Biosynthesis-Based Quantitative Analysis of 151 Secondary

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Biosynthesis-Based Quantitative Analysis of 151 Secondary Metabolites of Licorice to Differentiate Medicinal Glycyrrhiza Species and Their Hybrids Wei Song, Xue Qiao, Kuan Chen, Ying Wang, Shuai Ji, Jin Feng, Kai Li, Yan Lin, and Min Ye Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04919 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 19, 2017

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Analytical Chemistry

Biosynthesis-Based

Quantitative

Analysis

of

151

Secondary

Metabolites of Licorice to Differentiate Medicinal Glycyrrhiza Species and Their Hybrids

Wei Song†,∆, Xue Qiao†,∆, Kuan Chen†, Ying Wang‡, Shuai Ji†, Jin Feng†, Kai Li†, Yan Lin†, Min Ye†,*

Affiliations †

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical

Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China ‡

South China Botanical Garden, Chinese Academy of Sciences, 723 Xingke Road,

Guangzhou 510650, China

*

Corresponding author. Tel.: +86 10 82801516. Fax: +86 10 82802024. Email

address: [email protected] (M. Ye).

∆

The first two authors contributed equally to this work.

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ABSTRACT Secondary metabolites are usually the bioactive components of medicinal plants. The difference in the secondary metabolisms of closely related plant species and their hybrids has rarely been addressed. In this study, we conducted a holistic secondary metabolomics analysis of three medicinal Glycyrrhiza species (G. uralensis, G. glabra, and G. inflata), which are used as the popular herbal medicine licorice. The Glycyrrhiza species (genotype) for 95 batches of samples were identified by DNA barcodes of the internal transcribed spacer and trnV-ndhC regions, and the chemotypes were revealed by LC/UV- or LC/MS/MS-based quantitative analysis of 151 bioactive secondary metabolites, including 17 flavonoid glycosides, 24 saponins, and 110 free phenolic compounds. These compounds represented key products in the biosynthetic pathways of licorice. For the 76 homozygous samples, the three Glycyrrhiza species showed significant biosynthetic preferences, especially in coumarins, chalcones, isoflavanes, and flavonols. In total, 27 species-specific chemical markers were discovered. The 19 hybrid samples indicated that hybridization could remarkably alter the chemical composition and that the male parent contributed more to the offspring than the female parent did. This is hitherto the largest-scale secondary metabolomics study of medicinal plants and the first report on uniparental inheritance in plant secondary metabolism. The results are valuable for biosynthesis, inheritance, and quality control studies of licorice and other medicinal plants.

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INTRODUCTION Plant secondary metabolites are a huge group of small-molecule natural products with high structural diversity.1,2 For medicinal plants, secondary metabolites are usually their bioactive chemical components and serve as important sources of new drugs.3,4 Many herbal medicines such as licorice, ephera and astragalus are derived from several closely related plant species.5 Moreover, interspecies hybridization has been reported for a number of medicinal plants.6 However, little is known about the impact of species variation and hybridization on plant secondary metabolism. To guarantee safe and effective use of medicinal plants, a comprehensive understanding of their secondary metabolome is of significance.

Because of the large number, structural diversity, and content range (10% to sub-ppm level) of chemical compounds in plants, their secondary metabolomics analysis has been a great challenge.7,8 Although various methods combining analytical techniques with statistical calculations have been developed in recent years, most of these studies have used the untargeted approach, where all potential metabolites are semi-quantitatively analyzed.9-11 With this approach, the structures of the metabolites are not fully identified, and their contents cannot be accurately determined. These problems can be resolved by targeted secondary metabolomics, where a group of compounds are quantitatively determined using reference standards.3 Nevertheless, limited by the availability of pure reference standards and efficient analytical techniques, very few targeted secondary metabolomic studies on

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plants have been reported, and only tens of compounds can be determined for a given plant species.12

Licorice is one of the most popular herbal medicines worldwide. It is mainly used to treat cough, inflammation, and liver damage.13 In China and Europe, the roots and rhizomes of Glycyrrhiza uralensis, G. glabra, and G. inflata are used as licorice without discrimination, whereas only the former two species are used in the United States and Japan.5,14-16 The major bioactive secondary metabolites of licorice include saponins (e.g., glycyrrhizin), flavonoid glycosides (e.g., liquiritin), and various types of free phenolic compounds. Approximately 250 compounds have been reported from licorice, and their abundances could remarkably affect the therapeutic effects. However, the contents of no more than 20 compounds in licorice have been determined thus far.17,18 Moreover, no report is available to compare the secondary metabolomes of Glycyrrhiza species.

This study aims to reveal differences in the secondary metabolomes of the three medicinal Glycyrrhiza species. A holistic targeted secondary metabolomics analysis was conducted to quantitatively determine the contents of 151 bioactive secondary metabolites (Figure S1) in 95 batches of licorice samples using ultra-performance liquid

chromatography

with

ultraviolet

detection

(UPLC/UV)

or

ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC/MS/MS). The biosynthetic preferences and the

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uniparental inheritance in the secondary metabolism of Glycyrrhiza species were also discussed.

EXPERIMENTAL SECTION Plant Materials. A total of 95 batches of licorice samples were used. Among them, 58 batches of wild licorice roots and rhizomes were collected by one of the authors (Dr. Ying Wang) in northwestern China from 2010-2012. Their species were preliminarily identified by morphological identification and then confirmed by DNA barcoding analysis. The other 37 batches of cultivated licorice roots and rhizomes were collected from herb markets or pharmacies around China from 2007-2012. Sample information is given in Table S1. Voucher specimens were deposited at the School of Pharmaceutical Sciences, Peking University (Beijing, China).

Chemicals and Reagents. Plant genomic DNA kits, TransStart KD Plus DNA polymerase, and DNA Marker II were from TianGen Biotech Co. Ltd. (Beijing, China). Formic acid (Sigma-Aldrich, MO, USA), methanol, and acetonitrile (J. T. Baker, NJ, USA) were of HPLC grade. De-ionized water was prepared using a Milli-Q purification system (Millipore, MA, USA). Other reagents were purchased from Beijing Chemical Corporation (Beijing, China) unless otherwise specified.

DNA Barcoding Analysis. The dried licorice materials were wiped with 75% ethanol and ground into powder. Total DNA was extracted from approximately 100

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mg of the powder with the plant genomic DNA kit following the manufacturer instruction and was dissolved in 30 µL of sterile water. The internal transcribed spacer (ITS), trnH-psbA and trnV-ndhC sequences were amplified from genomic DNA by polymerase chain reaction (PCR) using the primers of ITS-G-F (5’-GAAGG ATCAT TGTCG ATGCC-3’), ITS-G-R (5’-GCGTT CAAAG ACGCC TATTG

G-3’);

trnH-forward

(5’-ACGGG

AATTG

AACCC

GCGCA-3’),

Gly-trnHR1 (5’-CATAT GACTT CACAA TGTAA AATC-3’); ndhC-F (5’-AGACC ATTCC AATGC CCCCT TTCGC C-3’), trnV-R (5’-GTTCG AGTCC GTATA GCCCT A-3’), respectively.19,20 Other primers for universal intergenic regions are listed in Table S2. Detailed PCR conditions are given in the note of Figure S2. Single nucleotide polymorphism analysis was performed using DNAman (version 8.0, Lynnon Biosoft, USA) and BioEdit (version 7.0.0, Ibis Biosciences, USA).

GenBank Accessions. ITS of Glycyrrhiza uralensis (AB280738), G. glabra (AB280739), and G. inflata (AB280740); chloroplast trnH-psbA intergenic region of G. uralensis (GU396734); chloroplast trnV-ndhC intergenic regions of G. uralensis (KU985183), G. glabra (KU985171), and G. inflata (KU985170); complete genome of G. glabra chloroplast (KF201590.1).

Targeted Secondary Metabolomics Analysis. A total of 151 reference compounds (1-151) were isolated by the authors from the roots and rhizomes of Glycyrrhiza uralensis (compounds 1-44, 47-54, 64-68, 79-84, 86-88, 92, 93, 98-103, 112-136,

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149), G. glabra (compounds 55-61, 77, 78, 85, 89-91, 94-96, 105-111, 147, 148, 150, 151), and G. inflata (compounds 45, 46, 62, 63, 69-76, 97, 104, 137-146), most of which had been reported in our recent publications.21 Structures for all the compounds were identified by 1D and 2D NMR, and their purities were above 98% (Figure S1). Considering their remarkably varied contents, UPLC/UV and UHPLC/MS/MS techniques were used to determine the major (1-10, 18-22) and minor (11-17, 23-151) compounds, respectively. Seven sets of methods were established to ensure accuracy and specificity for each of the 151 analytes (Table S3). The methods were optimized and validated in terms of linearity, sensitivity (limit of detection and limit of quantification), intra- and inter-day precision, repeatability, accuracy (except for method 3 due to limited amounts of pure reference standards), and stability, following FDA guidance (see Supporting Information).22

UPLC/UV. The contents of major compounds in licorice were determined by UPLC/UV. Method 1-a (for flavonoid glycosides) is described here as an example. Dried licorice roots and rhizomes were pulverized into a fine powder and sifted through an 80-mesh sieve. The powder (200 mg) was accurately weighed and extracted with 10 mL of 50% methanol in an ultrasonic bath (25°C, 30 min). The extract was filtered through a 0.22-µm membrane, and a 1-µL aliquot was injected for analysis. An Acquity H-Class UPLC system (Waters, Milford, MA, USA) was used. Samples were separated on an Acquity UPLC HSS T3 column (150×2.1 mm I.D., 1.8 µm) equipped with a VanGuard pre-column (5×2.1 mm I.D., 1.8 µm)

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(Waters). The mobile phase consisted of acetonitrile-methanol (4:1, v/v, A) and water containing 0.1% formic acid (v/v, B). The analytes were eluted using a linear gradient program: 0–1 min, 30% A; 1–8 min, 30–40% A; 8–16 min, 40–52% A; 16–16.2 min, 52–100% A; 16.2–17.6 min, 100% A. The flow rate was 0.40 mL/min. The column temperature was 55°C. The detection wavelength was 254 nm. The data were acquired and processed using an Empower™ 3 analytical workstation (Waters). Other UHPLC/UV analyses were conducted using similar methods with slight modifications (see Supporting Information).

UHPLC/MS/MS. The contents of minor compounds in licorice were determined by UHPLC/MS/MS. Method 2-b (for 45 free phenolic compounds) is described here as an example. The licorice powder (30 mg) was accurately weighed and extracted with 10 mL of the internal standard solution (chrysophanol, 2.90 µg/mL in methanol) in an ultrasonic bath (25°C, 30 min). The sample solutions were filtered through a 0.22-µm membrane before use, and a 2-µL aliquot was injected for analysis. A 1290 series UHPLC instrument was coupled with a 6495 triple quadrupole mass spectrometer via an ESI interface (Agilent Technologies, Waldbronn, Germany). The analytes were separated using a modified UPLC method as described above. The mass spectrometer was operated in negative ion mode, with sheath gas temperature at 350°C, gas flow at 11 L/min, and nebulizer gas at 25 psi. The capillary voltage was set at 3500 V, nozzle voltage 1500 V, and delta EMV 100 V. The analytes were detected in multiple reaction monitoring (MRM) mode. Two precursor-product ion

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MRM transitions were selected for each compound (one for quantitation and the other for qualification), as shown in Supporting Information. The data were acquired and processed by MassHunter (version B.06.00) and QQQ Quantitative Analysis (version B.03.02) software (Agilent Technologies, USA). Other UHPLC/MS/MS analyses were conducted using similar methods with minor modifications (see Supporting Information).

Data Processing and Statistics. The contents of 151 secondary metabolites in 95 batches of licorice samples were statistically analyzed using the Mann-Whitney U-test. To confirm the results, an independent-samples t-test was also performed for the same datasets on the basis of F-test (SPSS 13.0 for Windows, SPSS Inc., USA). For all statistical tests, a significance threshold of α = 0.05 was set. The boxplots were charted by Origin 8 (OriginLab Co., USA). Principal component analysis (PCA) was performed using SIMCA-P software (v11.5, Umetrics AB, Umeå, Sweden) with the weight power of square root of x.

RESULTS AND DISCUSSION Identification of Glycyrrhiza Species by DNA Barcoding. The macroscopic characteristics of the three Glycyrrhiza species are very similar, especially beyond the flowering and fruiting periods. Thus, we used DNA barcoding to identify the species of the 95 batches of licorice samples. DNA barcoding is a powerful tool to distinguish closely related species by amplifying short genetic markers.23 The

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barcodes could be located on nuclear ribosomal DNA (nrDNA) such as the ITS or could be located on chloroplast DNA (cpDNA) such as the matK gene, rbcL gene, and the trnH-psbA intergenic spacer.

Two DNA barcode regions, ITS and trnH-psbA, were amplified for the licorice samples.19,24 For the ITS region, we designed new primers (ITS-G-F and ITS-G-R) to improve PCR specificity (Figure S2). As shown in Figure 1, G. uralensis could be distinguished from the other species as the ITS genotype I-3, while G. glabra and G. inflata shared the same ITS genotype I-2. Previous studies had used three single nucleotide polymorphism (SNP) sites of the trnH-psbA spacer to differentiate G. glabra and G. inflata. However, approximately 10% of the samples could not be differentiated.24 In this work, we screened 15 loci in the chloroplast genome for genetic marker regions (Table S2). As a result, stable SNP site 487 was discovered in the trnV-ndhC spacer to distinguish G. inflata from G. glabra (Figure S2). G. glabra and G. uralensis shared the same trnV-ndhC genotype TN-2.

A DNA barcoding strategy combining nrDNA and cpDNA was established and applied to 95 batches of licorice samples (Figure 1, Table S1). In total, 60 batches of G. uralensis (U), 11 batches of G. glabra (G), and 5 batches of G. inflata (I) were identified. Unexpectedly, 19 samples showed overlapped ITS SNP signals at sites 159 and 383-385 (Figure 1). Since the ITS region was located on nrDNA and inherited from both parents, these samples should be hybrid species. Their parental

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species were identified by analyzing the SNP sites (72, 125 and 171) of trnH-psbA. Although maternal inheritance of chloroplast is widely accepted for higher plants,24 a recent publication clearly indicated paternal inheritance of Glycyrrhiza species.25 This deduction was adopted in our study. For samples of TP-3 and TP-4 types, the paternal species were identified as G. inflata and G. uralensis, respectively. For samples of TP-1 type, the paternal species was identified as G. uralensis, though a probability of G. glabra or G. inflata (8%) also existed (5 out of 65 samples in a previous report).19 For the TP-2 type, the paternal species could be G. glabra (75%) or G. uralensis (25%) (14 of 55 samples were G. uralensis according to a previous report)19 and was tentatively identified as the more possible G. glabra in this work.

Internal transcribed spacer (ITS)

C

Hybrid

I-1†

I-2

I-3 159

383-385

159

383-385

159

383-385

159

383-385

T G C

T

C A A

T

T G C

T C

C A A T G C

G. uralensis

SNP sites→

trnV–ndhC

Other species

←SNP sites

trnH–psbA

TN-2

TN-1

TP-1

TP-2

TP-3

TP-4

487

487

72 125 171 C A T

72 125 171 C A G

72 125 171 T A T

72 125 171 C G T

×U*

×G# ×U#

× In

×U

A

G. glabra

T

G. inflata

(Male parents of the natural hybrids)

Figure 1. Species identification of medicinal Glycyrrhiza species by ITS, trnV-ndhC, and trnH-psbA regions. ITS, internal transcribed spacer; SNP, single nucleotide polymorphism; × U, × G, and × In indicates hybrids whose male parents are G. uralensis, G. glabra, and G. inflata, respectively; * × U accounts for 92% probability, whereas × G and × In accounts for 5% and 3%; # × G and × U accounts for 75% and 25%, according to a literature;19 † I-1 type is from non-medicinal Glycyrrhiza species

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such as G. yunnanensis and G. pallidiflora.

Targeted Secondary Metabolomics. The contents of 151 secondary metabolites (1-151, Figure S1) were determined in the 95 batches of licorice samples. This is hitherto the largest-scale targeted secondary metabolomics study of medicinal plants. The analytes included 17 flavonoid glycosides, 24 triterpenoids (23 saponins and a major aglycone, glycyrrhetinic acid), and 110 free phenolic compounds. They covered approximately 60% of the 253 compounds hitherto reported from the three Glycyrrhiza species (isolated from the whole plants, not limited to roots).26 These pure compounds were isolated by our group, and the majority of them showed significant biological activities.21 More importantly, they represented the key products in the major biosynthetic pathways of licorice and could comprehensively reflect the secondary metabolism of Glycyrrhiza species.

The 151 analytes showed high diversity in chemical structure, polarity, and content level. Their precise quantitative analysis was a large challenge. For instance, many phenolic compounds sharing the same molecular weights and MS/MS fragment ions could compromise the specificity of MRM detection, and the wide concentration range from 10% to sub-ppm challenged the linearity of the analytical methods. To guarantee the accuracy of the results, the 151 analytes were divided into seven groups for quantitative analysis. Briefly, abundant flavonoid glycosides and saponins were quantified by UPLC/UV, and the minor free phenolics were determined by

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UHPLC/MS/MS (Figure S3). All the methods were fully validated according to FDA guidance.22 The accuracy, precision, repeatability, and stability complied with the FDA requirements (see Supporting Information).

From the results, the 151 compounds accounted for 11.6±5.0% of the dried herb materials of licorice, or approximately 65% of the methanol extract (the average methanolic extraction yield was 18%, as shown in Table S4). Among them, flavonoid glycosides (17 compounds, 2.8±1.5%) and triterpenoids (24 compounds, 8.2±4.1%) were the major components (Figure 2). The free phenolics (110 compounds, belonging to 11 structural types) only accounted for 0.7±0.4% of the herbal materials. Nevertheless, their significant bioactivities rendered their contents critical for the quality control of licorice.21

A 150

B G. uralensis (n=60)

4

120

Secondary metabolites (mg per g of crude drug material)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3

***

90

Homozygous samples (76 batches)

2 60

***

***

30

***

0 150

1 0

***

G. inflata (n=11)

4

120

3

90 2 60

*

30

***

0 150

▲ US

▲ UC

▲G

▲ In

1

Homozygous and hybrid samples (95 batches) 0 4

G. glabra (n=5)

120

3

90 2 60 30

1

*

0

0

▲ U× ×G

▲ US ▲ UC ▲ G ▲ In ×In ▲ O× ×In ▲ O× ×U ▲ In/G× ×U ▲ U×

Figure 2. General chemical differences for the three Glycyrrhiza species. 13



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(A) Contents of 15 structural types in the three species. The numbers of compounds for each type are shown in brackets. Other glycosides refer to flavone and isoflavone glycosides. *P < 0.05,

**

P < 0.01,

***

P < 0.005, Mann-Whitney U-test, compared

with the other two species. (B) Score plots from unsupervised PCA analysis. R2X[1]=0.313, R2X[2]=0.096 and R2X[1]=0.299, R2X[2]=0.110 for the upper and bottom plot, respectively. UC, wild-growing G. uralensis; US, cultivated G. uralensis; G, G. glabra; In, G. inflata; O, other non-medicinal Glycyrrhiza species; U×G indicates hybrids whose female parent is G. uralensis, and the male parent is G. glabra. This nomenclature applies to all hybrids.

Variation in Secondary Metabolomes for Homozygous Glycyrrhiza Species. The data were first processed by principal component analysis (PCA), using contents of the 151 compounds as variables. In the score plot, the three species could be readily differentiated by unsupervised PCA (Figure 2B). The closer distance between G. inflata and G. glabra was in line with their closer genetic relationship.19 In addition, the influence of growing area was much less than that of species variation (Figure S4). The contents of many compounds, particularly free phenolics, varied remarkably inter-species (Figure 3, Figure S5). In total, 67, 20 and 17 compounds in G. uralensis, G. inflata and G. glabra showed significant differences from the other two species (P < 0.005, Mann–Whitney U-tests), and 79%, 90% and 94% of them were free phenolics.

Discovery of Chemical Markers. Several studies have tried to reveal the chemical differences of the three Glycyrrhiza species. Kondo et al. determined 9 major compounds and found that glycycoumarin (82), glabridin (103), and licochalcone A 14


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(68) were specific markers for G. uralensis, G. glabra, and G. inflata, respectively.17 Other studies also discovered inter-species chemical variations by determining up to 19 compounds, although they did not indicate specific markers.18 Our study discovered 85 potential specific compounds (P < 0.005 from the other two species, Mann–Whitney U-tests), including the three known markers.17 The nonparametric test was employed due to remarkably different sample volumes for the three species (n = 60, 5, 11 for U, G, and In, respectively). We also used the independent-samples t-test to confirm the results. The number of potential specific compounds was similar (84, P < 0.05), and 67 specific compounds were common for the two tests, indicating the three Glycyrrhiza species contained a number of species-specific compounds. Finally, a total of 27 free phenolic compounds were discovered as species-specific chemical markers to differentiate the three Glycyrrhiza species (Figure 4). They fulfilled the following requirements: high inter-species variance (P < 0.005 by Mann–Whitney U-tests), relatively high contents (> 10 µg/g), and non-overlapped content ranges at 95% confidence interval. These markers were also supported by t-tests (Table S5). Recently, Rizzato et al. conducted an LC/MS-based untargeted metabolomics analysis of Glycyrrhiza species, and obtained similar results to our study.27 They also discovered new possible markers, which need to be confirmed using reference standards.

Biosynthetic Preferences of Glycyrrhiza Species. The 107 free phenolic compounds analyzed in this study belong to 10 structural types (excluding the 3 compounds of

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other types). They formed a comprehensive biosynthetic pathway for licorice, as shown in Figure 3.28,29 Interestingly, the three Glycyrrhiza species showed distinct biosynthetic preferences. The contents of 3-aryl-5-methoxyl coumarins (79-84) and flavonols (47-54) in G. uralensis were significantly higher than those in G. inflata or G. glabra (Figure 4, Figure S6). Likewise, G. inflata contained remarkably more abundant 2'-H chalcones (e.g., licochalcones A, C, E), and G. glabra contained more abundant 8-cyclized isoprenyl isoflavanes (e.g., glabridin and 4'-O-methylglabridin) than the other two species. Contents for all the phenolic compounds are shown in Figure S6. CHS

4-Coumaroyl-CoA + 3 x malonyl-CoA U U

In

In

G

In

eurycarpin A (137) angustone B (138) 2'-hydroxyl isoformonentin (112) glycyrrhiza-isoflavone C (113) luteone (114) isolupalbigenin (115) angustone A (116) IFS licoricone (117) glyurallin B (139) licoisoflavone B (118) glicoricone (119) licoisoflavone A (120) allolicoisoflavone B (121) 6,8-diprenylgenistein (140) semilicoisoflavone B (122) hirtellanine I (123) isoderrone (124) isoangustone A (125) 7-O-Methylluteone (126) 2'-hydroxyl formononetin (142) 7,8-OH-4’-OMe-6-IPY isoflavone (141) lupiwighteone (127) 6-C -prenylorobol (128) wighteone (129) 2'-hydroxyisolupalbigenin (130) pratensein (131) genistein (132) formononetin (133) glycyrrhisoflavone (143) abiochanin A (134) gancaonin H (144) glabrone (145) 3',4',7-trihydroxyisoflavone (146) gancaonin L (135) isoglabrone (147) daidzein (136) parvisoflavones-A (148)

G

IFR In

G glyasperin F (97) licoisoflavanone (98)

VR DMID

U

In

G 3-methoxy-9-hydroxy-pterocarpan (92) 1-methoxyphaseollin (93) licoagrocarpin (94) shinpterocarpin (95) dehydroglyceollin I (96)

U

In

PTR

8-cyclized IPY

G glycyuralin A (99) dehydroglyasperin C (100) 3,4-didehydroglabridin (105) licoricidin (101) glyasperin D (102) 7,4'-dihydroxy-3'-methoxyisoflavan (104) 8-prenyl-phaseollinisoflavan (106) glabrene (107) hispaglabridin B (108) 4'-O-methylglabridin (109) glabridin (103) 3'-hydroxy-4'-O-methylglabridin (110) hispaglabridin A (111)

U

fold of average

In

G liquiritin (5) liquiritigenin-7,4'-diGlu (1) glucoliguiritin apioside (2) liquiritin apioside (4) isoliquiritin (10) neoisoliquiritin (15) isoliquiritin apioside (9) isoliquiritigenin-4,4'-diGlu (8)

U

In

G euchrenone A5 (57) shinflavanone (58) glabrol (62) abyssinone II (59) (2S)-abyssinone I (60) paratocarpin L (63) naringin (61) liquiritigenin (64)

F3H U

In

G kanzonol Z (55) 3-OH glabrol (56)

FLS

U

In

G licoflavonol (47) uralenol (48) kaempferol (49) isolicoflavonol (50) 3-methoxyl isolicoflavonol (51) topazolin (52) kumatakenin (53) kaempferol-3-O-methyl ether (54)

In

G glycyrrhetinic acid (41)

U

4.0

CHI

U

U

0.05

licochalcone C (69) 3-OH licochalcone A (70) licochalcone E (71) licochalcone A (68) echinatin (65) licoagrochalcone C (72) licoagrochalcone A (73) kanzonol C (77) 2,3,4'-tri-OH-4-OMe-chalcone (74) corylifol B (75) isobavachalcone (78) 3,4,3',4'-Tetrahydroxychalcone (76) isoliquiritigenin (66) homobutein (67)

G

licoarylcoumarin (79) isoglycyrol (80) glycyrol (81) glycycoumarin (82) isoglycycoumarin (83) glycyrin (84) kanzonol W (85)

U

G

licoflavone B (45) 5,3’,4'-tri-OH-8,6'-di-IPY-flavone (46) licoflavone A (42) kumatakenin B (43) FNS genkwanin (44)

U

U

In

2’,6’-H, 4’-OH

G

glycyuralin E (86) licocoumarone (87) gancaonin I (88) kanzonol U (90) glyinflanin (91) 2'-O- demethybidwillol B (89)

5-OMe

CHR U

In

In

G 6'''-(3-OH-3-MG) isoviolanthin (14) sophoraflavone B (16) 4',7-Dihydroxyflavone 7-O-Glu (17) isoschaftoside (12) isoviolanthin (13) E-ring schaftoside (11) ononin (7) substituted vicenin (3) glycyroside (6)

In

G uralsaponin O (35) 22β-acetoxyl-glycyrrhaldehyde (29) uralsaponin Y (28) uralsaponin M (34) uralsaponin X (26) uralsaponin F (24) licorice-saponin E2 (20) uralsaponin C (23) 22β-acetoxyl-glycyrrhizin (19) uralsaponin P (27)

uralsaponin N (40) licorice-saponin A3 (18) licorice-saponin G2 (21) glycyrrhizin (22) licorice-saponin J2 (31) glycyrrhetic acid 3-O -gluA (36) ∆9,11-11-deoxo-glycyrrhizin (37) uralsaponin V (38) licorice-saponin B2 (33) uralsaponin T (25) uralsaponin W (39) uralsaponin B (30) araboglycyrrhizin (32)

Figure 3. Variation in the contents of 148 secondary metabolites in the three Glycyrrhiza species (compounds 149-151 were not included). The proposed biosynthetic pathways and the general structures are shown. U= G. uralensis, G= G. glabra, In= G. inflata. CHS, chalcone synthase; CHR, chalcone reductase; CHI, 16


Page 17 of 27

chalcone isomerase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; IFS, isoflavone synthase; FNS, flavone synthase; IFR, isoflavone reductase; VR, vestitone reductase; DMID, 7,2-dihydroxy-4-methoxyisoflavanol dehydratase; PTR, pterocarpan reductase. Ac, acetoxyl group; GluA, glucuronyl group; Glu, glucosyl group; IPY, isoprenyl group; MG, methylglutaroyl group; OMe, methoxyl group.

Glycyrol

Isolicoflavonol

Licoflavonol 180

47

450

50

300

81

Glycycoumarin 900

120

300

200

600

60

150

100

300

0

0

30

In UInxU G U GxU U UxIn/G In ×In G ×G U In/G×U

In InxU G GxU In U ×In G U×G U U UxIn/G In/G×U

Glycyuralin E

Glyasperin D

86

600

102

0

×In G ×G U In/G×U In U InxU G U GxU U UxIn/G

180

117

In UInxU G U GxU U UxIn/G In ×In G ×G U In/G×U

Glicoricone 90

400

120

60

10

200

60

30

In InxU In U ×In G GxU U×G U UxIn/G In/G×U

In In InxU U×In G G GxU U×G U U UxIn/G In/G×U

Isoangustone A

7-O-Methylluteone

6,8-Diprenylgenistein

125

90

200

Glicophenone 180

149

120

60

60

0

0

0

In InxU In U ×In G G GxU U×G U U UxIn/G In/G×U



Glabridin

Hispaglabridin B

4'-O-Methylglabridin

3'-Hydroxy-4'-O-methylglabridin

103

180

108

120

450

×In G U×G U UxIn/G In/G×U In U InxU G GxU

Hispaglabridin A

111

480 460 100

160 80 ×In G U×G U UxIn/G In/G×U In U InxU G GxU

0

3-Hydroxy-glabrol

56

3000

30 0

0 In In InxU U×In G G GxU U×G U U UxIn/G In/G×U


Parvisoflavones-A

Glycybridin C

Erybacin B

148

75

150

180

50

120

25

60

0

0




Licochalcone C

3-OH Licochalcone A

68

240

69

450

2000

160

300

80

1000

80

150

0

0


95th peroentile 75th peroentile median mean 25th peroentile 5th peroentile

180

151

Licochalcone A

160 0

110


50

0

90 60

150

0

0

109

300

60

400

240

140

120



800

240

180

30

100

1200

126

60

119

0


300

0

0

0

82

0

Licoricone

20 0

Content (µg/g in the raw material)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


70

0

In U ×In G GxU U×G U UxIn/G In/G×U In InxU



Licochalcone E

Licoagrochalcone C

3,4-Didehydroglabridin

71

450

72

240

120

300

160

60

150

80

0

0

In In InxU U×In G GxU U×G U UxIn/G In/G×U

105

0 In In InxU U×In G G GxU U×G U U UxIn/G In/G×U


Figure 4. Contents of 27 species-specific marker compounds for the three Glycyrrhiza species and their hybrids (µg/g in the raw herbal materials). Specific markers for G. uralensis, G. glabra, and G. inflata were colored in red, purple, and yellow, respectively. All marker compounds showed significant difference between homozygous species (P < 0.005, Mann–Whitney U-test, see Table S5). Sample volumes: In, n=11; U×In, n=2; G, n=5; U×G, n=10; U, n=60; In/G×U, n=3.

The variations of the flavonoid glycosides and triterpenoid saponins among the three species were not as significant as those of the free phenolic compounds. The

17



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Page 18 of 27

predominant saponin glycyrrhizin was evenly distributed in the three species (40.5±29.1, 49.3±15.6, and 40.6±26.4 mg/g in U, In, and G, respectively). E-ring-substituted

saponins

(e.g.,

22β-acetoxyl-glycyrrhizin)

and

11-deoxy-glycyrrhizins (33, 37, 38) were relatively abundant in G. uralensis and G. inflata, respectively (Figure S7). For flavonoid glycosides, the apiosylation ratio of liquiritin and isoliquiritin was significantly lower in G. uralensis (47±19%) than in G. glabra and G. inflata (74±13% and 82±16%, P < 0.005), whereas the content ratio of flavanone glycosides (1, 4, 5) / chalcone glycosides (8, 9, 10) was significantly higher in G. uralensis (2-5-fold, P < 0.005, Figure S7), which was consistent with previous report.24

These biosynthetic preferences allowed us to deduce the genetic differences corresponding to the biosynthesis of species-specific compounds. For example, flavones 42, 43 and 44 were distributed in all three species, whereas their 3-OH derivatives (flavonols 47, 54 and 53) were specific for G. uralensis. This result indicated the existence of specific genes encoding flavanone 3-hydroxylase and flavonol synthase or their regulators in G. uralensis.30 Similarly, genes encoding tailoring enzymes, such as coumarin monooxygenase, prenyl cyclase, and chalcone reductase may be distinctive for G. uralensis, G. glabra, and G. inflata, respectively.28 Meanwhile, the variations in the E-ring-substituted triterpenoids and apiosylated flavonoids could be related to tailoring enzymes such as β-amyrin 11-oxidase31 and apiosyltransferase. The mechanism for biosynthetic preferences based on the

18


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Glycyrrhiza genome and transcriptome warrants further study.

Inter-species Difference in Bioactivity. The content variation in chemical constituents, particularly bioactive ones, could be crucial for the bioactivity of medicinal plants. Here, we tested the Nrf2 activation activities of licorice samples derived from the three Glycyrrhiza species. Chalcones have been reported to be potent Nrf2 activators.32 As expected, G. inflata, which contained abundant chalcones, showed significantly stronger activities than the other two species (Figure S8 and Table S4). Nrf2 activation is related to anti-oxidation and liver protection. Thus, it could be speculated that G. inflata is the superior source when licorice is used for these activities. Likewise, coumarins and isoprenyl isoflavanes show significant anti-hepatitis C virus33 and anti-inflammatory34 activities, respectively. G. uralensis and G. glabra could be superior to the other species for the corresponding bioactivities.

Variation in Secondary Metabolomes for Hybrid Glycyrrhiza Species. The DNA barcoding analysis indicated that 33% of the wild-growing samples (19 out of 58 batches) were hybridized species. Among them, 15 batches were hybrids of the three medicinal species, whereas the other four batches involved hybridization with other Glycyrrhiza species, such as G. pallidiflora and G. aspera.19 The hybridization could have resulted from overlapped cultivation of Glycyrrhiza species and deficiency in species isolation.25 To our best knowledge, little attention has been paid to the impact

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of hybridization on plant secondary metabolomes. Among the few available reports, Cheng et al. determined the contents of pyrrolizidine alkaloids in hybridized Jacobaea species, though the impact of parental plants was not elucidated.35

Based on the quantitative analysis of 151 secondary metabolites, we found that hybridization remarkably changed the secondary metabolomes of Glycyrrhiza species. Furthermore, the paternal species contributed more than the maternal species to the metabolome of the offspring for licorice. Unsupervised PCA of the metabolomics data located the hybrid species between the maternal and paternal species, closer to the male parents than to the female parents (Figure 2B). This result was consistent when we analyzed the contents of the 27 species-specific markers. For instance, glycycoumarin (82, 343±225 µg/g) was a specific marker for G. uralensis. Its content in the In/G×U hybrids (121±154 µg/g) was remarkably higher than that in the U×In hybrids (14±18 µg/g). The same rule applied to all the 27 specific markers, with 50 and 126 as exceptions probably due to their relatively low abundances (Figure 4). The superior impact of paternal species on secondary metabolism could result from uniparental inheritance.36 In the case of licorice, certain organelles (plastid and mitochondria) may participate in secondary metabolism, and the hybrids obtained the biosynthesis-related organelle genes mainly from the paternal species.37,38

Implications to Quality Evaluation of Licorice. Given that species variation and

20


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hybridization could remarkably change chemical composition, it is necessary to improve the quality control of licorice. Currently, glycyrrhizin (22) and liquiritin (5) are widely used as quality control markers.5,14-16 Chinese Pharmacopoeia requires contents of glycyrrhizin and liquiritin not less than 2.0% and 0.5%, respectively. Among the 76 batches of homozygous samples we tested, 86% (65 batches) fulfilled the requirement for glycyrrhizin. However, only 19% of G. glabra and G. inflata samples contained the required amount of liquiritin, although 77% of G. uralensis samples were qualified. Based on our data, we suggest that glycyrrhizin (22, 2.0%) and liquiritin apioside (4, 0.4%), instead of liquiritin, be used as quality control markers for licorice. Liquiritin apioside showed a high content but low variation (0.94% ± 0.67% for the 76 samples) when compared to other major glycosides (Figure S7). In total, 80% (61/76) samples contained more than 0.4% of liquiritin apioside. On the other hand, HPLC fingerprinting could be used to identify Glycyrrhiza species, according to the presence of species-specific markers such as glycycoumarin, licochalcone A, and glabridin. In case some hybrids cannot be identified by HPLC, DNA barcoding may be used to accurately identify the plant species.

CONCLUSION In this study, we conducted a biosynthesis-based secondary metabolomics analysis of three medicinal Glycyrrhiza species and their hybrids. Geno- and chemotypes of 95 batches of licorice samples were revealed by DNA barcoding and LC/MS/MS

21



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quantitative analysis of 151 compounds, respectively. The three species showed significant difference in secondary metabolite profiles, especially for free phenolic compounds including coumarins, chalcones, isoflavanes, and flavonols. A total of 27 species-specific chemical markers were discovered. Moreover, we found that hybridization could remarkably alter the secondary metabolomes of licorice and that the paternal species contributed more to the offspring than the maternal species did. This is hitherto the largest-scale targeted secondary metabolomics study of medicinal plants and the first to reveal the uniparental inheritance in plant secondary metabolism.

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ACKNOWLEDGMENTS

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

We thank Dr. Zheng-xiang Zhang and Dr. Tao Bo (Agilent Technologies) for their technical help in LC/MS analysis, Dr. Qing-jun Yuan (China Academy of Chinese Medical Sciences) for providing part of the PCR primers, and Dr. Si-wang Yu (Peking University) for providing the HepG2C8 cells. This work was supported by National Natural Science Foundation of China (No. 81222054, No. 81470172).

ASSOCIATED CONTENT Supporting Information. Tables S1-S5; Figures S1-S8; Method development; Contents of 151 compounds in all samples. This material is available free of charge via the Internet at http://pubs.acs.org.

CONFLICT OF INTEREST DISCLOSURE The authors declare no competing financial interest.

26


Page 26 of 27

Page 27 of 27

For TOC Only 159 150 120

G. uralensis (n=60)

4

G. uralensis

C C

383-385 T G C T G

3

***

90

159

383-385

T C

C A A T G C

2 60

***

***

30

Contents (mg per g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

***

0 150 120

homozygous

hybrid

0

***

G. inflata (n=11)

G. inflata

4 3

90 2 60

*

30

1

***

0 150 120

0 4

G. glabra (n=5)

G. glabra

3

90

▲ U ▲× ▲×U hybrid ▲ G ▲×G ▲× hybrid ▲ In ▲×In ▲× hybrid

2 60 30

1

*

0

0

151 compounds of 15 types

Targeted Secondary Metabolomics

27


Biosynthesis-Based Quantitative Analysis of 151 Secondary

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