Agitated Vitex agnus castus L. shoot cultures were established to analyse the content of selected pharmaceutically important flavonoids and phenolic acids. Two variants (selected from nine ones) of MS medium were prepared: A (BAP 1 mg/L; NAA 0.5 mg/L; GA3 0.25 mg/L) and B (BAP 2 mg/L; NAA 0.5 mg/L). The biomass was harvested after 1, 2, 3,4, 5 and 6 weeks. Four‐week cultures (variant A) were selected to perform the precursor feeding experiment. The L‐phenylalanine dose of 1.6 g/L appears to be the most advantageous. Compared to the control cultures, the content of the individual compounds increased in a range from 1.4 to 17.3‐fold (e.g. p‐coumaric acid – 17.3 fold; casticin – 4.8‐fold). The biomass from in vitro cultures is richer in neochlorogenic acid (16‐fold), p‐coumaric acid (5.3‐fold), rutin (2.8‐fold), caffeic acid (1.5‐fold) and cinaroside (1.5‐fold) than the leaves of its parent greenhouse‐cultivated plants. Extracts contained 30 mg/100 g DW of casticin, but after the hydrolysis its amount increased up to 200 mg/100 g DW and twice exceeded the content in the greenhouse leaves. The results indicate that V. agnus castus agitated shoot cultures might be considered as a potential biotechnological source of some pharmaceutically important compounds, especially casticin, rutin, neochlorogenic and p‐coumaric acids.

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.

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The paper contains: problem definition, presentation of the measured data and the final analysis with the fuzzy regression approach. The benefits of such approach are shown in the case of small size samples. Both mentioned technological processes, the one from the automotive industry and the second from the biotechnological industry, have shown the significant benefits of using the fuzzy regression. In the case of small sample size problem, the results obtained from the fuzzy regression have significantly narrower spread in the comparison with the traditional confidence interval being very wide. The approach should also be useful for similar studies, when the probabilistic description of uncertainty is not possible for the reason of the time, the equipment or the financial limits. The developed method will be useful in eventual transition of the process from a laboratory scale to an industrial scale.