Natural Chalcones in Chinese Materia Medica: Licorice

Licorice is an important Chinese materia medica frequently used in clinical practice, which contains more than 20 triterpenoids and 300 flavonoids. Chalcone, one of the major classes of flavonoid, has a variety of biological activities and is widely distributed in nature. To date, about 42 chalcones have been isolated and identified from licorice. These chalcones play a pivotal role when licorice exerts its pharmacological effects. According to the research reports, these compounds have a wide range of biological activities, containing anticancer, anti-inflammatory, antimicrobial, antioxidative, antiviral, antidiabetic, antidepressive, hepatoprotective activities, and so on. This review aims to summarize structures and biological activities of chalcones from licorice. We hope that this work can provide a theoretical basis for the further studies of chalcones from licorice.


Introduction
Glycyrrhiza, one of the oldest medicinal plants, is often referred to as Chinese licorice, which belongs to the family of Leguminosae [1,2].
ere are 30 species of licorice distributed all over the world.
ree Glycyrrhiza species including Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., and Glycyrrhiza glabra L. are prescribed as licorice in the Chinese Pharmacopoeia [3,4]. Licorice, derived from the dried roots and rhizomes of genus Glycyrrhiza, is called "gancao" in China and was firstly recorded in Shennong's Classic of Materia Medica around 2100 BC [5]. It is widely distributed in many provinces of China, such as Xinjiang, Gansu, Inner Mongolia Autonomous Region, Ningxia, and Shanxi, as well as the Siberian region of Russia [5]. As a kind of crucial Chinese materia medica (CMM), licorice has a great many pharmacological effects and is widely used in clinical practice. Licorice is capable of tonifying qi, invigorating spleen and harmonizing stomach, clearing heat, detoxifying, reducing phlegm, relieving cough, alleviating pain, etc. [3,6]. Licorice, which not only has significant efficacy but also has mild and nontoxic properties, can reconcile plenty of CMMs. erefore, it can be combined with various CMMs to form a variety of Chinese herbal compound prescriptions, such as Guizhi Gancao decoction, Shaoyao Gancao decoction, and Xuefu Zhuyu decoction, thus exerting different effects [3].
Licorice is not only a bulk medicine for traditional Chinese medicine but also a major raw material for Mongolian medicine, or an important component of many folk prescriptions. In addition, the chemical composition of licorice is very complicated. To date, more than 20 triterpenoids and 300 flavonoids have been isolated from licorice [4]. e total flavonoids of licorice are a large class of compounds. At present, many flavonoids have been isolated from licorice, including dihydroflavones, chalcones, isoflavones, and other types of flavonoids. Chalcones, the vital class of secondary metabolites of plant [7], are not only an important synthetic precursor of flavonoids but also a major form of the natural products [8]. Currently, about 42 chalcones have been isolated from licorice. ese chalcones play a pivotal role when licorice exerts its pharmacological effects. Numerous studies have indicated that these bioactivities include anticancer [9], antiinflammatory [10], antimicrobial, antiviral [5], antioxidative, hepatoprotective [11], antidiabetic [12], antidepressant activities [13], and so on.
Chalcone compounds and their derivatives are important organic synthesis intermediates. Highlighting the broad range of biological activities of chalcones in numerous reports has caused extensive attention. In recent years, people have increasingly studied the components and activities of chalcones in licorice.
is work aims to sum up the structures and pharmacological activities of chalcones that have been identified from licorice. is review might provide some dependable experiment gist and a theoretical basis for further research on chalcones in licorice.

The Structures of Chalcones in Licorice
Chalcones are 1,3-diphenyl-2-propene-1-ones, in which two aromatic rings named as A and B ring are linked by a threecarbon α,β-unsaturated carbonyl system [7]. Chalcones belong to the group of flavonoids, including chalcones and dihydrochalcones ( Figure 1). ey are not only widespread in nature but also obtained by biosynthesis and chemical synthesis. Since 1988, 42 chalcones including 33 chalcones and 9 dihydrochalcones have been reported in licorice, which were proved to possess a large number of biological activities.
To date, many researchers have analyzed the ingredients in licorice with a variety of methods. For example, based on ultra-performance liquid chromatography-tandem mass spectrometry ( To date, about 42 chalcones in licorice have been reported (Table 1). e structures of these compounds were shown in Figure 2.

The Biological Activities of Chalcones in Licorice
Chalcones in licorice have been reported to manifest different biological activities, including anticancer, anti-inflammatory, antibacterial, antiviral, antioxidative, hepatoprotective, antidiabetic, antidepressant, and other activities ( Figure 3). e following is the summary of the biological activities of chalcones in licorice (Table 2).
3.1. Anticancer Activity. Cancer, the enemy of humanity, seriously endangers human life and health. With the aging of the social population structure, environmental pollution, and the prevalence of unhealthy living behaviors such as smoking and unreasonable diet, the situation of cancer will become more intense in a few years. In recent years, phytochemicals from herbal medicine remain a main source of drug discovery [31]. ere is no doubt that chalcones in licorice have great influence on the treatment of cancer. Chalcones have the property of electrophiles that interact with nucleophiles in proteins to inhibit the growth of tumor cells. At present, several chalcones from licorice have been demonstrated to possess anticancer property by induction of cell cycle arrest, inhibition of cancer cell metastasis, apoptosis, and autophagy [114].
Isoliquiritigenin is an impactful cancer chemopreventive agent in vitro as well as in vivo. According to the reports, isoliquiritigenin was resistant to multiple tumor cells. Recent study suggested that isoliquiritigenin induced apoptosis in A549 non-small-cell lung cancer cells by crosstalk between protein 53 (p53), B-cell lymphoma-2 (Bcl-2) family proteins, caspase cascades, and protein kinase B (Akt) survival pathways [32]. It also caused DNA damage and inhibited ataxia-telangiectasia mutated (ATM) expression leading to G2/M phase arrest and apoptosis in oral squamous cell carcinoma [33]. Isoliquiritigenin induced apoptosis in renal cell carcinoma Caki cells via the generation of ROS, resulting in induction of p53 expression and inhibition of the signal transducer and activator of transcription 3 (STAT3) signaling pathway [34]. Moreover, isoliquiritigenin affected WIF1 gene and downregulated κ-catenin signal, resulting in the undergoing arrest of breast cancer cells at the G0/G1 phase [35]. In prostate cancer, it affected expression of protein B1, cyclin-dependent kinase 1 (CDK1), and other related molecules in the G2/M cell cycle of PC-3 and 22RV1 cells [31].
Licochalcone A, a characteristic chalcone isolated from licorice roots [36], was proved to be resistant to many types of cancer cells through different mechanisms. Induction of mitochondrial dysfunction is one of the main pathways leading to apoptosis by licochalcone A. It was found that it induced activation of caspases via the mitochondrial pathway and then mediated its antiproliferative and apoptotic effects of oral squamous cell carcinoma by inhibiting Sp1 and Sp1-mediated signaling pathways [37]. Licochalcone A inhibited glioma cell growth by mediating cell cycle arrest at G0/G1 and G2/M phases and induced cell death by inducing mitochondrial dysfunction [38,39]. Licochalcone A induced apoptosis of gastric cancer cells via the caspase-dependent mitochondrial pathway and exerted G2 cell cycle arrest through the regulation of G2/M phase checkpoint proteins [40]. Additionally, it handled activation of the LC3-II signaling pathway while inhibiting the phosphatidylinositol 3kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) signaling pathway that promoted autophagy and apoptosis in cells [41]. Licochalcone A caused G2 and late-G1 arrests in androgen-independent PC-3 prostate cancer cells by affecting the expression of proliferating cell nuclear antigen (PCNA), DNA polymerase δ, Rb and E2F, cyclins B1 and D1, and so on, which led to apoptosis [42].
Licochalcone B led to S-phase arrest, decreased the expression of cyclin A, CDK1, CDK2 mRNA, Bcl-2, survivin, and cell division cycle 25 (Cdc25A and Cdc25B) proteins, induced downregulation of antiapoptotic proteins (Bid, Bcl-xl, and Mcl-1), and also caused the loss of  Evidence-Based Complementary and Alternative Medicine 3 mitochondrial membrane potential (MMP) [43,44]. However, it enhanced Bax expression, activated caspase-3, and cleaved poly ADP-ribose polymerase (PARP) protein [44]. In addition, it attenuated migration, adhesion, and invasion of cancer cells accompanied with the downregulated protein expression of MMP-9 and nuclear translocation of nuclear factor-κB (NF-κB) [115]. Similar to licochalcone B, licochalcones C and D induced apoptosis by altering the expression of Bcl-2 family member genes and activating the caspase-mediated cell death signaling pathway [45,46]. Meanwhile, licochalcone D induced apoptosis through mitochondrial pathway and blocked cell migration and invasion into surrounding tissues by reducing the activity and expression of MMP-2 and MMP-9 [46]. In addition to increasing the expression of proteins (proapoptotic factors, caspase-3, and PARP), suppressing the constitutive NF-κB activation, and downregulating Bcl-2 and Bax, licochalcone E reduced tumor growth and metastasis through inhibiting tube formation of vascular endothelial cells [47][48][49]. Same as licochalcone D, kanzonol C could inhibit MMP-2 secretion from tumor cells [50]. Unlike the preceding compounds, isobavachalcone induced apoptosis through increasing the expression of tumor necrosis factor-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2) [51].

Anti-Inflammatory Activity.
e inflammatory reaction is a common clinical pathological process that could be born in various parts of body tissues and organs [116]. According to the duration of inflammation, it is divided into two stages: acute and chronic inflammation [56]. In the more serious inflammatory diseases, especially when the pathogenic microorganisms spread in the body, there is obvious systemic reaction [60]. So far, the treatment of inflammation encounters a lot of difficulties. How to safely and effectively treat inflammation is also an issue that should be solved urgently. As a commonly used CMM for the treatment of inflammation, licorice has notable anti-inflammatory effect. Triterpenes and flavonoids from licorice might cure different types of inflammation, especially chalcones [6]. NF-κB and mitogen-activated protein kinase (MAPK) pathways were proved to be important for chalcones to exert anti-inflammatory activity.
Licochalcone C played a vital role in sepsis-induced inflammation through repressing NF-κB translocation and several kinds of downstream molecules, including iNOS, intercellular adhesion molecule-1 (ICAM-1), as well as vascular cell adhesion molecule-1 (VCAM-1). Meanwhile, it might upregulate the PI3K/Akt/eNOS signaling pathway  [112,113] Evidence-Based Complementary and Alternative Medicine [76]. Similar to licochalcone C, licochalcone E reduced expression of inducible enzymes (iNOS and COX-2) and proinflammatory cytokines via the suppression of NF-κB and AP-1 transcriptional activity [77]. Isobavachalcone and kanzonol B suppressed the production of PGE 2 and NO and downregulated the expression of iNOS and COX-2 through suppressing IκBα degradation in LPS-activated microglia [79]. Histone deacetylase enzymes (HDACs), potential drug targets for natural chalcones, were employed to treat cancer and inflammation. Isoliquiritigenin and homobutein might suppress both NF-κB and HDAC activities [68]. rough inhibiting the binding of LPS to TLR4 on immune cells and increasing the polarization of M1 macrophages to M2 macrophages, 4-hydroxylonchocarpin attenuated colitis in mice [81]. e anti-inflammatory mechanism of morachalcone A was related to the activation of MAPKs (p38, ERK, and JNK) and NF-κB pathways, particularly decrease of nuclear translocation of NF-κB p65 subunit [80]. Kanzonol B, DTM, and licoagrochalcone C revealed efficacious inhibitory activity on NF-κB transcription [10].

Antibacterial Activity.
ere are many antibacterial drugs in the market today. However, with the abuse of antibacterial drugs, the resistance of bacteria is increasing. erefore, it is crucial to find the new, safe, and effective antibacterial drugs. Some compounds from licorice were proved to have antibacterial activity.
On the basis of previous research studies, isoliquiritigenin had obvious inhibitory effect on methicillin-resistant Staphylococcus aureus and Ralstonia solanacearum [82,83]. Licochalcone A, as one of the vital antibacterial components in licorice, was resistant to both bacterial and fungal infections [118,119]. e study also showed promising antifungal activity of licochalcone A against Trichophyton rubrum via inhibiting important antifungal targets of ergosterol synthesis, cell wall synthesis, and glyoxylate cycle [119]. It has been reported to display good activity against Staphylococcus aureus biofilm [84]. Licochalcone A exhibited in vitro inhibitory effect on human pathogenic Mycobacteria species (Mycobacterium tuberculosis, M. bovis, M. kansasii, M. xenophii, and M. marinum) and Legionella species (Legionella bozemanni, L. dumoffii, L. feelei, L. longbeacheae, L. wadsworthii, L. gormanii, and L. micdadei), which might be a candidate for treating severe lung infections [85]. Besides, licochalcone A revealed the effects of resisting to all Gram-positive bacteria tested, including spore-forming bacteria, such as genera Bacillus and Clostridium, and toxin-producing bacteria, such as Bacillus cereus and Ralstonia aureus [118]. And it affected the growth of Streptococcus suis by inhibiting biofilm formation and hemolysin lyase secretion [86]. Licochalcone A also inhibited the growth of Helicobacter pylori [87]. Furthermore, it was an effective antifungal agent that might act in synergy with nystatin to inhibit the growth of Candida albicans so as to treat candidiasis [88].

Antiviral Activity.
Licorice is a universal CMM from several prescriptions, which has been proved to inhibit viral infection [121]. At present, there are some antiviral studies on the chalcone compounds from licorice. e components studied include isoliquiritigenin, licochalcone A, isobavachalcone, echinatin, and kanzonol Y. Isoliquiritigenin and licochalcone A evinced anti-HCV activity [92,93]. Isobavachalcone had potent antiporcine reproductive and respiratory syndrome virus (PRRSV) activity in vitro by inhibiting PRRSV replication at the postentry stage of PRRSV infection [94]. Echinatin and isoliquiritigenin showed strong inhibitory effects on influenza viral strains, H1N1, H9N2, and novel H1N1 [28]. Kanzonol Y had antidengue virus (DENV) activity [95].

Antioxidative Activity.
Excessive free radicals will accelerate human aging and bring on other diseases [122]. erefore, enhancing the body's antioxidant capacity is critical to human health. e phenolic hydroxyl structure of chalcones is a good proton donor, which can terminate the oxidative damage by combining with a radical [123]. ere are a few chalcone compounds in licorice with antioxidant capacity.
Isoliquiritigenin, as an abundant and forceful antioxidant toward low-density lipoprotein (LDL) oxidation, could attenuate atherosclerosis [96]. Isoliquiritigenin and paratocarpin B were found to be the most powerful antioxidant agents by an authentic peroxynitrite antioxidant assay [26]. Licochalcones A, B, and D, DTM, and isobavachalcone inhibited NADPHinduced microsomal lipid peroxidation [10,22]. Isobavachalcone also suppressed ascorbate-, t-BuOOH-, and CCl 4 -induced microsomal lipid peroxidation, and NADHdependent and ascorbate-induced mitochondrial lipid peroxidation [100]. Studies manifested that echinatin and licochalcone B possessed the strong scavenging activity toward ABTS ·+ radical [10]. Licochalcones B and D strongly suppressed superoxide anion production and expressed powerful scavenging activity on DPPH radical [22]. Licochalcone A protected skin 8 Evidence-Based Complementary and Alternative Medicine cells from oxidative stress by activating Nrf2-signaling, resulting in increased expression of HO-1 and glutamatecysteine ligase regulatory subunit (GCLM) and reduction of intracellular ROS concentration [97]. Moreover, licochalcones A and C increased the expression of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) proteins that were closely related to antioxidant mechanisms [98,99]. Glypallichalcone, the inhibitor of LDL oxidation, reduced cholesterol levels through modulation of β-1 adrenergic receptor [101]. Dihydroisoliquiritigenin was used as a neuroprotectant against glutamate-induced oxidative stress in a mouse-derived hippocampal neuronal cell line (HT22) [102].
3.6. Hepatoprotective Activity. Liver is a vital organ that performs many metabolic functions in the body such as detoxification, glycogen storage, and protein synthesis [124]. CCl 4 is a chemical substance that is severely toxic to hepatocytes [125]. Also, overuse of acetaminophen (APAP) could cause drug-induced liver toxicity [126]. e natural ingredients of CMMs are considered to be an effective and safe alternative way to treat hepatocyte damage [11]. A few chalcone constituents in licorice have been proved to possess significant hepatoprotective activity. For example, echinatin and licochalcones A and B showed Nrf2 activation activities on HepG2C8 cells in AREluciferase reporter assay and a promising hepatoprotective effect on CCl 4 -induced acute liver injury in the ICR mice model [104]. Additionally, isoliquiritigenin, licochalcone B, and licoagrochalcone A possessed a remarkably potent protective effect against both CCl 4 -and APAP-induced HepG2 cell injuries. 3,4,3′,4′-Tetrahydroxychalcone also showed significantly potent activity against CCl 4 -induced liver injury [103]. Moreover, studies have demonstrated that licochalcone B might protect hepatocytes from alcohol-induced cell damage by reducing apoptosis, inhibiting oxidative stress, and upregulating Erk-Nrf2 [105]. Licochalcone E basically exhibited its protective role for treating hepatotoxicity through the peroxisome proliferator-activated receptor-c (PPAR-c)/NF-κB-mediated pathway [106].

Antidiabetic Activity.
Diabetes is a group of metabolic diseases characterized by hyperglycemia [127]. Longstanding hyperglycemia in diabetes leads to various tissue dysfunctions [128]. At present, drugs for the treatment of this disease are urgently needed to be developed. As confirmed by the experiment, extracts from licorice have a protective effect on diabetic nephropathy [129].
rough the oral glucose tolerance test, the hypoglycemic effect of isoliquiritigenin on normal Swiss albino male mice was reported [107]. Another finding demonstrated that isoliquiritigenin diminished high glucose-induced mesangial matrix accumulation through retarding transforming growth factor (TGF)-β1-SMAD signaling transduction [108]. Angiotensin-converting enzyme (ACE) plays a prominent role in hypertension, heart failures, myocardial infarction, and diabetic nephropathy. e study showed that echinatin has been proved to show a certain inhibitory effect on ACE in vitro [109]. Furthermore, licochalcone E enhanced expression of PPAR-c through irritating Akt signals as well as functions as a PPAR-c partial agonist, which improved hyperlipidemia and hyperglycemia under diabetic conditions [12]. Kanzonol C, licoagrochalcone A, and isobavachalcone as inhibitors of protein tyrosine phosphate 1B (PTP1B) were potential candidates for treating type II diabetes [21,110].
3.8. Antiobesity Activity. Obesity is a globally epidemic chronic metabolic disease, and the proportion of obese people continues to rise due to changes in lifestyle and diet. Obesity poses a series of potential safety hazard, so the use of antiobesity drugs can help improve the health of patients. Kanzonol C, licoagrochalcone A, and isobavachalcone were found to be PTP1B inhibitors for treatment of obesity [21,110]. Isoliquiritin apioside and isoliquiritigenin, as sources of pancreatic lipase (PL) inhibitors for preventing obesity, could lower the plasma total triglycerides and total cholesterol [111]. Licochalcone A had an inhibitory effect on adipocyte differentiation and lipogenesis via the downregulation of PPAR-c, CCAAT/enhancer binding protein α (C/EBPα), and sterol regulatory element-binding protein 1c (SREBP-1c) in 3T3-L1 preadipocytes [112]. And other results demonstrated that licochalcone A was effective to reduce obesity and could recover metabolic homeostasis by inducing adipocyte browning [113].

Conclusion
Phytochemical constituents especially flavonoids are largely considered to be beneficial for human health and disease prevention. As a category of nontoxic and effective natural ingredients, chalcones are proved to possess lots of biological activities and medicinal properties. To date, about 42 chalcones in licorice have been isolated and identified, and more new structures will be unveiled. Meanwhile, most of chalcones in licorice have been widely and deeply studied for their various activities, such as anticancer, anti-inflammatory, antimicrobial, antiviral, antioxidative, hepatoprotective, antidiabetic, and antiobesity activities. However, it will be a long way to further validate the pharmacological action and develop new drug. As chalcones in licorice are deeply explored and fully utilized, it will be served as a broad prospect for development and utilization of licorice.