Abstract
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 μM), ethephon (Eth, 50 μM) 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, α-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.
Highlights
Harpagoside (8-O-cinnamoyl ester of harpagide–Fig 1) and harpagide are the major iridoid constituents of the secondary roots of Harpagophytum procumbens (Burch.) DC. ex Meisn.–a medicinal plant currently listed in the European Pharmacopoeia [1]
To the best of our knowledge we present for the first time the results of the quantitative UPLC-Murashige and Skoog (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
To the best of our knowledge, it is the first time that the analysis of harpagide and 8-O-acetylharpagide in M. melissophyllum shoot cultures has been reported
Summary
Harpagoside (8-O-cinnamoyl ester of harpagide–Fig 1) and harpagide are the major iridoid constituents of the secondary roots (tubers) of Harpagophytum procumbens (Burch.) DC. ex Meisn.–a medicinal plant currently listed in the European Pharmacopoeia [1]. Ex Meisn.–a medicinal plant currently listed in the European Pharmacopoeia [1]. The demand for harpagoside and harpagide containing plant medicines is high but the availability of H. procumbens roots is limited. This is the reason for searching for other species containing harpagide and its derivatives or developing biotechnological methods to obtain these compounds [3]. It is possible to scale up plant organ cultures (up to 10-20 L) because numerous types of temporary immersion systems are developed and used Numerous biotechnological strategies, such as elicitation, the precursor feeding and others [15,16,17,18,19,20], have been developed to enhance secondary metabolite accumulation in the cultures
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