6-chloro-9h-fluoren-2-amine

Harpagide inhibits neuronal apoptosis and promotes axonal regeneration after spinal cord injury in rats by activating the Wnt/beta-catenin signaling pathway.[Pubmed:30940474]

Brain Res Bull. 2019 Mar 30;148:91-99.

The neuronal apoptosis program associated with spinal cord injury (SCI) has a severe impact on spinal cord function, which leads to further secondary and permanent neuronal damage that may cause irreparable damage to the central nervous system. Activation of the Wnt/beta-catenin signaling pathway is effective in reducing apoptosis and preventing SCI. Harpagide is one of the main active constituents of the iridoid class of molecules, which have neuroprotective effects after SCI. In this study, we demonstrated that harpagide attenuated neuronal apoptosis via activation of the Wnt/beta-catenin signaling pathway. This resulted in a promotion of axonal regeneration and an inhibition of glial scar formation, which ultimately improved functional behavioral recovery after SCI in rats. Specifically, the administration of harpagide after SCI increased the expression levels of beta-catenin, c-myc and cyclin D1 proteins in spinal cord neurons, as well as increased the number of motor neurons and reduced the size of the SCI lesion area. In addition, the administration of harpagide after SCI also decreased the protein expression levels as well as the number of cells immuno-stained for the pro-apoptotic proteins Bax and cleaved-caspase 3. The expression level of the anti-apoptotic protein Bcl-2 was also increased. When the Wnt /beta-catenin signaling pathway was inhibited, a weakened anti-apoptotic effect of harpagide was observed. Additionally, the application of harpagide led to an increase in NF200 staining and a reduction in GFAP staining in the SCI injury site. In summary, our study suggested that harpagide may be a promising drug for the treatment of SCI.

Enhanced accumulation of harpagide and 8-O-acetyl-harpagide in Melittis melissophyllum L. agitated shoot cultures analyzed by UPLC-MS/MS.[Pubmed:30133513]

PLoS One. 2018 Aug 22;13(8):e0202556.

Harpagide and its derivatives have valuable medicinal properties, such as anti-inflammatory, analgesic and potential antirheumatic effects. There is the demand for searching plant species containing these iridoids or developing biotechnological methods to obtain the compounds. The present study investigated the effects of methyl jasmonate (MeJa, 50 muM), ethephon (Eth, 50 muM) and L-phenylalanine (L-Phe, 2.4 g/L of medium), added to previously selected variant of Murashige and Skoog medium (supplemented with plant growth regulators: 6-benzylaminopurine 1.0 mg/L, alpha-naphthaleneacetic acid 0.5 mg/L, gibberellic acid 0.25 mg/L) on the accumulation of harpagide and 8-O-acetyl-harpagide in Melittis melissophyllum L. agitated shoot cultures. Plant material was harvested 2 and 8 days after the supplementation. Iridoids were quantitatively analyzed by the UPLC-MS/MS method in extracts from the biomass and the culture medium. It was found that all of the variants caused an increase in the accumulation of harpagide. In the biomass harvested after 2 days, the highest harpagide content of 247.3 mg/100 g DW was found for variant F (L-Phe and Eth), and the highest 8-O-acetyl-harpagide content of 138 mg/100 g DW for variant E (L-Phe and MeJa). After 8 days, in some variants, a portion of the metabolites was released into the culture medium. Considering the total amount of the compounds (in the biomass and medium), the highest accumulation of harpagide, amounting to 619 mg/100 g DW, was found in variant F, and the highest amount of 8-O-acetyl-harpagide, of 255.4 mg/100 g DW, was found in variant H (L-Phe, MeJa, Eth) when harvested on the 8th day. These amounts were, respectively, 24.7 and 4.8 times higher than in the control culture, and were, respectively, 15 and 6.7 times higher than in the leaves of the soil-grown plant. The total amount of the two iridoids was highest for variant F (0.78% DW) and variant H (0.68% DW) when harvested on the 8th day. The results indicate that the agitated shoot cultures of M. melissophyllum can be a rich source of harpagide and 8-O-acetyl-harpagide, having a potential practical application. To the best of our knowledge we present for the first time the results of the quantitative UPLC-MS/MS analysis of harpagide and 8-O-acetyl-harpagide in M. melissophyllum shoot cultures and the enhancement of their accumulation by means of medium supplementation with elicitors and precursor.