Perspective on Biotransformation and De Novo Biosynthesis of

Nov 27, 2017 - Licorice, an important herbal medicine, is derived from the dried roots and rhizomes of Glycyrrhiza genus plants. It has been widely us...
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Perspective Cite This: J. Agric. Food Chem. 2017, 65, 11147−11156

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Perspective on Biotransformation and De Novo Biosynthesis of Licorice Constituents Yujia Zhao,† Bo Lv,† Xudong Feng,* and Chun Li* Institute for Biotransformation and Synthetic Biosystem, Department of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, People’s Republic of China ABSTRACT: Licorice, an important herbal medicine, is derived from the dried roots and rhizomes of Glycyrrhiza genus plants. It has been widely used in food, pharmaceutical, tobacco, and cosmetics industries with high economic value. However, overexploitation of licorice resources has severely destroyed the local ecology. Therefore, producing bioactive compounds of licorice through the biotransformation and bioengineering methods is a hot spot in recent years. In this perspective, we comprehensively summarize the biotransformation of licorice constituents into high-value-added derivatives by biocatalysts. Furthermore, successful cases and the strategies for de novo biosynthesizing compounds of licorice in microbes have been summarized. This paper will provide new insights for the further research of licorice. KEYWORDS: licorice, glycyrrhizin, biological activities, biotransformation, bioengineering, synthetic biology

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INTRODUCTION Licorice, also called “Gan-Cao” in Chinese, is derived from the dried roots and rhizomes of Glycyrrhiza species, which belongs to the Leguminosae family.1 Currently, there are approximately 30 species in the Glycyrrhiza genus, which is natively distributed in most of Asia and southern Europe.2 On the basis of the Chinese Pharmacopoeia, only three species, Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata, are officially regarded as “Gan-Cao” in China. Among them, G. uralensis is the primary species, which constitutes more than 90% of the total licorice production. As one of the most famous and oldest herbal medicines, licorice has been recorded in the pharmacopoeias of many Asian and European countries, including China, Japan, and the United Kingdom.2 In China, licorice has been used as a traditional herbal medicine for thousands of years. It is an indispensable ingredient, which appears in approximately 60% of the traditional Chinese medicine prescriptions.2 Licorice exhibits a wide variety of bioactivities, including antiallergy,3 anti-inflammation,4,5 antivirus,6,7 antiasthma,8 antitumor,9−11 antidiabetes,12 antioxidant,13 hepatoprotective,14−16 neuroprotective,17 and immunomodulatory18,19 effects. These bioactivities are derived from the chemical constituents of licorice. Thus far, approximately 400 compounds20 have been identified in licorice, primarily including triterpenoid saponins and flavonoids. Licorice shows important economic value, besides the pharmaceutical industry, it is also widely used in the food, tobacco, and cosmetics industries.21 On the basis of Chinese customs data (http://www.customs.gov.cn/), the amount of the licorice trade (13021200 and 12119036) in 2016 was 34 424 020 kg with the value of 78 615 509 dollars. With the rapid growth of the world licorice trade, wild licorice resources have been overexploited, which is threatening the local ecology. To balance the economic development and ecological protection, scientists have tried to produce bioactive compounds of licorice by constructing special microbial cell © 2017 American Chemical Society

factories. In the recent 10 years, several key active ingredients of licorice have been successfully produced in microbial cell factories, such as isoliquiritigenin, liquiritigenin, and glycyrrhetinic acid (GA).22,23 However, their production is still too low for industrial application, and only a few kinds of compounds have been biosynthesized. Therefore, metabolic engineering strategies and synthetic biology methods in recent decades have been summarized to promote the biosynthesis of licorice bioactive compounds in microbes. In this perspective, a brief overview of the chemical constituents and the biotransformation processes of licorice is first provided. Then, the transcriptome and genomic information about G. uralensis is introduced to identify new synthetic pathways. Finally, biosynthesis of major bioactive compounds using microbial cell factories and the bioengineering strategies are summarized. This paper will provide new insights for the further research of licorice.

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BIOACTIVE COMPONENT AND BIOTRANSFORMATION PROCESS OF LICORICE As a traditional medicinal herb and an important industrial raw material, much attention has been paid to the isolation and identification of the bioactive compounds in licorice. Currently, approximately 400 kinds of compounds have been identified from licorice, including triterpenoid saponins, flavonoids, alkaloids, phenols, and polysaccharides.20 These compounds exhibit various bioactivities, especially triterpenoid saponins and flavonoids. All triterpenoid saponins24−39 (Figure 1) and significant flavonoids11,32,41−67 (Figure 2) in licorice are summarized in Table 1. Among them, glycyrrhizin (GL) is one of the most representative constituents of licorice. The content of GL is Received: Revised: Accepted: Published: 11147

September 26, 2017 November 23, 2017 November 27, 2017 November 27, 2017 DOI: 10.1021/acs.jafc.7b04470 J. Agric. Food Chem. 2017, 65, 11147−11156

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Figure 1. GA and triterpenoid saponins from licorice. GlcA, β-D-glucuronopyronosyl; Glc, β-D-glucopyronosyl; Xyl, β-D-xylopyronosyl; and Rha, α-Lrhamnopyronosyl.

Figure 2. Flavonoids from licorice. Api, β-D-apiofuranosyl; Glc, β-D-glucopyronosyl; and Rha, α-L-rhamnopyronosyl.

neuroprotective,29 hepatoprotective,30 and immunomodulatory31 effects, and anti-inflammation,32 antiallergy,33 antiasthma,34 and antitumor35 activities. Clinically, it is used as hepatoprotective for the treatment of chronic hepatitis B,

generally served as an indicator to characterize the quality of licorice with a minimum content of 2% in the Chinese Pharmacopoeia. As an important triterpenoid saponin of licorice, GL exhibits various pharmacological effects, including 11148

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Journal of Agricultural and Food Chemistry Table 1. Chemical Constituents and Application Isolated from Licorice number

compound

classification

1

glycyrrhetinic acid

triterpenoids

2 3

glycyrrhetinic acid 3-O-glucuronide glycyrrhizin

triterpenoid saponins triterpenoid saponins

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

uralsaponin C uralsaponin F 22-β-acetoxyl-glycyrrhizin licorice saponin G2 3-O-[β-D-glucuronopyranosyl-(1→2)-β-D-galactopyranosyl]glycyrrhetic acid uralsaponin M uralsaponin N uralsaponin P uralsaponin Q uralsaponin R uralsaponin S uralsaponin U uralsaponin V uralsaponin Y 24-hydroxy-licorice-saponin E2 licorice saponin E2 uralsaponin O uralsaponin T uralsaponin X licorice saponins A3 uralsaponin W licorice saponin J2 liquiritigenin

triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid flavanones

27

liquiritin

flavanones

28 29 30 31

liquiritigenin-7,4-diglucoside liquiritin apioside glabrol isoliquiritigenin

flavanones flavanones flavanones chalcones

32 33 34 35

isolisuiritin neoisoliquiritin licuraside licochalcone A

chalcones chalcones chalcones chalcones

36

licochalcone B

chalcones

37

glabridin

isoflavanes

38 39 40 41

2′-O-methylglabridin 4′-O-methylglabridin 2′,4′-O-dimethylglabridin licoricidin

isoflavanes isoflavanes isoflavanes isoflavans 11149

saponins saponins saponins saponins saponins saponins saponins saponins saponins saponins saponins saponins saponins saponins saponins saponins saponins saponins saponins saponins saponins saponins

application

reference

hepatoprotection anti-inflammation anticancer antioxidant sweetener neuroprotection hepatoprotection immunomodulatory anti-inflammation antianaphylaxis antiasthma anticancer a a a anti-HIV a a a a a a a a a a a a a a a a a a neuroprotection cardioprotection anticancer neuroprotection anti-inflammation anticancer a lung protection a anticancer antioxidant a a a anticancer anti-inflammation hepatoprotection anticancer cardioprotection anti-inflammation anticancer antiatherosis skin whitening a a a anticancer

24 26 25 27 28 29 30 31 32 33 34 35 36 36 36 37 38 39 39 39 39 39 39 39 39 39 36 36 39 39 39 38 38 36 40 41 42 43 32 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 62 62 11

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Journal of Agricultural and Food Chemistry Table 1. continued number 42 43 44 45 46 47 48 49 50 a

compound

classification

hispaglabridin A hispaglabridin B licoflavone A licoflavone B licoflavonol isolicoflavonol glycyrrhisoflavone licoricone licoisoflavanone

isoflavans isoflavans isoflavenes isoflavenes isoflavenes isoflavenes isoflavones isoflavones isoflavanones

application a a schistosomicidal schistosomicidal antimicrobe a anti-α-glucosidase a a

reference 62 62 63 63 64 65 66 65 67

Unreported.

Figure 3. Scheme of biotransformation of GL catalyzed by β-glucuronidase. GL, glycyrrhizin; GAMG, glycyrrhetinic acid 3-O-mono-β-glucuronide; GA, glycyrrhetinic acid; and GlcA, glucuronic acid.

hepatitis C, chronic hepatitis, liver fibrosis, liver cirrhosis, and hepatic carcinoma. Although GL exhibits various bioactivities, it is hard to be absorbed in the blood as a result of its hydrophilicity, thus disturbing ionic metabolic equilibrium, which may cause hypertension.68 GA can be derived from the complete hydrolysis of two glucuronidic acid moieties from GL. A further study has shown that GA has a higher efficacy compared to GL.68 Glycyrrhetinic acid 3-O-glucuronide (GAMG) is another important derivative of GL, which is obtained by hydrolyzing one glucuronic acid moiety. GAMG is a sweet-taste triterpenoid saponin, which imparts a strong sweetness that is approximately 941-fold of sucrose, and it has been widely used in the food and tobacco industries.28 Recently, biotransformation of GL to GAMG and GA catalyzed by β-glucuronidase has drawn much attention as a result of the mild reaction conditions and high substrate specificity. β-Glucuronidase (GUS, EC 3.2.1.31) hydrolyzes β69 D-linked glucuronides to yield various valuable derivatives. Currently, GUS from various organisms have been identified.70,71 Generally, they show three different reaction types in GL biotransformation categorized as follows: (1) GL−GAMG, (2) GL−GAMG + GA, and (3) GL−GA (Figure 3). For

example, Li and co-workers identified three GUSs from Aspergillus oryzae Li-3 (PGUS), Aspergillus terreus Li-20 (AtGUS), and Aspergillus ustus Li-62 (AuGUS), which could transform GL into GAMG and/or GA with different types.72 Structural modeling analysis showed that the different catalytic mechanisms were dependent upon the different shapes of the aglycone binding pocket caused by the bacterial loop.73 In addition to screening new GUSs and analyzing the catalytic mechanisms, some studies focus on improving functional and structural stabilities of β-glucuronidase to decrease the industrial costs. By N-glycosylation, the structure stability of PGUS has been significantly enhanced in diverse temperature, pH value, organic solvent, and detergent conditions.74

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SYNTHETIC PATHWAYS IDENTIFIED FROM GENOME AND TRANCRIPTOME ANALYSES With the fast growth of the world licorice trade, the wild licorice resource in China has been overexploited, which induced severe land desertification. The Chinese government legislated to prohibit exploitation of wild licorice in 2008 for the sustainable utilization. Currently, licorice in China is mainly harvested from artificial cultivation and imported from 11150

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Figure 4. Liquiritigenin biosynthesis pathway. PAL, phenylalanine ammonia-lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHR, chalcone reductase; and CHI, chalcone isomerase.

psc.riken.jp/Gur-genome/download.pl. On the basis of this information, 125 unigenes have been identified and annotated as enzymes involved in the flavonoid synthesis pathway, including 39 chalcone synthases (CHSs), 9 chalcone reductases (CHRs), 4 chalcone isomerases (CHIs), etc. Furthermore, G. uralensis-specific β-amyrin synthase (bAS) and 261 cytochrome P450 monooxygenases (CYP450s) have also been identified, which is beneficial for understanding the biosynthesis mechanism of licorice triterpenoids.

Kazakhstan, Uzbekistan, Afghanistan, and Azerbaijan. However, the content of bioactive constituents from the cultured licorice is much lower than that of the wild licorice. For example, the GL content from wild licorice has been reported to be approximately 4.43 ± 1.32%, while only 1.51 ± 0.49% from cultured licorice.75 In addition, the long cultivation periods for qualified roots (2−5 years) and the complicated extraction and purification processes make it hard to prepare bioactive constituents of licorice. In comparison to plants, microbes exhibit several advantages in producing chemicals, such as fast-growing, land-saving, and controllable culture conditions. With the development of synthetic biology and metabolic engineering, various valuable plant-derived compounds have been produced in microbial cell factories, including artemisinic acid,76−78 taxadiene,79,80 βamyrin,81,82 β-carotene,83,84 lycopene,85,86 astaxanthin,87,88 and ginsenoside.89−91 Notably, some major components in licorice, such as liquiritigenin23 and GA,22 have also been successfully biosynthesized in Escherichia coli and Saccharomyces cerevisiae, respectively. Transcriptome and Genome of Licorice. Bioengineering is a considerable solution to balance the large market demand and urgent plant protection. However, this strategy has long been limited by the lack of genomic information on Glycyrrhiza plants. With the development of gene sequencing technology, the transcriptome of G. uralensis was successively published in 201392 and 2015.93 The transcriptome of G. uralensis provided the expression profile of 43 882 assembled unigenes and the functional annotations. This information can be used to elucidate the biosynthetic mechanisms of important chemical constituents of licorice. All transcriptome information can be browsed through http://ngs-data-archive.psc.riken.jp/Gur/ index.pl. Recently, genomic information on G. uralensis has been published, including 34 445 predicted protein-coding genes and their annotation.94 The genome assembly and annotation data can be downloaded at http://ngs-data-archive.

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BIOSYNTHESIS OF MAJOR BIOACTIVE COMPOUNDS USING MICROBIAL CELL FACTORIES Biosynthesis of Liquiritigenin in E. coli. The biosynthesis pathway of liquiritigenin has been decoded, as shown in Figure 4. It starts with L-phenylalanin, the common precursor of the vast majority of flavonoids, and then L-phenylalanin is converted to trans-cinnamate by a phenylalanine ammonialyase (PAL). After that, cinnamic acid 4-hydroxylase (C4H), 4coumaroyl:CoA ligase (4CL), CHS, CHR, and CHI sequentially take effect to transform trans-cinnamate to liquiritigenin. In 2007, Koffas and co-workers successfully established the whole biosynthesis pathway of liquiritigenin in E. coli, and the final production was 16.9 mg/L.23 In this work, they first tested the in vitro enzyme activity of CHI from Petunia × hybrida and Medicago sativa expressed in E. coli. Furthermore, the in vivo enzyme activity of CHS from P. hybrida and M. sativa were also tested. Finally, by introduction of heterogeneous C4H from Petroselinum crispum, 4CL from P. crispum, CHS from P. hybrida, and CHR and CHI from M. sativa, liquiritigenin has been successfully biosynthesized in E. coli, with the production of 16.9 mg/L. Biosynthesis of GA in S. cerevisiae. Currently, critical genes involved in the biosynthesis of GA have been successfully cloned and characterized, including bAS (β-amyrin synthase), CYP88D6 (cytochrome P450 monooxygenase 88D6), and CYP72A154 (cytochrome P450 monooxygenase 72A154) or 11151

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Figure 5. Scheme for GA biosynthesis pathway in engineered S. cerevisiae. Single arrows represent one-step conversions, and triple arrows represent multiple steps. bAS, β-amyrin synthesis; CYP88D6, cytochrome P450 monooxygenase 88D6; CYP72A154, cytochrome P450 monooxygenase 72A154; CYP72A63, cytochrome P450 monooxygenase 72A63; CPR, cytochrome reductase; DMAPP, dimethylallyl pyrophosphate; IPP, isopentenyl pyrophosphate; and FPP, farnesyl diphosphate.

CYP72A63 (cytochrome P450 monooxygenase 72A154).22,95 As shown in Figure 5, squalene-2,3-oxide, the common precursor of triterpenoids, is converted to β-amyrin by bAS. After that, β-amyrin is oxidized into GA with 11-oxo-β-amyrin as an intermediate, catalyzed by CYP88D6 and CYP72A154/ CYP72A63. In 2011, Muranaka and co-workers first established the whole biosynthesis pathway of GA in S. cerevisiae, and the production was 15 μg/L. In this work, they first tested the in vitro enzyme activity of CYP72A154 from G. uralensis. The results demonstrated that CYP72A154 is capable of catalyzing three sequential oxidation steps at C-30 of 11-oxo-β-amyrin to produce GA. In addition, by introduction of bAS and CPR (cytochrome P450 reductase) from Lotus japonicas and CYP88D6 and CYP72A154 from G. uralensis under the control of Gal-inducible promoters (GAL10 or GAL1), GA has been successfully synthesized in S. cerevisiae.22 Although the production of GA is only 15 μg/L, this study shed light on the potential for using microbes to produce bioactive triterpenoids of licorice. Strategies for Improving the Production. Although liquiritigenin and GA have been successfully biosynthesized in microbes, their production is poorly low. Here, we summarize strategies for enhancing the production of licorice-derived compounds in microbes, which may be used to further increase liquiritigenin and GA production. Generally, balancing metabolic flux is a high-efficiency strategy. By improvement of the precursor supply and/or reduction of metabolic flow on competition pathways, production of plant-derived compounds can be increased. For example, the titer of β-amyrin, the precursor of GA, was improved up to 138.80 mg/L by overexpressing isopentenyl pyrophosphate (IPP) isomerase, farnesyl diphosphate (FPP) synthase, squalene synthase, and transcription factor UPC2, almost 185-fold of the initially engineered strain.82 The titer of sesquiterpenoid nerolidol was increased by 86% (to ∼100 mg/ L) by dynamic control of squalene synthase expression.96 Promoter engineering is widely used to balance metabolic flux.

Furthermore, transcription factors also act as a controller for regulating promoters. Yu and co-workers have developed a twolevel expression enhancement system to improve isoprene production in S. cerevisiae. The competition for GAL4p was avoided by the overexpression of GAL4, transcription activating factor, and deletion of the native GAL1/7/10 promoters. Meanwhile, GAL80, the transcription inhibitory factor, was disrupted to eliminate the dependency of gene expression on galactose induction. The IspS expression was obviously elevated, and the isoprene production was improved from 6.0 to 23.6 mg/L.97 Protein engineering is another efficient strategy to increase the target compound titer. By a semi-rational design of UGT51, a UDP-glucose:sterol glucosyltransferase from S. cerevisiae, its catalytic efficiency for converting protopanaxadiol to ginsenoside Rh2 was increased by approximately 1800-fold in vitro. Introducing the glycosyltransferase mutant gene into yeast increased the Rh2 yield up to 0.39 mg/g.90 Because the precursor was not fully consumed by CYP72A154-mediated oxidation, there is potential for yield improvements through protein engineering of CYP72A154 to enhance its catalytic activity and/or modulate its product specificity. Furthermore, CYP88D6 and CYP72A154 belonging to the P450 family are located on the endoplasmic reticulum membrane of S. cerevisiae.22 Goossens and co-workers have developed a novel yeast strain with engineered endoplasmic reticulum as a platform for overproduction of triterpenoids.81 They dramatically expanded the endoplasmic reticulum by disrupting PAH1 (phosphatidic acid phosphatase) of S. cerevisiae, which stimulated the expression of recombinant P450 enzymes and ultimately boosted triterpenoid accumulation. In comparison to the original strain, production of βamyrin and medicagenic acid were increased by 8- and 6-fold, respectively. Hence, this may be a promising strategy to increase the yield of GA and its derivatives in microbes. In summary, licorice is widely used in pharmaceutical, food, tobacco, and cosmetic industries, as a result of a great variety of bioactive constituents, such as triterpenoid saponins and 11152

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Journal of Agricultural and Food Chemistry flavanoids. With the fast growth of the international licorice trade, wild licorice resources have been overexcavated, which is threatening the local ecological environment. Bioengineering is considered to balance the large market demand and ecology protection. Recently, the transcriptome and genomic information of licorice has been published. This genetic information dramatically boosted producing bioactive compounds of licorice in microbes. Thus far, liquiritigenin and GA have been successfully biosynthesized in E. coli and S. cerevisiae, respectively. However, this strategy is still far from industrial production as a result of the low titer. With the rapid development of synthetic biology and metabolic engineering, further studies should be focused on improving yield through metabonomics, protein engineering, and cellular morphological engineering. In addition, more licorice bioactive compounds, such as GL and GAMG, may be biosynthesized in microbes. GL and GAMG are derived from the glycosylation of GA catalyzed by glycosyltransferase (GT). Although GT that can catalyze GA to GL/GAMG is still not yet available, there are 91 predicted protein-coding genes annotated as GT in the G. uralensis genome database, which may prompt the biosynthesis of GL and GAMG in G. uralensis.

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(7) Fukuchi, K.; Okudaira, N.; Adachi, K.; Odai-Ide, R.; Watanabe, S.; Ohno, H.; Yamamoto, M.; Kanamoto, T.; Terakubo, S.; Nakashima, H.; Uesawa, Y.; Kagaya, H.; Sakagami, H. Antiviral and Antitumor Activity of Licorice Root Extracts. In Vivo 2016, 30, 777−786. (8) Yang, N.; Patil, S.; Zhuge, J.; Wen, M. C.; Bolleddula, J.; Doddaga, S.; Goldfarb, J.; Sampson, H. A.; Li, X. M. Glycyrrhiza uralensis Flavonoids Present in Anti-Asthma Formula, ASHMITM, Inhibit Memory Th2 Responses in Vitro and in Vivo. Phytother. Res. 2013, 27, 1381. (9) Chung, W. T.; Lee, S. H.; Kim, J. D.; Sung, N. S.; Hwang, B.; Lee, S. Y.; Yu, C. Y.; Lee, H. Y. Effect of the extracts from Glycyrrhiza uralensis Fisch on the growth characteristics of human cell lines: Antitumor and immune activation activities. Cytotechnology 2001, 37, 55− 64. (10) Zeng, G.; Shen, H.; Tang, G.; Cai, X.; Bi, L.; Sun, B.; Yang, Y.; Xun, W. A polysaccharide from the alkaline extract of Glycyrrhiza inf lata induces apoptosis of human oral cancer SCC-25 cells via mitochondrial pathway. Tumor Biol. 2015, 36, 6781. (11) Ji, S.; Tang, S.; Li, K.; Li, Z.; Liang, W.; Qiao, X.; Wang, Q.; Yu, S.; Ye, M. Licoricidin inhibits the growth of SW480 human colorectal adenocarcinoma cells in vitro and in vivo by inducing cycle arrest, apoptosis and autophagy. Toxicol. Appl. Pharmacol. 2017, 326, 25. (12) Nakagawa, K.; Kishida, H.; Arai, N.; Nishiyama, T.; Mae, T. Licorice flavonoids suppress abdominal fat accumulation and increase in blood glucose level in obese diabetic KK-A(y) mice. Biol. Pharm. Bull. 2004, 27, 1775. (13) Chin, Y. W.; Jung, H. A.; Liu, Y.; Su, B. N.; Castoro, J. A.; Keller, W. J.; Pereira, M. A.; Kinghorn, A. D. Anti-oxidant constituents of the roots and stolons of licorice (Glycyrrhiza glabra). J. Agric. Food Chem. 2007, 55, 4691−7. (14) Son, M.; Lee, M.; Sung, G. H.; Lee, T.; Shin, Y. S.; Cho, H.; Lieberman, P. M.; Kang, H. Bioactive activities of natural products against herpesvirus infection. J. Microbiol. 2013, 51, 545−551. (15) Rasool, M.; Iqbal, J.; Malik, A.; Ramzan, H. S.; Qureshi, M. S.; Asif, M.; Qazi, M. H.; Kamal, M. A.; Chaudhary, A. G. A.; Al-Qahtani, M. H.; Gan, S. H.; Karim, S. Hepatoprotective Effects of Silybum marianum (Silymarin) and Glycyrrhiza glabra (Glycyrrhizin) in Combination: A Possible Synergy. Evidence-Based Complementary Altern. Med. 2014, 2014, 1−9. (16) Jung, J.-C.; Lee, Y.-H.; Kim, S. H.; Kim, K.-J.; Kim, K.-M.; Oh, S.; Jung, Y.-S. Hepatoprotective effect of licorice, the root of Glycyrrhiza uralensis Fischer, in alcohol-induced fatty liver disease. BMC Complementary Altern. Med. 2015, 16, 1−10. (17) Hosseinzadeh, H.; Nassiri-Asl, M. Pharmacological Effects of Glycyrrhiza spp. and Its Bioactive Constituents: Update and Review. Phytother. Res. 2015, 29, 1868. (18) Guo, A.; He, D.; Xu, H. B.; Geng, C.-A.; Zhao, J. Promotion of regulatory T cell induction by immunomodulatory herbal medicine licorice and its two constituents. Sci. Rep. 2015, 5, 14046. (19) Ayeka, P. A.; Bian, Y.; Mwitari, P. G.; Chu, X.; Zhang, Y.; Uzayisenga, R.; Otachi, E. O. Immunomodulatory and anticancer potential of Gan cao (Glycyrrhiza uralensis Fisch.) polysaccharides by CT-26 colon carcinoma cell growth inhibition and cytokine IL-7 upregulation in vitro. BMC Complementary Altern. Med. 2016, 16, 1−8. (20) Zhang, Q.; Ye, M. Chemical analysis of the Chinese herbal medicine Gan-Cao (licorice). J. Chromatogr. 2009, 1216, 1954. (21) Hayashi, H.; Sudo, H. Economic importance of licorice. Plant Biotechnol. 2009, 26, 101−104. (22) Seki, H.; Sawai, S.; Ohyama, K.; Mizutani, M.; Ohnishi, T.; Sudo, H.; Fukushima, E. O.; Akashi, T.; Aoki, T.; Saito, K.; Muranaka, T. Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin. Plant Cell 2011, 23, 4112−4123. (23) Yan, Y.; Huang, L.; Koffas, M. A. G. Biosynthesis of 5deoxyflavanones in microorganisms. Biotechnol. J. 2007, 2, 1250. (24) Zhu, X.; Tsendayush, A.; Yuan, Z.; Wen, J.; Cai, J.; Luo, S.; Yao, J.; Bian, J.; Yin, L.; Zhou, J.; Yao, J. Glycyrrhetinic acid-modified TPGS polymeric micelles for hepatocellular carcinoma-targeted therapy. Int. J. Pharm. 2017, 529, 451−464.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Bo Lv: 0000-0001-7879-0741 Chun Li: 0000-0003-2517-9644 Author Contributions †

Yujia Zhao and Bo Lv contributed equally in this work.

Funding

The authors kindly acknowledge financial support from the National Science Foundation for Distinguished Young Scholars of China (21425624) and the National Natural Science Foundation of China (21506011 and 21476026). Notes

The authors declare no competing financial interest.

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