Glycyrrhiza glabra is an important herb throughout the World. Glycyrrhizin is the major constituent of the rhizomes of Glycyrrhiza glabra. Fars and Khorasan provinces are the production poles of licorice in Iran. The present study (with a randomized factorial design) was performed in tree replications to determine the variation in phytochemical composition and antioxidant capacity on the licorice rhizomes (less than 1 and 1–2 cm diameters) of Fars (FA) and Khorasan (KH) populations in 2016 and 2017. Glycyrrhizin, saponin, phenolic compounds, and the flavonoid content, as well as antioxidant capacity of the rhizomes, were evaluated. Moreover, the metabolite profile of these two licorice populations was detected by GC–MS analysis. The results showed that these factors were significantly influenced by environmental factors and rhizome diameter. The highest glycyrrhizin (43 mg/g) and saponin (47 g/100 g DW) contents belonged to the thicker rhizomes of Fars population. Flavonoid showed a significant negative correlation with saponin and glycyrrhizin (r2 = − 0.92 and 1− 0.87, respectively) while exhibiting a significant positive relationship with antioxidant activities (r2 = 0.77). The high antioxidant capacity (by DPPH) or IC50 values (the concentration showing 50% antioxidant activity) (0.66 mg/ml) were observed in the thinner rhizomes of the Khorasan population. Moreover, the constituents were isolated from the hydroalcoholic extract of the rhizomes of two licorice populations whose GC–MS results were different. The most abundant phytochemical compound was dihydro-p-coumaric acid in both populations. The results of PCoA, PCA-biplot, and cluster analysis revealed a low diversity between all the individuals of FA and KH populations. Phenolic compounds and flavonoid of the two populations had the largest amount of difference of traits. Thus, the different environmental conditions can dramatically influence the production of secondary metabolites and antioxidant capacity of G. glabra.

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