**2.1. Cytotoxicity**

is worsened by hypersplenism, leucopenia or thrombocytopenia, and hypergammaglobulinemia are characteristic. Untreated disease in any age group in time can produce profound cachexia, multisystem disease, bleeding, susceptibility to secondary infections and death [6].

For visceral leishmaniasis, treatment is always recommended due to the fatal nature of the disease. The only treatment currently available for leishmaniasis relies on chemotherapy [7] as no vaccine has been successfully developed till date for humans. The first line of classical drug for treatment of leishmaniasis includes pentavalent antimonials, stibogluconate or meglumine antimonite. Pentavalent antimonials have been the drug of choice for more than 50 years [8–10]. Other common drugs are pentamidine, allopurinol, amphotericin B, imidazoles, miltefosine and paromomycin among others. Even though these drugs are effective other problems associated with these are (1) there lengthy regimens (weeks to months), (2) invasive modes of administration (intramuscular or intravenous) and (3) high drug-related toxicity along with the high cost of therapy which is beyond the capacity of many as this disease is known to be the 'disease of the poor'[10, 11]. All these factors together may lead to the patients discontinuing there therapies in the mid which have resulted in emergence of drug-resistant parasite strains adding to the list of problems associated with antileishma-

To add to these overwhelming situations, emergence of coinfection collaterally with increasing incidences of HIV has decreased the number of options available to patients. As we will see in this chapter that immunosuppression is a key for parasite survival drugs, which can modulate host immune defenses, should be considered for effective treatment of leishmaniasis. Development of vaccines has remained a challenge; however, a canine vaccine has been developed, and it is in use in South America [13]. At present, no immunization options are available against

Like any other successful pathogens, *Leishmania* has also developed strategies to evade host immune mechanisms in order to survive within the host. A significant number of virulence factors discovered in *Leishmania* are directed against circumventing the host immune response. Apart from these, the parasite also has the ability to maintain a chronic infectious state within its host by modulation of regulatory factors, which exemplifies the extent on its immune evasion potential. Indeed, the ongoing battle between the robust immune response mounted by a host and the counter evasion strategies by the parasite ultimately decides the fate of the

All these further establishes the fact that a chemotherapeutic alone is not enough to treat this intelligent parasite, and indeed an immunomodulator which can activate hosts own defense mechanism will be a better adjunct to the current line of treatment, which is relatively better

in terms of toxicity, efficacy and mode of administration.

**1.5. Current treatment options**

150 Biological Activities and Action Mechanisms of Licorice Ingredients

nial therapies [9, 11, 12].

leishmaniasis in humans.

disease.

**1.6. Subversion of host defense**

The first and foremost criteria for evaluation of a drug for its antiparasitic activity would be to evaluate its cytotoxic effects. The development of new drugs that has immunomodulatory properties requires both pharmacokinetic and toxicity studies to be carried out in conjunction to clinical verification. Our work so far has shown that GRA is relatively a nontoxic compound with up to 1.5 g/day consumption in humans [18].

GRA exhibited potent in vitro activity against intracellular *L. donovani* amastigotes (IC50, 4.3 μg/ml); it was devoid of any obvious cytotoxicity on macrophage host cells, because the cytotoxic concentration causing 50% cell death was ~100 μg/ml [19]. The infected mice treated with GRA were completely cured. Moreover, this therapy was seen to be effective in mice with established disease-progressive Th2 response. After treatment and resolution of parasitism, the cytokine profile indicated a switch to a protective Th1 pattern associated with upregulation of NO [19].

### **2.2. Mechanism**

#### *2.2.1. Macrophage activation by NF-κB-mediated nitric oxide and proinflammatory cytokine production*

Once the cytotoxic parameters were found satisfying the mechanistic profile was explored. The disease resolving property of GRA could be attributed to the production of NO and proinflammatory cytokines such as IL-12 [20, 21]. The most diversely studied mechanism that assists the *Leishmania* survival is modulating the macrophage cytokines production to bias the immune response to its benefit [22, 23]. *Leishmania* mainly inhibits the secretion of macrophage proinflammatory or disease resolving cytokines, which include interleukin 12 (IL-12), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ) and aids the secretion of antiinflammatory or disease promoting cytokines interleukin 10 (IL-10), transforming growth factor beta (TGF-β) for its own benefit.

Cytokine IL-12 is downregulated by *Leishmania via* ligation with macrophage receptors [22, 23]. One of the most important initial signaling events is the release of IL-12 by the infected macrophage, leading to subsequent priming of the Th1 response and production of IFN-γ [5, 24–26]. *Leishmania* also upregulates the production of anti-inflammatory cytokines such as IL-10. IL-10 is important in suppressing macrophage leishmanicidal activity by opposing IFN-γ [22, 24, 27], nitric oxide (NO) and IL-12 production [28].

The killing of intracellular *Leishmania* parasites by GRA correlated with the induction of the iNOS pathway. Nitric oxide (NO) is known to mediate many of the cytotoxic and immunological effects upon pathogenic challenge. This NO production has been shown to be dependent upon the inducible for of the nitric oxide synthases (iNOS) [29], whereas under normal physiological conditions only the constituitive forms of NOS (cNOS) are functional. Induction of iNOS in turn is under transcriptional regulation of NF-κB, a group of transcription factors that belong to the Rel protein family. The activated form of NF-κB is a heterodimer, which usually consists of two proteins, p65 (RelA) and p50 subunit [30]. In unstimulated cells, NFκB is found in the cytoplasm bound to an inhibitor, inhibitor of nuclear factor κB (IκB)α and IκBβ [31]. This association prevents nuclear translocation of NF-κB and hence iNOS transcription and NO production. Upon pathogenic challenge, the large multisubunit protein kinase, inhibitor of nuclear factor kappa-B kinase subunit beta(IKK) causes rapid proteasomal degradation of IκBα, thereby releasing NFκB and allowing its nuclear translocation. This event is inhibited in *Leishmania donovani* infection thereby promoting parasite survival [19] (**Figure 4**).

**Figure 4.** GRA promotes NF-κB-mediated parasite killing.

GRA, on the other hand, activates NF-κB through the regulation of genes essentially involved in encoding proinflammatory cytokines and inflammatory mediators such as NO. *L. donovani* infection suppressed NF-κB activation and translocation which was restored upon GRA administration via induction of IκBα phosphorylation. This in turn was achieved by modulating the upstream signal leading to IKK (inhibitor of nuclear factor kappa-B kinase subunit beta) activation, that is, without directly interfering with IKK (inhibitor of nuclear factor kappa-B kinase subunit beta). Overall this signaling activation by GRA led to degradation of IκBα leading to the translocation of NF-κB in the nucleus and transcriptional activation of iNOS and proinflammatory cytokines [19] (**Figure 4**). The antileishmanial activity of GRA was dependent on the iNOS activity and NO production was further justified by addition of a specific NOS inhibitor, NG-monomethyl-L-arginine (NMMA). Upon administration of NMMA along with GRA, a reduction in the parasite killing capability of GRA was seen and withdrawal of NMMA led to decreased parasite survival indicating a role of GRA-induced NO-mediated parasite killing.

#### *2.2.2. MAPK and phosphatases*

The killing of intracellular *Leishmania* parasites by GRA correlated with the induction of the iNOS pathway. Nitric oxide (NO) is known to mediate many of the cytotoxic and immunological effects upon pathogenic challenge. This NO production has been shown to be dependent upon the inducible for of the nitric oxide synthases (iNOS) [29], whereas under normal physiological conditions only the constituitive forms of NOS (cNOS) are functional. Induction of iNOS in turn is under transcriptional regulation of NF-κB, a group of transcription factors that belong to the Rel protein family. The activated form of NF-κB is a heterodimer, which usually consists of two proteins, p65 (RelA) and p50 subunit [30]. In unstimulated cells, NFκB is found in the cytoplasm bound to an inhibitor, inhibitor of nuclear factor κB (IκB)α and IκBβ [31]. This association prevents nuclear translocation of NF-κB and hence iNOS transcription and NO production. Upon pathogenic challenge, the large multisubunit protein kinase, inhibitor of nuclear factor kappa-B kinase subunit beta(IKK) causes rapid proteasomal degradation of IκBα, thereby releasing NFκB and allowing its nuclear translocation. This event is inhibited in *Leishmania donovani* infection thereby promoting parasite survival [19] (**Figure 4**).

GRA, on the other hand, activates NF-κB through the regulation of genes essentially involved in encoding proinflammatory cytokines and inflammatory mediators such as NO. *L. donovani* infection suppressed NF-κB activation and translocation which was restored upon GRA administration via induction of IκBα phosphorylation. This in turn was achieved by modulating the upstream signal leading to IKK (inhibitor of nuclear factor kappa-B kinase subunit beta) activation, that is, without directly interfering with IKK (inhibitor of nuclear factor kappa-B kinase subunit beta). Overall this signaling activation by GRA led to degradation of IκBα leading to the translocation of NF-κB in the nucleus and transcriptional activation of iNOS and proinflammatory cytokines [19] (**Figure 4**). The antileishmanial activity of GRA was dependent on the iNOS activity and NO production was further justified by addition of a specific NOS

**Figure 4.** GRA promotes NF-κB-mediated parasite killing.

152 Biological Activities and Action Mechanisms of Licorice Ingredients

Mitogen-activated protein kinase(MAPK) plays an important role in activation of NF-κB. MAPK signaling cascades are rather complex events which ultimately results in manifold increase in stimulus-mediated responses. However, if this response remains unchecked, it may be detrimental for the cells and thus a balance between the activity of the kinases and phosphatases play important roles in physiological scenario. In macrophages, the MAPK (mitogen-activated protein kinase) cascade and the NF-κB pathway play important roles in the regulation of functions involved in inflammation and host defense. *L. donovani* successfully sabotage these pathways creating an anti-inflammatory milieu by inhibiting production of NO and proinflammatory cytokines thereby strengthening their existence within the macrophages [32]. Thus, the agents that can activate NF-κB pathway creating a proinflammtory milieu potent enough to kill the parasites might prove attractive candidates to control *Leishmania* infection. Our studies also revealed that the Mitogen-activated protein kinase kinase - extracellular signal regulated kinases (MEK-ERK) pathway is compensated in infected cells [33]. GRA, the triterpenoid is an ideal candidate both because of its pharmacologically safe parameters and immunomodulatory properties [19]. The switching of the immunological response was found to be dependent upon the MAPK (mitogen-activated protein kinase) activation among which

**Figure 5.** The balance between kinases and phosphatases is restored upon GRA treatment.

only ERK and p38 were found to be regulated by GRA [34]. In *Leishmania* infection, these MAPK (mitogen-activated protein kinase) activation was severely compensated which was in turn is attributed to the fact that activity of MAPK (mitogen-activated protein kinase) phosphatases, which dephosphorylate and thereby inactivate MAPK (mitogen-activated protein kinase), significantly increased. This corroborated with the fact that inhibition of SHP-1, a MAPK (mitogen-activated protein kinase) phosphatase led to stronger proinflammatory responses against infection [35]. However, GRA treatment could potentially inhibit the activity of the phosphatases, thereby shifting the total kinase to phosphatase balance in favor of creating an antileishmanial milieu [34] (**Figure 5**).

**Figure 6.** GRA activates p38 via the TLR2/4 pathway.

#### *2.2.3. Downregulation of toll-like receptor (TLR) pathways*

Toll-like receptor (TLR) expressed on the cells of the innate immune system are critical for recognition of pathogen-associated molecular patterns (PAMP). Upon ligand binding, the TLR gets activated leading to downstream signaling cascade activation leading to NF-κB- and MAPK (mitogen-activated protein kinase)-mediated proinflammatory cytokine production. The TLR2 agonists used to activate TLR2 signaling pathway showed host protective immune response resulting in parasite clearance from *L. donovani* infected macrophages [36]. To subvert this inflammatory response *Leishmania* either recruits suppressors of the cytokine signaling (SOCS) family proteins, SOCS-1/3 [37], or activates host de-ubiquitinating enzyme A20 to negatively regulate TLR2/4-induced host protective response [38, 39]. *Leishmania* can alter TLR4 signaling to favor its establishment within the macrophages. TLR4-mediated macrophage activation was shown to be suppressed in *Leishmania* infection through the release of TGF-β [40]. *L. mexicana* capitalizes on TLR4 signaling to inhibit the production of IL-12 by infected macrophages and promotes parasite establishment [41]. Other TLRs involved in infection with *Leishmania* include TLR3 and TLR9.

only ERK and p38 were found to be regulated by GRA [34]. In *Leishmania* infection, these MAPK (mitogen-activated protein kinase) activation was severely compensated which was in turn is attributed to the fact that activity of MAPK (mitogen-activated protein kinase) phosphatases, which dephosphorylate and thereby inactivate MAPK (mitogen-activated protein kinase), significantly increased. This corroborated with the fact that inhibition of SHP-1, a MAPK (mitogen-activated protein kinase) phosphatase led to stronger proinflammatory responses against infection [35]. However, GRA treatment could potentially inhibit the activity of the phosphatases, thereby shifting the total kinase to phosphatase balance in favor of creating an

antileishmanial milieu [34] (**Figure 5**).

154 Biological Activities and Action Mechanisms of Licorice Ingredients

**Figure 6.** GRA activates p38 via the TLR2/4 pathway.

*2.2.3. Downregulation of toll-like receptor (TLR) pathways*

Toll-like receptor (TLR) expressed on the cells of the innate immune system are critical for recognition of pathogen-associated molecular patterns (PAMP). Upon ligand binding, the TLR gets activated leading to downstream signaling cascade activation leading to NF-κB- and MAPK (mitogen-activated protein kinase)-mediated proinflammatory cytokine production. The TLR2 agonists used to activate TLR2 signaling pathway showed host protective immune response resulting in parasite clearance from *L. donovani* infected macrophages [36]. To subvert this inflammatory response *Leishmania* either recruits suppressors of the cytokine signaling (SOCS) family proteins, SOCS-1/3 [37], or activates host de-ubiquitinating enzyme A20 to

The effect of GRA is mediated by means of MAPK (mitogen-activated protein kinase) activation and phosphatase downregulation. Deeper analysis revealed that the MAPK (mitogenactivated protein kinase) p38 activation was dependent on GRA-mediated canonical and noncanonical activation where numerous upstream molecules play important roles [42]. This study further highlighted the importance of p38 MAPK (mitogen-activated protein kinase) in GRA-mediated parasite elimination and its activation by upstream kinases like MKK3/6 which itself is dependent upon upstream signaling molecules (**Figure 6**).

**Figure 7.** Mode of Action of 18β-Glycyrrhetinic Acid-potent antileishmanial immunomodulator. *Leishmania* (i) inhibits p38 phosphorylation, (ii) activates phosphatases, (iii) inhibit activation of NFκB thereby leading to (iv) inhibition of proinflammatory cytokine reponses. 18β-Glycyrrhetinic Acid acts as an immunomodulator which in infection activates (a) canonical and (b) non canonical pathways leading to phosphorylation and activation of p38, (c) activates IκB Kinase which in turn results in IκBα phosphorylation and degradation leading to NFκB phosphorylation and activation, (d) inhibit phosphatases which would otherwise lead to dephosphorylation and inhibition of major kinases therby promoting proinflammatory cytokine response generation and (e) parasite killing.

Thus, the effective macrophage activation via NO and proinflammatory cytokines production in response to GRA treatment justifies the candidature of this potent immunomodulator as an antileishmanial. Furthermore, this compound may prove to be effective in terms of generating immunity not only in nonhealing leishmaniasis but also for the treatment of other chronic infectious diseases. A comprehensive model giving the overall mechanistic insight into the mode of action of GRA as antileishmanial agent will help in deducing its function in other intramacrophage pathogens which assume similar immunoevasive mechanisms to escape host defenses (**Figure 7**).
