5.1.3. Phytochemical Aspects

Table 3 presents a summary of phytochemical studies of *G. americana* extracts, as well as the structures of their phytochemical constituents.



*Molecules* **2020**, *25*,3879

The chemical characterization of *G. americana* fruits and leaves presents iridoids as major constituents, belonging to the class of terpenes. Genipin (**55**) is the main iridoid found in *G. americana* fruits and has considerable economic potential due to its pigmentation property. The extraction of genipin (**55**) can occur by three di fferent methods, enzymatic hydrolysis, extraction with solvents and ultrasound. A study performed the extraction of genipin (**55**) from *G. americana* fruits using the enzymatic method and quantified 7.85 mg/g of this phytochemical in the sample [119,145].

Pacheco et al. [146] carried out a study on the nutritional composition and energy value of the pulp of *G. americana* fruits. After triplicate analysis, 70% of moisture, 0.5% of proteins, 0.0% of lipids, 1.1% of ash, 22.1% of carbohydrates, 6.3% of dietary fiber, 0.0 mg/100g of beta-carotene, 22.5 mg/100g of vitamin C, 176 mg GAE/100g of phenolic compounds and 90.7 kcal/100g of total energy value were found.

In the methanolic extract of *G. americana* fruits, the following iridoid glycosides were identified, isolated and structurally elucidated: geniposidic acid (**40**), geniposide (**48**), gardenoside (**54**), genipin-gentiobioside (**49**) and 4 new iridoids not previously identified: genameside A (**50**), genameside B (**51**), genameside C (**52**) and genameside D (**53**) [140]. Also in *G. americana* fruits, iridoids geniposidic acid (**40**), gardenoside (**54**), genipin-1-β-gentiobioside (**49**), geniposide (**48**), 6---*O*-*p*-coumaroyl-1-β-gentiobioside geniposidic acid (**59**), 6---*O*-*p*-coumaroylgenipin gentiobioside (**60**), genipin (**55**), 6- -*O*-*p*-coumaroyl-geniposidic acid (**61**), 6- -*O*-feruloylgeniposidic acid (**62**) were found in the methanolic extract from the endocarp and mesocarp. In addition, possible antioxidant and antiproliferative properties have been attributed to the extract and, mainly, to genipin (**55**) [143].

After extraction by pressurized ethanol and analysis of the genipin (**55**) and geniposide (**48**) content in the whole fruit and its parts separately at 50 ◦C and pressure of 2 bars, 20.7 mg/g of genipin (**55**) and 59 mg/g of geniposide (**48**) were found in the mesocarp; 1.16 mg/g of genipin (**55**) and 0.06 mg/g of geniposide (**48**) were found in seeds; 7.5 mg/g of genipin (**55**) and 39.9 mg/g of geniposide (**48**) were found in fruit bark; 38.9 mg/g of genipin (**55**) and 0.01 mg/g of geniposide (**48**) were found in the endocarp; 22.9 mg/g of genipin (**55**) and 0.1 mg/g of geniposide (**48**) were found in the endocarp extract and seeds; and 37.2 mg/g of genipin (**55**) and 0.57 mg/g of geniposide (**48**) were found in the whole fruit [144].

For fruits, the presence of other classes of secondary metabolites was investigated in the hydroalcoholic extract. Mayer reagen<sup>t</sup> was used to detect alkaloids; for tannins, reaction with ferric chloride; for anthraquinones, reaction with ammonia; for flavonoids, Shinoda's reaction; for steroids and triterpenes, the Libermann-Burchard reagen<sup>t</sup> was used; the saponins test was carried out through agitation, observing the presence or absence of foam; and the coumarin test by fluorescence under UV light. The results for this qualitative analysis point to the presence of alkaloids, tannins, flavonoids, triterpenes, saponins and coumarins in the fruit pulp extract and absence of steroids and anthraquinones [147].

Silva et al. [139] identified 13 compounds in a hydroalcoholic extract of *G. americana* leaves. The following are among the isolated substances: (A) coniferin; (B) the iridoids asystasioside D (**39**), geniposidic acid (**40**), tarenoside (**41**) and teneoside A (**42**); (C) loganic, chlorogenic and 1,3-di-*O*-caffeoylquinic acids; and (D) flavonoids, first identified in this genus, kaempferol-3- *O*-hexoside-deoxyhexoside-7- *O*- deoxyhexoside (**43**), isorhamnetin-3- *O*-hexoside-deoxyhexoside-7- *O*-deoxy-hexoside (**44**), quercetin-3- *O*-hexoside-deoxyhexoside (**45**), kaempferol-3- *O*-hexoside-deoxyhexoside (**46**) and isorhamnetin-3- *O*-hexoside-deoxyhexoside (**47**). Other iridoids were also found in the hydroalcoholic extract of *G. americana* leaves such as genipin derivative (**55**), 1-hydroxy-7-(hydroxymethyl)-1 *H*,4a *H*,5 *H*, 7a *<sup>H</sup>*-cyclopenta[c]pyran-4-carbaldehyde (**56**) and 7-(hydroxymethyl)-1-methoxy-1 *H*,4a *H*,5 *H*,7a *H*cyclopenta[c]pyran-4-carbaldehyde (**57**) [142].

There are few studies on phytochemical screening and characterization of compounds for *G. americana* stem, roots, and seeds. A qualitative study that analyzed the ethanolic extract of leaves and stem bark determined the presence of flavonoids, xanthones, saponins and triterpenes in the stem

bark and saponins and triterpenes in leaves [148]. [149] isolated and characterized lectin present in *G. americana* stem bark, which was named GaBL and tested for hemagglutinating properties.

Neri-numa et al. [143] evaluated the antioxidant and antiproliferative activity of the methanolic extract from *G. americana* ripe and green fruits. The ability to eliminate DPPH radicals has been reported to be like that of ascorbic acid, and the extract from green fruits has higher concentration of iridoids and greater e fficiency to eliminate radicals. The *in vitro* antiproliferative activity was also observed with e fficiency in all tested cell lines, with greater activity for the extract from green fruits, which has high concentration of iridoid genipin (**55**), to which this property was attributed. In addition, the anticholinesterase activity of the ethanol extract from *G. americana* fruit bark, pulp and seeds was also observed and may be associated with the presence of chlorogenic acid, an acetylcholinesterase (AChE) inhibitor [139,150].

Genipin (**55**), a triterpene found in *G. americana* fruits was tested *in vitro* and *in vivo* for its anti-inflammatory property and its role on memory deficiencies induced by LPS. Microglia stimulation by LPS of gram-negative bacteria induces the production of inflammatory mediators, whose overproduction can cause neuronal damage. Genipin (**55**) inhibited the production of these mediators in the BV2 microglial cell line through the dose-dependent suppression of LPSe-induced NF-κB activation by activating the expression of erythroid nuclear factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Active NF-κB induces the production of inflammatory mediators such as PGE2, TNFα and IL-1β, while Nrf2 encodes antioxidant enzymes such as HO-1, which promote the elimination of ROS and, consequently, the inhibition of the NF-κB expression [151].

The anti-inflammatory role of genipin (**55**) is also associated to another mechanism, the inhibition of the activation of NLRP3 and NLRC4 inflammasomes by suppressing macrophage autophagy. Agonists of NLRP3 and NLRC4 inflammasomes promote the activation of autophagy in macrophages, which enhances the secretion of IL-1β and ASC oligomerization, while suppression of autophagy promoted by genipin (**55**) inhibits this e ffect [152].

The protective role of genipin (**55**) on LPS-induced acute lung injury has been investigated. Genipin (**55**) positively regulated the signaling of the phosphoinositide 3-kinase/phosphorylated protein kinase B (PI3K/p-AKT) pathway by increasing the levels of p-AKT. PI3K generates phosphatidylinositol-3,4,5-triphosphate (PIP3), which acts as a second messenger and facilitates the translocation of protein kinase B (AKT) to the plasma membrane, where it is activated by phosphorylation and can later be transported to the nucleus. AKT promotes the phosphorylation of some molecules, among them AMPc-responsive binding protein (CREB), whose activation is associated with increased Bcl-2 activity, which inhibits pro-apoptotic caspase-9, promoting a protective e ffect of cell survival [153,154].

In contrast, the action of genipin (**55**) on the PI3K/p-AKT pathway was also related to its inhibitory activity on the growth of human bladder cancer cells [155] and squamous cell carcinoma [156]. It was observed that genipin (**55**) induced the cell cycle to stop in G0/G1 phases, and promoted the apoptosis of cancer cells, with increase in the expression of pro-apoptotic protein Bax. Such cell growth suppressive effects have been associated with inactivation of the PI3K/p-AKT pathway, shown by the reduction of phosphorylated PI3K and AKT levels [155].

Zhao et al. [157] analyzed the protective role of genipin (**55**) against ischemia-reperfusion lesion associated with energy deficiency and oxidative stress, which are regulated by mitochondrial uncoupling protein 2 (UCP2) and NAD-dependent deacetylase sirtuin-3 (SIRT3), respectively. In this lesion, damage increases due to the increase in the ischemia duration, as well as the degree of energy deficiency and oxidative stress, with increase in UCP2 expression and SIRT3 activity. Genipin (**55**) acts as a specific inhibitor of UCP2. Therefore, in mice submitted to treatment with genipin (**55**), reduction in UCP2 expression and SIRT3 activity was observed, as well as a lower NAD +/NADH ratio and increased levels of adenosine triphosphate (ATP), reducing oxidative stress and energy deficiency and, consequently, mitigating damage.

The effects of genipin (**55**) on energy metabolism are also related to its anti-tumor property, capable of inhibiting the proliferation of several cancer cells in breast [158], colon [159], hepatocellular [160] cholangiocarcinoma [161] and gioblastoma [162]. UCP2 overexpression is observed in tumor cells, which gives genipin (**55**), a UCP2 inhibitor, a potential anti-tumor activity mechanism. UCP2 promotes the decoupling of the electron transport chain to oxidative phosphorylation, reducing energy availability and the production of O2<sup>−</sup>, a ROS.

Cancer cells are under oxidative stress and to protect themselves, they increase the UCP2 expression to reduce the formation of ROS. UCP2 inhibition by genipin (**55**) promotes an increase in ROS, triggers the nuclear translocation of glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), formation of autophagosomes and expression of LC3-II autophagy marker, leading to cell death or growth inhibition, invasion and migration of tumor cells. In addition, genipin (**55**) enhances autophagic cell death induced by gemcitabine, a clinically used chemotherapeutic agen<sup>t</sup> (Figure 5) [163,164].

**Figure 5.** Effects of genipin on energy metabolism: anti-tumor property: Transport mechanisms of geniposide and genipin, which are abundantly present in extracts from plants such as *Genipa americana*, involve converting geniposide into genipin in the intestinal lumen through bacterial enzymes β-glucosidases. Uncoupling protein 2 (UCP2) is a genipin target in the treatment of cancer. In mitochondria, the respiratory chain, formed by complexes I to IV, transfers electrons from NADH through oxidation-reduction reactions. Complexes I, II, and III contribute to the production of H+ ion gradient. The electrochemical gradient generated is coupled to the ADP phosphorylation process via ATP synthase. Oxygen is the final electron acceptor and is reduced to water by the electron transfer of complex IV. However, its early reduction into complexes I and III leads to the formation of O2•–. UCP2 is a protein widely expressed in tumor cells. Its function is to reduce ROS production and increase the survival of tumor cells by uncoupling the electrochemical gradient generated by the respiratory chain. For this purpose, UCP2 increases H<sup>+</sup> output from the intermembrane space to the mitochondrial matrix and reduces the mitochondrial membrane potential. This mechanism, present in tumor cells as a survival factor by reducing ROS generation, is the genipin target [165]. A, adenosine; CoQ, coenzyme Q; Cyt C, cytochrome C; FAD, flavin adenine dinucleotide; NAD, Nicotinamide adenine dinucleotide; ROS, reactive oxygen species; UCP2, uncoupling protein 2.

Another anti-tumor mechanism associated with genipin (**55**) occurs through negative regulation of the signal transducer and transcription activator (Stat)/cell differentiation protein of induced myeloid cell leukemia 1 (Mcl-1). Mcl-1 is a member of the Bcl-2 family and has anti-apoptotic activity, being associated with cell survival and is overexpressed in gastric cancer cells. The apoptotic mechanism of cancer cells promoted by genipin (**55**) is related to the negative regulation of Mcl-1, which can occur through the activation of the SHP-1 phosphatase and the suppressor of cytokine

signaling 3 (SOCS3). In addition, this phytoconstituent inhibits the activity of JAK2 enzymes of the Janus kinase family (JAK), responsible for the activation of the Stat3 transcription factor, which regulates the expression of genes related to cell survival. Thus, inactivating JAK2 enzymes, there is no activation of Stat3 and expression of the MCL1 gene, which encodes the anti-apoptotic protein Mcl-1 (Figure 6a,b) [166].

**Figure 6.** Apoptosis mediated by genipin through interference with myeloid cell leukemia-1 (Mcl-1) synthesis in gastric cancer cell lines: (**a**) Cytokine receptors without intrinsic protein kinase domain amplify extracellular signals through signal transduction via Janus Kinase (JAK) family (JAK1 to JAK3 and tyrosine kinase 2). After receptor activation, JAK2 phosphorylates the tyrosine residue of transcription factor Signal Transducer and Activator of Transcription 3 (STAT3), which enables its binding to the promoter of target genes related to survival and apoptosis. Subsequently, Mcl-1 is synthesized; (**b**) Genipin absorption by tumor cells induces mitochondrial dysfunction due to decreased Mcl-1 expression through the JAK2/STAT3 pathway. Δψm, mitochondrial membrane potential; JAK2, Janus Kinase 2; Mcl-1, myeloid cell leukemia-1; STA3, Signal transducer and activator of transcription 3 [166].

Inhibition of Sonic Hedgehog, one of three proteins in the signal family called hedgehog found in mammals by genipin (**55**), was also associated with its anti-tumor property. Genipin (**55**) binds to the protein of the Hedgehog Smoothened (SMO) signaling pathway through the drug-affinity-responsive target stability (DARTS), increasing the expression of p53 and NOXA, a protein of the Bcl-2 family that contributes to apoptosis promoted by p53. This mechanism occurs by inhibiting the expression of the GLI1 gene, a transcriptional activator of the Hedgehog pathway that reduces p53 expression. Thus, the binding of genipin (**55**) to SMO promoter induces a reduction in GLI1 activity and an increase in p53 expression [167].

Genipin (**55**) was used in cattle to increase corneal stiffness and induce corneal collagen cross-linking (CXL), which reduces the progression of ectasia, that is, corneal distention. Through a mechanism still unknown, genipin (**55**) induces CXL by up to 7%, a result superior to that induced by treatment with riboflavin and UV light applied in the control group, which presented only 5.6% cross-linking. To achieve 7% cross-linking, 140 μL of 0.5% genipin (**55**) was administered every 1 h for 2 h [168]. The role of genipin (**55**) investigated in cattle suggests further studies in humans. Furthermore, this cross-linking property of genipin (**55**) has also been used in the biotechnological production of hydrogels, gelatin biofilms and transdermal patches for the controlled release of drugs [169].

Also, for ophthalmic treatment, genipin (**55**) was used in posterior scleral contraction/reinforcement surgery (PSCR), which delays axial stretching of the eyeball, common in human myopia. However, despite the significant effect of the procedure, it was not sustained in the long term, which was related to the loss of sclera resistance to traction. The sclera is a layer of fibrous, opaque and dense tissue that lines the eye, on which the cross-linking capacity of genipin was tested (**55**), which doubled the sclera resistance and increased by 30 % resistance to enzymatic degradation, which could promote sclera weakening. The study points out the efficacy and safety of PSCR with sclera cross-linked with genipin (**55**) to restrict axial elongation of the eyeball [168].

Genipin (**55**) was tested for possible antiviral activity on Kaposi's sarcoma herpes virus (KSHV). Genipin (**55**) played a double and dose-dependent role. At lower concentration and administered for 48 h, the phytochemical significantly reduced the production of the nuclear antigen associated with KSHV latency (LANA) and increased the number of copies of the virus intracellular genome, favoring lytic replication of KSHV. Treatment with higher genipin (**55**) doses induced the activation of caspases 3 and 7 by reducing the expression of Bcl-2, promoting apoptosis, which is impaired, since the virus produces viral Bcl-2, approximately 60% identical to cellular Bcl-2, which makes the infected cell more resistant. New studies have been proposed to investigate the role of genipin (**55**) in modulating the KSHV life cycle and possibly prevent disorders associated with the virus [170].

Nonato et al. [171] evaluated the GABA-mediated anticonvulsant e ffect of the methanolic extract from *G. americana* leaves rich in polysaccharides. A heteropolysaccharide (PRE) with inhibitory and anticonvulsant e ffect on the central nervous system (CNS) was identified, which was reversed after the administration of the GABAergic flumazenil antagonist, indicating the participation of this receptor in the e ffect performed by PRE, which also reduced oxidative stress in the pre-frontal cortex, hippocampus and striated nucleus of animals that had induced seizures, observed by the increase of GSH levels and reduction of lipid peroxidation levels.

In *G. americana* leaves, a glycoconjugate rich in arabinogalactane and uronic acid was found, with anticoagulant, antiplatelet, and antithrombotic properties. Anticoagulant activity was observed in fraction containing uronic acid and occurs through the intrinsic and/or the common pathway of the coagulation cascade by a still unknown mechanism. Glycoconjugate inhibits platelet aggregation induced by adenosine diphosphate (ADP), but not collagen-induced aggregation. Antithrombotic action was observed in a model of rats with venous thrombosis and, similar to the antiplatelet activity, it was found in fraction rich in arabinogalactane [172].

The hydroalcoholic extract from *G. americana* stem bark was evaluated for its possible antimicrobial properties. Minimum Inhibitory Concentration (MIC) tests were carried out with *E. coli*, *S. aureus* and *P. aeruginosa* and MIC was ≥ 1024 μg/mL in all strains. E fficiency was not considered satisfactory, but the association of the extract with aminoglycoside drugs amikacin and gentamicin and the lincosamide clindamycin, increased the antimicrobial potential of these drugs. This property was attributed to tannins present in the extract with antimicrobial activity. The result of this association induced greater susceptibility of *P. aeruginosa* and *E. coli* to death by gentamicin and *S. aureus* by Amikacin in all strains submitted to this treatment [173].

The polysaccharide extract from *G. americana* leaves was tested against *Trypanosoma cruzi* epimastigotes, trypomastigotes and amastigotes. The results showed antiparasitic e ffect against the three forms of the protozoan with low toxicity to mammalian cells. In addition, it demonstrated potent activity even on amastigote forms, which are intracellular, suggesting that the compound responsible for this activity has access to the intracellular medium. After extract administration, ROS generation was observed, which causes damage to trypanothione reductase, an enzyme important for the oxidative balance of the protozoan. Morphological changes indicate cell death due to necrosis with rounding and shortening of the parasite, cytoplasmic leakage and membrane degradation [174].
