Antiparasitic, antibacterial, antioxidant, and cytotoxic activities of six Malaysian medicinal plants and identification of their active constituents

Nor Azman, Nadiah Syafiqah (2020) Antiparasitic, antibacterial, antioxidant, and cytotoxic activities of six Malaysian medicinal plants and identification of their active constituents. PhD thesis, University of Nottingham.

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Abstract

Increasing mortality and morbidity rates caused by Plasmodium, Leishmania, and drug-resistant bacteria worldwide have become a major problem. Current drugs do seem less effective, toxic, expensive, and require long periods of administration. WHO recommends the development of new antiparasitic and antibacterial agents. Therefore, research on Malaysian medicinal plants has been performed by our researchers in search of new natural products in the past five decades, offering them a great opportunity to discover new chemical structures in drug discovery.

In 2015, a survey was done to evaluate and document the use of medicinal plants used by local people in day-to-day practice in two preserved villages (Kuala Kangsar and Manong) in the State of Perak, Peninsular Malaysia, which afforded the collection of 6 medicinal plants from three different families in the class Magnoliopsida (Dicotyledons). The collected plants were identified as Chilocarpus costatus Miq. (Apocynaceae), Leuconotis eugeniifolia (Wall. ex. G. Don) A.DC (Apocynaceae), Tabernaemontana peduncularis Wall (Apocynaceae), Uvaria grandifolia Roxb. ex Hornem. (Annonaceae), Artabotrys suaveolens (Blume) Blume (Annonaceae), and Diospyros wallichii King & Gamble (Ebenaceae). To the best of our knowledge, detail pharmacological studies regarding antiparasitic, antibacterial, antioxidant, and cytotoxic activities reported on these six Malaysian medicinal plants are very scanty. Thus, the general objective of this study was to identify natural products from rare Malaysian medicinal plants with antiplasmodial, antileishmanial, antibacterial, antioxidant or cytotoxic activities.

Parasites such as Plasmodium falciparum and Leishmania donovani are responsible for infectious disease with over 200 million reported cases and thousands annual deaths. Therefore, in vitro antiparasitic activities and cytotoxic activities were performed on both chloroform extracts and compounds by the microtetrazolium test (MTT), Histidine-Rich Protein II (HRPII) assay, and malaria SYBR Green I-based fluorescence (MSF) assay with quercetin, vincristine, chloroquine, dihydroartemisinin, and miltefosine as positive controls. Based on the results obtained, 11 out of 12 extracts tested showed good antiplasmodial activities against P. falciparum K1 chloroquine-resistant strains with IC50 below 10 µg/mL.

The highest antiplasmodial activities against P. falciparum K1 chloroquine-resistant strains were recorded on both stems and leaves chloroform extracts of C. costatus with IC50 values of 1.15 and 0.85 µg/mL, respectively. The antileishmanial analysis on plant chloroform extracts revealed that 3 of the 12 extracts showed good antileishmanial activities against promastigote in vitro, which were stems A. suaveolens, stems C. costatus, and leaves D. wallichii with IC50 values of 17.33, 17.32, and 7.6 µg/mL, accordingly. The remaining exhibited moderate activities against L. donovani BHU-1251 promastigote strains. We also examined the potential toxicity of the extracts against mammalian cells prior to antileishmanial and antiplasmodial testing to assess the toxicity of these potential antiparasitic agents. Protozoan parasites such as L. donovani and P. falciparum can cause lung diseases (Vijayan, 2008). Thus, a cytotoxicity study of extracts against human lung epithelial cells (MRC-5) was performed. Four of the 12 extracts tested were toxic to human cells with CC50 below 20 µg/mL, which were stems T. peduncularis (11.5±3.8 µg/mL), stems L. eugeniifolia (15.5±3.4 µg/mL), stems D. wallichii (15.0±2.0 µg/mL), and stems U. grandiflora (10.0±6.1µg/mL).

Pathogenic bacteria have become more resistant to conventional antibiotics, leading to an increase of nosocomial bacterial infection worldwide. The most frequent nosocomial bacterial infections are bloodstream infection (BSI), urinary tract infection (UTI), surgical site infection (SSI), pneumonia, gastroenteritis, meningitis, and respiratory infection. Common pathogens found to be responsible for these infections include Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus epidermidis, Methicillin-resistant Staphylococcus aureus (MRSA), and Klebsiella pneumoniae (KP). Hence, antibacterial assessment on these seven pathogenic bacteria was done using the disc diffusion method, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) as well as synergy and time killing assay with cefotaxime and vancomycin as positive controls.

C. costatus extract had moderate antibacterial activity against S. epidermidis with MIC values of 187.5 ± 0.20 µg/mL and was inactive against the other six bacteria tested with MIC values above 1500 µg/mL. The remaining 11 chloroform extracts were inactive against all the bacterial strains tested. Since only stem C. costatus extract displayed antibacterial activities in MIC, therefore, MBC/MIC ratio, time-killing, and synergistic effects were studied only on this extract. C. costatus stem extract was found to be bactericidal. Time killing assay was done for 4 hours. In the time killing assay, C. costatus stem chloroform killed S. epidermidis bacteria faster than cefotaxime and vancomycin. It also was found that C. costatus stem chloroform reacts synergistically with cefotaxime (FICI 0.5), however, no synergistic effect was observed with vancomycin (FICI 1.1) when combined against S. epidermidis.

Free radicals are involved in the pathogenesis of many human diseases. The investigation of the antioxidant properties of plant extracts was achieved through the assessment of total phenolic content and iron reducing antioxidant power (FRAP) assay with gallic acid and FeSO4 as positive controls. All chloroform extracts had low total phenolic and FRAP content.

Two plant extracts (C. costatus stem chloroform and U. grandiflora leaves chloroform) were selected in the present study for further isolation and bioactivity, as they were very active and had good antiplasmodial, antileishmanial, and antibacterial activities. Therefore, isolation of the compounds was done by High Performance Liquid Chromatography (HPLC) and resulted in the isolation of three compounds: pinoresinol, zeylenol, and ferrudiol. To the best of our knowledge, this is the first report on the occurrence of pinoresinol and ferrudiol in C. costatus Miq. and U. grandiflora Roxb. ex Hornem, respectively.

These three compounds were then tested for their bioactivities. Pinoresinol displayed no antileishmanial and antiplasmodial activities, and was not toxic to human lung, MRC-5 cells. However, pinoresinol acted synergistically with cefotaxime against S. epidermidis. Meanwhile, both zeylenol and ferrudiol were inactive against L. donovani.

In conclusion, the findings of our study indicated that C. costatus stem chloroform and its major constituents, pinoresinol may be used as potential candidates for the development of antibiotic potentiators.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Wiart, Christophe
Koo, Teng Jin
Nissapatorn, Veeranoot
Raju, Chandramathi Samudi
Keywords: natural products, drug delivery, NMR, HPLC, antiparasitic, antibacterial, antileishmanial, antimalarial
Subjects: R Medicine > RS Pharmacy and materia medica
Faculties/Schools: UNMC Malaysia Campus > Faculty of Science > School of Pharmacy
Item ID: 59513
Depositing User: NOR AZMAN, NADIAH SYAFIQAH
Date Deposited: 22 Feb 2020 04:40
Last Modified: 06 May 2020 10:51
URI: http://eprints.nottingham.ac.uk/id/eprint/59513

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