Ethnopharmacological studies of natural product extracts of Bangladesh: anticancer evaluation of selected isolated natural products

Hossan, Md Shahadat (2019) Ethnopharmacological studies of natural product extracts of Bangladesh: anticancer evaluation of selected isolated natural products. PhD thesis, University of Nottingham.

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Ethnomedicinal studies play a pivotal role in the drug discovery arena. Humans have relied on nature to cater basic needs including new remedies for a wide spectrum of diseases based on the empirical findings of hundreds and thousands of years. Exploitation of medicinal plants for bioactive compounds could be an imperative source of providing new panoramas for novel drug discovery and development. Bangladesh has many rainforest plants enriched with medicinal values. Ethnomedicinal survey conducted on the Khyang tribe and local TMPs of Bangladesh. Khyang were observed to use 84 plant species in their treatment. Moreover, 69 different plant species were sequentially extracted with hexane, ethyl acetate and ethanol, obtained 197 extracts and subjected to preliminary antimicrobial and cytotoxic screening. Bioactivity-guided fractionation led to the isolation of embelin, cerberin, 062B-F2, furanodienone and semisynthetic Cardamonin-Cu (II) and subsequently investigated the mechanism of action.

Viral respiratory infections are escalating serious concern globally. The present study examines the activity of six plants against the influenza virus. MDCK cells infected with influenza virus A/Puerto Rico/8/34 (H1N1) were treated with increasing concentrations of ethyl acetate extracts, and their cytotoxicity (CC50), virus-inhibiting activity (IC50), and selectivity index (SI) were calculated. The ethyl acetate extract of fruits of Embelia ribes Burm. f. (Myrsinaceae) had the highest antiviral activity, with an IC50 of 0.2 μg/mL and a SI of 32. Its major constituent, embelin, was further isolated and tested against the same virus. Embelin demonstrated antiviral activity, with an IC50 of 0.3 μM and an SI of 10. Time-of-addition experiments revealed that embelin was most effective when added at early stages of the viral life cycle (0-1 h post infection). Embelin was further evaluated against a panel of influenza viruses including influenza A and B viruses that were susceptible or resistant to rimantadine and oseltamivir. Among the viruses tested, avian influenza virus A/mallard/Pennsylvania/10218/84 (H5N2) was the most susceptible to embelin (SI = 31), while A/Aichi/2/68 (H3N2) virus was the most resistant (SI = 5). In silico molecular docking showed that the binding site for embelin is located in the receptor-binding domain of the viral hemagglutinin. The results of this study provide evidence that E. ribes can be used for the development of a novel alternative anti-influenza plant-based agent.

Coxsackievirus B4 (CVB4) is the causative agent leading in humans to the development of type 1 diabetes, heart diseases, neurodegenerative pathologies and fatal meningoencephalitis. In the present study, we evaluated extracts of 12 medicinal plants against Coxsackie B4 virus in vitro. Among them, extract of Curcuma aeruginosa Roxb derived furanodienone had a remarkable antiviral activity with a mean IC50 of 7.2 μg/mL and an SI of 9. Docking studies suggest that furanodienone bind specifically to Coxsackie B4 capsid protein 1 to form stable complexes with a binding energy of 66.2 kcal/ mol.

Dengue and Japanese encephalitis virus are the most prevalent mosquito-born virus, causes morbidity in tropical countries. E. ribes, G. atroviridis and B. ramiflora possessed IC50 1.41 ±0.14 µg/mL, 2.35 ±0.06 µg/mL and 3.06 ±0.009 to inhibit the DENV-2 intracellular replicon by targeting non-structural protein respectively. Concurrently, investigated the anti-JEV activity, 7 extracts exhibited IC50 <5 µg/mL, where E. ribes exhibited the lowest IC50 value 0.14 (±0.04) µg/mL, followed by G. atroviridis and C. zeylanicum showed 0.4 (±0.11) and 0.517 (±0.02) µg/mL respectively. B. ramiflora, P. longum hexane extract, P. longum ethyl acetate extract and Z. alatum demonstrated IC50 value 1.3 (±0.10) µg/mL, 2.49 (±0.20) µg/mL, 3.82 (±0.35) µg/mL and 4.18 (±0.10) µg/mL respectively. Antiviral results provide evidence that E. ribes, G. atroviridis, B. ramiflor, C. aeruginosa and Z. alatum can be investigated further to develop a novel alternative anti-viral target.

The resistance of bacteria to antibiotics is a serious global concern. The antibacterial properties of medicinal plants utilised by the Khyang tribe in Bangladesh have not been investigated previously. In this present study investigated the antibacterial properties of 18 medicinal plants used by the Khyang tribe against human pathogenic bacteria. The corresponding 54 extracts were tested against six human pathogenic bacteria by broth microdilution assay. The hexane extract of the bark of Cinnamomum cassia (L.) inhibited the growth of MRSA, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii with MIC values below 100 mg/mL. From this plant, cinnamaldehyde evoked with vancomycin for MRSA with fractional inhibitory concentration index of 0.3. at 4 x MIC in 1h an irreversible decrease of MRSA count Log10 (CFU/mL) from 6 to 0 and was synergistic. Furthermore, the antiplasmodial test revealed that IC50 values ranged between 2.08 – 4.58 µg/mL (SI > 10) for 5 of 6 extracts indicating good activity against chloroquine-resistant Plasmodium falciparum where A. subulatum hexane extract (SI = 30.66) was the most active followed by O. americanum Hexane extract (SI > 19.33). At the same time from the anti-leishmanial test, A. europaeum hexane and ethyl acetate extract and M. fragrans extracts were exhibited SI of 12.01, 8.46 and 5.72, respectively, which showing a moderate effect and reporting for the first-time. Eugenol from M. fragrans, O. americanum, O. basilicum and A. subulatum; showed equipotency (IC50 value 4.4 ±0.42 µg/mL) compared to the standard anti-leishmanial drug miltefolsine (4.6 ± 2.5 µg/mL). At the same time phytoconstituents β-amyrin exist in C. chinense, C. zeylanicum and W. tinctoria exhibited IC50 15.4 ± 0.01 µg/mL which was moderately but selectively (SI > 13) active against L. donovani. Our study provides evidence that the medicinal plants in Bangladesh have high potential to improve the current treatment strategies for microbial infection.

Natural products play a significant role in anticancer drug discovery. Many currently available drugs are of natural origin. Preliminary growth inhibition (GI) investigation revealed that A. europaeum, A. subulatum, B. ramiflora, C. aeruginosa, C. caesia, C. chinense, C. odollam, C. zeylanicum, E. ribes, G. atroviridis, O. americanum and T. bracteata possessed significant growth inhibitory activity against a panel of the cancer cell line. In all the cells except MDA-231, C. odollam extract possessed GI50 ≤0.01 μg/mL which shows significance at the concentration of ≤ 0.5 μg/mL. T. bracteata ethyl acetate extract (062B) exhibited GI50 activity in the range of 1.6 - 10.84 μg/mL for all the ten cancer cells tested. Furthermore, Cerberin (CR)- a cardenolide isolated from the fruit kernel of Cerbera odollam, purified fraction 062B-F2 of T. bracteata and the semi-synthesized cardamonin-Cu (II) potently inhibited cancer cell growth at very low concentration: CR (GI50 values <90 nM); 062B-F2 (GI50 values < 0.08 μg/mL); and Cardamonin Cu (II) (GI50 values < 12 μM) in the tested cell line panel respectively. CR, 062B-F2 and Cardamonin-Cu (II) significantly inhibited cancer cell proliferation, migration and colony formation in human-derived pancreatic, triple negative breast, non-small lung carcinoma and nasopharyngeal cancer cell lines. Profound G2/M cell cycle arrest and disruption of cytoskeletal architecture were preceded time- and dose-dependent apoptosis-induction corroborated by dose-and time-dependent PARP cleavage and caspase 3/7 activation in addition to reduced Bcl-2 and Mcl-1 expression by CR, 062B-F2 and Cardamonin-Cu (II). CR, 062B-F2 and Cardamonin-Cu (II) potently inhibited PI3K/AKT/mTOR signalling and additionally depleted PLK-1, p-ERK 1/2, p-STAT3 by CR only; PTEN upregulation, inhibition of c-Myc and eIF4E expression by CR, 062B-F2 and Cardamonin-Cu (II) were also exerted. Additionally, CR, 062B-F2 and Cardamonin-Cu (II) significantly increased the generation of reactive oxygen species (ROS) producing and caused DNA double-strand breaks.

In conclusion, CR, 062B-F2 and Cardamonin-Cu (II) exerts potent, selective antitumor activity, targeting key signalling mechanisms pertinent to tumorigenesis supporting further preclinical evaluation.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Wiart, Christophe
Bradshaw, Tracey D
Collins, Hilary
Khoo, Teng Jin
Keywords: ethnopharmacological; natural product extracts; anticancer; human pathogenic bacteria; traditional medicine
Subjects: R Medicine > RS Pharmacy and materia medica
Faculties/Schools: UNMC Malaysia Campus > Faculty of Science > School of Pharmacy
Item ID: 57028
Depositing User: HOSSAN, Dr. Md Shahadat
Date Deposited: 29 Jul 2019 04:40
Last Modified: 07 May 2020 11:16

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