Annotation of secondary metabolites in the tested extracts

The UPLC-QqQ-MS/MS analysis (Figure S1) of raw, roasted, honey roasted and fermented licorice roots samples revealed many metabolites belonging to distinct chemical classes. A total of 133 chromatographic peaks were designated in the different samples, with saponins, flavonoids, chalcones, pterocarpans and coumarins being the most abundant groups (Table 1; Fig. 1). A significant amount of structural data was gathered by evaluating the chromatographic behavior of the annotated compounds, as well as related fragmentation pathways already published in the literature. Table 1 displays the full list of annotated compounds and their structural data, including retention time, protonated molecules [M + H]+, deprotonated molecules [M–H]–, diagnostic MS fragmentation patterns, and molecular formulas. Numbers were allocated to the compounds depending on the order in which they were eluted.

Table 1 Metabolites annotated in the different licorice root samples extracts using UPLC-MS/MS in positive and negative ionization modes Fig. 1

Relative quantitation of the total content of different chemical classes annotated in licorice samples expressed as mg Equivalents (Eq.)/ 100 g dry weight (A). Hierarchical analysis heat maps of all annotated constituents in the tested licorice samples. Brick red and blue indicate higher and lower abundances, respectively (B)

Tracking the effect of roasting and fermentation changes on the chemical profile of licorice roots via UPLC-QqQ-MS/MS analysis in combination with multivariate statistical analysis

Semi quantitation of the annotated compounds was carried out using representative standards of the identified chemical classes; quercetin, glycrrhizic acid, esculetin, licochalcone A, ellagic acid, trans-stilbene, and 7, 12-dimethoxy coumestan. Standard calibration curves were established by plotting peak areas of the standards as the analytical responses against their known concentration. Validation parameters like linearity, limit of detection (LOD) and limit of quantification (LOQ) were assessed based on FDA guidelines on bioanalytical method validation [23] (Table S2). Standard compounds were effectively used to compute the relative quantities of the detected metabolites. Each studied extract’s measured components were reported as mg standard Equivalents/g dry extract Table S3.

As depicted in Fig. 1. Triterpene saponins overwhelmingly dominated the secondary metabolites in the aqueous extracts of fermented, roasted, honey roasted and raw licorice roots samples, with fermented samples showing the highest relative amounts of saponins. Meanwhile, the ethanol extracts of the tested samples showed significant amounts of chalcones and chalcone glycosides followed by isoflavones. Flavanones and flavanone glycosides showed significant accumulation in the ethanol extracts of honey roasted samples while melanoidins were only detected in the ethanol and aqueous extracts of roasted and honey roasted samples.

Semi-quantitative data of annotated compounds was used to create an unsupervised hierarchical heat map for the investigated samples (Fig. 1B). Licoumarin A, trihydroxy chalcone and polypodoside B were only detected in the ethanol extracts of roasted roots while glycyrrhizol B and kanzonol U were detected only in the ethanol extracts of honey roasted roots. Meanwhile the main licorice saponin glycyrrhizin was mainly detected in the aqueous extracts of raw as well as fermented roots and in lesser amounts in the aqueous extracts of roasted and honey roasted samples. Licorice saponins A and B as well as uralsaponin F were only detected in the aqueous extracts of raw root samples while yuanganosides G1 and G2, uralsaponin E, trihydroxy coumestan glycoside and dihydroxy benzoic acid were detected in the fermented root samples only. The resorcinol 2-(Methyl-butenyl)-5-(phenylethyl)-benzenediol as well as the melanoidins phenol, dimethoxy-4-(2-propenyl) and Pyran-4-one, dihydro-dihydroxy-methyl were only detected in the aqueous extracts of honey roasted samples.

MetaboAnalyst 5.0 was used to process the data from the various root samples which were subjected to unsupervised self-organizing map (SOM) analysis, a neural network-based dimensionality reduction approach. Within the samples, PC1 and PC2 explained 62.3% and 15.1% of variation, respectively. As shown in Fig. 2A, samples were divided into three primary clusters, one comprising the aqueous extracts of raw and fermented root samples, the other comprising the aqueous extract of roasted and honey roasted roots and finally a cluster containing the ethanol extracts of raw, roasted and honey roasted samples.

Fig. 2

Unsupervised self-organizing map (SOM) of the tested licorice samples (A). Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) score scatter plot (B)

The in-between and within-class discrimination of samples using an OPLS-DA (Orthogonal projection to latent structure-discriminant analysis) model based on their phytochemical profiles, as well as markers that chemically distinguish each class was attempted (Fig. 2B). The first component was responsible for 61.3% of sample variation, while the second component was responsible for 14.1%. The correlation coefficient (R2 = 0.997) and the redundancy value of cross validation (Q2 = 0.981) values were used to determine the predictability and reliability of the created OPLS-DA model, which demonstrated the model’s predictability and reliability, respectively. The ethanol extracts of the samples showed in-between class discrimination from aqueous extracts while within-class discrimination was observed between the aqueous extracts of fermented and raw and the roasted and honey roasted ones. Variable of importance (VIP) plot (Fig. 3) showed the main chemical features responsible for the discrimination of samples.

Fig. 3

Variables of importance (VIP) plot of the annotated secondary metabolites in the tested licorice samples

Determination of discriminatory metabolites between roasted, honey roasted and raw licorice roots samples

The up-accumulated and down-accumulated secondary metabolites with roasting and honey-roasting of licorice root samples were visualized using volcano and coefficient plots compared to ethanol and aqueous extracts of raw roots. Figure 4 A shows that after roasting of the licorice samples, 21 metabolites were up-accumulated (red scatter points), 28 were down-accumulated (blue scatter points), and 22 metabolites showed no change when comparing the ethanol extracts of the raw and roasted roots. Dihydroxy-dimethoxy prenylisoflavan, dihydroxy methoxy-prenylisoflavon, dihydroxy-dimethoxyflavone, 3-(4-Hydroxyphenyl)-phenyl-propenone, phenethanamine, methyl-N-vanillyl and licocoumarin A were the main metabolites which showed high accumulation with roasting of the roots samples while hydroxyisoflavone methoxy-O-glucopyranoside, licorice glucoside D1, flavestin B, dihydroxyflavanone-O-rutinoside, shinpterocarpin, vitexin-O-rhamnoside and amorfrutin were the main compounds showing down-accumulation with roasting of the samples.

Fig. 4

Volcano and coefficient plots of ethanol extracts of raw and roasted samples (A), ethanol extracts of raw and honey-roasted samples (B)

Meanwhile, honey roasted samples showed an increase in 11 compounds with the decrease in the relative concentrations of 19 compounds while 48 compounds showed no significant changes with honey roasting (Fig. 4B). Dihydroxy-diprenylflavanone, tetrahydroxy-diprenylflavanone, dihydroxy-dimethoxyflavone, dihydroxy-dimethoxy prenylisoflavan, licocoumarin A, glycyrrhizol B were the main metabolites which showed higher accumulation with honey roasting of the roots samples while trihydroxyflavanone-O-pentosyl hexoside, flavestin B, shinpterocarpin, vitexin-O-rhamnoside, hydroxyisoflavone methoxy-O-glycoside were the main compounds showing down-accumulation with honey roasting of the samples.

On the other hand, volcano, and coefficient plots of the aqueous extracts of roasted and honey roasted samples compared to raw ones (Fig. 5A and B) depicted a much significant reduction in the accumulation of secondary metabolites where 46 and 48 metabolites were significantly down-accumulated in the roots with roasting and honey-roasting, respectively. Hydroxyisoflavone methoxy-O-glucopyranoside, amorfrutin 1/A, dihydroxyflavanone-O-rutinoside, dihydroxyflavone-O-pentosyl hexoside, vitexin-O-rhamnoside, licorice saponin J2, uralsaponin C and uralsaponin F were among the main secondary metabolites that were significantly reduced with roasting of the roots while 2,3-Dihydro-3-methylfuran, 2-(Methyl-butenyl)-5-(phenylethyl)-benzenediol, and 1-(4-Hydroxyphenyl)-3-(7-methoxybenzofuran-6-yl) propanone glabraisoflavanone A showed significantly higher accumulation in the aqueous extracts of roasted roots. Meanwhile, arabino-glycyrrhizin, tetrahydroxyflavan-O-pentoside, trihydroxychalcone diglycoside, dihydroxy flavanone-O-hexoside, amorfrutin and dihydroxyflavanone-O-rutinoside showed significant down accumulation with honey roasting while 7,8-Dihydro-methylpyrrolopyrimidinone, hydroxy methoxyisoflavone and tetrahydroxy-prenylflavanone displayed significant up-accumulation in the aqueous extracts of honey roasted roots.

Fig. 5

Volcano and coefficient plots of ethanol extracts of aqueous extracts of raw and roasted sample (A), aqueous extracts of raw and honey-roasted samples (B) and aqueous extracts of raw and fermented licorice roots samples (C)

Comparing the volcano and coefficient plots of the fermented roots to the raw ones revealed (Fig. 5C) significant increase in the relative concentration of 20 compounds, significant decrease in 16 compounds, where 35 compounds showed no significant change. The oleanane-type triterpene saponins Yunganoside P, Yunganoside G1, Yunganoside G2 as well as the polyhydroxylated derivatives of flavones like trihydroxychalcone diglycoside, trihydroxyflavone-O-Rhamnopyranoside (afzelin), trihydroxyflavanone-O-hexoside, trihydroxyflavanone-O-pentosyl hexoside and trihydroxycoumestan were the main up-accumulated secondary metabolites in fermented roots samples while flavestin G, amorfrutin, dihydrolicoisoflavone A, licoagroisoflavone, glabrene, glabraisoflavanone A and glabridin showed significant down-accumulation with fermentation of licorice roots extracts.