| dc.description.abstract | Saraca asoca, commonly known as Asoka is the primary component in Asokarishta, an
Ayurvedic polyherbal preparation for treating gynecological conditions in women. Various
pharmacological and biological properties including antioxidant, antibacterial, anti-
inflammatory, anti-keratinization and others have been reported in this plant.
Kingiodendron pinnatum, known as Malabar mahogany is used as an alternative for S.
asoca in Asokarishta, has shown similar phytochemical composition and efficacy. While,
K. pinnatum has been shown to possess antioxidant and anti-inflammatory qualities, its
other biological properties remain underexplored. Therefore, the present study sought to
investigate the anticancer potential of S. asoca and K. pinnatum with a special emphasis on
breast cancer.
The crude methanol extracts of S. asoca and K. pinnatum bark demonstrated cytotoxic
effect on murine DLA and EAC tumour cells and exhibited significant antitumour
properties on mouse solid and ascites tumour models. In MTT assay, the extract shows
potent antiproliferative effect on cell lines of breast cancer expressing ERβ, including
MDA-MB-231, MDA-MB-468 and SK-BR-3. Similar results were observed on prostate,
colorectal and cervical cancer cell lines, which also express ERβ. However, despite at high
concentrations, the extracts didn’t exhibit anti-proliferative effects on the ERα expressing
cell line, MCF-7. Given the low efficacy of conventional chemotherapy in treating triple-
negative breast cancer, this particular cytotoxicity against ERβ expressing breast cell lines
is noteworthy. Phytochemical study of both plant extracts revealed the existence of several
compounds like phenols, alkaloids, saponins, flavonoids, terpenoids, phytosterols and
tannins. Numerous functional groups, including aldehydes, alkenes, amines, aromatics,
carboxylic acids, esters, phenols, and others, were identified by FT-IR analysis.
Chromatographic techniques, such as HPTLC, HPLC, and LC-MS, disclosed the existence
of phytoestrogenic compounds like β-sitosterol, quercetin, kaempferol, and others. This
raises the possibility that the phytoestrogens identified in the plants may have an agonistic
effect on cells expressing ERβ. Consequently molecular docking of phytoestrogens with
targeted estrogen receptors were done and results shows that phytoestrogens such as
quercetin and kaempferol bind to the ERβ more deeply than the ERα.
The in vitro anti-inflammatory capabilities of plant extracts was demonstrated through theinhibition of 5-lipoxygenase enzyme activity and scavenging of NO radicals in LPS-
activated RAW 264.7 cells. The extracts effectively decreased inflammation in acute and
chronic paw edemas in mice induced by carrageenan and formalin, respectively. The
antiestrogenic ability of extracts was evaluated using the estrogen screen assay revealing
proliferative effect of extracts on ERα-expressing cells and modest antiproliferative effect
on ERβ-expressing breast cancer cell lines. Furthermore, the in vivo rodent uterotropic
assay shows suppressed growth of the endometrial lining and reduced serum estrogen
levels indicating the antiestrogenic effects of extracts. The antioxidant potency of the plants
was assessed through in vitro and in vivo models. The extracts effectively inhibited or
scavenged free radicals such as ABTS, DPPH, superoxide and hydroxyl, in a
concentration-dependent manner. Additionally, the plant extracts considerably hindered
AAPH-induced lipid peroxidation and hemolysis in human erythrocytes. Significant
augmentation of the endogenous antioxidant system has been seen in the in vivo sodium
fluoride intoxication model, as demonstrated by increased levels of SOD, catalase, and
GSH, coupled with a reduction in lipid peroxidation. Both plant extracts substantially
mitigated the structural changes in the liver tissue exposed to NaF.
Considerable protective effects were shown by the extracts against DMBA induced
mammary carcinogenesis in vivo. The elevation in liver marker enzymes by DMBA was
mitigated by the extracts, and the histology of the mammary gland exhibited fewer invasive
cancer cells and reduced cell proliferation. The extracts decreased the mRNA expression of
oncogenes ER- α 1, BCL2 and c-MYC, which were elevated by DMBA except for PIN1
expression. The extract also demonstrated potent inhibition of both tumour growth and
metastasis in 4T1 cell induced breast cancer in mice. Histological analysis of the mammary
glands exhibited a marked reduction in malignant cell proliferation, thereby effectively
preventing metastasis in the group administered with the plant extract.
The possible mechanism of action of S. asoca and K. pinnatum extracts on TNBC cells was
analysed in vitro by assessing cell death patterns and the results point to apoptotic-mediated
cell death. Fluorescent staining with EB/AO reveals structural alterations with dispersed
chromatin and perforated cells, which are indications of apoptosis. In the FRET-based
caspase-3 activation assay, treatment of MDA-MB-231 cells with plant extracts activatesexecutioner caspases like caspase 3, and flow cytometry-based cell cycle analysis
demonstrates arrest in the G0/G1 phase, reflected by percentage of MDA-MB-231 cells in
the sub-G0/G1 phase, indicating apoptosis. In conclusion, morphological and biochemical
assays indicate that the cytotoxicity exerted by S. asoca and K. pinnatum extracts,
particularly against TNBCs, is through an apoptotic mechanism.
The results suggest that the activation of caspase 3 likely played a significant role in the
anticancer ability of both plant extracts on triple-negative breast cancer cells, by inducing
apoptosis. This opens a promising avenue for further research addressing TNBCs, and
additional exploration is warranted to understand the influence of phytoestrogens on
cancers expressing ERβ. Compounds capable of binding to ERβ have the potential to
prevent or treat malignancies such as TNBCs. Consequently, the development of
antiestrogenic drugs is crucial for impeding the growth of this subtype of cancer. | en_US |
