Effects of Payena dasyphylla (miq.) on hyaluronidase activity and metalloproteinases expression in interleukin-1B stimulated human chondrocytes cells

Hyaluronidase is one of the target enzymes in the development of osteoarthritis (OA) disease that results from the disruption of the extracellular matrix structure in the cartilage. Although various treatments for OA have been identified, they suffer from numerous drawbacks. The drawbacks are signif...

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Main Author:	Citalingam, Kamini
Format:	Thesis
Language:	English
Published:	2012
Subjects:	Hyaluronic acid Metalloproteinases
Online Access:	http://psasir.upm.edu.my/id/eprint/48333/1/FBSB%202012%2055R.pdf
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id	my-upm-ir.48333
record_format	uketd_dc
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Hyaluronic acid Metalloproteinases
spellingShingle	Hyaluronic acid Metalloproteinases Citalingam, Kamini Effects of Payena dasyphylla (miq.) on hyaluronidase activity and metalloproteinases expression in interleukin-1B stimulated human chondrocytes cells
description	Hyaluronidase is one of the target enzymes in the development of osteoarthritis (OA) disease that results from the disruption of the extracellular matrix structure in the cartilage. Although various treatments for OA have been identified, they suffer from numerous drawbacks. The drawbacks are significant as OA patients require the treatment for their entire lifetime. While there is still no curative treatment for this disease, recent studies for the prevention or treatment on OA were focused on the efficacy of natural products which are capable of ameliorating the symptoms with limited side effects. Therefore, this research aimed to study the effect of Payena dasyphylla, a selected Malaysian plants on the hyaluronidases activity, and certain matrix metalloproteinases (MMPs) expression. Prior to this, a total of 20 methanolic crude extracts (bark and leaf) were screened using a colorimetric anti-hyaluronidase enzymatic assay. Out of the plants tested, bark extracts of Palaquium gutta, Pauteria obovatta and Payena dasyphylla (100 μg/ml) showed the highest inhibitory activity against bovine testicular hyaluronidase with 88.82 ± 0.15, 90.47 ± 0.09 and 91.63 ± 0.21 percent respectively. Due to insufficient amount of samples, only Payena dasyphylla with an IC50 value of 48.75 ± 8.97 μg/ml was further studied for its effect on hyaluronidase activity in interleukin-1β (100 ng/ml) stimulated human chondrocytes cells (NHAC-kn) using the zymography method. Payena dasyphylla was then fractionated into aqueous (Aq), ethyl acetate (EA) and hexane fractions where the EA fraction which showed the highest inhibitory activity against hyaluronidase was selected for further evaluation on its capability inhibiting the gene expressions of HYAL1 and HYAL2 using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technique. Besides evaluating the effect of Payena dasyphylla on the hyaluronidases, this plant extract was also tested on the expression of other mediators mainly on MMP-3 and MMP-13 using Western blotting method. Payena dasyphylla was then further investigated for its total phenolic and flavonoid content as well as antioxidant activity. The antioxidant activity was measured using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay whereas total phenolic content was determined using the Folin-ciocalteu’s method. Results showed that Payena dasyphylla methanolic extract (100 μg/ml) inhibited hyaluronidase activity in the cultured human chondrocyte cells in response to IL-1β (100 ng/ml) stimulation. Similarly, treatment with Payena dasyphylla EA fraction (100 μg/ml) also inhibited the HYAL1 and HYAL2 gene expressions. Cytotoxicity results revealed that both Payena dasyphylla methanolic and EA fraction did not show any concomitant effect on the NHAC-kn cell line at all concentrations tested. On the other hand, the Payena dasyphylla EA fraction also inhibited MMP-3 and MMP-13 protein expression. Interestingly, treatment without IL-1β (10 ng/ml) stimulation revealed a significant difference as MMP-3 and MMP-13 protein expressions were seen only with the presence of IL-1β inducer. In addition, Payena dasyphylla EA fraction showed the highest amount of phenolic and flavonoid content with 168.62 ± 10.93 mg GAE/g and 95.96 ± 2.96 mg RE/g respectively compared to aqueous and hexane fractions. Besides, Payena dasyphylla EA extract exhibited antioxidant activity of 82.19% at the concentration of 100 μg/ml with IC50 value of 14.10 ± 1.11 μg/ml. As a conclusion, these findings showed that Payena dasyphylla contained potential classes of compound inhibiting hyaluronidases, HYAL1 and HYAL2 gene expressions as well as MMP-3 and MMP-13 protein expressions that are responsible for the degeneration of the cartilage tissue. Therefore, this plant might serve as an alternative in formulating new generation of drugs in the management of cartilage damage.
format	Thesis
qualification_level	Master's degree
author	Citalingam, Kamini
author_facet	Citalingam, Kamini
author_sort	Citalingam, Kamini
title	Effects of Payena dasyphylla (miq.) on hyaluronidase activity and metalloproteinases expression in interleukin-1B stimulated human chondrocytes cells
title_short	Effects of Payena dasyphylla (miq.) on hyaluronidase activity and metalloproteinases expression in interleukin-1B stimulated human chondrocytes cells
title_full	Effects of Payena dasyphylla (miq.) on hyaluronidase activity and metalloproteinases expression in interleukin-1B stimulated human chondrocytes cells
title_fullStr	Effects of Payena dasyphylla (miq.) on hyaluronidase activity and metalloproteinases expression in interleukin-1B stimulated human chondrocytes cells
title_full_unstemmed	Effects of Payena dasyphylla (miq.) on hyaluronidase activity and metalloproteinases expression in interleukin-1B stimulated human chondrocytes cells
title_sort	effects of payena dasyphylla (miq.) on hyaluronidase activity and metalloproteinases expression in interleukin-1b stimulated human chondrocytes cells
granting_institution	Universiti Putra Malaysia
publishDate	2012
url	http://psasir.upm.edu.my/id/eprint/48333/1/FBSB%202012%2055R.pdf
_version_	1747811972803461120
spelling	my-upm-ir.483332017-03-28T07:39:39Z Effects of Payena dasyphylla (miq.) on hyaluronidase activity and metalloproteinases expression in interleukin-1B stimulated human chondrocytes cells 2012-08 Citalingam, Kamini Hyaluronidase is one of the target enzymes in the development of osteoarthritis (OA) disease that results from the disruption of the extracellular matrix structure in the cartilage. Although various treatments for OA have been identified, they suffer from numerous drawbacks. The drawbacks are significant as OA patients require the treatment for their entire lifetime. While there is still no curative treatment for this disease, recent studies for the prevention or treatment on OA were focused on the efficacy of natural products which are capable of ameliorating the symptoms with limited side effects. Therefore, this research aimed to study the effect of Payena dasyphylla, a selected Malaysian plants on the hyaluronidases activity, and certain matrix metalloproteinases (MMPs) expression. Prior to this, a total of 20 methanolic crude extracts (bark and leaf) were screened using a colorimetric anti-hyaluronidase enzymatic assay. Out of the plants tested, bark extracts of Palaquium gutta, Pauteria obovatta and Payena dasyphylla (100 μg/ml) showed the highest inhibitory activity against bovine testicular hyaluronidase with 88.82 ± 0.15, 90.47 ± 0.09 and 91.63 ± 0.21 percent respectively. Due to insufficient amount of samples, only Payena dasyphylla with an IC50 value of 48.75 ± 8.97 μg/ml was further studied for its effect on hyaluronidase activity in interleukin-1β (100 ng/ml) stimulated human chondrocytes cells (NHAC-kn) using the zymography method. Payena dasyphylla was then fractionated into aqueous (Aq), ethyl acetate (EA) and hexane fractions where the EA fraction which showed the highest inhibitory activity against hyaluronidase was selected for further evaluation on its capability inhibiting the gene expressions of HYAL1 and HYAL2 using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technique. Besides evaluating the effect of Payena dasyphylla on the hyaluronidases, this plant extract was also tested on the expression of other mediators mainly on MMP-3 and MMP-13 using Western blotting method. Payena dasyphylla was then further investigated for its total phenolic and flavonoid content as well as antioxidant activity. The antioxidant activity was measured using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay whereas total phenolic content was determined using the Folin-ciocalteu’s method. Results showed that Payena dasyphylla methanolic extract (100 μg/ml) inhibited hyaluronidase activity in the cultured human chondrocyte cells in response to IL-1β (100 ng/ml) stimulation. Similarly, treatment with Payena dasyphylla EA fraction (100 μg/ml) also inhibited the HYAL1 and HYAL2 gene expressions. Cytotoxicity results revealed that both Payena dasyphylla methanolic and EA fraction did not show any concomitant effect on the NHAC-kn cell line at all concentrations tested. On the other hand, the Payena dasyphylla EA fraction also inhibited MMP-3 and MMP-13 protein expression. Interestingly, treatment without IL-1β (10 ng/ml) stimulation revealed a significant difference as MMP-3 and MMP-13 protein expressions were seen only with the presence of IL-1β inducer. In addition, Payena dasyphylla EA fraction showed the highest amount of phenolic and flavonoid content with 168.62 ± 10.93 mg GAE/g and 95.96 ± 2.96 mg RE/g respectively compared to aqueous and hexane fractions. Besides, Payena dasyphylla EA extract exhibited antioxidant activity of 82.19% at the concentration of 100 μg/ml with IC50 value of 14.10 ± 1.11 μg/ml. As a conclusion, these findings showed that Payena dasyphylla contained potential classes of compound inhibiting hyaluronidases, HYAL1 and HYAL2 gene expressions as well as MMP-3 and MMP-13 protein expressions that are responsible for the degeneration of the cartilage tissue. Therefore, this plant might serve as an alternative in formulating new generation of drugs in the management of cartilage damage. Hyaluronic acid Metalloproteinases 2012-08 Thesis http://psasir.upm.edu.my/id/eprint/48333/ http://psasir.upm.edu.my/id/eprint/48333/1/FBSB%202012%2055R.pdf application/pdf en public masters Universiti Putra Malaysia Hyaluronic acid Metalloproteinases

Effects of Payena dasyphylla (miq.) on hyaluronidase activity and metalloproteinases expression in interleukin-1B stimulated human chondrocytes cells

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