Liquorice, a Unique “Guide Drug” of Traditional Chinese Medicine: a Review of Its Role in Drug Interactions

Home , Glycyrrhizin, Herb, Zhang Zhongjing

Journal of Ethnopharmacology 150 (2013) 781–790

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Journal of Ethnopharmacology

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Review Liquorice, a unique “guide drug” of traditional Chinese medicine: A review of its role in drug interactions

Xiaoying Wang a,c, Han Zhang b,c, Lili Chen b,c, Lihua Shan a, Guanwei Fan b,c, Xiumei Gao b,c,n a College of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China b Institute of Traditional Chinese Medicine Research, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China c Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae, Ministry of Education, Tianjin 300193, China article info abstract

Article history: Ethnopharmacological relevance: Liquorice is the root of Glycyrrhiza uralensis Fisch. or Glycyrrhiza glabra L., Received 23 June 2013 Leguminosae. It is a widely used herbal medicine native to southern Europe and parts of Asia and has Received in revised form beneficial applications in both the medicinal and the confectionery sectors. Unlike its usage in Europe, 24 September 2013 liquorice in traditional Chinese medicine is commonly combined with other herbs in a single prescription, Accepted 24 September 2013 as a unique “guide drug” to enhance the effectiveness of other ingredients, to reduce toxicity, and to Available online 5 November 2013 improve flavor in almost half of Chinese herbal formulas. A review on phytochemical and pharmacological Keywords: research to explain this unique “guide” effect is suggested for future investigations. Liquorice Materials and methods: The information was collected from scientific journals, books, and pharmacopeia. The Cytochrome P450 studies about the traditional uses, randomized controlled trials, chemical, pharmacological and pharmaco- Drug interactions kinetic data related to liquorice–herb/drug interaction or combination were included in the review. Traditional Chinese medicine Results: According to recent reports, the “guide” effect of liquorice is partially through components Detoxification transformed in liquorice–drug interaction; altering enzyme activity of P450 isoforms, as evidenced by induction of model probe substrates; and modulation of drug transporter proteins such as intestinal P-glycoprotein. Conclusion: The overview and comparison of traditional uses of liquorice with recent pharmacological studies and randomized controlled trials provide new insights into this ancient drug for future investigations and clinical use, especially in drug combination. & 2013 Elsevier Ireland Ltd. All rights reserved.

Contents

1. Introduction ...... 782 2. Liquorice in TCM theories ...... 782 3. Modern pharmacological experiments and clinical trials of liquorice...... 782 4. Component analysis in liquorice–drug interaction ...... 783 5. The effects of liquorice and its active components on model probe substrates of CYP isoforms ...... 785 6. The effects of liquorice/active ingredients on drug transporter proteins ...... 786 7. Adverse effects and toxicological studies of liquorice ...... 788 8. Conclusions ...... 788 Acknowledgements ...... 788 References...... 788

Abbreviations: CYP, cytochrome; DGLL, diammonium glycyrrhizinate lipid ligand; GA, glycyrrhetic acid; GZ, glycyrrhizin; HSYA, hydroxysafﬂor yellow A; IC50, half maximal inhibitory concentration; P-gp, P-glycoprotein; TCM, traditional Chinese medicine n Corresponding author at: Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae, Ministry of Education, Tianjin University of Traditional Chinese Medicine, # 312 Anshanxi Road, Nankai District, Tianjin 300193, China. Tel.: þ86 22 59596586. E-mail address: [email protected] (X. Gao).

1. Introduction liquorice is based on the ability to tonify the qi (life energy) of the heart and spleen. Liquorice is also useful for relieving the follow- Herbal products are becoming popular as alternative medicines ing conditions: phlegm, cough, dyspnea, spasms, pain, and toxicity worldwide. Herb–drug and herb–herb interactions are a current and cooperates with other medicines. As a tonic herbal medicine topic of debate, while combination therapies have been validated in TCM, liquorice is a high-grade herb, non-toxic and sweet, and it and show potential clinical benefits. Traditional Chinese Medicine is slightly “cold” in nature. It is combined in various formulas due (TCM) has focused on this issue because most herbs are combined to its ability to harmonize the characteristics of other herbs. with others in a single prescription. The most notable of these is Through detoxification, it can reduce the toxic properties of certain liquorice (Glycyrrhiza), which appears in approximately 60% of herbs. Through harmonizing, it can improve the synergy between TCM prescriptions (Xing et al., 2011). “cold” and “heat” herbs in a formula, alleviate the toxicity of herbs, Liquorice (Radix Glycyrrhizae, Liquiritiae radix) is the root of and modulate the taste of herbs due to its sweet flavor. Glycyrrhiza uralensis Fisch. or Glycyrrhiza glabra L. or Glycyrrhiza After the mention of liquorice in Shen Nong Ben Cao Jing, all inflata Bat., Leguminosae. It is a widely used herbal medicine subsequent TCM literature describes liquorice as an essential herb native to southern Europe and parts of Asia and has beneficial with a variety of usages. In 283 herbal prescriptions in “Shang Han applications both in the medicinal and the confectionery sectors. Za Bing Lun” (which is among the oldest complete clinical text- The sweet-tasting compound makes the liquorice root in popular books in the world and compiled by Zhang Zhongjing prior to 220 candies or sweets in Europe, and meanwhile a history of its A.D.), liquorice was used in 140 formulas. In Tai Ping Hui Min He Ji various therapeutic applications also has been documented Ju Fang, the first official formula book in China (Song Dynasty, 960- (Fiore et al., 2005). Unlike its usage in Europe, in China, liquorice 1279), liquorice appeared in 261 prescriptions among 788 listed is one of the most widely used herbal medicines, as it appears in herbal formulae. Therefore, classical texts state that “nine out more than half of TCM prescriptions. According to TCM theory, of ten herbal formulas contain liquorice” (Wang et al., 2002). liquorice is primarily effective for fatigue and debilitation, asthma A summary of historical references having important formulas that with coughing, excessive phlegm and for relieving drug toxicity. contain liquorice is shown in Table 1. Moreover, it has a unique effect on moderating and complement- We analyzed the frequency of herbs that were combined with ing the characteristics of other herbs, leading to a special guide liquorice in Shang Han Za Bing Lun. The ten herbs with the highest role in many Chinese herbal prescriptions (Commission, 2010). frequencies are the following: Fructus jujubae (Da Zao, 63 times), Unlike European herbal medicine, in which herbs are often Ramulus Cinnamomi (Gui Zhi, 63 times), Rhizoma Zingiberis used alone, TCM uses several herbs in one prescription. According Recens (Sheng Jiang, 60 times), Radix Paeoniae Alba (Shao Yao, to the theories of TCM, the prescriptions are classified as monarch 43 times), Herbal Ephedrae (Ma Huang, 28 times), Radix Ginseng (main) drugs, minister drugs, assistant drugs, and guide drugs (Ren Shen, 27 times) Rhizoma Pinelliae (Ban Xia, 26 times), (Steven and Yue, 1992). Monarch drugs are those that exert the Rhizoma Zingiberis (Gan Jiang, 26 times), Radix Aconiti Lateralis primary effect on the health condition. Assistant drugs cooperate Praeparata (Fu Zi, 20 times), Poria (Fu Ling, 17 times), and Gypsum with the major ingredient in a prescription to enhance its effect. Fibrosum (Shi Gao, 17 times), which comprised approximately 50% Minister drugs synergistically increase the effect of the monarch of the drugs combined with liquorice (Fig. 1). Of these top ten drug. Meanwhile, the "guide drug" is added to enhance the herbs, six are warm in nature, and two are hot in nature which effectiveness of other ingredients, to reduce toxicity or to improve refers medicine's effect on our bodies. In TCM theory, all medicinal taste. Although it has multiply pharmacological effects, liquorices are ascribed a nature, cold, cool, neutral, warm, or hot. “Cool” and is more commonly used as a unique “guide drug” in more than half “cold” herbs treat heat diseases like high fever, strong sweating, of all traditional and modern formulas and prescriptions, which and big thirst. “Warm” and “hot” herbs treat cold diseases like cold accounts for its unique effect on moderating the characteristics of limbs, aversion to cold, watery diarrhea (Ergil and Ergil, 2009). other herbs in low dosages (Wang and Gong, 2013). The moderat- According to TCM theory, “warm”/“hot” drugs are more likely than ing effects of liquorice on other herbs include a marked detoxify- “cold” drugs to lead to serious adverse reactions of the respiratory ing effect that can treat drug and food poisoning and/or inhibit system and to cause cardiovascular damage. “Hot” drugs, such as adverse effects, and this “guide” effect has been examined in a Rhizoma Zingiberis and Radix Aconiti Lateralis Praeparata, are number of formulas (Chen and Xu, 2006; Jin, 2000). The exact more likely to cause systemic numbness, a reduction in reflexes, mechanism of this action is not entirely clear, but is believed to be difficulty in swallowing, insomnia, and blurred vision (Zhao et al., related to the chemical composition changes that occur during co- 2011b). Therefore, the “cold” nature of liquorice may help to decoction and its regulation of both cytochrome (CYP) P450 harmonize other herbs in a formula to reduce these adverse enzymes and drug transporter proteins when interacting with reactions. It is the most common of the “protecting medicinals” other drugs (Liu et al., 2012a; Pan et al., 2008). The purpose of this (Ergil and Ergil, 2009). Radix Aconiti Lateralis Praeparata is a toxic review is to summarize and merge traditional knowledge and herb (Wong et al., 2002); combining it with liquorice has shown contemporary evidencesknowledge of liquorice, especially on key significant detoxification in clinical usage (Yang et al., 2009). pharmacokinetic steps which, taken together, can give some Furthermore, the sweetness helps liquorice to play a role in explanation of its traditional use as a “guide drug” to enhance ameliorating the strong effects of certain medicines, for example the effectiveness of other ingredients and to reduce toxicity in a the intensively purgative action of Radix et Rhizoma Rhei and number of formulas. The information collected here will help in Natrii Sulfas (Chen, 2007). clinical use and developing further indications for this ancient Liquorice is indexed in the 2010 version of the Chinese Pharm- herb. acopoeia, with more than 320 formulations and concoctions containing liquorice as a principal or assistant drug among a total of 1062 recorded prescriptions. This number has increased from 166 in the previous version (2005) of Chinese Pharmacopoeia, 2. Liquorice in TCM theories indicating a sustained development in modern clinical application of liquorice. The theory and written discussion of TCM are difficult The first documented medical use of liquorice in China can be to understand and may restrict the elucidation of the pharmaco- traced back to the oldest source on remedies, the Shen Nong Ben logical actions of liquorice. However, since the 1970s, modern Cao Jing, which was created in 200 B.C. The major medicinal use of research has made great progress in characterizing the chemical X. Wang et al. / Journal of Ethnopharmacology 150 (2013) 781–790 783

Table 1 Summary of liquorice formulas in historical references.

Apparatus Period Formulas Effects Involved TCMs except liquorice Books

Digestive system I–II century A.D. Gancao Xiexin Tang Restorative, regulates stomach, relieves Rhizoma Pinelliae, Radix Scutellariae, Jin Gui Yao Lue fullness and vomiting Rhizoma Zingiberis, Radix Ginseng, Rhizoma Coptidis,Fructus Jujubae Digestive system X century A.D. Si Jun Zi Tang Restorative, tonifying spleen, Radix Ginseng, Rhizoma Atractylodis Tai Ping Hui Min promoting digestion Macrocephalae, Poria He Ji Ju Fang Digestive system I–II century A.D. Dahuang Gancao Purgative Radix et Rhizoma Rhei Jin Gui Yao Lue Tang Respiratory system I–II century A.D. Shaoyao Gancao Spasmolysis, relieve pain Radix Paeoniae Alba Shang Han Lun Tang Respiratory system I–II century A.D. Gancao Ganjiang Pulmo-peritoneal fistula Rhizoma Zingiberis Shang Han Lun Tang Respiratory system I–II century A.D. Ma Xing Gan Shi Relieving syndrome of influenza, Herba Ephedrae, Semen Armeniacae Shang Han Lun Tang relieving asthma Amarum, Gypsum Fibrosum Respiratory system I–II century A.D. Mahuang Tang Antipyretic, relieving syndrome of Herba Ephedrae, Ramulus Cinnamomi, Shang Han Lun influenza Semen Armeniacae Amarum Respiratory system X century A.D. Liangge San Antipyretic, antibechic Radix et Rhizoma Rhei, Natrii Sulfas, Tai Ping Hui Min Fructus Gardeniae, Herba Menthae, He Ji Ju Fang Radix Scutellariae, Fructus Forsythiae, Herba Lophatheri, Mel Respiratory system X century A.D. San Ao Tang Relieving syndrome of influenza and Herba Ephedrae, Semen Tai Ping Hui Min asthma Armeniacae Amarum He Ji Ju Fang Cardiovascular system I–II century A.D. Gan Mai Dazao Nourishing heart and quieting spirit Fructus Tritici Levis, Fructus Jujubae Jin Gui Yao Lue Tang Cardiovascular system I–II century A.D. Zhigancao Tang Restorative, nourishing blood, restoring Radix Ginseng, Radix Rehmanniae, Shang Han Lun the circulation of blood. Ramulus Cinnamomi, Colla Corii Asini, Radix Ophiopogonis, Frutus Cannabis, Rhizoma Zingiberis Recens, Fructus Jujubae Circulation system I–II century A.D. Gancao Mahuang Treating edema Herba Ephedrae Jin Gui Yao Lue Tang Circulatory system I–II century A.D. Pai Nong Tang Apocenosis Radix Platycodonis, Rhizoma Zingiberis, Jin Gui Yao Lue Fructus Jujubae

Fig. 1. Top ten herbs in Shang Han Za Bing Lun that are combined with liquorice. composition and mechanism of action of liquorice and in defining acid. Liquorice roots are comprised of approximately 3–5% 18β- its drug interactions. glycyrrhizic acid (18β-glycyrrhizin), which is considered to be the primary active ingredient (Yu et al., 2012). Chemical changes in glycyrrhizin upon roasting involve its conversion to 18-glycyrrhetic 3. Modern pharmacological experiments and clinical acid 3-omonoglucuronide and glycyrrhetic acid (Sung and Li, 2004). trials of liquorice Approximately 50 other minor oleanane-type saponins have been isolated from liquorice (Zhang and Ye, 2009). Other constituents Despite being used for several centuries, with its effectiveness include triterpenoid saponins (glabridin, glycyrrhetol and glabrolide), noted in the ancient Chinese and European materia medica, the isoflavones (formononetin, neoliquiritin and glabrone), triterpene mechanisms of action of liquorice and its constituents remain sterols (onocerin, beta-amyrin and stigmasterol), and coumarins unknown. In recent years, the chemical constituents of liquorice (herniarin and umbelliferone). The pharmacological actions of the have been investigated extensively (Kitagawa, 2002). The primary primary active components of liquorice are summarized in Table 2. constituents of liquorice are the triterpenoid saponin, glycyrrhizin, Several randomized controlled trials of liquorice efficacy were and a mixture of calcium and potassium salts of glycyrrhizinic reported in recent years (Table 3). Either the extract of liquorice or 784 X. Wang et al. / Journal of Ethnopharmacology 150 (2013) 781–790

Table 2 Pharmacological actions and possible mechanisms of the primary active components of liquorice.

Active components Effects Experimental Dose range Minimal active Mechanism of liquorice model tested concentration

18β-Glycyrrhetinic Anti-inﬂammation Mice 75 mg/kg 75 mg/kg Reduces MIP-1-alpha expression (Xiao et al., 2010) acid Anti-cancer Mice 10–80 μg/mL 20 μg/mL Reduces T cell apoptosis and regulatory T cell expression (Kuang et al., 2013) MCF-7 cells 25–200 μM100μM Induces apoptosis through Akt/FOXO3a/Bim pathway (Sharma et al., 2012) SiHa cells 1–100 μM10μM Induces Bax and cytochrome c levels, reduces Bcl-2 level, caspase-3 and the loss of the mitochondrial transmembrane potential (Lee et al., 2008) Anti-obesity Mouse 3T3-L1 1–40 μM1μM Alters fat mass by directly affecting adipogenesis (Moon et al., 2012) cells Inhibits cholestasis Rats 10 mg/kg 10 mg/kg Preserves the bile ﬂow and the accumulative bile excretion of ketoprofen glucuronide (Zhai et al., 2007) Antiallergic effect Rats 5–50 mg/kg 50 mg/kg Inhibits the production of IgE (Shin et al., 2007)

Glycyrrhizin Immunomodula- Mouse splenic 0.1–200 μg/mL 1 μg/mL Up-regulates the expression of CD40, CD86 and MHC-capital I; (Glycyrrhizic acid) tory dendritic cells enhanced proliferation of allogenic T cells (Bordbar et al., 2012) Anti-ocular Rabbits 25 mg (half 25 mg Decreases intraocular pressure and the amplitudes of flash hypertension Glycyron electroretinogram and flash visual evoked potential tablets) (Song et al., 2011) Protective effect on HEK293 cells 1–30 μM10μM Against beta(2)–AR agonist-induced receptor internalization and respiratory system apoptosis (Shi et al., 2011) Antidiabetic effect Rats 100 mg/kg 100 mg/kg Against hyperglycemia, hyperlipidemia and associated oxidative stress (Sen et al., 2011) Liver protection Rats/Kupffer cells 60 mg/kg 60 mg/kg Decreases serum HMGB1 levels and lipo-MDP, prevents liver injury after I/R (Ogiku et al., 2011) Mice 10–50 mg/kg 10 mg/kg Inhibits ConA-induced phosphorylation of JNK, ERK and PI3K/AKT (Tu et al., 2012) Anti-inflammation Mice 10–50 mg/kg 10 mg/kg Against LPS-induced ALI, suppresses COX-2 and iNOS expression (Ni et al., 2011) Neuroprotection Rats 10 mg/kg 10 mg/kg Anti-inflammatory by inhibiting HMGB1 secretion, anti-excitotoxic, and anti-oxidative (Kim et al., 2012a) Inhibits cholestasis Rats 100 mg/kg 100 mg/kg Preserves the bile flow and the accumulative bile excretion of ketoprofen glucuronide (Zhai et al., 2007) Antiallergic effect Rats 5–50 mg/kg 50 mg/kg Inhibits the production of IgE (Shin et al., 2007)

Liquiritigenin Anti-cancer Mice 10–40 mg/kg 10 mg/kg Downregulates VEGF (Liu et al., 2012b) A549 cells 10–100 μM25μM Reduces the expression of promatrix metalloproteinase-2 through suppression on PI3K/Akt signaling pathway (Wang et al., 2012b) Anti-inﬂammation D10.G4.1 cells 1.56–25 μg/mL 3.12 μg/mL Suppresses IL-4 and IL-5 production (Yang et al., 2012) Angiogenesis HeLa cells 25–100 μM25μM Inhibits HIF-1 alpha and VEGF expression via the AKT/mTOR-p70S6K signaling pathway (Xie et al., 2012)

Isoliquiritin Antigenotoxicity human 5.96–191 μM 11.93 μM Decreases the SOS inducing potency of hydrogen peroxide and NQO peripheral blood (Kaur et al., 2009) lymphocytes Antidepressant Mice 10–40 mg/kg 10 mg/kg Increases 5-HT and NE in the mouse hippocampus, hypothalamus and cortex (Wang et al., 2008) Anti-inﬂammation RAW 264.7 cells 0.4–1.6 μM 0.4 μM Down-regulates iNOS, COX-2, TNF-alpha, and IL-6 due to NF-kappaB inhibition via the suppression of IKK, ERK1/2 and p38 phosphorylation (Kim et al., 2008)

Licochalcone A Antiobesity Mice 5–10 mg/kg 10 mg/kg Suppresses the differentiation of 3T3-L1 preadipocytes, reduces body weight, triglyceride, cholesterol, and non-esterified fatty acid levels (Quan et al., 2012) Osteogenic activity MC4/C2C12 cells, 2.5–10 μM5μM Enhances BMP-2-stimulated expression by the activation of ERK zebrafish, mice (Kim et al., 2012b) Antiangiogenic HUVECs/rat 2.5–20 μM5μM Blocking VEGF/VEGFR-2 signaling (Kim et al., 2010b) effect aortic rings/mice Antitumor and Mice 5–30 mg/kg 15 mg/kg Reduces tumor formation, increases cell nuclear antigen, antimetastatic beta-catenin, COX-2, and iNOS, decreases proinflammatory effects cytokines and chemokines (Kim et al., 2010a) Anti-inflammation NIH 3T3 Cells 10–30 μM10μM Inhibits nuclear factor-kappaB activation through the inhibition of IkappaB kinase complex activation (Funakoshi-Tago et al., 2009)

Licochalcone E Neuroprotection neuronal and 0.1–5 μM1μM Activates Nrf2/antioxidant response element signaling pathway microglial cells (Kim et al., 2012c) Antimicrobial effect Bacterial strains 1/32–1/4 MIC 1/32 MIC Reduces the production of alpha-toxin (Zhou et al., 2012) (MIC 1–4 mg/ml) Antidiabetic effect 3T3-L1 5–20 μM5μM Increases PPAR-gamma expression, adipocyte differentiation and the preadipocyte and population of small adipocytes (Park et al., 2012) C3H10T1/2 stem cells Induces apoptosis ECV304 cells 5–50 μM15μM Induces endothelial apoptosis by modulating NF-kappaB and the Bcl-2 family (Chang et al., 2007) X. Wang et al. / Journal of Ethnopharmacology 150 (2013) 781–790 785

Table 3 Randomized controlled trials of liquorice efﬁcacy.

Indication Year Patients Drug/formula Conclusion

Metabolic syndrome 2012 106 Extractives from red yeast rice, bitter gourd, Improve total cholesterol, low-density lipoprotein cholesterol, chlorella, soy protein, and licorice and triglyceride (Lee et al., 2012) Non-alcoholic fatty 2012 66 Aqueous licorice root extract Decreasing transaminase activities liver disease (Hajiaghamohammadi et al., 2012) (NAFLD) Functional Dyspepsia 2012 50 Extract of Glycyrrhiza glabra (GutGard) Decrease in symptoms scores in concordance with improvements in almost all individual symptoms (Raveendra et al., 2012) Adiposity 2011 22 and 23 Licorice flavonoid oil (LFO) Little effect of LPO on anthropometric and biochemical markers of individually health and adiposity (Bell et al., 2011) Bronchial asthma 2010 63 A combination of Boswellia serrata, licorice root Pronounced effect on anti-inflammatory and inhibit leukotriene (Glycyrrhiza glabra) and Tumeric root (Curcuma longa) (Houssen et al., 2010) Asthma 2009 20 A novel three-herb antiasthma herbal medicine ASHMI appeared to be safe and well tolerated by subjects with (phase I trial) intervention (ASHMI, includes Ganoderma lucidum, asthma (Kelly-Pieper et al., 2009) Sophora flavescens, and Glycyrrhiza uralensis) Chronic liver diseases 2009 480 Magnesium isoglycyrrhizinate Effective and safe treatment for chronic liver diseases (Mao et al., 2009) Recurrent aphthous 2009 15 Licorice patches Reduce of pain and the inflammatory halo and necrotic center of stomatitis aphthous ulcers (Moghadamnia et al., 2009) Mental arithmetic 2008 10 The combination of ginseng, oriental bezoar and Reduce mental stress by autonomic nervous activity and immune stress glycyrrhiza system (Zheng and Moritani, 2008) Chronic B-type 2002 80 α and β Glycyrrhizic acid individually β Glycyrrhizic acid had a better clinical effect than α type Hepatitis (Cao et al., 2002)

the combination with other herbs showed the effect mainly in Semen Strychni is an extremely toxic herbal medicine. How- metabolic syndrome, asthma, and chronic liver diseases. ever, it has superior therapeutic action in the treatment of arthritis, rheumatoid arthralgia, numbness and paralysis (Jia et al., 2009). The toxicity of strychnine and brucine were significantly decreased when Semen Strychni was combined with 4. Component analysis in liquorice–drug interaction liquorice, providing the scientific basis for the detoxification action of liquorice (Yan et al., 2007, 2009). In the process of the co-decoction of prescribed herbs, chemical Changes in other chemical compounds following liquorice– constituents may change due to the solvent or heating, which is herb combinations have also been reported. For example, by considered to be the basic mechanism for their pharmacological HPLC–TOF–MS, 13 more chemical compounds were observed after effects. Liquorice, when combined with toxic herbs, such as Radix a combination of liquorice and Rhizoma Coptidis (Huang Lian) in Aconiti Lateralis Praeparata (Fu Zi), Rhizoma Dioscoreae Bulbiferae comparison to single herb decoction (Li et al., 2007). The combi- (Huang Yao Zi) and Semen Strychni (Ma Qian Zi), was shown to nation of Ramulus Cinnamomi (Gui Zhi) with liquorice did not reduce toxic intermediates. Moreover, in certain formulas, liquor- result in the production of novel substances but caused a sig- ice may increase the activity of components of other herbs to nificant increase in the concentration of primary component, improve the efficacy. cinnamaldehyde (Zhou et al., 2009). Radix Aconiti Lateralis Praeparata is an herb that is used in As previously discussed, liquorice is commonly used in TCM emergency situations for its cardiovascular effects. However, it is formulas. Many research efforts on herb interactions have focused used with great caution because of its relatively narrow therapeutic on alterations in chemical constituents. In the research of Chai Hu 7 index. Aconitines from Radix Aconiti Lateralis Praeparata were Shu Gan powder, an L8 (2 ) orthogonal design method was used to proven to have cardiac toxicity (Wang and Xu, 2009). Liquorice determine the content of saikosaponin A in compatibility samples and Radix Aconiti Lateralis Praeparata are commonly combined for using HPLC/DAD. The results showed that liquorice, when used as clinical use. According to the prescription database analysis platform a guide drug in the prescription, could significantly increase the of Radix Aconiti Lateralis Praeparata, out of a total of 3188 prescrip- dissolution of the monarch drug (radix bupleuri, Chai Hu). The tions, 29.52% are combined with liquorice. Liquorice significantly effect was more pronounced than that observed with minister and reduces the toxicity of aconite when the two herbs are decocted assistant drugs (Wang et al., 2012a). In the decoction of the herbal together. Contents of aconitines from Radix Aconiti Lateralis Prae- mixture Xue Fu Zhu Yu with liquorice as a guide drug, the half life parata and flavonoids from liquorice have varying degrees of of absorption and elimination was shortened, and the area under reduction during decoction. Research showed that upon hydrolysis, the curve (AUC) was significantly reduced for hydroxysafflor the glycyrrhizic acid converts to the aglycone glycyrrhetinic acid and yellow A (HSYA, the primary component of the monarch drug two molecules of glucuronic acid. The detoxification action of Flos Carthami, Hong Hua) after drug combination, which demon- liquorice is attributed to glucuronic acid, which can combine with strates that liquorice exerts its harmonizing effect by enhancing the hydroxyl group of aconitine in vivo, generating glucuronide absorption and elimination of HSYA (Luo et al., 2012). derivatives that are excreted in the urine (Yang et al., 2003). Rhizoma Dioscoreae Bulbiferae is used clinically to treat endemic goiter, hyperthyroidism and cough. Hepatotoxicity and nephrotoxi- 5. The effects of liquorice and its active components on model city were demonstrated and were attributed to diosbulbin B, dioscin probe substrates of CYP isoforms and diosgenin (Tan et al., 2003; Wang et al., 2009a). Co-boiling of Rhizoma Dioscoreae Bulbiferae with liquorice produced a markedly Drug metabolism via the CYP450 system has emerged as an different chemical content than the pooling of individually boiled important determinant in the occurrence of several drug inter- extracts, including diosbulbin B (Ma, 2011). actions. Different CYP isoforms are related to liquorice–herb/drug 786 X. Wang et al. / Journal of Ethnopharmacology 150 (2013) 781–790 interactions. CYP3A4, CYP2C9 and CYP2E1 have been the most CYP2C9 plays a major role in the oxidation of both xenobiotic studied CYP isoforms related to liquorice. The principal CYP iso- and endogenous compounds. CYP2C9 makes up approximately forms regulated by active components of liquorice are shown in 18% of the CYP450 protein in liver microsomes and metabolizes

Fig. 2. Half maximal inhibitory concentration (IC50) or inhibitory/ certain therapeutic drugs, including those with a narrow thera- inducing rate of CYP isoforms regulated by liquorice is shown in peutic index, such as warfarin (Rettie and Jones, 2005). GA Table 4. inhibited the 4-hydroxylation of diclofenac by CYP2C9 with an

CYP3A4 is one of the most important CYP isoforms involved in IC50 value of 16.21 μM; this concentration was 8-fold higher than the metabolism of xenobiotics in the human body (Li et al., 1995). that observed following specific inhibition with sulfaphenazole This enzyme is involved in the metabolism of approximately half (SUL) (Liu et al., 2011). CYP2C9 was competitively inhibited (50% the drugs that are in use today. CYP3A4 is involved in the ratio) but not inactivated by 100 μM glabridin (Kent et al., 2002). oxidation of the largest range of substrates of all the CYPs (Evans CYP2E1 is involved in the metabolism of xenobiotics in the and Relling, 1999). Extracts and various components from liquorice body (Kolble, 1993) and mediates many important drug interac- were reported to regulate CYP3A4 enzymes, with inhibiting tions (Touw, 1997). Liquorice and its active components are CYP3A4 enzymatic activity. Alcoholic licorice root extract (1.4 to capable of regulating the effects of CYP2E1 on drugs and toxins. 69 μg/mL) and glabridin (0.625 to 40 μM), an isoflavan purified Isoliquiritigenin (20 μM) inhibited CYP2E1 activity and mRNA from the extract, inactivated CYP3A4 in a time- and concentration- expression by 75.7% and 78.7%, respectively. This inhibition was dependent manner. The inactivation was NADPH-dependent and partly responsible for decreased acetaminophen toxicity in rats not reversible by extensive dialysis, which was also demonstrated (Zhang et al., 2008). A water extract of liquorice (0.5 g/kg) to be correlated with the loss of the P450-reduced CO spectrum significantly inhibited the overexpression of CYP2E1 induced by and the intact heme moiety (Kent et al., 2002). Glycyrrhetic acid Rhizoma Dioscoreae Bulbiferae (air potato) (Zhao et al., 2011a). (GA) at a concentration of 50 μmol/L inhibited CYP3A4-catalyzed Glycyrrhetinic acid and liquiritin downregulated brucine-induced testosterone 6β-hydroxylation with an IC50 of 25.96 μmol/L CYP2E1 activity and mRNA expression by 51.4% and 41.1%, respec- (Liu et al., 2011). The IC50 values of liquorice extract dissolved in tively (Xing et al., 2011). Diammonium glycyrrhizinate lipid ligand ethanol and glycyrrhizin (GZ) with respect to CYP3A4-mediated (DGLL) inhibited CYP2E1 mRNA expression in the treatment of metabolism were 140.95 and 174.62 μg/mL, respectively. These nonalcoholic steatohepatitis (Liu et al., 2010). Conversely, other values are higher than the value for the positive control ketoco- studies reported a lack of inhibition of CYP2E1 by liquorice or its nazole (5.74) (Pandit et al., 2011). The results showed that the active components (He et al., 2009; Liu et al., 2011; Paolini et al., interaction potential of the extract of liquorice was greater than for 1998). the pure compound and demonstrated that liquorice and glycyr- The regulation of metabolism by liquorice and its active compo- rhizin exhibited a weak interaction potential with drug-metabolizing nents through other CYP isoforms has also been investigated. The enzymes when co-administered with conventional medicines. In induction of CYP1A2 (He et al., 2007; Paolini et al., 1999; Xiao et al., Tsukamoto' research (Tsukamoto et al., 2005), glycyrrhizin, the 2006), CYP3A1/2 (Dai et al., 2005; Paolini et al., 1999; Xiao et al., primary constituent of liquorice, did not inhibit CYP3A4. However, 2006; Xu et al., 2007), and CYP2D6 (Cai et al., 2003) were reported, other constituents separated from the methanol extract of liquorice as was the inhibition of CYP2C19 (Liu et al., 2011). were demonstrated to be CYP3A4 inhibitors, including (3R)-vestitol, Similar to the discrepancies between various studies of CYP3A4 liquiritigenin 7,4′-diglucoside and 4-hydroxyguaiacol apioglucoside, and CYP2E1, researchers have reported varying results regarding with IC50 values of 3.6, 17 and 20 μM, respectively, contributing to the effects of liquorice on other CYP isoforms and have attempted the potent CYP3A4 inhibitory activity of the extract (IC50 0.022 mg/ to explain these differences. For example, the intragastric admin- mL). Contrary to these results, recent research has demonstrated that istration of glycyrrhizic acid resulted in self-induced metabolism glycyrrhizin (100 μM) and its major metabolite GA (25 μM) signifi- in mice, while intraperitoneal administration did not have this cantly increased CYP3A4 activity by 25.5% and 30.2% individually effect (Diao et al., 1999). Similarly, a study of the effect of the (Hou et al., 2012). The apparently contrasting results on CYP3A4 of isomers of glycyrrhizic acid (18α-GA and 18β-GA) on CYP3A glycyrrhizin and GA might be related to different concentrations or activity showed that 18α-GA (25–100 μM) induced CYP3A mRNA model systems. expression, while 18β-GA (25–100 μM) markedly inhibited the

Fig. 2. The principal CYP isoforms regulated by active components of liquorice. Table 4 IC50 or inhibitory/inducing rate of CYP isoforms regulated by liquorice.

Effect Component Method/model system IC 50 or inhibitory/inducing rate of CYPs References

CYP3A4 CYP2C9 CYP2E1 CYP1A2 CYP2D6 CYP2C19 CYP2B6

Inhibition Glycyrrhetic acid (GA) HPLC 25.96 μM 16.21 μM – 219.40 μM N/A 43.18 μM N/A Liu et al. (2011) Hydro-alcoholic extract CYP450–CO complex assay 140.95 μg/mL N/A N/A N/A 132.49 μg/ N/A N/A Pandit et al. (2011) mL – μ μ Glycyrrhizin CYP450 CO complex assay 174.62 g/mL N/A N/A N/A 156.25 g/ N/A N/A Pandit et al. (2011) 781 (2013) 150 Ethnopharmacology of Journal / al. et Wang X. mL Glycyrrhizin Nifedipine oxidation Inhibits less than 50% N/A N/A N/A N/A N/A N/A Tsukamoto et al. (2005) at 1.2 mM (3R)-vestitol Nifedipine oxidation 3.6 μM N/A N/A N/A N/A N/A N/A Tsukamoto et al. (2005) Liquiritigenin 7,4′-diglucoside Nifedipine oxidation 17 μM N/A N/A N/A N/A N/A N/A Tsukamoto et al. (2005) 4-Hydroxyguaiacol apioglucoside Nifedipine oxidation 20 μM N/A N/A N/A N/A N/A N/A Tsukamoto et al. (2005) Alcoholic extract 7BFC assay 6.9 μM after 15 min N/A N/A N/A N/A N/A N/A Kent et al. (2002) Glabridin 7BFC/7EFC assay 7 μMin5min 100μM – N/A N/A N/A 12 μM Kent et al. (2002) Isoliquiritigenin RT-PCR/Enzymatic analysis N/A N/A Inhibits 75.7/78.7% N/A N/A N/A N/A Zhang et al. (2008) at 20 μM N/A N/A N/A He et al. (2007) 7 Induction Water extract HPLC N/A N/A N/A 1.01 /min0.50 nmol/ mgat 754 mg/kg Glycyrrhizin Vivid CYP449 screening kits Increases 25.5% at 100 μM N/A N/A N/A N/A N/A N/A Hou et al. (2012) Gycyrrhetic acid (GA) Vivid CYP450 screening kits Increases 30.2% at 25 μM N/A N/A N/A N/A N/A N/A Hou et al. (2012)

N/A, currently not available; –, cannot be determined; 7BFC assay: 7-benzyloxytrifluromethylcoumarin O-debenzylation activity; 7EFC assay: 7-ethoxy-4-(trifluoromethyl) coumarin O-debenzylation activity. – 790 787 788 X. Wang et al. / Journal of Ethnopharmacology 150 (2013) 781–790 expression in a concentration-dependent manner (Wang et al., potentiate the effects of warfarin therapy (Heck Am Fau—DeWitt 2009b). Although different methods of administration and chemi- et al., 2000; Miller, 1998). cal isomers may contribute to the inconsistent effects of liquorice Few toxicological studies on liquorice and liquorice–herb/drug on CYP isoforms, the explicit mechanism is unclear. However, interactions was were reported. A research in US proposed an according to current research reports, the intake of liquorice will acceptable daily intake of 0.015–0.229 mg glycyrrhizic acid/kg/day without a doubt regulate important CYP isoforms like CYP3A4, is based on the in vivo and clinical evidence (Isbrucker and CYP2C9 and CYP2E1. This is likely to have an impact over Burdock, 2006). In the 2010 version of the Chinese Pharmacopoeia, metabolic processes of other drugs or foods. Therefore, no matter it has been suggested the standard daily dosage for liquorice its use as a food additive or herbal medicine, potential safety issues ranges from 2 g to 10 g daily. In EMEA reports from 2012 (EMEA on herb/drug interactions should be addressed. (2012) Community herbal monograph on Glycyrrhiza glabra L. and/or Glycyrrhiza inflata Bat. and/or Glycyrrhiza uralensis Fisch., radix. European Medicines Agency), cases of overdose have been 6. The effects of liquorice/active ingredients on drug reported with prolonged use (more than 4 weeks) and/or intake of transporter proteins high amount of liquorice, with symptoms such as water retention, hypokalaemia, hypertension, cardiac rhythm disorders, hyperten- Of the various causes of drug interactions, pharmacokinetics sive encephalopathy. Recent research reported glycyrrhetinic acid

(including drug metabolism and transport) is considered to be the with LD50 value 553.09 mg/kg, 95% confidence interval 468.08– most frequent and the most clinically important. Several reviews 653.54 mg/kg (Cao, 2012). Considering the potential of drug–drug on herb–drug interactions have discussed liquorice (Fugh-Berman, interaction and widely usage of liquorice either as herbal medicine 2000; Yu et al., 2012; Zhou et al., 2003). For example, licorice root or food supplement, systematic safety evaluation of liquorice is was shown to inhibit P-glycoprotein (P-gp) in an in vivo ATPase needed for future direction. assay (Satoh et al., 2009). In intestinal mucosa, liquorice slightly inhibited P-gp function and increased the intestinal absorption of rhodamine 123 (Yao et al., 2009). As the inhibitory action of 8. Conclusions P-glycoprotein was enhanced by a combination of liquorice and Kansui (Radix Euphorbiae Kansui), the inhibition of P-gp may be Liquorice and formulated products containing liquorice or its the mechanism that underlies the toxicity of the mixture (Sun active components are widely used in China and other countries. et al., 2010). Similarly, the action of P-gp was also inhibited by co- As a unique "guide drug" that enhances the effectiveness of other administration of liquorice and Daphne genkwa (Huang et al., ingredients and reduces toxicity in a multi-herb prescription, the 2008). The activation of P-gp by glycyrrhetic acid (GA), the major role of liquorice in drug interactions should be considered. Recent metabolite of GZ, reduced the oral bioavailability of CsA (Hou et al., research has demonstrated a wide range of pharmacological 2012). The discrepancy with liquorice and GA might related to effects of liquorice. Based on chemical and metabolic studies, it other chemicals in liquorice, different concentrations and inspec- has been confirmed that the major ingredients of liquorice may tion methods. Because liquorice is one of the most commonly used cause metabolism-based interactions involving CYPs. Certain com- ingredients in TCM, it is essential to consider the interaction ponents of liquorice were also demonstrated to act on drug between liquorice and herbs/drugs that are transported by P-gp. transporter proteins, such as P-gp, during drug interactions. The effects of the active components of liquorice on other Therefore, the "guide" effect of liquorice may be partly explained transporter proteins have also been investigated. Glycyrrhizin was by the regulation of CYPs and P-gp. No matter it is used as a food demonstrated to increase hepatic glutathione content, possibly additive or herbal medicine, the potential safety issues on herb/ by inhibiting Mrp2 (multidrug resistance-associated protein 2), drug interaction should be attended. However, different sources of an effect that then reduced the biliary excretion of glutathione (Xu liquorice, multitudinous model system, and various research levels et al., 2012). lead to some contrasting results especially on the effect on CYPs, which is one of the most decisive parameters in drug-interactions. It is therefore important to continue investigate consider how CYP 7. Adverse effects and toxicological studies of liquorice or transporter proteins are related to liquorice–herb/drug interactions for clinical use. Meanwhile, the systematic safety evaluation According to TCM theories, liquorice is contraindicated in on liquorice—herb/drug interactions is still needed. combining with Sargassum (Hai Zao), Herba Cirsii Japonici (Da Ji), Euphorbia kansui (Gan Sui) and Flos genkwa (Yuan Hua). The combination of these four herbs with liquorice might attenuate Acknowledgements liver function and cause cardiac toxicity (Huang et al., 2001). In suspected adverse reactions associated with herbal laxatives This work was supported by the National Program for Key Basic in Italy from 2002 to 2011, liquorice was evolved in 27% of total 26 Research Projects (2012CB518404); NSFC (81102840 and 81125024); cases (Vitalone et al., 2012). A number of side effects including Doctoral Fund of Ministry of Education of China (20101210120012); cardiac dysfunction, oedema and hypertension have been reported Key Project of Chinese Ministry of Education (211013) and Tianjin by high doses (400 mg/day) of glycyrrhizic acid (Stormer et al., Research Program of Application Foundation and Advanced Technol- 1993). Consumed liquorice in doses of 50–200 g/day (correspond- ogy (12JCQNJC08200 and 10JCYBJC27100). ing to 75–540 mg/day glycyrrhetinic acid) can cause a significant rise in blood pressure (Sigurjonsdottir et al., 2001). Aqueous liquorice extract has fewer adverse effects than pure glycyrrhizin (Bernardi References et al., 1994; Cantelli-Forti et al., 1994)whichmightberelatedto differences in bioavailability and the resulting pharmacokinetics of Bell, Z.W., Canale, R.E., Bloomer, R.J., 2011. A dual investigation of the effect of glycyrrhizin and glycyrrhetic acid (Cantelli-Forti et al., 1997). 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