Review of Constituents and Biological Activities of Triterpene Saponins from Glycyrrhizae Radix et Rhizoma and Its Solubilization Characteristics

Glycyrrhizae Radix et Rhizoma is regarded as one of the most popular and commonly used herbal medicines and has been used in traditional Chinese medicine (TCM) prescriptions for over 2000 years. Pentacyclic triterpene saponins are common secondary metabolites in these plants, which are synthesized via the isoprenoid pathway to produce a hydrophobic triterpenoid aglycone containing a hydrophilic sugar chain. This paper systematically summarizes the chemical structures of triterpene saponins in Glycyrrhizae Radix et Rhizoma and reviews and updates their main biological activities studies. Furthermore, the solubilization characteristics, influences, and mechanisms of Glycyrrhizae Radix et Rhizoma are elaborated. Solubilization of the triterpene saponins from Glycyrrhizae Radix et Rhizoma occurs because they contain the nonpolar sapogenin and water-soluble sidechain. The possible factors affecting the solubilization of Glycyrrhizae Radix et Rhizoma are mainly other crude drugs and the pH of the decoction. Triterpene saponins represented by glycyrrhizin from Glycyrrhizae Radix et Rhizoma characteristically form micelles due to amphiphilicity, which makes solubilization possible. This overview provides guidance regarding a better understanding of Glycyrrhizae Radix et Rhizoma and its TCM compatibility, alongside a theoretical basis for the further development and utilization of Glycyrrhizae Radix et Rhizoma.


Introduction
The Glycyrrhiza genus belongs to the Fabaceae family, comprising approximately 20 species primarily distributed across Asia, Europe, North America, and South America, with eight distributed throughout China [1]. Glycyrrhizae Radix et Rhizoma, also named glycyrrhiza or "Gan-Cao" in China and licorice or liquorice in Europe, is the dry root and rhizome of three official Glycyrrhiza species, namely, Glycyrrhiza uralensis Fisch, Glycyrrhiza glabra L., and Glycyrrhiza inflata Batal [2,3]. It is extensively used in traditional Chinese medicine (TCM) to treat hepatitis, influenza, cough, and gastric

Biological Activities
Modern pharmacological studies revealed that glycyrrhiza shows a variety of pharmacological effects against inflammation, oxidative stress, immunoregulation, viral infection, and cancer [3]. These bioactivities are attributed to the chemical constituents of glycyrrhiza. In this section, the main pharmacological activities of saponin monomers, including hepatoprotective, anti-inflammatory, antimicrobial, antiviral, and antitumor activities, are summarized (Table 2). GL (1) is the most commonly reported monomer with extensive activities.

Hepatoprotective Activities
There are many reports about possible mechanisms in vitro and vivo by which saponins from glycyrrhiza are hepatoprotective. GL (1) was proven to relieve liver disease and prevent druginduced liver injury through multitargeting therapeutic mechanisms, including antisteatosis, antioxidative stress, anti-inflammation, immunoregulation, antifibrosis, anticancer, and drug-drug interactions [3]. Nakamura et al. [39] reported that GL (1) prevented soluble enzyme release from primary cultured rat hepatocytes induced by CCl4. Sato et al. [40] found that GL (1) could modify the expression of hepatitis B virus (HBV)-related antigens on the hepatocytes and suppress sialylation of hepatitis B surface antigen (HBsAg) in PLC/PRF/5 cells. Tsuruoka et al. [41] showed that GL (1, 10.5 mg/kg) suppressed increases in aspartate aminotransaminase (AST) and alanine aminotransaminase (ALT), inhibited inducible nitric oxide synthase (iNOS) mRNA expression, and reduced protein and cell infiltration and the degeneration of hepatocytes in the liver of concanavalin A (Con A)-treated BALB/c mice. Lee et al. [42] reported that GL (1) alleviated carbon tetrachloride (CCl4)-induced liver injury in ICR mice, probably by inducing heme oxygenase-1 and downregulating proinflammatory mediators. Lin et al. [43] found that a three-day pretreatment with GL (1) exhibited a protective effect on retrorsine-induced liver damage in Sprague Dawley rats. GL (1) is able to provide partial protection of the liver against ischemia-reperfusion damage in Wistar rats [44]. Orazizadeh et al. [45] showed that GL (1) effectively protects against NTiO2-induced hepatotoxicity in Wistar rats.
Licorice-saponin M3, and uralsaponin T were previously reported as new oleanane-type triterpene saponins, but they are the same compounds.

Biological Activities
Modern pharmacological studies revealed that glycyrrhiza shows a variety of pharmacological effects against inflammation, oxidative stress, immunoregulation, viral infection, and cancer [3]. These bioactivities are attributed to the chemical constituents of glycyrrhiza. In this section, the main pharmacological activities of saponin monomers, including hepatoprotective, anti-inflammatory, antimicrobial, antiviral, and antitumor activities, are summarized ( Table 2). GL (1) is the most commonly reported monomer with extensive activities.

Hepatoprotective Activities
There are many reports about possible mechanisms in vitro and vivo by which saponins from glycyrrhiza are hepatoprotective. GL (1) was proven to relieve liver disease and prevent drug-induced liver injury through multitargeting therapeutic mechanisms, including antisteatosis, antioxidative stress, anti-inflammation, immunoregulation, antifibrosis, anticancer, and drug-drug interactions [3]. Nakamura et al. [39] reported that GL (1) prevented soluble enzyme release from primary cultured rat hepatocytes induced by CCl 4 . Sato et al. [40] found that GL (1) could modify the expression of hepatitis B virus (HBV)-related antigens on the hepatocytes and suppress sialylation of hepatitis B surface antigen (HBsAg) in PLC/PRF/5 cells. Tsuruoka et al. [41] showed that GL (1, 10.5 mg/kg) suppressed increases in aspartate aminotransaminase (AST) and alanine aminotransaminase (ALT), inhibited inducible nitric oxide synthase (iNOS) mRNA expression, and reduced protein and cell infiltration and the degeneration of hepatocytes in the liver of concanavalin A (Con A)-treated BALB/c mice. Lee et al. [42] reported that GL (1) alleviated carbon tetrachloride (CCl 4 )-induced liver injury in ICR mice, probably by inducing heme oxygenase-1 and downregulating proinflammatory mediators. Lin et al. [43] found that a three-day pretreatment with GL (1) exhibited a protective effect on retrorsine-induced liver damage in Sprague Dawley rats. GL (1) is able to provide partial protection of the liver against ischemia-reperfusion damage in Wistar rats [44]. Orazizadeh et al. [45] showed that GL (1) effectively protects against NTiO 2 -induced hepatotoxicity in Wistar rats.

Anti-Inflammatory Activities
In a study, Li et al. [46] suggested that the anti-inflammatory mechanism of total saponins of glycyrrhiza may be related to a reduction in the release of inflammation factors in macrophages and inhibition of the key enzymes in the arachidonic acid (AA) metabolism pathway of prostaglandin E2 (PGE 2 ) synthesis, as observed through an inflammatory model of mouse macrophage RAW264.7 cells induced by lipopolysaccharide (LPS). Wang et al. [47] investigated the anti-inflammatory effect of GL (1) on LPS-stimulated mouse endometrial epithelial cells (MEEC), demonstrating that GL (1) inhibited LPS-induced inflammatory response by inhibiting TLR4 signaling pathway in MEEC.
Akamatsu et al. [48] found that GL (1) inhibited reactive oxygen species (ROS) generation by neutrophils, which were potent inflammatory mediators in the in vitro study.
In addition, GL (1) may inhibit high-mobility group protein B1 (HMGB1) expression and subsequent production of inflammatory cytokines to prevent cerebral vasospasm (CVS) following subarachnoid hemorrhage (SAH) in Sprague-Dawley rats [49]. Pang et al. [50] demonstrated that inhibiting HMGB1 with GL (1) alleviated brain injury after diffuse axonal injury (DAI) via its anti-inflammatory effects in SD rats.

Antimicrobial and Antiviral Activities
Saponins of G. glabra L. have broad-spectrum antimicrobial activities and can be used as natural antimicrobial agents [51]. GL (1) is an effective antiviral component against hepatitis C virus (HCV), human immunodeficiency virus (HIV), coxsackie virus B3 (CVB3), duck hepatitis virus (DHV), enterovirus 71 (EV71), coxsackievirus A16 (CVA16), herpes simplex virus (HSV), and H5N1 by weakening viral activity and enhancing host cell activity [52]. GL (1) is also shown to inhibit varicella zoster virus (VZV) and the severe acute respiratory syndrome coronavirus (SARS-CoV) replication in vitro [53,54]. In another study, Wolkerstorfer et al. [55] found that GL (1) inhibited influenza A virus (IAV) uptake into the cell. In detail, Sun et al. [56] summarized the antiviral effects of GL (1) in their research regarding progress and mechanism in recent years.

Cytotoxic and Antitumor Activities
Deng et al. [58] showed that GL (1) profoundly reduced expression of thromboxane synthase (TxAS), as well as proliferating cell nuclear antigen (PCNA), and rescued liver and kidney damage in tumor-bearing mice, the effect of which is possibly through suppression of the TxA2 pathway. It was shown that GL (1) has protective effects against Aflatoxin B1 (AFB 1 )-induced cytotoxicity in human hepatoma cell line (HepG2) [59]. In addition, dipotassium glycyrrhizinate (DPG), a dipotassium salt of GL, presented antitumoral effects on glioblastoma (GBM) cell lines through decreased proliferation and increased apoptosis. The DPG antitumoral effect is related to NF-κB suppression, where IRAK2and TRAF6-mediating miR16 and miR146a, respectively, might be potential therapeutic targets of DPG [60].
In the cytotoxic assay, GL (1), licorice-saponin G2 (9) and uralsaponin D (21), showed no cytotoxic activity on tested cancer cell lines, whereas their corresponding aglycones exhibited potently cytotoxic activities against human cervical cancer HeLa cells and human breast adenocarcinoma MCF-7 cells [29]. In vitro-primary cultured rat hepatocytes induced by CCl 4 Prevent soluble enzyme release [39] In vitro-PLC/PRF/5 cells Modify the expression of hepatitis B virus (HBV)-related antigens on the hepatocytes and suppress sialylation of HBsAg [40] In vivo-BALB/c mice Suppress increases in AST and ALT, inhibit inducible nitric oxide synthase (iNOS) mRNA expression, and reduce protein and cell infiltration and the degeneration of hepatocytes [41] In vivo-ICR mice Alleviate CCl 4 -induced liver injury [42] In vivo-Sprague Dawley rats Exhibit protective effect on retrorsine-induced liver damage [43] In vivo-Wistar rats Provide partial protection of the liver against ischemia-reperfusion damage [44] In vivo-Wistar rats Protect against NTiO 2 -induced hepatotoxicity [45] Anti-inflammatory activities

In vitro-lipopolysaccharide (LPS)-stimulated mouse endometrial epithelial cells (MEEC)
Inhibit LPS-induced inflammatory response by inhibiting TLR4 signaling pathway [47] In vitro-neutrophil Inhibit reactive oxygen species (ROS) generation by neutrophils [48] In vivo-Sprague Dawley rats Inhibit HMGB1 expression and subsequent production of inflammatory cytokines to prevent cerebral vasospasm (CVS) following subarachnoid hemorrhage (SAH) [49] In vivo-SD rats Alleviate brain injury after diffuse axonal injury (DAI) via its anti-inflammatory effects [50] Antimicrobial and antiviral activities In vitro Inhibit varicella zoster virus (VZV) [53] In vitro Inhibit severe acute respiratory syndrome coronavirus (SARS-CoV) replication [54] In vitro Inhibited influenza A virus (IAV) uptake into the cell [55] In vitro Reduce the severity of an infection with COVID-19 at the two stages of the COVID-19 induced disease process, 1. To block the number of entry points and 2. provide an ACE2 independent anti-inflammatory mechanism. [57] The commercial NA inhibitory screening kit Possess moderate influenza NA inhibitory activity [31] Cytotoxic and antitumor activities In vivo-tumor-bearing mice Reduce expression of TxAS, as well as proliferating cell nuclear antigen (PCNA), and rescue liver and kidney damage [58] In vitro-HepG2 Display protective effects against Aflatoxin B1 (AFB 1 )-induced cytotoxicity [59] Other activities -   [32] 1 , Columns 1 and 2 is the same in Table 1. It should be noted that the empty rows with triterpene saponins are grouped together or not listed in Table 2.

Other Activities
Saponin monomers of glycyrrhiza were shown to have various other physiological and pharmacological activities. GL (1) also possesses immunomodulatory, and neuroprotective effects [61] and antioxidant activities [62,63]. Furthermore GL (1) can be used in the clinical treatment of bronchitis, peptic ulcers, skin diseases, and oral diseases [56,64,65].
In addition, GA (1) may have a therapeutic effect on allergic rhinitis, partly by modulation of the Th1/Th2 balance through suppression of OX40 and by increasing the activity of regulatory T cells [66].

Solubilization Characteristics
In nature, saponins are distributed in 90 plant families from 500 genera [67]. Some of them have the potential to be used as natural surfactants because they contain the nonpolar sapogenin and water-soluble sidechain [68]. Glycyrrhiza is the most frequently used TCM in TCM formulae, with the function of harmonizing all kinds of TCMs. Research on the chemistry, pharmacological effects, clinical applications et al. of glycyrrhiza has been very extensive in recent decades. Besides, the saponins from glycyrrhiza have also significant solubilizing effects [69]. Interestingly, the solubilization characteristics of glycyrrhiza and saponins from glycyrrhiza were studied extensively over recent years. This part of review will deal with the solubilization characteristics, influences, and mechanisms regarding glycyrrhiza and triterpene saponins from glycyrrhiza (Table 3).

Solubilization Characteristics of Glycyrrhiza
Shi et al. [70] reported that glycyrrhiza has solubilization effects in TCM formulae, including sijunzi decoction, huangqi dazao decoction, and baishao gancao decoction, further explaining that the solubilizing components in glycyrrhiza are triterpene saponins. Meng et al. [71] studied and analyzed the decoctions of ephedra and glycyrrhiza, demonstrating that, compared to that of a single decoction, the contents of GL (1), ephedrine (including pseudoephedrine), and methephedrine (including methylpseudoephedrine) in the combined decoction of ephedra and glycyrrhiza were increased by 13.50%, 14.52%, and 64.0%, respectively. Nie et al. [72] demonstrated that after administration of a combined decoction of epimedium and glycyrrhiza, the contents of some chemical constituents, such as icariin in epimedium, were increased. Han et al. [73] reported that when extracted with 30% ethanol (v:v) with a 1:1 ratio of glycyrrhiza to curcuma longa, the extractive rate of curcumin doubled. At the same time, other studies also found that glycyrrhiza increased the contents of active ingredients in codonopsis, poria, atractylodes [74], Baphicacanthus cusia [75], Paeoniae Radix Alba [76], Isatidis Radix [77], and Scutellaria baicalensis [78].
Glycyrrhiza plays a significant role in solubilizing insoluble components, improving the bioavailability of active components and enhancing efficacy, reducing toxicity, and improving taste [79,80]. It should be noted that not all glycyrrhiza compounds in TCM formulae have surface activity, because solubilization is also affected by some crude drugs, such as Schisandra chinensis [70]. As for the mechanism of solubilization, most researchers demonstrated that the saponins of glycyrrhiza significantly reduced surface tension to play a solubilizing role, and further agreed that GL (1) is the main surfactant in glycyrrhiza [73][74][75]78,81,82]. [82]

baicalin
The dissolution rate of baicalin increases - [84] 10 pueraria The solubility of pueraria increases Another possible factor affecting the solubilization is the pH value of the solution [85] 11 pachymic acid Increase the solubility of pachymic acid Improve the bioavailability of pachymic acid [69] 12 Puerarin-glycyrrhizic acid dispersible tablets Improve the dissolution of puerarin GL (1) possesses solubilization effect [86] 3.

Solubilization Characteristics of GL (1)
Sasaki et al. [83] found that the water solubility of saikosaponin-a, the active principle of Bupleurum root, is increased in the presence of water extract or the saponin fraction of glycyrrhiza and that this solubilizing effect is due to GL (1). Du [82] demonstrated that glycyrrhiza exhibits solubilization on Ben Lamge granules, thereby proving that GL (1) possesses solubilization activity. In detail, the solubility of Ben Lamge granules increases with the addition of GL (1), whereas the surface tension of GL (1) decreases. Experimental results also indicated that GL (1) exists in micelles in aqueous solution, where the critical micelle concentration (CMC) is 1.188 mg/mL. Lu et al. [84] showed that GL (1) exhibits certain solubilization on baicalin, and the dissolution rate of baicalin increases gradually as the concentration of GL (1) increases. The optimal CMC of GL (1) is 0.22 mg/mL regarding compatibility between Scutellaria baicalensis and glycyrrhiza, at which time the solubilization of GL (1) is at its highest. Yang et al. [85] reported that when the ratio of pueraria and glycyrrhiza was 5:3, the dissolution of puerarin reached its maximum and the CMC of GL (1) was 0.18 mg/mL, which was affected by the structure of the drug and the pH value of the solution. Cai et al. [69] demonstrated that GL (1) increased the solubility of pachymic acid in an aqueous solution, thereby improving the bioavailability of pachymic acid. Liu et al. [86] demonstrated that puerarin-glycyrrhizic acid dispersible tablets could improve the dissolution of puerarin in vitro due to the solubilization effect of GL (1).
GL (1) was reported to possess amphiphilic components consisting of one triterpenoid aglycone molecule and two glycosyl groups. With its inward hydrophobic group (triterpenoid aglycone) and outward hydrophilic group (two glucuronic acids), GL (1) spherical micelles can form in aqueous solution to increase the solubility of hydrophobic drugs. Petrova et al. [87] showed that NMR chemical shifts of the protons of the GL (1) glucuronic moiety were sensitive to solution pH and not sensitive to GL (1) concentration changes during GL (1) aggregation. At the same time, the protons of the triterpene moiety were shown to be sensitive to the nearest environment, and micelles formed via hydrophobic interaction between the triterpene moieties of GL (1).

Conclusions
This review provides an up-to-date summary concerning the phytochemistry and pharmacology of glycyrrhiza. Three species of the genus Glycyrrhiza-G. uralensis, G. glabra, and G. inflata-are considered to have a shared botanical origin of Glycyrrhizae Radix et Rhizoma. By the end of 2020, 77 triterpene saponins were discovered and identified from these plants. Previous phytochemical investigations revealed that triterpene saponins are one of the major constituents contributing either directly or indirectly to the biological effects of glycyrrhiza.
Over recent decades, total saponins or saponin monomers from glycyrrhiza were found to possess various biological activities, such as hepatoprotective, anti-inflammatory, antimicrobial, antiviral, antitumor, antioxidant, and neuraminidase-inhibitory activities. Currently, these pharmacological studies on glycyrrhiza are limited to bioassays of only a few saponin monomers. Hence, further studies are needed to investigate the biological activities of more triterpene saponins via in vitro/vivo models. In addition, the phytochemistry of glycyrrhiza requires further study, with new compounds or degradation products possibly showing new pharmacological activities.
Another aim of this review was to summarize the solubilization characteristics, influences, and mechanisms regarding triterpene saponins from glycyrrhiza. Glycyrrhiza is widely used in TCM formulae and plays an important role in solubilizing insoluble components, thereby improving the bioavailability of active components, enhancing efficacy, and reducing toxicity, as well as demonstrating various pharmacological effects. The solubilization of glycyrrhiza with ephedra, epimedium, curcuma longa, codonopsis, poria, atractylode, Baphicacanthus cusia, Paeoniae Radix Alba, Isatidis Radix, and Scutellaria baicalensis was previously explored and the possible factors affecting the solubilization of glycyrrhiza were discussed, including some crude drugs and the pH of decoctions. Therefore, not all glycyrrhiza in a TCM formula possess solubilization activity. The solubilization of GL (1), the main solubilizing component from glycyrrhiza, with multiple active components from some other TCMs was also explored. In the discussion of solubilization mechanisms, the triterpene saponins represented by GL (1) from glycyrrhiza were shown to characteristically form micelles due to their amphiphilicity, thereby showing solubilization ability. The optimal CMC of micelle formation is different when GL (1) is combined with different TCMs or active ingredients. The pH of the solution was also shown to be critical to the formation of micelles. This review provides guidance regarding the better understanding of TCM compatibility and a theoretical basis for the further development and utilization of glycyrrhiza.

Conflicts of Interest:
The authors declare no conflict of interest.