OrthogonalAnalysis Underscores the Relevance of Primaryand Secondary Metabolites in Licorice

Large-ScaleCPC Fractionationand Sample Recovery

Ingeneral, the use of pH-zone refining in HSCCC and CPC is attractivein terms of high loading capacity and the ability to isolate largequantities (∼100 mg) of target metabolites.¹⁸ A large-scale CPC method that allowed targeted isolationof substantial amounts of the 2°Ms, 1 and 2, while simultaneously concentrating the polar 1°Msinto a separate fraction was developed. Ideally, such a one-step CPCmethod would accommodate crude licorice extract and have a relativelyhigh loading capacity (>10 g), greater than that previously achievedby pH-zone refining CPC (2 g per injection, V_tot = 300 mL).¹⁸ Isolation of 1 and 2 has been achieved with EtOAc and H₂O,^18,19 both of which were used as startingpoints for the optimization of the two-phase solvent system (SS) conditions.However, considering the known pH-dependent interconversion of chalconesand flavanones,²⁷ pH-zone refining wasruled out, as it involves a pH gradient, which would alter the characteristicchalcone–flavanone pattern in G. uralensis. Therefore, the SS used in the present study was free of any acidor base to minimize pH-induced chemical alteration.

The stationaryphase retention volume ratio, S_f, canbe predicted based on the settling time (separation of the two phases)²⁸ and is important when optimizing conditionsfor larger loading. Two intermediate solvents, MeOH and MeCN, wereexamined (Table 1). The settling times of theEtOAc/MeOH/H₂O SS were extended with the addition of G. uralensis crude extract (from 20 to more than 60 s).When MeCN was used as an intermediate solvent, the settling timeswere also increased in the presence of the crude extract, but to alesser extent. On the basis of an optimized settling time, and takinginto account the K value distribution of the screenedmetabolites (Table 1), the SS composed of EtOAc/MeCN/H₂O (5:6:9, v/v) was chosen for the fractionation of G. uralensis crude extract. The calculated selectivity factorfor 1 and 2, α = K₂/K₁ (where K₂ > K₁), was >2, indicatingan achievable baseline separation. Even though the separation efficiencyof the target analytes was predicted, impurity profiling was stillnecessary to assess potential overlap and/or coelution with otherconstituents exhibiting similar K values.

The CPCwas performed in reversed phase mode (15 g of crude extract,aqueous [LP] mobile, S_f=71.8%) to achieve rapid elution of the polar 1°Ms. Phase I ofthe elution-extrusion (EECCC)¹⁷ mode wascompleted after 129 min, immediately followed by sweep elution andextrusion (phases II and III, respectively), which completed the stationaryphase recovery after 2 h and 40 min (Figure 1A). A total of 171 fractions were pooled according to their UV andTLC profiles, yielding 13 final fractions. The weight recovery was96.5%, confirming that CS can essentially recover the entire injectedsample, even when working with relatively large loadings (Figure 1B).

Figure 1

CPC chromatograms and weight distribution of collectedfractions.(A) Large-scale CPC chromatograms recorded at 330 and 360 nm as afunction of elution time in hours. The fractionation of G.uralensis crude extract (15 g) was performed in less than3 h using the elution-extrusion (EECCC) mode, leading to a recoveryof 96.5% of the injected material. The elution of the major flavanones,liquiritin (1) and liquiritin apioside (2), is visualized at 330 nm. (B) The weight distribution of each pooledCPC fraction highglights that fraction 1 accounts for 85.2% w/w ofthe crude extract.

The dried fractions wereanalyzed using orthogonal methods (UHPLC-UVand qHNMR) to comprehensively evaluate their composition (FiguresS2 and S3, Supporting Information). Theweight distribution varied throughout the CPC run, with the fractionscontaining the two target compounds, 1 and 2, representing only 0.62% and 3.40% w/w of the extract load, respectively.In contrast, the early eluting polar fraction containing the majorityof the polar 1°Ms accounted for 85.2% w/w of the extract.

PurityDetermination of 1 and 2

In orderto evaluate the efficiency of the large-scale CPC method,the purity and RC of the isolated compounds were determined by orthogonalanalysis using UHPLC-UV/LC-MS-MS and qHNMR. Both target flavanoneswere shown to be present in fractions 4 to 6 as follows: 2 was identified in fractions 4 (vials 13 and 15) and 5 (vials 16to 28); 1 was predominant in fraction 6 (vials 29–53),from which (vials 35–42) compound 1 crystallizedspontaneously to give fraction 6a. The MS and ¹H NMR profilesof both isolates were in agreement with published data.^7,29,30 Together with NMR, LC-MS-MS contributedto the structural identification of both the target metabolites andcoeluted impurities. Purity determination was performed with UHPLC-UVand qHNMR, using the 100% method in both instances (Table 2, Figure 2).^31,32 It should be noted that, in general, UHPLC-UV overestimates puritywhenever UV-transparent metabolites such as sugars are present. Incontrast, ¹H NMR offers nearly universal detection of protonsand bears the specific advantage of simultaneous access to both qualitativeand quantitative information. The 100% method reflects the relativeconcentration of a targeted compound compared to its coeluted impurities.

Figure 2

Puritydetermination of liquiritin (1) and liquiritinapioside (2) by qHNMR. (A, B) The analysis of the qHNMRspectra of liquiritin (1) and liquiritin apioside (2) enabled the structural identification of major impurities: p-hydroxybenzylmalonic acid (9, blue), sucrose(10, gray), lactic acid (La), and isoliquiritinapioside (4, green). Together, these impurities constitutethe RC of the isolated flavanone materials. (C) Using the 100% qHNMRmethod, the relative concentration (% w/w) of 1 and 2 in each fraction was determined. Such results indicate thepurity level and associated RC of each isolate. In fraction 6, liquiritigenin-7-O-glucoside (orange box) represented 2.9%, whereas isoliquiritinapioside (4) and licuraside (8, green) werethe most predominant impurities.

One impurity (9) was detected by comparingthe qHNMRspectra of fractions 4–6 (Figure 2Aand B). The spectrum of fraction 4 provided evidence of the presenceof a phenolic ring exhibiting a characteristic AA′/XX′pattern centered at 6.665 and 6.890 ppm (2H each, J = 8.29 Hz) and two up-field signals, a doublet at 3.010 ppm (2H, J = 5.10 Hz) and a triplet at 2.895 ppm (1H, J = 5.10 Hz). LC-MS analysis using negative ion electrospray revealeda deprotonated molecule [M – H]⁻ of m/z 209.0459, suggesting a molecular formulaof C₁₀H₁₀O₅ (Figure 3B). The fragmentation pattern of 9 was similar to that of benzylmalonic acid (reference no.100773, Sigma-Aldrich). In addition, both compounds produced extensiveloss of CO₂ during in-source fragmentation, further suggestinga structural similarity. Collectively, these results indicated thatthe common impurity, 9, should bear a p-hydroxyphenolic ring and two carboxyl functions. These observationswere compatible with 9 being p-hydroxybenzylmalonicacid (CAS no. 90844-16-9). It is noteworthy that due to extensivein-source decarboxylation, 9 could have been easily misidentifiedas phloretic acid. This possibility could only be ruled out by meticulouscomparison of the ¹H NMR profiles of fractions 4–6,as well as careful consideration of the MS spectra of benzylmalonicacid (Figures S5–S7, Supporting Information). Malonic acid derivatives are known to spontaneously decarboxylateat temperatures near 170 °C.³³ Assuch, the phenomenon of in-source decarboxylation of dicarboxylicacids has to be carefully considered during MS analysis.

Figure 3

Structuralidentification of p-hydroxybenzylmalonicacid in fraction 4 and comparative ¹H NMRprofiles of fractions 2 and 7. (A, C) By evaluating the differencebetween the HSQC (A) and HMBC (C) spectra of pure 2 (black,and 2D correlations in gray) and fraction 4, all the 2D NMR signalsof p-hydroxybenzylmalonic acid (9, inred) could be assigned in fraction 4. (B) The MS-MS spectrum of 9 indicated the presence of two carboxylic acid functionsand a molecular formula of C₁₀H₁₀O_5. (D) NMR analyses of neighboring fractions revealed the presenceof 9 in fractions 2 and 7 (SupportingInformation S8 and S9).

The second major impurities detected by qHNMR profiling werethe1°Ms, sucrose (10) (anomeric proton δ_H 5.174, d, J = 3.74 Hz) in fractions 4 to6 and lactic acid (methyl protons δ_H 1.019, d, J = 6.08 Hz) in fraction 4. Other detected impurities infractions 5 and 6 were mostly chalcone and flavanone isomers (e.g.,isoliquiritin apioside (4) and licuraside (8), Table 2), which had partition coefficientssimilar to those of compounds 1 and 2.

The accuracy of the purity determination of target isolates, 1 and 2, depends on the identity and the molecularweight of the impurities. In CS, these impurities result from possiblestationary phase loss, coelution (similar K values)with the target analyte(s), and the comparatively wide Gaussian peak(s)of highly abundant constituents eluting earlier (“tailing effect”).The relative concentration (% w/w) of 9 vs 10 assessed by qHNMR was determined to be 15.9, 9.2, and 5.0 vs 12.1,7.6, and 6.1% in fractions 4, 5, and 6, respectively (Table 2, Figure 2).

The single-stepCPC produced 90 mg of purified 1 (95.8%by qHNMR) through spontaneous crystallization occurring in fraction6 (K = 0.72), a phenomenon frequently observed inCS due to the physicochemical nature of multicomponent SSs. The motherliquor of fraction 6 yielded an additional 408 mg of 1 with 70.2% qHNMR purity (Table 2, Figure 2), compared to 89.9% by UHPLC-UV. The purity levelof target compound 2 was determined by qHNMR to be only65.0% and 80.3% in fractions 4 and 5, respectively, due to the coelutionof 1°Ms. These observations led to the conclusion that polar1°Ms were carried over with the aqueous mobile phase during thefirst 820 mL of elution (up to K = 0.72). In suchcases, a second CS step, even with the same solvent system, wouldincrease the purity of the isolates.

The presence of polar UV-transparentmetabolites such as sucrosewould have been missed if the purity control was performed only byUHPLC-UV and/or LC-MS analyses, as they generally elute at the solventfront on reverse phase and are usually diverted to waste prior toMS detection. Furthermore, they are not susceptible to ionizationunder general MS conditions.²⁴ Collectively,these results underscore the necessity of orthogonal analytical methodscombining LC-UV/MS with qHNMR to accurately define the purity levelof isolated metabolites and determine the structure of impurities.

Further Characterization of p-HydroxybenzylmalonicAcid (9) in Mixtures

Interestingly, the naturaloccurrence of p-hydroxybenzylmalonicacid 9 has not been described previously. However, thisphenolic acid has already been assigned a CAS number (90844-16-9)following chemical synthesis.³⁴ Moreover, 9 can be employed as a reagent in the synthesis of thyroxinderivatives.³⁵ A patent covering the useof any structural derivatives of 9 as stabilizers forplastic material (US patent 4,148,783) has been issued. Therefore,the question arises as to whether the presence of 9 inthe CPC fractions represented a potential chemical contamination.In order to confirm the genuine nature of the presence of 9 in licorice fractions, additional 1D and 2D NMR experiments includingCOSY, HSQC, and HMBC were carried out on the mixtures, as well ason isolated 9. Furthermore, the influence of the chemicalenvironment on the structural elucidation was investigated. Finally,the identification and quantification of 9 were performedon crude extracts from different Glycyrrhiza speciesand extracts.

COSY experiments of fraction 4 clearly displayedshort distance correlations between the methylene protons H-3 at 3.010ppm from 9 and its methine proton H-2 at 2.895 ppm. Onthe other hand, short distance correlations were observed betweenthe aromatic protons H-2′/6′ at 6.890 and H-3′/5′at 6.665 ppm (Figure S9, Supporting Information). Moreover, HMBC experiments revealed long-range correlations betweenaromatic protons H-2′/6′ and the benzyl carbon C-3 (δ_C 53.31, δ_H 2.895), along with the C-4′phenolic carbon (δ_C 155.78) (Figure 3C). Interpretation of the HMBC spectrum indicated that theH-3 benzylic proton correlated with the carboxylic acid functions(δ_C 174.02). Overall, 2D NMR analyses performed onthe mixtures (fractions 4–6) also led to the structural identificationof 9 as p-hydroxybenzylmalonic acid.Subsequent NMR analyses of the neighboring fractions indicated that 9 was concentrated in fraction 7, but was also present infractions 2 and 3 (Figure 3D, Figures S8 andS9, Supporting Information). Consideringthat the CPC SS used deionized water (pH 6) and was not buffered, 9 may have eluted either as a partially negatively chargedspecies or as a diacid. The potential occurrence of multiple formsof 9 explains its large volume of elution (>1.54 L)and,therefore, its presence in six consecutive fractions.

Compound 9 purified by semipreparative HPLC from fractions2 and 4 was also subjected to extensive NMR analysis (Figure S11, Supporting Information). The ¹H chemicalshifts of such a malonic acid derivative can be influenced by thechemical composition and, more importantly, the pH of the fractionfrom which it is characterized (Figure 3D andFigure S12, Supporting Information). Interestingly,the pH values of both fraction 7 and purified 9 werefound to be ∼4, while the pH of fractions 2–6 was determinedto be ∼5.5. Under acidic conditions (pH ∼4, fraction7 and purified 9), the aromatic protons, as well as thatof H-2, were deshielded (Δδ = 0.07 ppm for H-2′/6′and H-3′/5′, Δδ = 0.48 for H-2), while theH-3 resonance was shielded (Δδ = 0.09 ppm). Clearly, thecomposition influences the final pH of the fractions and ultimatelyinfluences the form in which compound 9 exists (Figure 3D). This explains why the ¹H NMR signalpattern of pure 9 differed from those observed in fractions2–5.

In order to ascertain its natural occurrence, compound 9 was quantified in the original G. uralensis crudeMeOH extract by qHNMR (Figure 3D) to be 1.66%.Further investigations (Figure S13, SupportingInformation) by qHNMR indicated that 9 could bedetected in the crude extracts of all three Glycyrrhiza species at concentrations between 1.42% and 2.67%. The quantificationswere performed utilizing the resonances of H-3′/5′ at6.556 ppm.

Interestingly, Farag and co-workers had previouslyreported theubiquitous presence of an “unknown compound” in Glycyrrhiza species.²⁴ This moleculealso produced a molecular ion [M – H]⁻m/z = 209.0453, and we conclude that itis likely the same compound as 9. In summary, our resultsconfirm the identity and presence of p-hydroxybenzylmalonicacid (9) in licorice roots and indicated that 9 is neither a minor metabolite nor the result of a chemical contamination.

Identification and Quantification of Major 1°Ms

Afterthe large-scale CPC fractionation, polar 1°Ms were assumedto be concentrated in the first fraction. Therefore, metabolite profilingof fraction 1 was performed by qHNMR in order to identify and quantifythese 1°Ms. Interestingly, the UHPLC-PDA profile revealed thepresence of only glycyrrhizin (5) at a concentrationof 5.0%. However, further investigation by 1D/2D NMR (gCOSY, HSQC,HMBC) and qHNMR spectra led to the identification and quantificationof major UV-transparent 1°Ms (Figure 4A and C).

Figure 4

Comparative qHNMR spectra (A) and UHPLC-UV profiles (C) of fraction1 and G. uralensis crude extract for identificationof the major 1°Ms (B). (A) Comparative qHNMR spectra (600 MHz,DMSO-d₆) using an internal calibrant (3,5-dinitrobenzoicacid TraceCERT at 2.12 mM) enabled the identification and quantificationof major 1°Ms in fraction 1 (Figures S14 and S15, Supporting Information). Sucrose (10), proline (11), and asparagine (12), butalso glycyrrhizin (5), were defined as being the mostabundant. (B) Structure and concentration of each identified 1°Mdetermined by qHNMR in fraction 1. (C) The UHPLC-UV profile of fraction1 displays only the presence of glycyrrhizin 5 (determinedat 5% w/w) along with other minor saponins. The crude extract wasprepared at 10 mg/mL, whereas fraction 1 was analyzed at 2.5 mg/mL(Kinetex C18-XB; 50 × 2.1 mm, 1.7 μm; gradient composedof H₂O and MeCN + 0.1% FA).

Sucrose (10) was identified and quantified,utilizingits nonoverlapped anomeric proton resonance, a doublet (J = 3.74 Hz) at 5.174 ppm in DMSO-d₆.Proline (11) was also identified based on its diagnostic ¹H NMR resonances at 1.671 ppm (1H, J = −12.88/7.92/7.50/7.40/0.71Hz, H-4a), 1.770 ppm (1H, J = −12.88/7.55/7.28/6.00/5.57Hz, H-4b), 1.928 ppm (1H, J = −12.74/7.50/6.00/5.55Hz, H-3a), and 2.003 ppm (1H, J = −12.74/8.87/7.92/7.28Hz, H-3b). The H-2 resonance observed at 3.618 ppm (1H, ddd, J = 8.87/5.55/0.71 Hz) was partially obscured by resonancesfrom 10. The proton H-5 resonated at 3.201 ppm (1H, dddd, J = −11.21/7.40/5.57 Hz) and 2.994 ppm (1H, ddd, J = −11.20/7.55/7.40 Hz). Furthermore, the aminoacid asparagine (12) was identified by its two doubledoublet resonances at 2.343 ppm (1H, dd, J = 16.49/8.89Hz, H-2a) and at 2.724 ppm (1H, dd, J = 16.49/4.04Hz, H-2b). The resonances of H-3 at 3.373 ppm (1H, dd, J = 8.89/4.04) were also partially obscured by hydroxy resonancesfrom 10. Lastly, trigonelline (13) was identifiedby its characteristic downfield proton at 9.923 ppm (1H, s, H-2),8.891 ppm (1H, d, J = 5.90, H-6), 8.768 ppm (1H,d, J = 7.91, H-4), and 8.032 ppm (1H, dd, J = 7.91/5.90, H-5). The presence and identity of compounds 10 to 13 in fraction 1 were also confirmed byMS analyses, as described previously.³⁶ The ¹H NMR spectrum of fraction 1 was compared with thespectrum from commercial reference compounds (Figures S14 and S15, Supporting Information) and were in agreementwith previously published data.^23,37,38 All identified metabolites were quantified in fraction 1 using 3,5-dinitrobenzoicacid (Fluka, TraceCERT) as the internal calibrant (IC, 2.12 mM). Byfar, 10 was the most abundant metabolite (71.7% in fraction1, qHNMR, anomeric proton), followed by 11 (10.3% offraction 1, qHNMR, H-3 resonance), 12 (2.91% of fraction1, qHNMR, H-3 resonance), and 13 (0.16% of fraction 1,qHNMR, H-4 and H-6). Hence, the sum of all identified and quantified1°Ms represented 85.1% of fraction 1 and therefore around 72%of the crude extract (Figures 4 and 5). The present results are in agreement with thework by Farag and co-workers, who indicated that saccharides, especiallysucrose, were the most abundant 1°Ms, followed by amino acidsand fatty acids.²⁴

Figure 5

UHPLC-UV and qHNMR quantitativeresults of major identified 2°Msand 1°Ms in G. uralensis crude extract. (A)Quantitative ¹H NMR was carried out in the crude MeOH extractfrom the roots of G. uralensis. All 1°Ms (gray)together with compound 9 were quantified by qHNMR. All2°Ms (red) were quantified by a previously established UHPLC-UVmethod.⁷ Results are expressed as masspercentages of the crude extract and highlight that sucrose (10) and proline (11) were the most concentratedof all quantified metabolites. Liquiritin (1) (3.81%)and glycyrrhizin (5) (3.18%) were the second most concentratedmetabolites. (B) Sum of all quantified major 1°Ms (74.80%; gray)compared to the sum of all quantified 2°Ms (11.91%, red). Compounds:sucrose (10), proline (11), asparagine (12), trigonelline (13), p-hydroxybenzylmalonicacid (9), liquiritin (1), liquiritin apioside(2), liquiritigenin (6), isoliquiritin (3), isoliquiritin apioside (4), neoisoliquiritin(7), licuraside (8), glycyrrhizin (5).

Concentrations of 1°Msvs 2°Ms in G. uralensis Crude Extract

qHNMR and UHPLC-UV were used to confirm thequantitative results previously obtained for fraction 1 and to assessthe relative abundance of major 1°Ms vs 2°Ms in the crudeextract. As such, all major UV–visible 2°Ms were quantifiedby UHPLC-UV using a previously validated method⁷ (Supporting Information S1),whereas all major UV-transparent 1°Ms, plus the phenolic acid 9, were quantified by qHNMR (Figure 5A). Combining these orthogonal analytical methods enabled the characterizationand quantitation of 86.8% of G. uralensis crude extract.Compounds 10 (61.9%) and 11 (10.2%) wereby far the most prevalent 1°Ms, thereby confirming the quantitativeresults obtained from fraction 1. Together, these compounds represented93% of all quantified 1°Ms. In plant physiology, 10 and 11 are known to be natural osmoprotectants,³⁹ capable of maintaining the moisture level insidethe roots in order to protect the plant cells against dehydration.⁴⁰ The third most concentrated 1°M was foundto be 12 (2.45% of the crude extract), one of the 20most common amino acids, which is also a nitrogen transport metaboliteintensely synthesized in the legume roots.⁴¹ The presence of excess 12 has been reported to be amarker of protein degradation under stress, such as drought, salinity,and nutrient deficiency.^42,43 The production of compounds 10, 11, and 12 has been reportedto increase in the roots of various plant species, including thosefrom the Fabaceae family, during a period of drought.^43,44 Therefore, the abundance of such metabolites (10–12) in licorice roots may be an indicator of drought stressat harvest time. Compound 13, commonly found in seedsand root exudates of various Fabaceous plants and reported to be a Nod gene inducer at molar concentrations,^45,46 represented 0.28% of the crude extract. Interestingly, its occurrencein licorice roots is reported here for the first time.

Fromthe perspective of botanical use, 10 contributes to thesweetness of the roots, together with the natural sweetener 5. Moreover, the amino acid 11 is known to enterinto the biosynthesis of proteins and collagen.⁴⁷ As a free amino acid, proline (11) has beendescribed as a protein-solubilizing solute and as a protein foldingagent both in vitro and in vivo.^48,49 The alkaloid 13 has also been attributed multiple health properties linkedto the prevention of central nervous system diseases and diabetes.^50,51

The flavanone, 1 (3.81%), and the saponin, 5 (3.18%), were found to be the second most prevalent metabolites.Interestingly, the dicarboxylic acid, 9 (1.66%), wasmore abundant than the flavanone apioside, 2 (1.47%),confirming the importance of this phenolic acid in licorice roots.From a structural perspective, compound 9 may be regardedas a chemical analogue of tyrosine, where the amino function is replacedby a carboxylic acid. The question remains whether 9,like tyrosine, is a biogenetic precursor of various hydroxycinnamicacids and, thus, of licorice chalcones and flavanones.^52,53 Interestingly, in the root–rhizosphere environment, flavonoidscan be degraded to phenolic derivatives by symbiotic rhizobia, mainlythrough the fission of the C-ring.⁵⁴⁻⁵⁶ This group of compoundsis considered as growth substrates for rhizobia. Therefore, 9 could be the result of either the plant or the rhizobiametabolism, highlighting its potential importance as a marker of plant–rhizobiametabolism.⁵⁵ From a pharmacological perspective,the only reported activity for 9 was in 1991, when Shechterand co-workers demonstrated that 9 inhibited the proteintyrosine kinase implicated in proliferative cell cycles as well asinsulin-dependent lipogenesis (IC₅₀ of 3.8 ± 0.1 mM).⁵⁷ Considering its natural occurrence in licoriceroots, the pharmacological properties of 9 are deservingof further investigations.

Finally, the most abundant 2°Ms,including 9,accounted for 11.9% of the crude extract, whereas the major 1°Msalone represented 74.8% (Figure 5B). The combinedquantitative results demonstrated that the 1°Ms were at leastsix times more concentrated than the 2°Ms in the crude MeOH extract.

In conclusion, the CPC method developed in this study enables thelarge-scale purification of the liquiritin derivatives in less than3 h, producing reference materials with qHNMR purities of 70.2–95.8%for 1 and 65.0–80.3% for 2. At thesame time, the major 1°Ms, which represent 74.8% of the crudeextract, were concentrated in a distinct fraction and subsequentlyidentified and quantified by qHNMR. Additionally, orthogonal analysesby UHPLC-UV/LC-MS and qHNMR led to a comprehensive evaluation of theRC of the reference materials of 1 and 2. One of the major coeluting impurities was identified as p-hydroxybenzylmalonic acid (9), isolated forthe first time from a natural source, representing 1.66% of the G. uralensis extract. The identification of 9 along with the detection of UV-transparent 1°Ms as coelutingimpurities illustrated the importance of orthogonal analyses (LC-UV/MS/qHNMR)for chemical profiling and purity determination. Finally, comparativequantitative results, obtained by UHPLC-UV for the major UV–visible2°Ms and by qHNMR for the major 1°Ms, (Figure 5) enabled 86.8% of the original G. uralensis crude extract to be described and clearly underlined the abundanceof these 1°Ms. This group of metabolites is implicated in thetaste, sweetness, nutritional value, and potentially health effectsof licorice preparations.^49−51,58 Consequently, qualitative and quantitative determination of both1°Ms and 2°Ms, including p-hydroxybenzylmalonicacid (9), should be considered for the quality controlof licorice botanicals. From a broader perspective, the existing butrelatively sparse knowledge about the biological effects of (designated)1°Ms also indicates thatthese small polar molecules warrant consideration as marker compounds,which may impact the overall biological profile of a plant extract.

General Experimental Procedures

UHPLC analyses wereperformed on a Shimadzu UPLC equipped with a Kinetex XB-C18 (2.1 ×5.0 mm, 1.7 μm, 00B-4498-AN, Phenomenex) column and using adiode array detector (DAD, Shimadzu SPD-M20-A). The autosampler temperaturewas set at 4 °C, and the column oven temperature was set at 40°C. Postrun data analyses were done with the Shimadzu LabSolutionsoftware package. Countercurrent separation was carried out on a hydrostaticcentrifugal partition lab-scale instrument SCPC-1000 system, equippedwith a 1000 mL column, and coupled with an Armen Spot Prep II system(Armen Instrument SAS, Saint Avé, France). The latter is equippedwith a quaternary valve, a mixing chamber, a 50 mL/min pump, an automaticinjection loop, and an automated back flush ascending/descending valve,coupled with a UV-DAD detector and a fraction collector all controlledby Armen CPC software. The injection capacity of the defined CPC instrumentranges from 100 mg to 30 g. All collected fractions were dried usinga Thermo-Fischer Savant SC250 EXP speed vacuum equipped with an RVT4104refrigerator vapor trap. Freeze-drying was performed on a LabconcoFreezone 4.5 (Kansas City, MO, USA). NMR spectra were acquired ona Bruker AVANCE 600 MHz spectrometer equipped with a 5 mm TXI cryoprobeand on a Bruker AVANCE 900 MHz. Off-line data processing was performedusing the Mnova NMR software package (v.6.0.2, MestreLab ResearchS.L., A Coruña, Spain). Standard NMR tubes of 3 mm, 7 in.,were from Norell (part no. S-3-HT-7, Norell Inc., Landisville, NJ,USA). A precise Mettler Toledo XS105 dual range analytical balancewas used to prepare all extracts and fractions for UPHLC and qHNMRanalyses.

Reagents

All chemicals and reagents including HPLC-gradesolvents were obtained from Fisher-Scientific (Hanover Park, IL, USA)or Sigma-Aldrich (St. Louis, MO, USA). For NMR acquisition, DMSO-d₆ (D 99.9%) was purchased from Cambridge IsotopeLaboratories Inc. (Andover, MA, USA).

Plant Material and Extraction

Dried roots of G. uralensis Fisch ex. DC werepurchased from a local supplier(Chicago, IL, USA). The plant material (voucher codes BC 624 and BC689) was identified through a series of microscopic analyses, comparingit to a voucher specimen from the Field Museum (Chicago, IL, USA, G. uralensis × glandulifera FM 2174544).DNA authentication was also performed according to Kondo and co-workers.⁵⁹ Other Glycyrrhiza species usedfor the quantification of 9 were identified followingthe same procedure. The powdered roots of BC 624 (1109.55 g) wereexhaustively extracted by percolation with MeOH at room temperature(weight/volume ratio: 1:15). Freeze-drying yielded 282.76 g of crudeextract, which represented 25.5% (w/w) of the powdered roots.

Selectionof the CPC Solvent Systems

The compositionof a two-phase solvent system (SS) was selected according to the partitioncoefficient (K) of the target compounds in the crudeextract, as well as the settling time of the selected SS. Approximately5 mg of the crude extract was weighed in a 4 mL vial, to which 1.0mL of the selected SS was added. The tube was shaken vigorously toequilibrate the sample thoroughly between the two phases. The upperphase (UP) and lower phase (LP) were separated and dried overnightin a Savant SC250 EXP speed vacuum. Each dried and separated UP andLP was diluted in 200 μL of HPLC-grade MeOH for further UHPLC-UVanalysis. The K value was expressed as a peak areaof the target compound in the UP divided by that in the LP.

Fractionationand Isolation by CPC

The fractionationwas performed with EtOAc/MeCN/H₂O (5:6:9 v/v/v) in an isocraticand descending mode. The SCPC-1000 (column volume 1030 mL) was filledwith the organic UP at a flow rate of 50 mL/min. The rotation speed,adjustable from 0 to 1500 rpm, was set at 1200 rpm. The system wasequilibrated at a flow rate of 20 mL/min with an S_f of 71.8% (V_S = 739.54 mLand V_m = 290.46 mL). G. uralensis crude extract (15.003 g), diluted in 21 mL of UP and 21 mL of LP,was injected into the column. Fraction collection was set up for 20mL/fraction and started after 300 mL of mobile phase elution. Theextrusion after 2.6 column volumes and during 30 min was performedat a flow rate of 50 mL/min, while the fraction collection was setfor 25 mL/fraction. CPC chromatograms were obtained by UV detection,using a UV-DAD detector at 254, 330, and 350 nm, as well as UV-scanfrom 200 to 600 nm. CPC fractions were pooled according to their UVand TLC profiles, leading to a total of 13 final fractions. TLC wasperformed on Alugram silica gel plates (SiO₂ F₂₅₄, Macherey-Nagel Gmbh & Co.), eluted with EtOAc/MeOH/AcOH/H₂O (8:1:1:2) and revealed with 5% H₂SO₄/vanillin reagent. The fractions were pooled as follows: Fraction1: vials 1 to 6 (from 0 to 80 mL after the first 300 mL), fraction2: vials 7 to 10, fraction 3: vials 11 to 12, fraction 4: vials 13to 15, fraction 5: vials 16 to 28, fraction 6: vials 29 to 53, fraction7: vials 54 to 76, fraction 8: vials 76 to 79, fraction 9: vials 80to 105, fraction 10: vials 106 to 120, fraction 11: vials 121 to 130,fraction 12: vials 131 to 156, fraction 13: vials 157 to 171.

PreparativeHPLC for the Purification of p-HydroxybenzylmalonicAcid

Preparative HPLC was performed on a Waters 600 apparatusequipped with a YMC-Pack-ODS-AQ column (250 × 10 mm, 5 μM),a PDA detector (Waters 2996), and an autosampler (Waters 717plus).Each of the fractions 2 and 4 was prepared at 50 mg/mL in 50% HPLC-gradeMeOH, and 50 μL was injected. The column was eluted with a gradientcomposed of (A) H₂O + 0.1% formic acid (FA) and (B) ACN+ 0.1% FA as follows: from 25% during 2 min, to 40% B in 20 min, andduring 5 min back to 25% B in 5 min (flow rate 2 mL/min). The retentiontimes (t_R) were 12.35 min for 9 and 15.00 min for compound 2. After concentration,samples were freeze-dried for 12 h and placed under vacuum prior toNMR analyses. A total of 10 injections were performed, leading tothe isolation of 5 mg of 9 (purity: 88.3% by qHNMR).

UHPLC-UV Analyses of Fractions and Crude Extract

G. uralensis crude extract and fractions obtained by CPCwere prepared at 10 and 2.5 mg/mL, respectively, and isolated compoundswere prepared at 0.5 mg/mL. Samples were diluted in 100% MeOH or MeOH/H₂O (70:30) HPLC grade (Fisher Co. Ltd.) and filtered (filterAcrodisc CR 13 mm, 0.45 μm PTFE membrane) prior to injection(2 μL). UHPLC analyses were performed following a publishedmethod.⁷G. uralensis crudeextract was examined in triplicate.

LC-MS-MS of Fractions 4to 7

LC-MS-MS analysis wascarried out using a Waters 2695 solvent delivery system connectedto a Water SYNAPT quadrupole/time-of-flight (Q/TOF) mass spectrometeroperated in the negative ion electrospray mode. Separations were carriedout using a Waters XBridge C₁₈ column (2 × 50 mm,2.5 μm particle size), eluted with a mobile phase consistingof 0.1% FA (solvent A) and 95% MeCN + 0.1% FA (solvent B) with a lineargradient from 10% to 60% B in 10 min. The flow rate was 0.25 mL/min,and the column was thermostated at 30 °C. High-resolution accuratemass measurements (HR-MS) were performed at the resolving power of10 000 fwhm using Leu-enkephaline as the lock mass. Production spectra were recorded at 15 or 25 eV using argon as the collisiongas. For identification of compounds, molecular compositions and tandemmass spectra were compared with the standard spectra from public (MassBank)and in-house generated databases as well as with spectra publishedin the primary literature.

qHNMR Analyses of Fractions and Purity Determination

The NMR samples of G. uralensis crude extractandthe CPC fractions were prepared by precisely (0.01 mg) weighing 8mg of the extract and 3–5 mg of the fractions, followed bythe addition of 300 μL of DMSO-d₆ using a Pressure-Lok gas syringe (VICI Precision Sampling Inc.,Baton Rouge, LA, USA) for accurate volume delivery. From this solution,200 μL measured with calibrated pipets (cat. no. 2-000-200,Drummond Scientific, Broomall, PA, USA) was added into 3 mm standardNMR tubes. All 1D ¹H NMR spectra were acquired at 298 Kunder quantitative conditions using a 90° single-pulse experiment(DE = 39.71 μs, D1 = 60.00 s, P1 = 8.75 μs, ds = 4, ns= 32) at 600 MHz. The 1D ¹³C NMR spectra of 9 and fractions 2 and 7 were acquired at 900 MHz (DEPT-Q experiment). ¹H and ¹³C chemical shifts (δ) were expressedin ppm with reference to the residual solvent signal of DMSO-d₅ (¹H spectrum: 2.500 ppm, and ¹³C spectrum: 39.525 ppm). The following processing schemewas used: a mild Lorentzian-to-Gaussian window function (line broadening= −0.3 Hz, Gaussian factor = 0.01) was applied, followed byzero filling to 256k acquired data points before Fourier transformation.After manual phasing, a fifth-order polynomial baseline correctionwas applied. The purity was calculated by qHNMR using the 100% method.^31,32 For absolute quantitation of 1°Ms in the crude extract andin fraction 1, qHNMR was performed using 3,5-dinitrobenzoic acid (Fluka,TraceCERT, purity P = 99.54% w/w, lot no. BCBH8381V) as internal calibrant(IC). A stock solution of the IC was prepared at 6.35 mM in a mixtureof D₂O/DMSO-d₆ (1.5:7). NMRsamples of fraction 1 (∼5 mg) and of crude extract (∼8mg) were prepared by precision weighing as described above, exceptthat their dilution was performed in 200 μL of DMSO-d₆ and 100 μL of IC stock solution (2.12mM final concentration). The pH of each solution was determined usingpH strips (Whatman, Panpeha) after diluting 100 μL of the remainingDMSO-d₆ solution into 300 μL ofdeionized H₂O. The 1D ¹H NMR spectra were acquiredunder quantitative conditions using 64 transients (DE = 39.71 μs,D1 = 60.00 s, P1 = 9.20 μs, rg = 64). The same data processingwas performed as described above. The signal-to-noise ratio (S/N)was ≫200:1, leading to an uncertainty level of 1% for the integrationprocedure.^60,61