioxidant activity of the plants under study.

Total antioxidant activities of plant essential oils were determined using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and the result was in agreement to the total antioxidant activities of plant extracts, which A. occidentale having the highest amount. The highest antioxidant activity exhibited by A. occidentale oil was attributed to the presence of high amounts of γ-terpinene (28%) and terpinen-4-ol (4.2%), both of which were shown to have strong radical scavenging activity.

The high phenolic content, antioxidant activity and occurrence of volatile components exhibited by A. occidentale has led to the final objective of this study, which is presented in Chapter 5. This was to screen for antimicrobial activities of A. occidentale extracts and essential oil against selection of Gram-positive (Enterococcus faecalis, Staphylococcus aureus, Meticillin-resistance Staphylococcus aureus (MRSA), coagulase negative Staphylococci (CoNS) and Lactobacillus acidophilus), Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) and fungi (Candida albican) using disc diffusion and minimum inhibitory concentration (MIC) methods. Investigation of the modes of action was determined using growth inhibition curve, scanning (SEM) and transmission (TEM) electron microscopy. A. occidentale was shown to have promising effects at 25 mg/ml with regard to inhibiting the growth of Gram-positive bacteria including MRSA. The essential oil and its major component, γ-terpinene at only 2.5% (v/v) inhibited the growth of all Gram-positive and Gram-negative bacteria. None of the A. occidentale extracts or oil exhibited antibacterial activities against Lactobacillus acidophilus, an important strain of bacteria found in the human gut. This indicates selective effects of A. occidentale.

A. occidentale extract and oil inhibited the growth of S. aureus cells within a 2-hour incubation observed in time-kill experiments. SEM and TEM examination revealed that the oil and its component, γ-terpinene, inhibited the bacteria through bacteriostatic and bactericidal effects which damaged the bacterial cell wall. Testing the oil and γ-terpinene against epidemic-MRSA (EMRSA) biofilms indicated an anti-adhesive effect, which disrupted the bacterial colonies in the biofilms to produce more extracellular polysaccharides (EPS). The effects of A. occidentale oil were comparable with tea tree oil, a widely used topical antiseptic.

All the Malaysian traditional vegetables under study are claimed to have medicinal properties and health effects. The results in the present study have provided some information on phytochemical and nutritional properties of Malaysian traditional vegetables, and as a consequence provide a sound scientific base for promoting their consumption particularly in Malaysia.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Malaysian vegetables, phytochemical analysis, phenolics, antioxidant, antibacteria, biofilm
• Subjects: Q Science > QR Microbiology; Q Science > QD Chemistry; S Agriculture > S Agriculture (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Crozier, Professor Alan and Williams, Dr Craig
• Date of Award: 2008
• Depositing User: MR MOHD SHUKRI MAT ALI
• Unique ID: glathesis:2008-158
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 15 Sep 2008
• Last Modified: 10 Dec 2012 13:16
• URI: https://theses.gla.ac.uk/id/eprint/158