METHODS: A total of 20 methanolic crude extracts (bark and leaf) from ten different plants were screened using a colorimetric hyaluronidase enzymatic assay. The active plant extract (Payena dasyphylla) was then studied for its hyaluronidase inhibitory activity in the interleukin-1β (IL-1β) stimulated human chondrocytes cell line (NHAC-kn) using zymography method. The Payena dasyphylla methanolic bark extract was then fractionated into several fractions in where the ethyl acetate (EA) fraction was evaluated for its inhibitory effects on the HYAL1 and HYAL2 gene expressions using reverse transcription-polymerase chain reaction (RT-PCR) technique. While the MMP-3 and MMP-13 protein expressions were evaluated using western blot method. The phenolic and flavonoid contents of the three fractions as well as the antioxidant property of the EA fraction were also evaluated.
RESULTS: Bark extract of Payena dasyphylla (100 μg/ml) showed the highest inhibitory activity against bovine testicular hyaluronidase with 91.63%. The plant extract also inhibited hyaluronidase expression in the cultured human chondrocyte cells in response to IL-1β (100 ng/ml). Similarly, treatment with Payena dasyphylla ethyl acetate (EA) fraction (100 μg/ml) inhibited the HYAL1 and HYAL2 mRNA gene expressions as well as MMP-3 and MMP-13 protein expression in a dose dependent manner. Payena dasyphylla EA fraction has demonstrated 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 as compared to water and hexane fractions. In addition, the Payena dasyphylla EA fraction showed strong antioxidant activity with IC₅₀ value of 11.64 ± 1.69 μg/mL.
CONCLUSION: These findings have shown that Payena dasyphylla might contained potential phenolic compounds that inhibiting the key enzyme in osteoarthritis development, which is the hyaluronidase enzyme through interruption of HYAL1 and HYAL1 gene expressions. The degradation of cartilage could also be inhibited by the plant through suppression of MMP-3 and MMP-13 protein expressions. We also reported that the inhibitory effect of Payena dasyphylla on hyaluronidase activity and expression might be due to its anti-oxidant property.
METHODS: The transcriptomes were sequenced on the Illumina HiSeq platform, assembled and followed by transcript clustering and annotations for gene expression and function. Pairwise or multiple sequence alignments were conducted on the toxin genes expressed. Substitution rates were studied for the major toxins co-expressed in NK-M and NK-T.
RESULTS AND DISCUSSION: The toxin transcripts showed high redundancy (41-82% of the total mRNA expression) and comprised 23 gene families expressed in NK-M and NK-T, respectively (22 gene families were co-expressed). Among the venom genes, three-finger toxins (3FTxs) predominated in the expression, with multiple sequences noted. Comparative analysis and selection study revealed that 3FTxs are genetically conserved between the geographical specimens whilst demonstrating distinct differential expression patterns, implying gene up-regulation for selected principal toxins, or alternatively, enhanced transcript degradation or lack of transcription of certain traits. One of the striking features that elucidates the inter-geographical venom variation is the up-regulation of α-neurotoxins (constitutes ∼80.0% of toxin's fragments per kilobase of exon model per million mapped reads (FPKM)), particularly the long-chain α-elapitoxin-Nk2a (48.3%) in NK-T but only 1.7% was noted in NK-M. Instead, short neurotoxin isoforms were up-regulated in NK-M (46.4%). Another distinct transcriptional pattern observed is the exclusively and abundantly expressed cytotoxin CTX-3 in NK-T. The findings suggested correlation with the geographical variation in proteome and toxicity of the venom, and support the call for optimising antivenom production and use in the region. Besides, the current study uncovered full and partial sequences of numerous toxin genes from N. kaouthia which have not been reported hitherto; these include N. kaouthia-specific l-amino acid oxidase (LAAO), snake venom serine protease (SVSP), cystatin, acetylcholinesterase (AChE), hyaluronidase (HYA), waprin, phospholipase B (PLB), aminopeptidase (AP), neprilysin, etc. Taken together, the findings further enrich the snake toxin database and provide deeper insights into the genetic diversity of cobra venom toxins.