ANTINEOPLASTIC ACTIVITY OF BIOSYNTHESISED BAICALEIN AND PIPERLONGUMINE NANOPARTICLES
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Date
2021-07-29
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR
Abstract
The research was undertaken with the objective of studying the
antineoplastic activity of biosynthesised baicalein and piperlongumine
nanoparticles in Daltons Lymphoma Ascitis (DLA) cells. The study was carried
out in three phases. In Phase 1, silver nanoparticles (AgNPs) synthesised using
baicalein and piperlongumine were characterised by UV-Vis spectroscopy, X Ray Diffraction (XRD) and Field emission Scanning Electron Microscopy
(FESEM). The characterisation revealed that the particles synthesised were
within the range of nanoscale (1-100 nm) and were of crystalline nature and had
spherical shape. In Phase II, both the pure compounds and nanoparticles were
screened for in vitro cytotoxic activity in DLA cell lines-by MTT assay. Based
on the per cent inhibition assayed using MTT, IC50 value for baicalein (BCLN)
was 59.41 µg/ mL whereas for AgNPs synthesised from BCLN (B-AgNPs), the
value reduced to 50.22 µg/ mL. Similarly, the IC50 value for piperlongumine
(PPLM) was 4.028 µg/ mL while the value reduced to 1.25 µg/ mL for AgNPs
synthesised from PPLM (P-AgNPs). But reference drug, 5-FU and chemically
synthesised C-AgNPs (C-AgNPs) imparted minimal cytotoxicity, with IC50
values calculated as 326.2 and 743.6 µg/ mL respectively. Cell viability assessed
after incubation with IC50 of test compounds for three hours using trypan blue
exclusion assay revealed that all test substances showed an average of 49.07 per
cent cell viability in three hours.
Acridine orange/Ethidium bromide (AO/EB) staining was done to
assess the apoptotic changes in cells exposed to IC50 of test compounds for 24 h.
The cells collected after exposure to IC50 of test compounds for 24 h were
subjected to DCF DA assay for intracellular ROS generation, DNA
fragmentation assay and JC-1 staining to analyse mitochondrial transmembrane
potential (MMP). The relative expression of Bcl-2, Caspase-3 and p53 was also
assayed in the cells exposed to treatments keeping GAPDH as reference gene. In
vitro studies revealed that among the treatments, PPLM and P-AgNPs showed
significant apoptotic changes, ROS generation, DNA fragmentation and MMP changes. BCLN and B-AgNPs also showed in vitro antineoplastic activity. In
both the cases, biosynthesised nanoparticles showed remarkable in vitro
antineoplastic potential than pure compounds.
In Phase II, acute oral toxicity test of biosynthesised nanoparticles
as per OECD guidelines-420 was performed and in vivo antineoplastic properties
of nanoparticles was evaluated in DLA induced solid tumour in mice. No toxicity
symptoms or loss in body weight were observed in animals that undergone acute
toxicity test. Solid tumour was induced by injecting viable DLA cells (1 × 106
cells/mouse) subcutaneously into the right hind limb of Swiss albino mice. Fifty
four tumour positive mice were selected and randomly allocated into nine groups
with six animals in each group. Normal control (Group I) also comprised of six
animals. Group II served as tumour control. Group III was administered with 5-
FU at 20 mg/kg. Group IV and V received B-AgNPs at 50 mg/kg and 100 mg/kg
respectively. Piperlongumine nanoparticle (P-AgNPs) was given at 50 mg/kg
and 100 mg/kg to Group VI and VII respectively. Group VIII and IX received
pure compounds, BCLN and PPLM respectively at 100 mg/kg. Chemically
synthesised nanoparticles (C-AgNPs) at 100 mg/kg was administered to Group
X. All the treatments were given orally for 10 days.
Progression of the tumour was assessed by measuring the tumour
volume once in four days. Animals were sacrificed and tumour masses were
collected on day 11 to assess ratio of tumour weight to body weight, tumour
volume, to estimate levels of lipid peroxidation and reduced glutathione.
Standard staining using Haematoxylin and Eosin, special staining using AO/EB,
relative gene expression studies of Bc-l2, Caspase-3 and p53 using Real time
PCR were also carried out in tumour masses.
The outcome of the tumour growth response studies suggested that
biosynthesised nanoparticles (P-AgNPs and B-AgNPs) exerted a pronounced
inhibitory effect on the tumour growth than that produced by the respective pure
compounds, PPLM and BCLN. All treatment groups showed an increase in lipid
peroxidation and decrease in reduced glutathione levels. There was a down
regulation of Bcl-2 and upregulation of Caspase-3 and p53 in treated groups
compared to tumour control group. As revealed in in vitro study, PPLM and P AgNPs showed more significant in vivo antineoplastic property than other
treatments.
From the study, it can be concluded that biosynthesised
nanoparticles produced more antineoplastic activity than the respective pure
compounds from which they were synthesised and in the current study, P-AgNPs
at 100 mg/kg was showing the most potent antineoplastic action against DLA
cells.
Description
Submitted in partial fulfilment of the requirement for the degree of
Doctor of Philosophy in Veterinary Pharmacology and Toxicology