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

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.

Fig 1. Chemical structures of harpagide and two related iridoid glycosides.

Harpagide (R = H), 8-O-acetyl-harpagide (R = Ac) and harpagoside (R = cinnamoyl).

Fig 2. Harpagide content in M. melissophyllum shoot cultures.

Biomass collected after 1, 2, 3, 4, 5 and 6 weeks cultivated on agar MS media. Variants previously selected (Table 7) as the best growth media: MS 2/1 (BAP– 2 mg/L, NAA– 1 mg/L), MS 1/0.5/0.25 (BAP– 1 mg/L, NAA– 0.5 mg/L, GA₃−0.25 mg/L), MS 2/1/0.5 (BAP– 2 mg/L, NAA– 1 mg/L, GA₃−0.5 mg/L).

Fig 3. 8-O-acetyl-harpagide content in M. melissophyllum shoot cultures.

Biomass collected after 1, 2, 3, 4, 5 and 6 weeks cultivated on agar MS media. Variants previously selected (Table 7) as the best growth media: MS 2/1 (BAP– 2 mg/L, NAA– 1 mg/L), MS 1/0.5/0.25 (BAP– 1 mg/L, NAA– 0.5 mg/L, GA₃−0.25 mg/L), MS 2/1/0.5 (BAP– 2 mg/L, NAA– 1 mg/L, GA₃−0.5 mg/L).

Fig 4. Dry weight of the biomass.

Material collected after 1, 2, 3, 4, 5 and 6 weeks from M. melissophyllum shoot cultures cultivated on agar and liquid MS medium supplemented with BAP– 1 mg/L, NAA– 0.5 mg/L and GA₃−0.25 mg/L.

Fig 5. Iridoid productivity in M. melissophyllum agar and submerged shoot cultures.

Biomass collected after 1, 2, 3, 4, 5 and 6 weeks (MS medium supplemented with BAP– 1 mg/L, NAA– 0.5 mg/L, GA₃−0.25 mg/L).

Fig 6. Dry biomass collected from Melittis melissophyllum agitated shoot cultures.

Different variants of supplementation A-H (see Table 2); harvesting time: I– 2^nd day and II– 8^th day after supplementation.

Fig 7. Harpagide content in Melittis melissophyllum agitated shoot cultures and ground plant.

Shoot cultures with different variants of supplementation A-H (see Table 2) vs. a ground plant (P). Other symbols: M–medium; T–tissue = biomass of shoots; harvesting time: I– 2^nd day and II– 8^th day after supplementation.

Fig 8. 8-O-acetyl-harpagide content in Melittis melissophyllum agitated shoot cultures and ground plant.

Shoot cultures with different variants of supplementation A-H (see Table 2) vs. a ground plant (P). Other symbols: M–medium; T–tissue = biomass of shoots; harvesting time: I– 2^nd day and II– 8^th day after supplementation.

Fig 9

Total content of iridoids in Melittis melissophyllum agitated shoot cultures and ground plant (a), and iridoids productivity (b). Shoot cultures with different variants of supplementation A-H (see Table 2) vs. a ground plant (P). Other symbols: M–medium; T–tissue = biomass of shoots; harvesting time: I– 2^nd day and II– 8^th day after supplementation. Productivity expressed as mg of harpagide and 8-O-acetyl-harpagide (sum of iridoids) per liter of a medium per day of the culture.

Fig 10. Pareto plot of standardized effects for the dry weight outcome.

Four factors (HTime, L-Phe, MeJa, Eth) and their interaction up to the 4^th order were considered. Significance level α = 0.05, t = 2.042.

Fig 11. The Pareto plot of standardized effects for the reduced model of DW outcome.

Two significant factors (HTime, L-Phe) and their interaction were considered. Significance level α = 0.05, t = 2.018.

Fig 12. The probability plot of residuals for the reduced model of dry weight outcome.

95% confidence band 95%, Anderson-Darling test of normality p = 0.534.

Fig 13. The predicted dry weight for four treatments of significant factors HTime and L-Phe.

HTime codes: -1 = 2^nd day, +1 = 8^th day; L-Phe codes: -1 = lack of L-Phe, +1 = 2.4 g/L of L-Phe.

Fig 14. The Box-Cox transformation plot of harpagide outcome.

The selected best value of λ parameter was 0 i.e. logarithmic transformation was selected.

Fig 15. Pareto plot of standardized effects for the transformed harpagide outcome.

Four factors (HTime, L-Phe, MeJa, Eth) and their interaction up to the 4^th order were considered. Significance level α = 0.05, t = 2.042.

Fig 16. The probability plot of residuals for the reduced model of the transformed harpagide outcome.

95% confidence band, Anderson-Darling test of normality p = 0.315.

Fig 17. The Box-Cox transformation plot of 8-O-acetyl-harpagide outcome.

The selected best values of λ parameter is -0.5 i.e. reciprocal square root transformation is selected.

Fig 18. Pareto plot of standardized effects for the transformed 8-O-acetyl-harpagide outcome.

Four factors (HTime, L-Phe, MeJa, Eth) and their interaction up to 4^th order were considered. Significance level α = 0.05, t = 2.042.

Fig 19. The probability plot of residuals for the reduced model of the transformed 8-O-acetyl-harpagide outcome.

95% confidence band, Anderson-Darling test of normality p = 0.375.

Fig 20. Box-Cox transformation plot of the total of harpagide and 8-O-acetyl-harpagide outcome.

The The selected best values of λ parameter was -0.5 i.e. reciprocal of square root transformation was selected.

Fig 21. Pareto plot of standardized effects for the transformed total of harpagide and 8-O-acetyl-harpagide outcome.

Four factors (HTime, L-Phe, MeJa, Eth) and their interaction up to the 4^th order were considered. Significance level α = 0.05, t = 2.042.

Fig 22. The probability plot of residuals for the reduced model of the transformed total of iridoids outcome.

95% confidence band, Anderson-Darling test of normality p = 0.252.

Fig 23. The Box-Cox transformation plot of the total productivity of the process (iridoids outcome per flask).

The selected best values of λ parameter was -0.5 i.e. reciprocal of square root transformation was selected.

Fig 24. Pareto plot of standardized effects for the transformed total productivity of the process (iridoids outcome per flask).

Four factors (HTime, L-Phe, MeJa, Eth) and their interaction up to the 4^th order were considered. Significance level α = 0.05, t = 2.042.

Fig 25. The probability plot of residuals for the reduced model of the transformed total productivity of the process.

Productivity defined as iridoids outcome per flask. 95% confidence band, Anderson-Darling test of normality p = 0.770.