Lawsonia Inermis Markedly Improves Cognitive Functions in Animal Models and Modulate Oxidative Stress Markers in the Brain

Background and Objective: Medicinal plants represent an important source of alternative medicine for the management of various diseases. The present study was undertaken to assess the potential of Lawsonia inermis ethanol (Li.Et) and chloroform (Li.Chf) extracts as memory-enhancing agents in experimental animals. Materials and Methods: Li.Et and Li.Chf were phytochemically characterized via gas chromatography-mass spectroscopy (GC-MS). Samples were tested for nootropic potentials at doses of 25, 50, 100, 200 mg/kg (per oral in experimental animals (p.o.)). Swiss albino mice of either sex (n = 210) were divided into 21 × 10 groups for each animal model. Memory-enhancing potentials of the samples were assessed using two methods including “without inducing amnesia” and “induction of amnesia” by administration of diazepam (1 mg/kg, intraperitoneally. Piracetam at 400 mg/kg (i.p.) was used as positive control. Cognitive behavioral models including elevated plus maze (EPM) and the passive shock avoidance (PSA) paradigm were used. Biochemical markers of oxidative stress such as glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) levels were analyzed in the brain tissue of treated mice. Results: In 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals scavenging assay, Li.Et and Li.Chf exhibited 70.98 ± 1.56 and 66.99 ± 1.76% inhibitions respectively at 1.28 mg/mL concentration. GCMS results revealed the presence of important phytochemicals. Both samples (Li.Et and Li.Chf) at 25 mg/kg (p.o.) dose significantly (p < 0.05) improved learning and memory as indicated by decline in transfer latency and increase in step down latency in EPM and PSA models respectively. Li.Et and Li.Chf at 25 mg/kg (p.o.) showed considerable increase in GSH (2.75 ± 0.018 ***), SOD (2.61 ± 0.059 ***) and CAT (2.71 ± 0.049 ***) levels as compared to positive and negative control groups. Conclusions: This study provides the preliminary clue that L. inermis may be a potential source of memory-enhancing and anti-oxidant compounds and thus warrant further studies.

The elevated plus maze was used to evaluate the memory enhancing capacity of the test samples. EPM is composed of two open (16 × 5 cm) and two closed arms (16 × 5 ×12 cm). EPM arms are 5 × 5 cm apart from the central platform. The apparatus is elevated to the height of 30 cm above the floor [38].
For modifications, open arms were painted white and closed arms were painted black.

Training and Test Sessions
Training was done for nine consecutive days and the test session was conducted after 90 min of the last dose on 15th day. The animal was placed on the open arm. The time taken by the animal to enter the closed arm (TL) was observed. A cut-off time period of 90 s was chosen for the animals i.e., if animal did not enter the closed arm with in 90 s, the animal was replaced from the study. Memory-retaining capability of each mouse was recorded 24 h after the training session on the second day (16th day) [39].

Passive Shock Avoidance Paradigm
The PSA tool is composed of a square shaped container (27 × 27 × 27 cm) enclosed in triple wooden walls, along with a base of stainless steel having rods. The rods were 3 mm long and 6 mm separated from each other. The base of wood was (10 × 7 × 1.7 cm) wide. A 15 W bulb was used to illuminate the wooden box during the experiment. A current of 20 V AC was supplied to the grid floor for providing the shock to the animals [40].

Training and Test Sessions
Training was done for nine consecutive days and a test session was given to each mouse after 90 min of the last dose on the 15th day. Each mouse was placed on the wooden base at the center of grid. When the mouse stepped down on the grid floor with all its paws, a current was supplied to the grid floor for 15 s.
Step down latency was recorded i.e., the time taken by the mouse to come down from the wooden base to the grid floor [41].

Brain Tissue Preparation
After the experimental procedures, animals were sacrificed by giving mild anesthesia. The brain was removed and put in ice-cold normal saline solution to remove the debris. A 1/10 (w/v) tissue homogenate was prepared in (0.1 M phosphate buffer, pH 7.0) Tissue homogenates were centrifuged (T6-3A-US, Galavano Scientific, Lahore, Pakistan) at 600× g for 10 min at 4 • C. Supernatants were collected and used for biochemical estimation [42].

Estimation of Glutathione
Tissue homogenates (1 mL) were mixed with 0.02 mol/L ethylene diamine tetra acetic acid (EDTA) and tubes were placed in ice bath for 10 min to precipitate the tissue proteins. Subsequently, 2 mL distilled water and 0.5 mL of tri-chloroacetic acid (50%) were added in the test tube and again placed in an ice bath for 10-15 min and centrifuged at 3000-3500 rpm/min. Supernatant was collected and absorbance was recorded at 412 nm. A blank was prepared by the same method without the addition of analyte [43].

Estimation of Superoxide Dismutase (SOD)
Sodium phosphate buffer (0.025 mol/L, pH 8.3) 1.25 mL, tissue homogenate 200 µL, phenazine methosulphate (186 µmol/L) 1.25 mL and nitro blue tetrazolium chloride (NBT) (300 µL) were taken in test tube. Then, (780 µmol/L), 200 µL reduced nicotinamide adenine dinucleotide was added to the mixture to start the reaction. The reaction mixture was incubated at 30 • C for 90 s. Furthermore, 1 mL glacial acetic acid was added in the reaction mixture to stop the reaction. The reaction mixture was shaken with 4 mL of n-butanol and incubated for 10 min at room temperature. The n-butanol layer was removed and intensity of chromogen was measured at wavelength 560 nm. A blank was prepared by the same method without adding tissue homogenate [45].

Estimation of Catalase (CAT)
Tissue homogenate, 100 µL, phosphate buffer pH 7 and 1.9 mL freshly prepared H 2 O 2 were transferred in cuvette. Blank and standard were prepared similarly without addition of tissue homogenate respectively. Absorption of samples was measured against a blank at 240 nm [43]. Following formula was used to calculate CAT activity: where OD is change in absorbance/min, Ex is extinction coefficient of hydrogen peroxide (0.071 mmol/cm) [44].

In Vitro 2,2-diphenyl-1-picrylhydrazyl (DPPH) Free Radical Scavenging Assay
Different concentrations (0.02-1.28 mg/mL) of Li.Et and Li.Chf were used to study the anti-oxidant potentials. DPPH was prepared in methanol. DPPH (3 mL) and 1 mL of each concentration were poured into their respective test tubes. The mixture was incubated at ambient temperature for half an hour. Blank was prepared by the same method without the addition of analyte. Absorbance was recorded at wavelength of 715 nm. Lesser absorbance indicates more antioxidant activity [46]. Rutin was used as positive control.

Statistical Analysis
Data are represented as mean ± SEM (n = 10). One-way analysis of variance (ANOVA) followed by Dunnett's t test was applied on data by using graph pad prism version 5.0 Data were considered statistically highly significant at p < 0.001, moderately significant at p < 0.01, and significant at p < 0.05 [47].

Acute Toxicity
Results of acute toxicity studies showed that Li.Et and Li.Chf were safe up to 2000 mg/Kg. No mortality was observed within 24 hrs immediately or up to 14 days of observation. No symptoms like loss of body weight, salivation, paralysis, tremors were observed. The Li.Et and Li.Chf have LD 50 of greater than 2000 mg/Kg.

Acute Toxicity
Results of acute toxicity studies showed that Li.Et and Li.Chf were safe up to 2000 mg/Kg. No mortality was observed within 24 hrs immediately or up to 14 days of observation. No symptoms like loss of body weight, salivation, paralysis, tremors were observed. The Li.Et and Li.Chf have LD50 of greater than 2000 mg/Kg.

Effect of Lawsonia Inermis Ethanol (Li.Et) and Lawsonia Inermis Chloroform (Li.Chf) on Transfer Latency Using EPM Paradigm
The   Effect of Li.Et therapy on transfer latency (TL) of mice in the elevated plus maze (EPM) model. Piracetam (400 mg/kg, i.p.) was used as a positive control. Results are presented as mean ± standard error of the mean (SEM) (n = 10). One-way analysis of variance (ANOVA) followed by Dunnett's t test was used for statistical analysis of data. *** indicates p < 0.001, ** indicates p < 0.01, and * indicates p < 0.05 in comparison with control. a: indicates p < 0.001 in comparison with Piracetam. Li.Et (25mg/kg) in combination with diazepam (1 mg/kg) exhibited a steady decline in TL (15.06 ± 1.24 s in comparison to diazepam group only, with TL of 32.02 ± 1.20 s. Diazepam (1 mg/kg in combination with piracetam at 400 mg/kg was most effective in reducing the TL (13.77 ± 1.53 s) of rodents. Among the other group, a concentration-dependent effect on TL was observed ( Figure 4). Likewise, the TL was 44.90 ± 1.82 s in diazepam treated group, which was significantly reduced by Li.Chf (25 mg/kg) in combination with 1 mg/kg diazepam to 26.08 ± 2.33 s ( Figure 5). Piracetam at 400 mg/kg in combination with 1 mg/kg diazepam reduced TL to 27.11 ± 0.58 s in comparison to the control group. All other group animals at 50, 100 and 200 mg/kg exhibited a dose-dependent decline in TL. Li.Et (25mg/kg) in combination with diazepam (1 mg/kg) exhibited a steady decline in TL (15.06 ± 1.24 s in comparison to diazepam group only, with TL of 32.02 ± 1.20 s. Diazepam (1 mg/kg in combination with piracetam at 400 mg/kg was most effective in reducing the TL (13.77 ± 1.53 s) of rodents. Among the other group, a concentration-dependent effect on TL was observed ( Figure 4). Likewise, the TL was 44.90 ± 1.82 s in diazepam treated group, which was significantly reduced by Li.Chf (25 mg/kg) in combination with 1 mg/kg diazepam to 26.08 ± 2.33 s ( Figure 5). Piracetam at 400 mg/kg in combination with 1 mg/kg diazepam reduced TL to 27.11 ± 0.58 s in comparison to the control group. All other group animals at 50, 100 and 200 mg/kg exhibited a dose-dependent decline in TL.    Et on transfer latency (TL) in mice using the EPM model. Data is presented as Mean ± SEM (n = 10). One-way ANOVA followed by Dunnett's t test was used for statistical analysis of data. ### indicates p < 0.001, ## indicates p < 0.01 and # indicates p < 0.05 in comparison with diazepam.

Effect of Li.Et and Li.Chf on Step-Down Latency Using Passive Avoidance Paradigm
Li.Et at 25 mg/kg dose exhibited a highly significant increase of 201.62 ± 1.56 s (p < 0.001) in step down latency (SDL) of rodents as compared to control group animals with SDL of 30.78 ± 0.58 s. However, positive control piracetam exhibited SDL of 244.08 ± 2.33 s. Other concentrations of Li.Et showed dose dependent but less significant increase in SDL of animals ( Figure 6).     Figure 6. Effect of Li.Et on step down latency (SDL) in mice using passive avoidance model. Piracetam (400 mg/kg, i.p.) was used as a positive control. Results are presented as Mean ± SEM (n = 10). One-way ANOVA followed by Dunnett's t test was used for statistical analysis of data. *** indicates p < 0.001, ** indicates p < 0.01 and * indicates p < 0.05 in comparison with control. a, indicates p < 0.001 in comparison with Piracetam.

Estimation of Glutathione (GSH)
Li.Et at dose of 25 mg/kg, exhibited a highly significant (p < 0.001) increase in GSH in comparison with diazepam. However, the groups in which disease was induced by diazepam (1mg/Kg) along with the administration of dose (25 mg/kg, p.o.) exhibited a highly significant increase in GSH levels as compared to the disease group. The dose (50 mg/kg, p.o.) along with the administration of diazepam showed a moderately significant (p < 0.01) increase in GSH levels as compared to diazepam (Table 3).

Estimation of Glutathione (GSH)
Li.Et at dose of 25 mg/kg, exhibited a highly significant (p < 0.001) increase in GSH in comparison with diazepam. However, the groups in which disease was induced by diazepam (1mg/Kg) along with the administration of dose (25 mg/kg, p.o.) exhibited a highly significant increase in GSH levels as compared to the disease group. The dose (50 mg/kg, p.o.) along with the administration of diazepam showed a moderately significant (p < 0.01) increase in GSH levels as compared to diazepam (Table 3). Values are expressed as Mean ± SEM. n = 10; *** indicated p < 0.001, ** indicated p < 0.01 and * indicated p < 0.05 in comparison with diazepam. Parenthesis indicated % increase in SOD, CAT and GSH levels.

Estimation of SOD
The Li.Et and Li.Chf lowest dose (25 mg/kg, p.o.), showed a highly significant (p < 0.001) increase in SOD levels in comparison with diazepam. The disease-induced group along with the administration of dose 25 mg/kg, p.o.) displayed highly significant (p < 0.001) increase in SOD levels as compared to diazepam (Table 3).

Estimation of CAT
Li.Et and Li.Chf at 25 mg/kg, p.o. dose showed a highly significant (p < 0.001) increase in CAT levels in comparison with diazepam (Table 3).

DPPH Free Radicals Scavenging Activity
In DPPH radicals scavenging assay, Li.Et showed 74.1 ± 1.56%, 61.56 ± 0.034% inhibitions at the highest concentrations of 1.28 and 0.64 mg/mL, respectively. All other concentrations of Li.Et inhibited free radicals in a concentration-dependent manner ( Figure 9). Likewise, Li.Chf at concentrations of 1.28 and 0.64 mg/mL exhibited 66.99 ± 1.76% and 53.28 ± 1.13% inhibition of DDPH radicals, respectively. Rutin displayed 43.57 ± 1.77% inhibition at 1.28 mg/mL against DPPH radicals. Several phenolic compounds have been reported from this plant, which might act as free-radical scavengers. The flavonoids from dietary sources exhibit anti-oxidant properties [49]. The anti-oxidant activity of L. inermis might be due to the presence of flavonoids. A low dose (25 mg/kg, p.o.) of both extracts displayed a highly significant (p < 0.001) increase in memory. Further studies are required to study the exact mechanism of action. radicals, respectively. Rutin displayed 43.57 ± 1.77% inhibition at 1.28 mg/mL against DPPH radicals. Several phenolic compounds have been reported from this plant, which might act as free-radical scavengers. The flavonoids from dietary sources exhibit anti-oxidant properties [49]. The anti-oxidant activity of L. inermis might be due to the presence of flavonoids. A low dose (25 mg/kg, p.o.) of both extracts displayed a highly significant (p < 0.001) increase in memory. Further studies are required to study the exact mechanism of action.

Discussion
Loss of memory and cognitive dysfunctions lead to progressive neurodegenerative disorders like AD [50]. Neurodegenerative symptoms arise due to improper neurogenesis in the hippocampus [51]. Aging is also a cause of memory loss and its burden is increasing day by day in the public due to stressful lifestyles [52]. Several behavioral tasks including EPM, PAT are among the most extensively used tools to assess the cognitive performance of rodents. EPM comprising covered and open arms was utilized for the estimation of stress and nootropic effect in animals. Mice escaped from the exposed arm towards the covered one. The interval of time that the animal took to escape the exposed arm towards the covered (transfer latency) was recorded. The transfer latency on the 2nd day significantly declined as compared to the 1st day, indicating improvement in memory and reduction in stress. Transfer latency on the 2nd day was not reduced in mice treated with diazepam

Discussion
Loss of memory and cognitive dysfunctions lead to progressive neurodegenerative disorders like AD [50]. Neurodegenerative symptoms arise due to improper neurogenesis in the hippocampus [51]. Aging is also a cause of memory loss and its burden is increasing day by day in the public due to stressful lifestyles [52]. Several behavioral tasks including EPM, PAT are among the most extensively used tools to assess the cognitive performance of rodents. EPM comprising covered and open arms was utilized for the estimation of stress and nootropic effect in animals. Mice escaped from the exposed arm towards the covered one. The interval of time that the animal took to escape the exposed arm towards the covered (transfer latency) was recorded. The transfer latency on the 2nd day significantly declined as compared to the 1st day, indicating improvement in memory and reduction in stress. Transfer latency on the 2nd day was not reduced in mice treated with diazepam (1 mg/kg, i.p.) indicating impairment in memory. Its working principle includes the natural ability of rodents to find protective environments and their natural ability to reject bright, unprotected, and elevated places (shown by the open arms). Confinement to the open arms generates physiological signs of stress (increased corticosterone levels and defecation), whereas exposure to memory-enhancing drugs, such as pirecetam increases exploration of these arms [53]. Both of our test samples at dose of 25 mg/kg, p.o. significantly (p < 0.001) enhanced the memory as compared to control and standard groups on the 15th day. Decrease in TL indicated enhancement in memory. Li.Et and Li.Chf displayed dose independent nootropic effects as shown in  It is well documented that diazepam cause impairment in retrieval of memory in mice and such amnesia is associated with a significant increase in oxidative stress. Continuous stress can lead to amnesia that resulted in memory loss [54]. Those groups treated with diazepam along with administration of oral doses of test samples (25,50,100, 200 mg/kg) indicated reversal of amnesia induced by diazepam (Figures 2-5). The least dose (25 mg/kg, p.o.) produced a highly significant decrease in TL as compared to diazepam alone. The effect on low dose might be due to the attenuation of auto receptors of acetylcholine on presynaptic neurons that inhibited the negative feedback of neurotransmitter release, while at a high dose it might potentiate the post-synaptic receptor stimulation that destroyed the acetylcholine rapidly and resultantly decrease the nootropic potential of L. inermis [55].
Passive avoidance is mostly used to estimate the cognitive effects of test samples on rodents. It is a simple and flexible method for evaluation of nootropic potential in the field of neuropharmacology and other fields. It can be utilized to evaluate both the diminutive along with longstanding memory. In the present study low doses of Li.Et and Li.Chf at 25 mg/kg, p.o. significantly (p < 0.001) enhanced the SDL as compared to control and standard. Those groups treated with diazepam along with the administration of oral doses (25, 50, 100, 200 mg/kg) indicated reversal of amnesia induced by diazepam. The lowest dose (25 mg/kg, p.o.) displayed a highly significant (p < 0.001) increase in SDL as compared to diazepam alone.
Factors responsible for inducing the neuronal changes seem to be free radicals. Various enzymes are responsible for radical scavenging of free radicals; these include CAT, GSH and SOD. Impairment in the proper functioning of these enzymes gives rise to the augmentation of free radicals [56]. Previous studies have revealed that stress and free radicals are responsible for impairing memory and learning. Stress can lead to a disturbance in levels of certain neurotransmitters [57]. Glutathione plays a significant role in the detoxification of free radicals thus reducing stress and enhancing cellular functions. Glutathione levels are reduced in diseased conditions leading to oxidative stress [58]. Results demonstrated that the GSH level was increased significantly (p < 0.001) in group treated with Li.Et and Li.Chf (25 mg/kg, p.o.) as compared to diazepam. An increase in GSH level indicated a reduction in stress and an enhancement in memory (Table 3).
SOD plays a vital role in shielding the living cells against the toxicity generated by the free radicals due to their capability to scavenge O −2 [59]. SOD levels were determined from brain tissue homogenates of mice. The least dose (25 mg/kg, p.o.) displayed highly significant increase in SOD level as compared to diazepam. CAT activity was estimated and linked with the nootropic activity of L. inermis in mice. The lowest dose (25 mg/kg, p.o.) exhibited more pronounced increase in CAT activity as compared to diazepam. In the case of oxidative stress the levels of CAT, SOD and GSH are reduced which can lead to death of neurons leading to memory impairment. L. inermis (25 mg/kg, p.o.) of both extracts enhanced the levels of these enzymes. Thus, L. inermis can be used as a nootropic agent.
The role of free radicals in the etiology of neurodegenerative disorders has been understood for a long time and the role of antioxidants in the management of neurodegenerative disorders has been reported time and again in the last few decades. A lot of natural and synthetic compounds have been showing promising results in the scavenging of free radicals but almost all the compounds have been associated with certain shortcomings [60]. One of the most prominent limitations of the antioxidant compounds is their highly polar nature due to the presence of polar functional groups which confine the flow of the antioxidants either to the blood stream and do not allow their entry to the target point that is the central nervous system. Among the natural compounds, the flavonoids have been showing promising in vitro results against the free radicals but fail to decrease the oxidative stress in the central nervous system (CNS) due to the presence of phenolic groups in the flavonoids which make them highly polar and which even decreases their bioavailability [61]. Secondly, there are numerous antioxidant compounds which have the ability to capture the free electrons from the free radicals, but the problem is that these compound do not have the capacity to remain stable after gaining the free electrons from free radicals [11]. This may lead to the generation of other metabolites which may be more hazardous than the oxidative stress. Researchers are striving for the development of various drug-delivery systems and blood brain barrier models to cope with the bioavailability and CNS delivery issues. Moreover, there is no role for antioxidants in reversing nerve damage, albeit the antioxidant compounds could be able to halt further exacerbation. Likewise, the radical scavenging antioxidants are oxidant specific and can only be effective if the specific mechanism for neurode generation involves the reactive species to which they are targeted. Most importantly, the suppression of oxidants may be deleterious for the human body, as we know that the reactive species have an important role in physiological signalling within the body [62,63]. In the current study, our test samples considerably augmented the antioxidant activity of indigenous antioxidant enzymes in the brain homogenates of the animals, which indicates that they effectively cross the BBB to gain access to the brain.

Conclusions
GC-MS analysis of Li.Etand Li.Chf revealed the presence of phytochemical constituents that may be responsible for the nootropic activity of L. inermis. It was concluded from the study that Li.Etand Li.Chf of L. inermis had the ability to reverse the memory loss due to free radical-induced neurodegeneration. The exact mechanism of action is not known; however, the anti-oxidant property of L. inermis might be responsible for the nootropic potential of the plant. The isolation of active phyto-constituents, their structure elucidation, and the exact mechanism by which extracts modulate Alzheimer's disease are limitations of the current study. Funding: This study received no external funding.