A Study on Justicia flava (Forssk.) Vahl.: Pharmacognostic Characterization and Antiplasmodial and Anti-Inflammatory Properties of the Leaves

Justicia fava (Acanthaceae) is utilized in folk medicine for treating malaria, infammatory conditions, and respiratory infections. In this study


Introduction
Te genus Justicia, with over 600 species of scandent perennial herbs and shrubs, represents the largest genus in the family Acanthaceae [1].Its species are usually found in warm and temperate regions of the world including Africa, Asia, and America.Justicia spp.are recognized for numerous ethnomedicinal applications including their use for treating carefully washed with clean tap water, air-dried under shade for a week, and ground into powder using a mechanical grinder.Extraction was carried out by cold macerating approximately 1000 g of the powdered leaves in 70% ethanol for 72 h.Te fltrate was reduced to a green syrupy extract on a rotary evaporator and further dried in an oven (40 °C).A semisolid dried extract (JFE) weighing ∼87 g (percentage yield � 8.7%w/w) was obtained and stored in an airtight container till needed for use.

Macroscopic and Microscopic
Assessment.Te leaves of J. fava were examined for organoleptic characteristics including taste, colour, odour, and texture.Macroscopic features of the leaf such as the leaf type, leaf arrangement, and lamina characteristics were noted.Microscopic assessment was carried out with the aid of a light microscope (DM-700) with a camera attached.Free-hand sections of the cleared leaf lamina, midrib, and petiole were observed under the microscope, and photomicrographs of cell types and cell inclusions were taken at ×10 and ×40 magnifcations.Te epidermal number, stomatal number and index, palisade ratio, veinlet termination, and vein-islet numbers were calculated as previously described [25].

Phytochemical Screening.
Major classes of secondary metabolites were screened for, following previously described protocols [26].

Biological Activity Screening
2.4.1.Animals.Cockerels (Gallus gallus, strain Shaver 579) were obtained from Akropong farms (Kumasi, Ghana) oneday after hatch and transferred to the vivarium of the Department of Pharmacology, KNUST, Kumasi.Te chicks were housed in groups of 12 chicks per cage in stainless steel cages (34 × 57 × 40 cm 2 ).Tey were maintained at room temperature of 29 °C under overhead incandescent illumination granting a 12 h light-dark cycle.Te chicks were fed with chick mash (GAFCO, Tema, Ghana) and water.Te chicks were used for experiments at 7 days of age.
Swiss albino mice (16-28 g) and chloroquine-sensitive Plasmodium berghei (ANKA strain)-infected donor albino rats were obtained from the Animal Facility Centre (AFC) of the Noguchi Memorial Institute for Medical Research (NMIMR), Legon, Accra, Ghana.Te animals were kept in a vivarium at the Pharmacology Department, KNUST, where all experiments were carried out.Tey were housed in cages with wood shaven as bedding under a 12-hour natural light-dark cycle.Te temperature of the lab was maintained at 27 ± 1 °C with a relative humidity of 60-70%.Te animals were fed with rodent feed and supplied with water ad libitum.All procedures employed were permitted by the Animal Research Ethics Committee (AREC) of KNUST.All experimental animals were handled according to the instructions given by the National Institute of Health 2 Te Scientifc World Journal Guidelines for the Care and Use of Laboratory Animals (2011).

Animal Grouping and Dosing.
For all in vivo assays, animals were put into fve groups consisting of fve mice each.Tese included three test groups which received doses of the test extract orally, the vehicle-treated group, which was given normal saline, and the positive control group which was administered the standard drug.

Acute Oral Toxicity Assay.
Te acute oral toxicity assay of JFE extract was performed according to the fxed dose method as given by the Guideline 420 of the Organization of Economic Cooperation and Development (OECD) [27].Accordingly, Swiss albino mice, either male or female, were selected randomly and placed in two groups of fve animals each and fasted overnight.Group 1 received a dose of JFE 5000 mg•kg −1 bodyweight (solubilized in distilled water), and group 2 received 0.2 mL of normal saline.Te general behaviour/well-being of mice as well as any signs of toxicity (including change in skin colour, salivation, diarrhoea, lacrimation, change in fur, nostril discharge, lethargy, convulsion, tremor, and death) were observed continuously for an hour, then subsequently every 30 min for 4 h, then after 24 h, and daily for the next 14 days.
If three or more mice survived, the LD 50 was stated to be more than 5000 mg•kg −1 .

In Vivo Antiplasmodial Activity
2.5.1.Inoculation of Parasite.Healthy mice were infected with the Plasmodium parasite by intraperitoneal injection of 0.2 mL of inoculum (infected blood with the parasitaemia level: 1 × 10 7 parasitized erythrocytes) as described by Baah et al. [28].

Te 4-Day Suppressive Test (Prophylactic Assessment).
Te method described by Baah et al. [28] was followed in this assay.Briefy, mice infected with the Plasmodium parasite were selected randomly and grouped into fve groups (5 mice/group).Tree hours after parasite inoculation, the treatment groups received JFE 300, 100, and 30 mg•kg −1 •day −1 (p.o); the negative controls received normal saline, 0.2 mL (p.o.); and the positive controls were given artesunate, 4 mg•kg −1 •day −1 (i.p.).Treatment of mice was done at the same time each day and continued for the next four days (day 0 to day 3).After treatment on day 3, blood samples were obtained from the tails of mice in all treatment groups for analysis of parasitized RBCs.Tis was done without euthanizing the animals.Tin blood smears were fxed on glass slides with absolute MeOH, stained permanently with 10% Giemsa solution, and viewed under a light microscope at an objective magnifcation of ×100.
Infected RBCs were counted from 5 randomly selected felds of view, and the percentage parasitaemia was determined as follows: % parasitaemia � Number of parasitized RBC Total number of RBC counted × 100. (1) Percentage parasitaemia suppression was calculated as Here, A is the average % parasitaemia in the untreated group and B is the average % parasitaemia of the treated groups.

Rane's Curative Assay.
Tis assay investigated the curative potential of JFE following a previously described method by Baah et al. [28].Infection of mice was performed as described in Section 2.5.1.Tree days after infection, the mice were grouped into fve groups (5 mice/group).Groups 1, 2, and 3 (treatment groups) received JFE 30, 100, and 300 mg•kg −1 •day −1 (p.o), respectively, group 4 received 0.2 mL of normal saline orally, and group 5 received artesunate, 4 mg•kg −1 •day −1 (i.p.).Treatment continued for the next four days (day 3 to day 7).After treatment on day 3 and day 7, blood samples were obtained from the tails of mice in all treatment groups for analysis of parasitized RBCs.Tin blood smears were fxed on glass slides with absolute MeOH, stained permanently with 10% Giemsa solution, and viewed under a light microscope at an objective magnifcation of ×100.Te level of parasitaemia on days 3 and 7 was determined by the formulas in Section 2.5.2.

Monitoring Survival Time.
Te length of survival time (in days) for each mouse was recorded within a 30-day period of observation in both suppressive and curative assays.Te mice were observed from the day of infection to the day of death (i.e., if death occurs).Te mean survival time (MST) was then determined by the formula: MST � Sum of survival time of all mice in a group (days) Sum of mice in the group . (3)

Assessment of Body Weight and Body Temperature.
Te body weights of animals were taken on a digital balance and the rectal temperatures were taken with a digital thermometer.In the suppressive assay, these parameters were recorded on the day of infection (day 0) and after treatment on the 3 rd day.In the curative assay, these parameters were taken on the 3 rd and 7 th days of treatment.

Carrageenan-Induced Footpad Oedema Assay in Chicks.
Te anti-infammatory assay was performed as previously described [29].Foot pad oedema was induced by injecting the subplantar tissue of the right footpad of chicks with 10 µL of 2% carrageenan solution.Before the induction of oedema, the footpad diameter was measured.Exactly 1 hour after carrageenan injection, the chicks were treated with JFE Te Scientifc World Journal extract suspended in normal saline at doses of 300, 100, and 30 mg•kg −1 given orally.Diclofenac sodium (10, 30, and 100 mg•kg −1 i.p) was given as a reference drug and normal saline (2 mL, p.o.) as the negative control.Te diameter of the chick footpad was recorded hourly for 5 hours with the aid of vernier callipers.Te raw footpad diameters were used to determine the percentage change in foot pad size as follows: Here, f o is the foot pad diameter at time 0 and f t is the foot pad diameter at time "t." 2.7.Data Management Analysis.Te results of the experiments were analysed using GraphPad Prism (version 8 for windows, San Diego, USA).Te data are given as the mean ± standard error mean (SEM).One-way analysis of variance (ANOVA) was used to make comparisons between the untreated and treated groups.Dunnet's post hoc test was used for multiple comparisons between tests.

Organoleptic and Macromorphological Characteristics.
Te organoleptic and macromorphological examination of J. fava leaves revealed that J. fava has simple, green, petiolate leaves which are oppositely arranged on the stem (Figures 1(a) and 1(b)).Te leaves are lanceolate to broadly lanceolate with entire to slightly crenate margins, arcuate venation, and a slightly rough surface (Figures 1(c) and 1(d)).Te plant bears terminal fowers subtended in leafy bracts.Te powdered leaf sample has a bitter taste and nonspecifc odour.A summary of the organoleptic and macromorphological features of J. fava leaf is provided in Table 1.

Microscopic Characteristics of J. fava Leaf
3.2.1.Leaf Lamina.Microscopy of the cleared leaf laminar displayed wavy-walled anticlinal epidermal cells on both surfaces.Diacytic stomata, i.e., stomatal pores with two subsidiary cells positioned perpendicular to the guard cells having common walls obliquely positions in a C-shape (diallelocytic stomata), were abundant.Glandular trichomes with a multicellular head and a unicellular stalk were also observed.Vein-islets (Vi) and veinlet terminations (Vt) were observed (Figures 2(a)-2(d)).

Transverse Section (T/S) of the Midrib and Petiole.
Microscopy of the T/S of the midrib showed a planoconvexshaped midrib with one layer of polygonal-shaped epidermal cells externally covered by a thin cuticle.Several uniseriate multicellular clothing trichomes were found on the upper epidermal layer and few on the lower epidermis.About 4-5 layers of closely packed collenchyma were observed above the lower epidermis and below the upper epidermal layer.Several parenchyma cells flled the midregion, at the centre of which collateral conjoint vascular bundles in the open arch were located.Two smaller meristeles were observed in the upper region placed beneath the palisades on either side, slightly above the central meristele.
Te T/S of the petiole displayed a circular-shaped petiole with undulating outline having abundant multicellular uniseriate nonglandular (covering) trichomes as well as glandular trichomes on the outer surface.A thin layer of cuticle, followed by one layer of polygonal epidermal cells and collenchyma, was observed.Parenchyma flled the core.Vascular bundles were present as meristeles, radially arranged along the periphery of the petiole.Xylem was located toward the interior and phloem to the exterior (Figures 2(e)-2(j)).
Microscopy of the leaf powder displayed prismatic calcium oxalate crystals, pitted xylem vessels, and lignifed fbres (Figure 3).Leaf surface constants are presented in Table 2.

Acute Toxicity.
Te hydroalcoholic leaf extract of J. fava at an oral dose of 5000 mg•kg −1 did not result in any behavioural, neurological, or physical changes such as staggering gait, anxiety, sedation, agitation, emesis, excessive salivation, straub tail, or diarrhoea in mice during 24 hours of monitoring and for 14 days after treatment.No death was recorded.Te LD 50 was estimated to be more than 5000 mg kg −1 .

3.5.
In Vivo Antiplasmodial Activity of J. fava Leaf Extract 3.5.1.Four-Day Suppressive Test.Te suppressive test was employed to assess the ability of J. fava leaf extract (JFE) to suppress parasitaemia.JFE demonstrated a signifcant (p < 0.0001) dose-dependent inhibition of parasite growth at the doses tested when compared to the untreated group.Te most remarkable parasitaemia suppression (51.31%) was demonstrated by JFE 300 mg•kg −1 .Te extract also signifcantly prolonged the mean survival rate of mice.JFE 300 mg•kg −1 gave the longest survival time of 23.85 (±1.50) days.Te positive control, artesunate, however, produced a higher suppression of parasitaemia (92.14%) than all doses of the leaf extract.Te results are presented in Table 4.

Efect of J. fava on Body
Temperature and Body Weight in the Suppressive Test.All treated and untreated mice infected with P. berghei experienced a decrease in body temperature from the day of infection till treatment was completed.Nevertheless, the change in rectal temperature recorded in infected mice in the negative control group was greater than that of the JFE and artesunate-treated groups (Table 5).Treatment with JFE also prevented a decline in the body weight of P. berghei-infected mice and rather caused 4 Te Scientifc World Journal a slight increase in weight of mice.Contrary to this, the normal saline-treated group experienced a decrease in weight after the induction of parasitaemia (Figure 4).
Te duration of survival of infected mice treated with JFE and artesunate was also substantially prolonged at all doses contrary to the untreated group (Table 6).

Efect of J. fava on Body Weight and Body
Temperature in the Curative Test.All doses of J. fava averted loss of weight in infected mice between days 3 and 7 (p < 0.0001).Te saline-treated group, however, experienced a substantial loss in weight by day 7 (Figure 5).Treatment with artesunate also prevented weight loss in infected mice.A slight reduction in temperature was observed for all P. berghei-infected mice from the 3 rd to the 7 th day (Table 7).Te change in body temperature for JFE-and artesunate-treated mice was, however, less pronounced compared to the temperature change in the negative control group.

Efect of J. fava Leaf Extract on Carrageenan-Induced
Footpad Oedema.Subcutaneous injection of 2% carrageenan into the chicks' footpads caused moderate acute infammation observed as foot oedema.Figures 6(a) and 6(c) represent the time course curves, showing the progression of infammation in treated and untreated groups, observed over a 6 h period.Figures 6(b) and 6(d) illustrate the total calculated area under the curve for each treatment group, which depicts the total foot oedema that occurred over the 6 h period of the experiment.For the treated groups, i.e., JFE-and diclofenac-treated groups, an increase in oedema was produced between the 1 st and 2 nd hour after which a sharp decrease in foot pad volume was recorded from the 3 rd to the 6 th hour.Te percentage footpad oedema reduction in chicks treated with JFE was dependent on the dose-dependent and signifcant (p < 0.0001) when compared to the negative control group.Te most remarkable inhibition of oedema was 54.04 ± 2.52%, produced by JFE 300 mg•kg −1 (Table 8).Te reference drug, diclofenac, also signifcantly (p < 0.0001) reduced oedema by 73.88 ± 5.33% dose at 100 mg•kg −1 .In the negative control group, however, footpad oedema heightened between the 2 nd and 3 rd hour and slightly decreased from the 4 th hour with substantial oedema present even at the 6 th hour.

Discussion
In this study, the antiplasmodial and anti-infammatory activities of the 70% hydroalcoholic extract of J. fava leaves were studied.Te LD 50 of J. fava leaf extract (JFE) was determined to be more than 5000 mg•kg −1 based on the results of the acute oral toxicity test.Tis report is congruent with the report by Bafor et al. who also reported an LD 50 above 5000 mg•kg −1 for J. fava leaf extract [30].Te antiplasmodial activity of JFE was investigated by the suppressive and curative tests [31].In both assays, the antiplasmodial efect is based on the percentage suppression of parasitaemia and the average survival period of infected mice in the treated group relative to the untreated group.J. fava leaf extract demonstrated the potential to reduce parasitaemia in both early (∼17-52%) and late or established malaria infection (∼57-71%).Te extract demonstrated a better curative ability than suppression, suggesting that the extract may be a better option for treating an established  Te Scientifc World Journal   6 Te Scientifc World Journal    Te Scientifc World Journal malaria infection than in prophylaxis.Deharo et al. classifed in vivo antiplasmodial activity of extracts as being very good, good, or moderate if extract caused parasitaemia suppression of greater than 50% at a dose of 100, 250, and 500 mg•kg −1 •day −1 , respectively [32].Based on this criterion, J. fava leaf extract can be said to have a very good antiplasmodial activity.Moreover, the extract signifcantly extended the survival period of mice afrming the overall reduction of parasitaemia [33].Treatment with JFE also averted signifcant weight loss and hypothermia in Plasmodium-infected animals.Te results obtained implied that J. fava leaf extract has the ability to suppress parasitaemia and attenuate the pathological events associated with P. berghei malaria infection.
Te anti-infammatory activity of J. fava leaves was evaluated by the carrageenan-induced footpad oedema assay, which is a suitable experimental model for infammation using chicks or rodents [37].Te infammatory response

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Te Scientifc World Journal elicited by carrageen follows a biphasic phenomenon: frst, the production of serotonin, histamine, and bradykinins within the frst 2 hours and a latter phase (3-6 h) depicted by the of prostaglandins and activation of cyclooxygenase-2 (COX-2) [38].From the experiment, JFE reduced chick foot pad oedema from the 3 rd to the 5 th hour of the experiment, implying that the extract may be active in the 2 nd phase of infammation.In a previous study, extracts from J. fava leaves inhibited 5-lipooxygenase (5-LOX), the enzyme responsible for the production of leukotrienes from arachidonic acid [24].Lignans, coumarins, favonoids, phytosterols, glycosides, and triterpenoids previously isolated from several Justicia species have shown promising anti-infammatory activity in both in vitro and in vivo models [7,[39][40][41][42].Some mechanisms of action suggested for the anti-infammatory efect of Justicia species include the downregulation of COX-2 and intracellular nitric oxide (iNOS) through the suppression of the NF-κB signalling  Te Scientifc World Journal pathway [43], inhibition of the synthesis, or action of tumor necrosis factor-alpha (TNF-α) and cytokine interleukin-1β (IL-β) [44,45].

Conclusion
Te antiplasmodial and anti-infammatory efects of the leaves of Justicia fava were verifed in this study.Te reduction in parasitaemia coupled with the remarkable weight recovery and prolongation of mice survival period approves the folkloric use of J. fava for the treatment of malaria.Furthermore, the attenuation of infammatory response recorded as a decrease in chick foot pad oedema justifes the use of the plant in infammatory conditions.Tis fnding also points to J. fava as a promising source of bioactive antimalarial and anti-infammatory constituents.Te diagnostic pharmacognostic characteristics obtained for the leaves of J. fava are important for the correct identifcation of the plant.Tis study highlights the signifcance of bridging traditional knowledge with modern scientifc exploration for the improvement of health and well-being.

Data Availability
Te raw data/results from experiments used to arrive at the fndings of this study are available from the corresponding author upon request.Previous reports that were used to support this study are cited at relevant places within the text as references.
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Figure 1 :
Figure 1: (a) J. fava plant in its natural habitat; (b) oppositely arranged simple leaves on a stem; (c) upper surface; (d) lower surface.

Figure 4 :
Figure 4: Relative change in body weight of P. berghei-infected mice in the 4-day suppressive test.Values are presented as the mean ± SEM, n � 5. Values are signifcantly diferent at * * * * p < 0.0001, compared to the vehicle-treated group.NC: negative control, JFE: J. fava leaf extract, and ART: artesunate 4 mg•kg −1 .

Figure 5 :
Figure 5: Relative change in body weight of P. berghei-infected mice in the curative test.Values are presented as the mean ± SEM, n � 5. Values are signifcantly diferent at * * * * p < 0.0001, compared to the vehicle-treated group.NC: negative control, JFE: J. fava leaf extract, and ART: artesunate 4 mg•kg −1 .

Figure 6 :
Figure 6: Efect of J. fava leaf extract (JFE) and diclofenac sodium (DIC) on the time course curve (a, c) and the total oedema response (AUC) (b, d) in carrageenan-induced oedema in chicks.Data are expressed as the mean ± SEM (n � 5).* * * * * p < 0.0001 compared to negative control (saline) (one-way ANOVA followed by Dunnett's post hoc test).

Table 1 :
Organoleptic and macromorphological characteristics of J. fava leaves.

Table 2 :
Leaf surface constants of J. fava.
Values are presented as a mean ± SEM (n � 5).

Table 4 :
Level of the parasitaemia level, % suppression, and mean survival time of the P. berghei-infected mice treated with JFE in suppressive test.Values are presented as the mean ± SEM, n � 5; NC � vehicle-treated group; ART �artesunate.Values are signifcantly diferent at * p < 0.05, * * p < 0.005, and * * * * p < 0.0001 compared to the negative control group.

Table 5 :
Te efect of J. fava on the rectal temperature in the 4-day suppressive assay.
Values presented as the mean ± SEM, n � 5; NC: negative control; ART: artesunate; ∆ temperature � relative change in rectal temperature.

Table 3 :
Preliminary phytochemical screening of J. fava leaves.

Table 7 :
Te efect of J. fava on the rectal temperature in the curative test.Values presented as the mean ± SEM, n � 5; NC: negative control; ART: artesunate; ∆ temperature: change in rectal temperature.

Table 8 :
Percentage inhibition of oedema in the carrageenan-induced paw oedema assay.