Overview of the Justicia Genus: Insights into Its Chemical Diversity and Biological Potential

The genus Justicia has more than 600 species distributed in both hemispheres, in the tropics and temperate regions, and it is used in the treatment of numerous pathologies. This study presents a review of the biological activities of plant extracts and isolated chemical constituents of Justicia (ACANTHACEAE), identified in the period from May 2011 to August 2022. We analyzed over 176 articles with various biological activities and chemical compound descriptions present in the 29 species of Justicia. These have a variety of applications, such as antioxidant and antimicrobial, with alkaloids and flavonoids (e.g., naringenin) the most frequently identified secondary metabolites. The most observed species were Justicia gendarussa Burm., Justicia procumbens L., Justicia adhatoda L., Justicia spicigera Schltdl, and Justicia pectoralis Jacq. The frontier molecular orbitals carried out using density functional theory (M062X and basis set 6-311++G(d,p) indicate reactive sites for naringenin compound and a chemical reaction on phytomedicine activity. The energy gap (206.99 kcal/mol) and dimer solid state packing point to chemical stability. Due to the wide variety of pharmacological uses of these species, this review points toward the development of new phytomedicines.


Introduction
The Cerrado is a highly heterogeneous landscape, and some of it is subject to severe threats and deserves special attention, including the Cerrado-Amazon transition, which coincides with an "arc of deforestation", and rupestrian fields [1]. The region called Cerrado, located in the central portion of Brazilian territory, has changed abruptly in environmental, social and economic aspects. These changes were caused by the intense process of human occupation to which this ecosystem has been subjected, due to a sum of political interventions, natural features of the landscape and technological advances in agriculture [2]. The Cerrado is considered a Biodiversity Hotspot, which supports high species richness and thousands of endemic species [3]. Part of this huge biodiversity can be associated with the diversity of native vegetation types (e.g., grasslands, shrublands, typical savannas, and woodland savannas) that differ in grass cover, percentage of canopy cover, and dominant plant species, as well as fire dynamics and water availability [4]. Among the plant biodiversity, the ACANTHACEAE family has pantropical distribution, reaching some temperate areas, with approximately 240 genera and approximately 3250 species [5]. In Brazil, it is estimated that there are approximately 40 genera and 449 species, of which at least 254 are endemic, with a high concentration of species in the southeast and central-west regions [6].
Many of the medicines currently available are derived from natural sources. In additional to their physiological roles in plants, flavonoids are important components of the human diet, although they are not considered nutrients. Flavonoids are an important class of plant secondary metabolites that serve several functions, including pigments and antioxidant activity. The biological activities from flavonoids make Cerrado plants good candidates for phytochemical studies, mainly naringenin, which belongs to the class of chalcones [69]. Naringenin is a naturally occurring flavonone (flavonoid) known to have a bioactive effect on human health, and it is found primarily in fruits (grapefruit and orange) and vegetables. Naringenin has several biological functions, such as antidiabetic, antiatherogenic, antidepressant, immunomodulatory, antitumor, anti-inflammatory, DNA protective, hypolipidemic, antioxidant, activator of peroxisome proliferator-activated receptors (PPARs) and memory enhancer. Several molecular mechanisms      (19 mm) and MIC = 1024 µg/mL. The same activity showed by alkaloids 8 (vasicoline) (has greater inhibitory capacity in the biosynthesis of fatty acids and stops the activity of the mtFabH enzyme of Mycobacterium tuberculosis, being able to interrupt the infection in its initial stage) and 9 (vasicine) [inhibiting the growth of K. pneumoniae (10.2 mm) and MIC = 6.25 µg/mL, E. coli (12.5 mm) and MIC = 3.125 µg/mL, P. aeruginosa (6 mm), S. pyogenes (9.8 mm) and MIC = 25 µg/mL, S. aureus (12.8 mm) and MIC = 12.5 µg/mL, S. marcescens (8.2 mm) and MIC = 3.125 µg/mL, when compared to ofloxacin (8.8 mm, 9.1 mm, 2 mm, 9.5 mm and 7.8 mm, respectively) and A. flavus (10.5) and MIC = 3.125 µg/mL, C. albicans (14.2) and MIC = 12.5 µg/mL and C. neoformans (11.5 mm) and MIC = 25 µg/mL when compared with amphotericin (12 mm, 11 mm and 10 mm, respectively)], obtained from the extract of leaves of J. adhatoda L. Compound 9 (vasicine) also showed antioxidant effects (protecting deoxyribose from the action of free radicals with IC 50 539.64 µg/mL and having a strong chelating activity) and anticancer effects [inhibitory effect on the growth of prostate cancer cells (IC 50 81.11 µg/mL)].
Etamine (10), a nitrogen compound, from the ethanolic extract of the leaves of J. gendarussa Burm., exhibited DPPH radical scavenging activity with IC 50 = 22.55 µM, and Quercetin as positive control, IC 50 = 18.56 µM. Pyrrolidines 11 (Secundallerone B) and 12 (Secundallerone C), along with acid 13 (2-caffeoyloxy-4-hydroxy-glutaric acid) showed antidiabetic effects, such as α-glucosidase inhibitors when extracted from leaves of J. secunda Vahl., using the methanolic extract. There are compounds that have various biological activities. An example is kaepferitrin (14), an alkaloid that has shown antinociceptive, cytotoxic effects against cancer cells (against human cervical carcinoma cells, inducing apoptosis of these cells by 35% and inhibiting their growth by 53%), antidiabetic and anticonvulsant. Another alkaloid 15 (gendarussin A), isolated from the ethanolic extract of the leaves of J. gendarussa Burm., has an anti-HIV cytotoxic effect, decreasing viral load, increasing anti-HIV activity (reverse transcriptase inhibition), with an IC 50
The crystal structure of naringenin makes a conjugated six-membered ring, forming strong O3-H3···O2 intramolecular interactions, as shown in Table 5. The crystal packing for naringenin is formed by dimers, which are responsible for generating O4-H4···O5 intermolecular interactions, which can be described as R 2 2 (24) [192] (Figure 4a). In a twodimensional hydrogen-bonding arrangement, there is a chain appearing in a zigzag and growing along the c-axis, which is formed by the O5-H5···O2 intermolecular interactions and can be described as C 1 1 (9) (Figure 4b). Additionally, the C15-H15···O4 intermolecular interactions also form a zigzag chain, which grows along the b-axis and can be described as C 1 1 (10) (Figure 4c). The crystal packing is formed by the dimers (involving hydroxyl groups), and the zigzag chains, which generate a two-dimensional crystalline network, as shown in Figure 4d.   Figure 5a correspond to a dimer formed by O4-H4···O5 intermolecular interaction. In addition, the red dots in Figure 5b are related to the O5-H5···O2 intermolecular interaction. Finally, the non-classical C15-H15···O4 intermolecular interaction is represented by the red dots on the HS, as shown in Figure 5c.  Figure 5a correspond to a dimer formed by O4-H4···O5 intermolecular interaction. In addition, the red dots in Figure 5b are related to the O5-H5···O2 intermolecular interaction. Finally, the non-classical C15-H15···O4 intermolecular interaction is represented by the red dots on the HS, as shown in Figure 5c.
The 2D fingerprint plot of naringenin is shown in Figure 6. The 2D fingerprint plots (d i vs. d e ) quantify the types of intermolecular contacts in the solid-state arrangement [193]. These H···H contacts make up 35.0% of the HS of naringenin because it is an organic compound [194]. The red spots represent O···H/H···O contacts, which are the second largest contributions, with 31.8% of the HS of naringenin, and it is shown as the spikes at the bottom of the 2D fingerprint plot. Finally, C···H/H···C contacts represent 23.5% of the HS of naringenin. The 2D fingerprint plot of naringenin is shown in Figure 6. The 2D fingerprint plots (di vs. de) quantify the types of intermolecular contacts in the solid-state arrangement [194]. These H···H contacts make up 35.0% of the HS of naringenin because it is an organic compound [195]. The red spots represent O···H/H···O contacts, which are the second largest contributions, with 31.8% of the HS of naringenin, and it is shown as the spikes at the bottom of the 2D fingerprint plot. Finally, C···H/H···C contacts represent 23.5% of the HS of naringenin.   The 2D fingerprint plot of naringenin is shown in Figure 6. T (di vs. de) quantify the types of intermolecular contacts in the solid-s These H···H contacts make up 35.0% of the HS of naringenin becau pound [195]. The red spots represent O···H/H···O contacts, which contributions, with 31.8% of the HS of naringenin, and it is sho bottom of the 2D fingerprint plot. Finally, C···H/H···C contacts rep of naringenin.  Naringenin has a molecular weight of 272.257 g/mol, resulting from the addition of three hydroxyl groups 4 , 5 and 7 carbons in the backbone of flavonoids, and its molecular formula is C 15 H 12 O 5 [195,196]. This compound is found in high concentrations, especially in grapefruit (43.5 mg/100 mL), followed by orange juice (2.13 mg/100 mL) and lemon juice (0.38 mg/100 mL) [197]. Naringenin has a range of biological effects on human health, which include a reduction in lipid peroxidation markers, defense of metabolism, increase in antioxidants, reduction of reactive carbohydrate species, as well as modulation of the immune response [198,199]. In vitro and in vivo animal studies have reinforced evidence of the diversity of pharmacological effects of naringenin; among them, we highlight hepatoprotective, antiatherogenic, anti-inflammatory, antimutagenic, anticancer and antimicrobial activity [200]. Although we have identified in the literature that there is an enormous amount of data on the in vitro biological effects of naringenin, there are still few studies available on its therapeutic potential [201], and thus, further clinical studies are needed, aiming at the safety, efficacy and bioavailability of naringenin in humans.
The frontier molecular orbitals (FMO) taken from the natural bond orbital (NBO) analysis for compound 5 (naringenin) were carried out at the M062X/6-311+G(d,p) level of theory, and this is shown in Figure 7. The HOMO appears as a π bonding orbital, and it is localized on the phenyl π bonding region, which is characteristic of the nucleophilic region with an energy value of −194.44 kcal/mol. The LUMO orbital appears as a π antibonding orbital, and it is localized on the π region of the pyrone ring with an energy value of 12.55 kcal/mol. The energy gap (206.99 kcal/mol) shows that compound 5 (naringenin) is chemically stable. Naringenin has a molecular weight of 272.257 g/mol, resulting from the addition of three hydroxyl groups 4′, 5 and 7 carbons in the backbone of flavonoids, and its molecular formula is C15H12O5 [196,197]. This compound is found in high concentrations, especially in grapefruit (43.5 mg/100 mL), followed by orange juice (2.13 mg/100 mL) and lemon juice (0.38 mg/100 mL) [198]. Naringenin has a range of biological effects on human health, which include a reduction in lipid peroxidation markers, defense of metabolism, increase in antioxidants, reduction of reactive carbohydrate species, as well as modulation of the immune response [199,200]. In vitro and in vivo animal studies have reinforced evidence of the diversity of pharmacological effects of naringenin; among them, we highlight hepatoprotective, antiatherogenic, anti-inflammatory, antimutagenic, anticancer and antimicrobial activity [201]. Although we have identified in the literature that there is an enormous amount of data on the in vitro biological effects of naringenin, there are still few studies available on its therapeutic potential [202], and thus, further clinical studies are needed, aiming at the safety, efficacy and bioavailability of naringenin in humans.
The frontier molecular orbitals (FMO) taken from the natural bond orbital (NBO) analysis for compound 5 (naringenin) were carried out at the M062X/6-311+G(d,p) level of theory, and this is shown in Figure 7. The HOMO appears as a π bonding orbital, and it is localized on the phenyl π bonding region, which is characteristic of the nucleophilic region with an energy value of −194.44 kcal/mol. The LUMO orbital appears as antibonding orbital, and it is localized on the π region of the pyrone ring with an energy value of 12.55 kcal/mol. The energy gap (206.99 kcal/mol) shows that compound 5 (naringenin) is chemically stable. The MEP is a physicochemical tool that helps to predict the reactive sites to be targeted in a chemical reaction and gives information about molecular interactions. The electrostatic potential at a given point in the vicinity of a molecule can be calculated by Equation (1).
where is the potential energy by a positive unit charge at point ; is the nuclear charge of the atom located at position , and is the electron density. The tridimensional molecular electrostatic potential (3D-MEP) representation for compound 5 (naringenin) shows that the oxygen atom of the carbonyl group localizes the most negative region (red), with the value of −26.85 kcal/mol (Figure 8). On the other hand, the positive region (blue) is around the hydroxyl hydrogen atom with a value of 45.11 kcal/mol. In conclusion, due to the presence of interactions within the hydroxyl group O4-H4···O5 in the crystal structures, we can assume a nucleophilic attack within this hydroxyl region. The MEP is a physicochemical tool that helps to predict the reactive sites to be targeted in a chemical reaction and gives information about molecular interactions. The electrostatic potential at a given point ρ(r) in the vicinity of a molecule can be calculated by Equation (1).
where V(r) is the potential energy by a positive unit charge at point r; Z α is the nuclear charge of the atom α located at position R α , and ρ r ' is the electron density. The tridimensional molecular electrostatic potential (3D-MEP) representation for compound 5 (naringenin) shows that the oxygen atom of the carbonyl group localizes the most negative region (red), with the value of −26.85 kcal/mol (Figure 8). On the other hand, the positive region (blue) is around the hydroxyl hydrogen atom with a value of 45.11 kcal/mol. In conclusion, due to the presence of interactions within the hydroxyl group O4-H4···O5 in the crystal structures, we can assume a nucleophilic attack within this hydroxyl region. The root of the mean squared (RMS) value between experimental geometries and theoretical calculation was 0.0135, predicted by Mercury software. The overlapping of the X-ray (black) and M062X/6-311+G(d,p) level of theory (green) is shown in Figure 9a. The comparative graphs for the bond lengths and angles obtained for experimental geometries and theoretical calculation are shown in Figure 9b,c. The mean absolute percentage deviations (MAPD) were calculated and defined by Equation (2):

100
. ( where and represents the geometric parameters for experimental geometries and theoretical calculation data, respectively. The MAPD values for bond lengths and angles were 0.86 and 0.64 for experimental geometries and theoretical calculation data of naringenin. The R 2 values for bond lengths were 0.9771 and 0.9670, for experimental geometries and theoretical calculation data of naringenin, respectively. The root of the mean squared (RMS) value between experimental geometries and theoretical calculation was 0.0135, predicted by Mercury software. The overlapping of the X-ray (black) and M062X/6-311+G(d,p) level of theory (green) is shown in Figure 9a. The comparative graphs for the bond lengths and angles obtained for experimental geometries and theoretical calculation are shown in Figure 9b,c. The mean absolute percentage deviations (MAPD) were calculated and defined by Equation (2): where χ XRD and χ DFT represents the geometric parameters for experimental geometries and theoretical calculation data, respectively. The MAPD values for bond lengths and angles were 0.86 and 0.64 for experimental geometries and theoretical calculation data of naringenin. The R 2 values for bond lengths were 0.9771 and 0.9670, for experimental geometries and theoretical calculation data of naringenin, respectively. The conformation analysis for naringenin was performed by Ávila and coworkers [202] showing two stable conformers (conformer 1 and conformer 2) obtained by molecular dynamics simulation in a DMSO solution. The conformation found in the solid state is approximate to conformer 2. Conformer 2 has the phenol ring in an equatorial position and it is 2.39 kcal/mol more stable than conformer 1. In addition, the free energy barrier is 3.75 kcal/mol for converting the conformer 1 to conformer 2 direct process and 6.15 kcal/mol for the reverse process, so a suggested conformation equilibrium can occur in the DMSO solution at 298.15 K. The conformation analysis for naringenin was performed by Ávila and coworkers [203] showing two stable conformers (conformer 1 and conformer 2) obtained by molecular dynamics simulation in a DMSO solution. The conformation found in the solid state is approximate to conformer 2. Conformer 2 has the phenol ring in an equatorial position and it is 2.39 kcal/mol more stable than conformer 1. In addition, the free energy barrier is 3.75 kcal/mol for converting the conformer 1 to conformer 2 direct process and 6.15 kcal/mol for the reverse process, so a suggested conformation equilibrium can occur in the DMSO solution at 298.15 K.

Systematic Review
The present study was carried out through a systematic review of articles, dissertations and theses published between May 2011 and August 2022. The searched electronic databases were ISI Web of Science and Scholar Google, using the following keywords: ACANTHACEAE, Justicia and Medicinal plants. The collected data were screened by analyzing titles, keywords, abstract and full texts. The literature containing information on isolation and property of different phytochemical compounds from species of the genus Justicia were included, too. More than 6500 articles, dissertations and theses were found on databases. Figure 10 shows the search and selection processes.

Systematic Review
The present study was carried out through a systematic review of articles, dissertations and theses published between May 2011 and August 2022. The searched electronic databases were ISI Web of Science and Scholar Google, using the following keywords: ACANTHACEAE, Justicia and Medicinal plants. The collected data were screened by analyzing titles, keywords, abstract and full texts. The literature containing information on isolation and property of different phytochemical compounds from species of the genus Justicia were included, too. More than 6500 articles, dissertations and theses were found on databases. Figure 10 shows the search and selection processes.

Molecular Modeling Analysis
The (R,S)-naringenin structure was extracted from the Cambridge Crystallography Data Centre (CCDC) with the code 1143928. Platon (2009) [203] and Mercury (2020) [204] were followed to analyze and draw the crystal supramolecular arrangement. Hirshfeld surface analysis (HS) (2009) [205] is a useful tool to understand the intermolecular contacts among atoms and crystal packing. HS is calculated based on the distances between the internal nucleus of the HS within the molecule (d i ) and the external nucleus of the HS within the molecule (d e ) [206]. The normalized contact distance (d norm ), which combines the normalized de and d i with the van der Waals radius, is used to identify the most important contacts present in the molecule. Moreover, the 2D fingerprint plots provide the frequency and quantitative information about the calculated intermolecular contacts. For this purpose, we used Crystal Explorer 21.5 [207] software to generate this HS surface and to calculate the 2D fingerprint plots. The electronic structure calculations were carried out with the Gaussian 16 [207] program package for compound 5 (naringenin). Full geometry opti-mization was carried out using density functional theory (DFT), with exchange-correlation functional M062X and basis set 6-311++G(d,p) [206], and the electronic properties, such as the highest occupied molecular orbital (HOMO), the lowest unoccupied molecular orbital (LUMO) and the molecular electrostatic potential (MEP), were calculated [207].

Molecular Modeling Analysis
The (R,S)-naringenin structure was extracted from the Cambridge Crystallography Data Centre (CCDC) with the code 1143928. Platon (2009) [204] and Mercury (2020) [205] were followed to analyze and draw the crystal supramolecular arrangement. Hirshfeld surface analysis (HS) (2009) [206] is a useful tool to understand the intermolecular contacts among atoms and crystal packing. HS is calculated based on the distances between the internal nucleus of the HS within the molecule (di) and the external nucleus of the HS within the molecule (de) [207]. The normalized contact distance (dnorm), which combines the normalized de and di with the van der Waals radius, is used to identify the most important contacts present in the molecule. Moreover, the 2D fingerprint plots provide the frequency and quantitative information about the calculated intermolecular contacts. For this purpose, we used Crystal Explorer 21.5 [208] software to generate this HS surface and to calculate the 2D fingerprint plots. The electronic structure calculations were carried out with the Gaussian 16 [208] program package for compound 5 (naringenin). Full geometry optimization was carried out using density functional theory (DFT), with exchange-correlation functional M062X and basis set 6-311++G(d,p) [207], and the electronic properties, such as the highest occupied molecular orbital (HOMO), the lowest unoccupied molecular orbital (LUMO) and the molecular electrostatic potential (MEP), were calculated [208].

Conclusions
There were 29 species of the genus Justicia studied all of which presented information regarding chemical information, with 28 biological activities presented: 19 had their compounds identified, and 10 species had their compounds isolated. Alkaloids and flavonoids (e.g., naringenin) were the compounds of the active extracts that had the highest frequency of identification among the researched data. The secondary metabolites that most frequently showed biological effects were lignans. The most researched species

Conclusions
There were 29 species of the genus Justicia studied all of which presented information regarding chemical information, with 28 biological activities presented: 19 had their compounds identified, and 10 species had their compounds isolated. Alkaloids and flavonoids (e.g., naringenin) were the compounds of the active extracts that had the highest frequency of identification among the researched data. The secondary metabolites that most frequently showed biological effects were lignans. The most researched species were Justicia gendarussa Burm, Justicia adhatoda L., Justicia procubens L., Justicia spicigera Schltdl, and Justicia secunda Vahl., with frequency values of articles surveyed of 40, 20, 19, 18 and 16, respectively. Species of the genus Justicia have a range of biological uses, identified as antioxidant, antimicrobial and anticancer, among others. The first two are the most representative; however, we would suggest the need for further research. The FMO taken from NBO analysis indicates reactive sites for compound 5 (naringenin) to be targeted in a chemical reaction on phytomedical activity. The energy gap (206.99 kcal/mol) and dimer solid state packing (R 2 2 (24) symmetry) indicates that naringenin is chemically stable.

Acknowledgments:
The authors would like to thank Ademir João Camargo (UEG) for fruitful discussion on molecular modeling of naringenin.