Linoleic acid and β-glucan quantification of tiger milk mushroom and its effect on BEAS-2B lungs epithelial cell line

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 phytoch...

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Main Author:	Sayuti, Siti Nurshazwani Muhamad
Format:	Thesis
Language:	English
Published:	2018
Subjects:	R Medicine
Online Access:	http://eprints.usm.my/48539/1/Dr.%20Siti%20Nurshazwani%20Muhamad%20Sayuti-OCR.pdf
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spelling	my-usm-ep.485392021-03-08T04:23:11Z Linoleic acid and β-glucan quantification of tiger milk mushroom and its effect on BEAS-2B lungs epithelial cell line 2018-06 Sayuti, Siti Nurshazwani Muhamad R Medicine 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. 2018-06 Thesis http://eprints.usm.my/48539/ http://eprints.usm.my/48539/1/Dr.%20Siti%20Nurshazwani%20Muhamad%20Sayuti-OCR.pdf application/pdf en public masters Universiti Sains Malaysia Pusat Pengajian Sains Perubatan
institution	Universiti Sains Malaysia
collection	USM Institutional Repository
language	English
topic	R Medicine
spellingShingle	R Medicine Sayuti, Siti Nurshazwani Muhamad Linoleic acid and β-glucan quantification of tiger milk mushroom and its effect on BEAS-2B lungs epithelial cell line
description	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.
format	Thesis
qualification_level	Master's degree
author	Sayuti, Siti Nurshazwani Muhamad
author_facet	Sayuti, Siti Nurshazwani Muhamad
author_sort	Sayuti, Siti Nurshazwani Muhamad
title	Linoleic acid and β-glucan quantification of tiger milk mushroom and its effect on BEAS-2B lungs epithelial cell line
title_short	Linoleic acid and β-glucan quantification of tiger milk mushroom and its effect on BEAS-2B lungs epithelial cell line
title_full	Linoleic acid and β-glucan quantification of tiger milk mushroom and its effect on BEAS-2B lungs epithelial cell line
title_fullStr	Linoleic acid and β-glucan quantification of tiger milk mushroom and its effect on BEAS-2B lungs epithelial cell line
title_full_unstemmed	Linoleic acid and β-glucan quantification of tiger milk mushroom and its effect on BEAS-2B lungs epithelial cell line
title_sort	linoleic acid and β-glucan quantification of tiger milk mushroom and its effect on beas-2b lungs epithelial cell line
granting_institution	Universiti Sains Malaysia
granting_department	Pusat Pengajian Sains Perubatan
publishDate	2018
url	http://eprints.usm.my/48539/1/Dr.%20Siti%20Nurshazwani%20Muhamad%20Sayuti-OCR.pdf
_version_	1747821942566551552

Linoleic acid and β-glucan quantification of tiger milk mushroom and its effect on BEAS-2B lungs epithelial cell line

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