Sayuti, Siti Nurshazwani Muhamad
(2018)
Linoleic acid and β-glucan quantification of tiger milk mushroom and its effect on BEAS-2B lungs epithelial cell line.
Masters thesis, Universiti Sains Malaysia.
Abstract
Lignosus rhinocerus, a polypore found in the tropical forests of Malaysia is
traditionally used by the locals to treat various diseases such as chronic cough,
asthma, breast cancer as well as a health tonic to strengthen immune system. The aim
of the current study is to identify potential phytochemicals and investigate their
effects on BEAS-2B bronchial epithelial lung cell line. L. rhinocerus was extracted
using soxhlet extraction, maceration and ultrasound assisted solvent extraction.
Water was used as solvent in all methods. Hexane and petroleum ether were also
used in ultrasound assisted solvent extraction. The extracts were then quantified for
the presence of linoleic acid using high performance liquid chromatography (HPLC)
and β-glucan using Megazyme Yeast and Mushroom β-glucan Assay Kit. These
extracts were then subjected to cytotoxicity assay by 3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS). Apoptosis
assay was determined using a flow cytometry while cytokine profile analysis of
interleukin-2, -4, -5, -6, -10, -12, -13, -17A, interferon gamma, tumor necrosis factor
alpha, transforming growth factor and granulocyte colony-stimulating factor was
conducted by an enzyme-linked immunosorbent assay (ELISA). In this study,
a simple HPLC method was optimized for detection of linoleic acid in L. rhinocerus
extracts. Separation was achieved on a reversed-phase C18 column (250 × 4.6 mm, 5
μm) and the detection was done by an ultraviolet detector at 208 nm. The mobile
phase used was a combination of acetonitrile, methanol and hexane in the ratio of
90:8:2 with the addition of 0.2% acetic acid at a flow rate of 1.0 ml/min. Generally,
non-polar extracts such as hexane and petroleum ether yielded a significantly higher
amount of linoleic acid as compared to water-based extracts. Analysis of β-glucan in
L. rhinocerus extracts indicated that the hexane residue yielded the highest β-glucan
level compared to the other extracts. In this study, the majority of the extracts (hot
water, hexane, hexane residue and petroleum ether residue) were not cytotoxic
against BEAS-2B cell line since there was only < 30% reduction in cell viability.
Cold water and petroleum ether extracts exhibited mild cytotoxic effect at the
concentration of 62.5-250.0 g/ml. Statistical analysis (α < 0.05) showed that the
treatment with L. rhinocerus extracts did not disturb the apoptosis activity of BEAS-
2B cells indicating that it is not harmful since dysregulation apoptosis in nontargeted
cell will lead to serious health problem such as cancer. These extracts also
demonstrated no effect on the cytokines secretion suggesting that it does not play an
important part in inflammatory response in BEAS-2B. In conclusion, L. rhinocerus is
a harmless traditional remedy. Further investigation should be done on this
mushroom which showed good potential in order to have a better understanding on
its therapeutic potential on various diseases.
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