Tyrosine-feeding in the cell suspensions cultures of Randia echinocarpa to increase the production of bioactive phenolic compounds

The fruit of Randia echinocarpa, an endemic plant to Northwest Mexico, is commonly used in the Mexican traditional medicine to treat different diseases and ailments. Different biological activities such as antioxidant, antimutagenic, immunomodulatory, and antidiabetic had been previously demonstrated. However, wild populations of R. echinocarpa have diminished due to high deforestation rates for agriculture expansion. In this regard, in vitro plant tissue culture (PTC), especially cell suspension cultures (CSC), could be an attractive and viable alternative for the sustainable conservation and production of bioactive compounds from this plant. However, some limitations, mainly related with low yields of metabolite production and prolonged fermentation times, result in difficult and expensive experimental procedures. Thus, the application of suitable kinetic models is necessary for optimal process control and simulation of plant cell cultures to assure the economic feasibility of these processes. The addition of precursors, like tyrosine (Tyr), is an effective strategy to increase the biomass and specialized metabolite production in plant cell suspensions. These approaches could be efficient to produce natural molecules with important biological effects. The identification of these compounds can be addressed by metabolomic analyses which will allow to understand the metabolic pathways involved in their synthesis. For all mentioned above, the objective of this work was to evaluate the effect of exogenous application of Tyr (0, 50, 100, and 200 mg/L) on the metabolic profile of R. echinocarpa suspension cells, as well as the inhibitory activity of the cell extracts on the enzymatic activity of α-amylase and α-glucosidase. For this, the growth kinetics of the CSC of R. echinocarpa was first estimated using a Logarithmic growth model. CSC of R. echinocarpa reached a dry cell biomass concentration of 15.16 g/L at day 20 of culture. The maximum specific growth rate (𝜇𝑚𝑎𝑥) was 0.15 d-1, with a duplication time (𝑡𝑑) of 4.62 d. The Logistic model adequately predicted the cell growth changes during the culture and the maximum dry cell content that the culture medium could sustain (≈ 13.63 g/L). Ten phenolic compounds were identified in the biomass and four in the supernatants. The major phenolic compound in the biomass was chlorogenic acid (CA), with a concentration of 828.6 μg/g at day 20. In the lyophilized supernatant, the major phenolic compound was salicylic acid (SA) with a concentration of 172.7 μg/g at day 30. The production of CA was a growth-dependent process in contrast to the concentration of SA in the media. Once the kinetic parameters of the CSC were determined, and a preliminary identification of phenolic compounds was obtained, the analysis on the metabolomic profile due to Tyr treatments and the enzyme inhibitory activities of the cell extracts was assessed. Methanolic extracts (1 mg/mL) of R. echinocarpa cell suspensions inhibited the activity of α-amylase similarly to acarbose at 50 μM. Nevertheless, no inhibition of α-glucosidase by the extracts was observed. Further purification of the methanolic extracts is required to prevent antagonist effects of the compounds. Four specific chemical profiles were determined by Hierarchical Cluster and Principal Components Analysis. Galactose metabolism and starch/sucrose metabolism were among the main modulated metabolic pathways. Molecular docking showed that compounds from Tyr_100 and 200 treatments had an estimated free binding energy of -2.4 to -5.6 kcal/mol and can interact with key amino acids involved with the catalytic activity of α-amylase. The results herein obtained demonstrated that CSC of R. echinocarpa were able to produce and accumulate compounds with biological effects. Moreover, the addition of Tyr to the cell suspensions of R. echinocarpa can be used to produce α-amylase inhibitory extracts. The implementation of kinetic models would also allow the scale-up and prediction of the growth of R. echinocarpa cell cultures, parameters necessary for the control of the production process of bioactive compounds.

https://orcid.org/0000-0003-1056-7126