Determination of in vivo biological activities of Dodonaea viscosa flowers against CCL4 toxicity in albino mice with bioactive compound detection

Dodonaea viscosa L.Jacq. is an evergreen shrub and native to Asia, Africa, and Australia. It has been used as traditional medicine in different countries. The foremost objective of the current study was to discover the protective potential of D. viscosa flowers Methanol (DVM) and Chloroform (DVC) extracts against CCL4 induced toxicity in mice. This study was intended to identify phytochemicals through HPLC, GCMS, and FT-IR, as well as in vitro antioxidant and in vitro anti-tuberculosis activity. Our comprehensive findings indicate that Dodonaea viscosa is valuable and widespread herbal medicine through therapeutic potentials for curing various ailments. Dodonaeaviscosa flowersare found to have a protective effect against oxidative stress produced by CCL4 in the liver, kidney, and spleen. The intake of DV extracts restored the level of hepatic enzymes (ALP, AST ALT, and Direct bilirubin), hematological parameters (RBCs, WBCs, and Platelets), total protein, and liver antioxidant enzymes (SOD, GPx, and CAT) after a decline in levels by CCL4. Histopathological results discovered the defensive effect of 300 mg/kg of DVM extract against CCL4 induced damage, thus having an improved protective effect compared to DVC and control. As a result of metabolite screening, the total flavonoids and total phenolics were present in abundance. A phytochemical investigation by HPLC identified gallic acid, epicatechin, cumeric acid, flavonoids, while GCMS estimated oleic acid (Octadecenoic acid) (C18H34O2), Stearic acid (C18H36O2), Ricinoleic acid (C18H34O3), and Cedrol (C15H26O). DVM extract exhibited resistance against in vitro Mycobacterium tuberculosis strains. So this study proposed that the protective effect of DV against oxidative damage induced in the liver, kidney, and spleen can be correlated to the antioxidant compounds.

In vivo study. Selection and purchase of animals. Fifty male albino mice (body weight 55.2 ± 2.5 g) were purchased from NIH (National Institute for health) Islamabad. The experimental study was approved by the National Veterinary Lab Islamabad ethical committee adhering to the institution's guidelinesand also in compliance with ARRIVE guidelines. All the protocols having animal study were in acquiescence with the guidelines of the ethics committee. The mice were housed under controlled conditions and had free access to mouse chow (Feed Mills, Islamabad) and water ad libitum. Animals were cautiously monitored and kept up in standard house conditions.
Acute oral toxicity study. An acute toxicity study was done to select suitable doses of plant extracts for animals, as earlier reported by 27 . The bodyweight of animals was recorded before and after thestudy. Plant extracts were orally given to mice at the dose of 100-300 mg/kg body weight. After the dosage, animals were meticulously observed after 24, 48, and 72 h for the development of any toxicological symptoms. Animals were euthanized on 21 days of the experiment.
Experimental design. Animals of the same age were divided randomly into 10 groups, and each group contains 5 animals. At 20 days of the experiment, mice were kept for fasting for 12 h and animals were anesthetized and euthanized with sodium pentobarbiton. After anesthesia (21 days) whole blood was obtained from the heart by cardiac puncture. To get the serum, place the blood sample tube to clot for 30 minand subjected to centrifugation (3000 rpm for 10 min). Animals were sacrificed through cervical dislocation, and organs were collected and then rinsed using ice-cold saline solution and kept at − 20 °C for further analysis. The weight of collected organs from all the groups was recorded. For biochemical analysis, phosphate buffer saline was used to the homogenized liver (one part), then centrifuged at 3000 rpm for 20 min and supernatant was stored at − 20 °C. For the histopathological study, the liver, kidney, and spleen were stored in formalin solution (10%).
Analysis of blood samples. The serum biomarkers alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and bilirubin were examined by using an auto-analyzer with AMS diagnostic kits (Italy). RBC (red blood cells), WBC (White blood cells), and platelets in blood samples were estimated with the method reported by 28 .
Antioxidant enzymes. Liver homogenates were used toevaluate oxidative defense markers (antioxidant enzymes). Catalase (CAT) and superoxide dismutase (SOD) activity was measured by using the protocol 29 , Glutathione peroxidase (GPx) was calculated by the method of 30 , and total protein was evaluated by the method suggested by 31 .
Histopathological study. Mice organs (liver, kidney, and spleen) were removed carefully after sacrifice and preserved in formalin (10%). Test specimen were fixed, dehydrated in alcohol, cleaned in xylene, and then inserted into molten paraffin wax. Paraffin sections were cut into 5 µm thickness by using a microtome, and obtain tissues were mounted on slides and deparaffinized. Tissue sections were processed to staining with Ehrlich's hematoxylin and eosin counterstained (H&E) and examined under a light microscope 32,33 . Determination of total phenolic and total flavonoid content. The total phenolic content of extracts was measured by using Folin-Ciocalteu reagent 34 . Results were expressed as grams of gallic acid equivalents per 500 g/dry weight. The total flavonoid content of the extracts was measured using colorimetric assay 35 . Results were expressed by using grams of quercetin equivalents per 500 g/dry weight. Antioxidant activity. Chemicals required. Methyl alcohol, ethyl alcohol, chloroform, DPPH, ABTS, Hydrogen peroxide, EDTA, Formalin, Xylene, Heamotoxylin, Essen, KH 2 PO 4 buffer, ALT Alanine aminotransferase, AST Aspartate aminotransferase, ALP alkaline phosphatase, and bilirubin were obtained. Analytical grade solvents and reagents were purchased from local dealers of Sigma Aldrich and Merck.
DPPH assay. This process is used to calculate the scavenging capacity of the sample by the protocol with some modifications 36 . 2 ml aliquot of DPPH (2,2-diphenyl-1-picrylhydrazyl) was poured into each concentration of plant sample ranged from 20 to 100 µg/ml. This mixture was incubated at 37 °C for 30 min in darkness. Standard or Positive controls were Ascorbic and Gallic acid. DPPH solution was taken as a negative control. Reading was taken recorded at 517 nm, and results were expressed in ascorbic acid equivalent AAE and gallic acid equivalent GAE. The experiment was done in a triplicate manner, and the inhibition percentage is obtained by the following formula A a -absorbance of reaction mixture exceptfor plant extract. A h -absorbance of a reaction mixture comprising plant extract. IC 50 (µg /mg) was measured by plotting scavenging percentage against extract concentration.
Iron chelating assay. The Iron Chelating method was described by 37 . Antioxidant potential of plant extract was assessed through incubation of reaction mixture that comprises ofdifferentconcentrations of plant sample extracts (20-100 µg/ml), 2 mM ferrous sulfate (1 ml), and 0.25 mM Ferrozine (1 ml). After stirring, let the mixture stand for10 min, and absorbance was read at 517 nm.
A a -absorbance of control lacking plant extract. A h -absorbance of mixture with plant extract. Standard solutions or Positive controls (Ascorbic and Gallic acid) were used to make the calibration curve. IC 50 was stated as µg AAE/mg and GAE/mg.

Analysis of plant with high-performance liquid chromatography (HPLC).
Crude extracts analysis was performed using a Shimadzu HPLC (high-performance liquid chromatography) system, Tokyo, Japan, equipped with a C18 column (250 mm × 4.5 mm, 5 m) used for the separation at the flow rate of 1 ml/min. The column temperature was sustained at 40•C followed by gradient pump and UV/Visible detector. HPLC grade methanol was used for extraction of the crude plant to prepare the tested sample. Before injection, the filtration of samples was done using a 0.2 μm PTFE filter, and the injection volume was 10 µl. The compounds were eluted usinga gradient elution of mobile phases A and B (Acetonitrile and 0.1% phosphoric acid; 36:64). Separation steps are as follows: 0 min-5% B, 15 min-15% B, 15 min-45% B, 5 min-90%B, and Conditioning cycle for 5 min along with the analysis of the following initial conditions. The UV-Vis detection was recorded at 280-285 nm at a current rate of 1 ml/min per 20 min retention time. Quercetin was used as standard, and all data were done in triplicates.

GC-MS.
Gas chromatography and mass spectrometry analysis. GC-MS system QP2010 model (Shimadzu®) equipped with Mass selective Detector and split-a split-less system of injection. The instrument was fitted with capillary column RTx-5MS (cross bond 5% diphenyl-95% dimethylpolysiloxane) with 30 m × 0.25 mm with Scavenging activity % = A a −A h /A a × 100 www.nature.com/scientificreports/ 0.25 μm film thickness. At the rate of 1.2 ml/min, helium was being used as carrier gas. The temperature program of the column was started at 150 °C (1 min) then programmed at 4 °C/min to 150 °C (10 min). The temperature of the injector was 275 °C while the detector was at 250 °C. 0.2 µl volume was injected in split mode. A split ratio was 1:50, and the mass spectra were operated electron ionization at 70 eV in Selected Ion Monitoring (SIM) mode were maintained. The run time of the machine was 40 min. The relative percentage of the plant extract compounds was expressed in percentage with normalization of peak area.
Compounds identification. GC mass spectrum interpretation was conducted by employing the database of the National Institute Standard and Technology (NIST). The compound name, molecular weight, and structure of the test materials were determined. The percentage (%) of each compound was calculated by comparison of the average area to total area. The spectrum of unknown constituents was compared with the version 2005, software, and Turbo mass 5.2. The aim was to discover the individual compound or group of compounds that might show its current commercial and traditional roles 45,46 .
Fourier transform infrared spectrophotometer analysis. The plant extract was analyzed for infrared spectrum analysis by FT-IR (Fourier transform Infrared) spectroscopy Shimadzu machine, IR affinity 1, Japan. At first, loaded samples were grounded by KBr (1:100 w/w) with scan range (400-4000 cm) and 4 cm −1 resolution. Samples components were subjected to structural characterization and indicated functional groups with chemical bond types 47 .
Anti-tuberculosis activity. The anti-mycobacterial activity of Dodonaea viscosa L. was measured using the REMA method 48 . Mycobacterium tuberculosis strains bug 206 and bug 1972 and H37Rv were grown in 7H9 broth. Sample stock solutions were diluted using DMSO to get the final concentration ranging from 0.98 to 250 μg/mL. Rifampicin was used as positive control drug ranges 0.004 to 1 μg/mL. Add the bacterial culture (5 × 10 5 CFU/mL) to each well of the 96-well plate and incubate at 37 °C. Viability was tested using resazurin, and the color change and fluorescence were examined in plates by using SPECTRAfluor Plus microfluorimeter (TECAN). Experiments were performed in triplicates. The lowest concentration resulting in 90% growth inhibition of M. tuberculosis. The MIC was defined as the lowest concentration results in the inhibition of 90% growth of M. tuberculosis.

Statistical analysis.
All the data were obtained in a triplicate manner, and results are presented as mean ± standard deviation. One-way ANOVA was used for the processing of results. Statistical analysis (Mean, standard deviation, probability, and Pearson coefficient correlation) was obtained via statistical software (Prism pad 7). The level of significance was considered at < 0.05.

Results
In vivo study. Acute toxicity and effect on weight. According to the results, acute toxicity manifested significant noticeable signs on the mice's body weight. The observed change was shown in Tables 1 and 2. In the present study, DV flowers with methanol and chloroform extracts found no devastating effect on mice. No mortality was found at the highest dose of 2000 mg/kg as it is considered the highest dose by OECD guidelines for any acute toxicity assay. Three plant concentrations 100 mg/kg, 200 mg/kg and 300 mg/kg were selected for the study.
Liver enzymes. DV flower extracts were tested for protective effect on liver enzymes in albino mice. Group 1 and 2 showed normal enzymes level with no treatment. As a result of CCL 4 treatment in (Group 3) slight to elevated changes were observed after 1, 2, 3, and 21 days in liver enzymes while compared with other groups.
(Group 4-9) DVM showed a more restorative effect on liver enzymes than chloroform extract, and group 10 indicated hepatoprotective effect by Silymarineffect (Table 3).
Hematological parameters. Induction of CCL 4 resulted in a decline in RBCs, WBCs and plateletslevel. DV methanol and chloroform extract significantly increase the level of parameters (Table 4). DV methanol depicted greater RBCs and WBCs values closer to the normal control with significant results (p < 0.05). www.nature.com/scientificreports/ Antioxidant enzymes. The results showed that the normal level of enzymes getsaltered due to CCL 4 administration. After carbon tetrachloride induction, hightoxicity reduced the antioxidant enzymes. Silymarin drug (positive control) and plant extracts revealed positive results. Results obtained in a triplicate manner along with coefficient variation were < 0.05 (Table 5).
Histopathology. Morphological changes in organ (Liver, kidney, and spleen) tissues were investigated by histopathological microscopy. CCL 4 intoxication damaged the normal architecture of cells, and after 21 days of DV treatment, the cell structure of mice tissues was integrated. Several concentrations of plant extracts exhibited a  www.nature.com/scientificreports/ protective effect on tissues by attenuating injuries, while methanol extract presented more supportive evidence on the cellular organization than DVC extract (Fig. 1). The normal liver architecture depicts central vein, hepatic sinusoids as well as portal veins with normal appearance. Fibrosis and vascular irregularities, for instance, liver sinusoids alteration and central vein congestion, were seen in carbon tetrachloride mice. Renal histology revealed normal features like intact glomerular, tubular structure, bowman's space and capillary tufts. Treated spleen showed white pulp containing normal lymphoid masses followed by extremely vascular red pulp, which was similar to normal mice histology.  Figure 1. "a" showed the normal mice liver tissues, followed by "b" (arrow) showed the degeneration of hepatocytes. "c" presents recovery of degenerated hepatocytescaused by CCL 4 induction. "d" CCL 4 treated cellular structure of kidney. "e" DVM treated kidneyshowed slight recovery of renal tubules. "f" presents a normal spleen, while "g" arrow depicts normal lymphoid masses and red pulp in DVC-treated spleen. www.nature.com/scientificreports/ Phytochemical analysis and antioxidant assays. Dodonaea viscosa flower possessed total phenolic content 174 ± 4 mg/g dry weight and total flavonoid content 98 ± 7 mg/g, where the results are significantly different p < 0.05. DVM extract was evaluated against different antioxidant activities. The lowest IC 50 was found in hydrogen peroxide assay 11.37 ± 0.4 mg/g dry weight and hydroxyl radical scavenging assay 19 ± 0.56 mg/g dry  www.nature.com/scientificreports/  www.nature.com/scientificreports/   www.nature.com/scientificreports/ weight, which is greater than Ascorbic and Gallic acid. DVM also showed good activity to quench free radicals in DPPH free radical scavenging assay (Table 6).

High-performance liquid chromatography.
For the detection of some important medicinal compounds, HPLC analysis was carried out. Identification of peaks wasmade by comparing the retention time of the Dodonaea viscosa flower with standard compounds. The resulting peaks correspond to each compound were proportioned (Fig. 2). HPLC quantification of DVM flower extract identified the presence of gallic acid, epicatechin, cumeric acid, and quercetin compound. Whereas catechin has not been quantified in DV extract (Table 7). Gallic acid was the highest content observed with the quantity of 196.78 mg/kg.

Gas chromatography-mass spectroscopy (GC-MS) analysis. DVM extract is composed of vola-
tile-based organic compounds, mainly fatty acids. Numerous compounds were identified by GC-MS, and the compound list followed by the corresponding GC-MS spectrum was presented in Table 8 (Fig. 3). Among all compounds, the most significant were Ascorbic acid (C 38  Fourier transform infrared spectroscopy. The most notable peaks in DVM were observed between 3500 and 2800 nm. A peak at 2926.68 nm belongs to O-H stretch, Carboxylic acids and then at 3399.20 nm correspond to O-H stretch, H-bonded which signifies Alcohols, phenols. Peaks ranging from 2900 to 700 nm belong to C-H stretch, C=O stretch, C-N stretch, and -C=C-stretch (Table 9; Fig. 4).

Anti-tuberculosis assay of Dodonaea Viscosa flowers.
Anti-tuberculosis assay of DVM extract was screened out against Mycobacterium tuberculosis 3 strains bg 1972, bg 206 and H37Rv. 5 mg, 10 mg, and 50 mg/ ml concentrations were used, and tuberculosis % inhibition was increased with the rise in concentrations. Plant extract showed resistance against all strains, but the highest activity was found against the H37Rv strain (

Discussion
The present study demonstrated the in vitro and in vivo biological activities of Dodonaea viscosa flowers. The liver, kidney, and spleen are important parts of our body and are involved in different pivotal functions. The liver is one of the most important organs and played an important role in the detoxification of toxins 49,50 . CCl 4 has been used to illuminate some mechanisms regarding various toxicities, i.e., lipid peroxidation, and cause necrosis, fibrosis, and apoptosis of cells 2 . CCL 4 is the toxin well known to produce chemical stimulated liver injurywhen it get metabolized into trichloromethyl radical (CCl 3 ). This radical damages important cellular process by altering lipid metabolism and quantities of protein and then also induce mutations and produce hepatocellular carcinoma (HCC). Moreover, CCl 3 oxygenation resulted in trichloromethylperoxy radicals (CCl 3 OO) thatlead to lipid peroxidation, polyunsaturated fatty acidsdestruction and lowered the permeability of the cellular membrane, and cause hepatic damage that is distinguished by fibrosis, cirrhosis, HCC, and Inflammation 51 .
Mice weight was reduced with the variation in weight of organs (Liver, Kidney, and Spleen). An increase in body weight owing to Dodonaea viscosa exhibited its protective effect. Dodonaea viscosa showed innocuous and protective to the mice, as reported earlier 52 . Level of liver enzymes ALT, ALP, AST, and direct bilirubin aimed to determine the sternness of damagedtissue 53 . Biochemical markers get altered by carbon tetrachloride and then restored by the treatment with plant extracts and standard drug Silymarin, indicating the usage of Dodonaea viscosa flowers against liver injury. CCL 4 has been reported to increaseliver enzyme levels in some biochemical studies 54,55 . When there areunstablechangesobserved in the ALP level, it causes liver diseases 56 . Harmful changes in liver enzymes reflected several conditions likethe development of tissue necrosis, the decline in liver capacity (biosynthetic and catabolic), and alter normal structure of hepatocytes 57 .
Hematological parameters such as RBCs, WBCs, and platelets were also disturbed by CCL 4 administration. Methanol and chloroform extracts of DV flower revealed positive effects on hematological parameters, which specify its suitability for managing blood cell disorders 58 . An endogenous enzyme CAT, SOD, and GPx involved in scavenging free radicals and declining normalenzyme levels indicate hepatic damage 58 . CCL 4 reduces the level of antioxidant enzymes (CAT, SOD, and GPx) and total protein compared with the normal group and confirmsliver injury 59 , whereas the above factors were reinstated after administrating plant extracts. Intoxication of CCL 4 in antioxidant enzymes can be improved using Medicinal plants 60,61 . Natural products have been investigated for the source of antioxidants that are being used for the hepato-protective activity. For the management of different diseases, flavonoids rich plants have shown protective effectsby decreasing serum markers with antioxidant and anti-inflammatory processes 62 .
Exposure to CCL 4 leads to liver damage such as necrosis, fibrosis, and central vein alteration. In the kidney, it caused renal fibrosis, glomerular and tubular changes, while in the spleen, deterioration in white and red pulp occurred [63][64][65] . As a result of CCL 4 toxicity, the cellular structure and function of the kidney rely onthe functional state of the liver 66 . Similar studies reveal that at high doses of extracts, liver, kidney, and spleen seemed nearly normal, with no observable gross morphological and histopathological modifications, supporting present findings 67 . Studies signify the use of Dodonaea viscosaagainst toxicity inan animal model and found to have revealeda protective effect for liver enzymes and attenuated the injury by diminishing the production of reactive oxygen species in hepatocytes 68  www.nature.com/scientificreports/ Methanol solvent was selected for further activities based onthe best results in the in-vivo study compared to chloroform. Preliminary screening of secondary metabolites resulted ina significant amount of total phenolic compounds and total flavonoid content. Phenolics and Flavonoids are considered singlet oxygen quenchers, radical scavengers, reducing agents, and hydrogen donors 69 . So, the analysis of the plant's total flavonoids and phenolic compounds is important to measure its antioxidant capacity. The results of the experiment presented strong antioxidant activities of D. viscosa flowers. The highest antioxidant activity of DVM was shown against hydrogen peroxide assay. DVM manifested great radical scavenging ability as follows Hydroxyl radical assay > iron chelating assay > DDPH assay > Reducing power assay > ABTS radical assay > Superoxide assay. In the current study, the reducing capacity of D. viscosa significantly decreases the complex of ferric cyanide to ferrous. The occurrence of antioxidants was determined by evaluating the ability of plant extract to form ferrous by reducing the ferric cyanide complex 70 . Reducing the power of plant compounds specifies its potential antioxidant capacity. High reducing power in a sample hasa great ability to donate the electron and free radicals and produce stable elements by accepting donated electrons, which terminates the free radical reaction 71 . Hydroxyl radicals are highly reactive free radicals in biological systems, and there are no specific enzymes present in humans to protect against them. Their presence in the human body causes oxidative DNA damage. Therefore, there is a need for a solution to scavenge ROS with natural products having scavenging activity. Due to the high reactivity of OH radicals, the antioxidant activity of scavenging hydroxyl radicals is important 72,73 . The most commonly used method for the evaluation of antioxidants is the DDPH assay. The quenching of DPPH measurement relies on the discoloration of the purple-colored 2,2-diphenyl-2-picryl-hydroxyl compound by antioxidant. Donor antioxidant decolorized DPPH radical by electron acceptance and measured quantitatively from variations in absorbance 74 . Furthermore, D. viscosa expressed significant radical scavenging activity against ABTS assay with a low value of IC 50. All the essays are positive as well as significantly correlated with phenols and flavonoids.
HPLC quantified four compounds in DVM, i.e., gallic acid, epicatechin, quercetin, and cumeric acid. Quercetin is an iso flavonoid and flavonoid content (rutin and quercetin) was identified in the stem of dodonaea viscosa. The remedial aptitudes of Dodonaea viscosaare associated using pharmacological effects brought through the synergistic action of numerous constituents, i.e., flavonoids, saponins, di, and triterpenes, along with a combination of phenolics existing in the plant 75 . Flavonoids and diterpenoids are the richest secondary metabolites that werepreviously identified and isolated from Dodonaea 76 . These phenolic and flavonoid compounds revealed anticancer, antiallergic, antibacterial, antiviral, and anti-inflammatory activities 77 . The chemical compounds elucidated by GCMS were Oleic acid (Octadecenoic acid), Ascorbic acid, Ricinoleic acid, Stearic acid, Carboxylic acid, Cyclopentanone, and Cedrol. Fatty acids (Oleic, linoleic and linolenic acids) enriched food showed pleiotropic effects and used for the management of inflammation, hypertension, cardiovascular diseases, hyperlipidemia, reproductive ailments, immune system, and aggregation of platelets 78,79 . Research studies showed that Oleic acid exerts remedial effects on the human body, such as cancer, anti-inflammatory and autoimmune diseases, anda vital role in wound healing 80 . Ricinoleic acid is a significant unsaturated and hydroxylated fatty acid that depicts antipathogenic activity by deterring bacteria, viruses, mold, and yeast 81 . DVM showed very good activity against Tuberculosis strains. Mycobacterium tuberculosis is responsible for tuberculosis, which is among the fatal diseases. DodonaeaViscosa has been locally used in traditional medicines to treat tuberculosis 82,83 . Tested plant extract of DV flowers exhibited stronger resistance from all tested strains of Mycobacterium tuberculosis owing to the occurrence of bioactive components among the different concentrations of plant methanol extract that are probably anti-mycobacterial metabolites. Tuberculosis remains accountable for numerous mortalities around the world. During treatment, TB patients require extensive chemical analysis and eventually generate antagonistic effects on patient wellbeing. To diminish the use of unnatural resistant drugs, medicinally important plants contribute to great sureness as a potential reason for bioactive anti-mycobacterial metabolites 84 . A limited distinct species of genus Dodonaea have been extensively examined both chemically and pharmacologically. The most known species of genus Dodonaea is D. viscosa in literature 85 .

Conclusion
Dodonaea viscosa is well-known plant species and widely possesses so many biological activities. Results showed the potential pharmacological effect of Dodonaea Viscosa against acute toxicity in albino mice, specifying its use against different diseases, most of all liver diseases. This plant showed significant biological activities such as antioxidant and anti-tuberculosis. The chemical composition of the plant is rich in antioxidant compounds, flavonoids, and phenols, and a rich source of Fatty acids, mainly oleic acid. These compounds could probably protect elevated hepatic enzymes caused by carbon tetrachloride and chronic tuberculosis. These curative effects are linked with the traditional use of this plant against different diseases. This plant might be used to extract promising drugs for the management of liver and multiple organs injury. The active compounds and their action mechanism, pharmacokinetics, toxicology, efficacy, and molecular mechanisms still need to be explored to attain integration into remedial practice.