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INTRODUCTION
Neurodegenerative disease is a term applied to a variety of conditions which result from a chronic breakdown and deterioration of neurons, particularly, those of Central Nervous System [CNS]. Neurodegenerative Diseases [ND] such as Parkinson’s Disease [PD] and Alzheimer’s [AD] and Multiple Sclerosis [MS] are characterized by a gradual onset of progressive symptoms including loss of memory and tremor, difficulty in learning or retaining information, inability to handle complex tasks, impaired spatial orientation and abilities, language deficits and behavioral changes (Adewusi et al., 2010). The greatest risk factor for ND is aging, which carries mitochondrial dysfunction, chronic immune inflammatory response, and oxidative stress (Angelopoulou et al., 2020; Angelopoulou et al., 2019), the major causes of neuronal damage and death.
Parkinson’s Disease [PD] is considered as the most common neuronal destructive disease after Alzheimer’s Disease [AD] and is characterized by the dopaminergic neuronal loss in the Substantia Nigra pars compacta [SNpc], as well as the accumulation of Lewy bodies and Lewy neurites in the degenerating neurons, mainly consisting of α-synuclein. PD patients display motor [bradykinesia, rigidity, resting tremor] and nonmotor symptoms [depression, dementia, autonomic dysfunction, sleep disorders]. Overall, the disease may be characterized by mixed phenotypic characteristics (Abayomi et al., 2013). Although, its exact etiology remains unrevealed, a complex interplay between mitochondrial dysfunction, oxidative damage, autophagy impairment, excessive neuroinflammation, and dysregulation of apoptosis has been shown to contribute to its pathogenesis (Ahmed et al., 2018). In addition, there are several environmental factors [like pesticides and neurotoxicants] and genetic factors [including mutation in the α- synuclein [SCNA], leucine rich repeat kinase 2 [LRRK2], DJ1, Parkin, PTEN induced kinase 1 [PINK-1], GBA1 genes, etc.] that have been associated with the development of PD (Atala et al., 2009).
From thousands of years a remarkable number of modern drugs have been obtained from natural sources, particularly from the plants. Plant based medicines have played an important role in primary health care needs of human as well as animals (Aziz-ul-Ikram et al., 2015). Variety of plants exhibit antimicrobial, larvicidal, anti-inflammatory and antioxidant activities due to the presence of some active compounds like essential oils, flavonoids, terpenoids, tri-terpeniods, glycosides, alkaloids and other natural phenolic compounds play a dominant role in the maintenance of human health since ancient times (Baker et al., 1995). Natural products play on important role in drug development programmes in the pharmaceutical industry (Brahmi et al., 2013).
Recently, a great number of natural medicinal plants have been tested for their therapeutic properties, showing that the raw extracts or isolated pure compounds from them have more effective properties than the whole plant as an alternative for the treatment of neurodegenerative diseases. These properties are due mainly to the presence of polyphenols, alkaloids, and terpenes, among others, that are micronutrients produced by plants as secondary metabolites (Calabrese et al., 2008).
Torilis nodosa is a species of flowering plant in the family Apiaceae known by the common names knotted hedgeparsley and short sock-destroyer. It is an annual herb producing a hairy stem up to half a meter in maximum height. The alternately arranged leaves are each divided into several pairs of smooth-edged lance-shaped or linear leaflets and bipinnate. Peduncles sessile or very short, styles erect. Calyx teeth triangular, fruit ovoid and 2-4 mm long, distributed in Europe, Asia, North and South America (Breithaupt et al., 2002).
Traditionally Torilis nodosa is used for the treatment of gastrointestinal ailments, liver problems, respiratory tract infections like cold and cough. Literature revealed that there no such activities are reported. It is therefore selected to evaluate the Phytochemical screening and their role in prevention ND especially PD. Therefore, this project was designed to examine its antiparkinsonian properties.
MATERIALS AND METHODS
Plant collection
Torilis nodosa fully matured whole plant was collected from Goriwala areas of District Bannu. Khyber-Pakhtunkhwa, Pakistan after identification by Dr. Fizan Ullah, Chairman Department of Botany, UST-Bannu and confirmation with literature. The voucher specimens with Accession No. 140-Bot were submitted at herbarium in the Department of Botany UST Bannu. All the dust particles and impurities were swashed with running tap water. The plant was shed dried and processed into a fine mesh using grinding machine.
Extraction and Fractionation
Fine-grained powder of Torilis nodosa was sodden in 80% methanol and incubated for one-week at room temperature. After incubation the solution was filtered through Whatman filter paper No. 45. The filtrate was evaporated using a rotary evaporator and got crude methanolic extract.
The crud extract was first diluted in distilled water to create an aqueous extract, which was used to prepare different fractions based on polarity. Different solvents, such as n-hexane, ethyl acetate, chloroform, and butanol were used and various fractions were obtained through solvent-solvent partition technique. Different fractions were evaporated using a rotary vacuum evaporator and collected separately. The dried fractions were stored at 4oC for further investigations.
Phytochemical Analysis
Torilis nodosa plant various fractions were processed for phytochemical qualitative investigation using standard procedures. Various qualitative tests were carried out including tannins, saponins, flavonoids, terpenoids, alkaloids, phlobatannins, cardiac glycosides, coumarins, and anthraquinone for assessment of bioactive groups of phytoconstituents.
Determination of Total Phenolic Contents
Standard protocol of Atala et al. (2009) was adopted to find out the total phenolic constituents in various fractions of Torilis nodosa plant extract. Various fraction samples and gallic acid used as standard were mixed with 10 mL of the folin-Ciocalteau solution and incubated for 10 min. The mixture was added with 0.115 mg/mL Na2CO3 solution. Optical Density of the mixture was checked at 765 nm to measure the absorbance spectrum. The amount of total phenolic compounds was calculated as mg of gallic acid equivalents/g of dried material.
Test to Determine the Presence of Total Flavonoids
Standard procedures of Pompilio et al. (2022) were followed to investigate the total flavonoids constituents. According to this protocol 0.25 mL of each fraction and 15-250 g/mL rutin concentrations were used. Various fractions of Torilis nodosa and rutin used as standard were mixed with 1.5 mL of deionized water and 5% NH4NO3 and were gestated for 6 min. Then 0.2 mL AlCl3 (w/v), 1.0 M Sodium Hydroxide (NaOH) were mixed and again incubated for 5 min. Optical Density of the mixture was find out at 510 nm. The dried fraction of rutin equivalent mg/g extract was used to calculate the total contents of flavonoids.
In vitro Antioxidant Tests
Evaluation of Plant Extracts for DPPH Radical Scavenging test
1,1-diphenyl di picrylhydrazine was used to assess the in vitro anti-oxidant activity of Torilis nodosa plant various fractions and ascorbic acid which were used as a standard (Khan et al., 2010). A stock solution of reagents was arranged by melting 0.006 mg of DPPH in 100 mL of methanol. 0.2 mL various plant fractions were assorted 2.8 mL DPPH solution and gestated for 30 min, using a spectrophotometer, the optical density was noted and considered at 517 nm. According to the formula below, the percentage of DPPH inhibition was premeditated as follows:
ABTS Cation Radical Test
The standard protocol of Brahmi et al. (2013) was used to assess the scavenging capability of ABTS free cation radicals with a minor amendment. According to this protocol we have mixed 7 mM of ABTS reagent with 2.45 mM potassium per sulphate reagent and incubated in the dark room for 8 hr. Then 50% methanol solution was used to dilute the sample after proper incubation and optical density was recorded at 745 nm of the mixture. A diluted reagent having 2.8 mL volume was mixed with various fraction and standard ascorbic acid separately and incubated for 6 min at room temperature. Optical density of each mixture was checked at 745 nm using spectrophotometer and percentage inhibition was calculated using the formula:
Superoxide Radical Scavenging Test
In this procedure each fraction of Torilis nodosa and ascorbic acid used as standard was mixed with Nicotinamide Adenine Dinucleotide Reduced (NADH) reagent and 0.5 mL of Nitro Blue Trizol. PMS was added, and then incubated for 15 min at 25ºC. Calculations of the absorbance spectrum was carried out at 530 nm (Kakkar et al., 1984; Gyamfi et al., 1999).
Assay for Detection of H2O2 Radicals Reducing Effect
Standard procedure was used to determine the H2O2 free radical activities of ascorbic acid and various fractions of plant extract Gutteridge and Halliwell (2000). Samples prepared from various fractions and standard ascorbic acid was mixed with 0.4 mL H2O2 (50 mM phosphate buffer, pH 7.4), to test the reduction of the H2O2 free radical. Phosphate buffer was used as a blank to measure the absorbance of mixtures at 230 nm. The experiment was repeated in replicates to determine the hydrogen per oxide scavenging ability.
Hydrogen peroxide scavenging percentage=[Control absorbance - sample absorbance] /Control absorbance] *100
Total Antioxidant Activity Assessment of Various Fractions
Total antioxidant activity of various fractions and ascorbic used a standard was calculated using the standard protocol of Umamaheswari and Chatterjee (2008). 4 mM ammonium molybdate, 28 mM sodium phosphate and 0.6 M sulfuric acid was combined to form the reaction mixture. From this mixture, 0.1 mL of the reagent solution take off and mixed with 0.1 mL of the aliquot solution. The mixture was placed in a test tube lined with silver foil and incubated at 90ºC for 90 min in a water bath. The optical density spectrum was noted at 765 nm using spectrophotometer. The following formula was used to compute the compound's antioxidant capacity:
Determination of Hydroxyl Radical Inhibition
The protocol of Gutteridge and Halliwell, (2000) was followed to check hydroxyl radical scavenging efficacy. Various reagents including 500 mL of 2-deoxyribose (2.8 mM), 100 mL of 300 mM ascorbate and 100 mL ferric chloride (100 mM) was combined to form the reaction mixture. To catalyze the process 200 mL of sodium phosphate buffered water (pH 7.4) was used. The whole mixture was placed for 1h at 37ºC. 1 mL of an aqueous solution containing 2.8% (w/v) TCA, NaOH, and TBA was heated for 15 min. After cooling, the OD was measured at 532 nm, and the percent inhibition was used to determine the formula below:
In vivo anti-parkinson animal model
Approval of Ethical Recommendations
The research work was carried out in accordance with the international guideline for animal care and handling. The ethical guidelines were approved by ethical committee of Faculty of Biological Sciences, University of Science and Technology Bannu with written permission letter no. ustb/ethic/1231.
Experimental design
30 adults male Wistar rats were used for in vivo anti-Parkinson activities. Rats were obtained from National Institute of Health (NIH) Islamabad. Standard protocols for induction of Parkinson disease were used for the treatment of animal. Rats were randomly divided into 5 groups and rationale of the doses were selected on the basis previous published reports.
- Group I (Control): Received 0.5% N/saline p.o. every day.
- Group II (Disease control group): Rotenone in 1% DMSO (1.5 mg/kg per body weight s.c.) after every two days.
- Group III (Standard drug control group): Stalevo (5 mg/kg b.w.) after 24 hr.
- Group IV (Low dose extract group): Torilis nodosa methanol extract (150 mg/kg b.w.,) after 24 hr.
- Group V (High dose extract group): Torilis nodosa methanol extract (300 mg/kg b.w.,) after 24 hr.
The experiment was conducted for 21 days. During these experiment various parameters were checked to assess the behavioral effects of various groups of rats. At the end of experiment the rats were sacrificed by an overdose of xylazine and ketamine anesthesia at day 22 by cervical dislocation. Brain was removed, cleaned with phosphate buffer and kept for biochemical and histological examination at -20ºC in freezer. Serum samples were collected through heart puncture. The collected serum was processed for various biochemical tests.
Evaluation of behavioral effects
During the experiment various behavioral tests were performed to investigate the protective effects of plant extract against rotenone induced Parkinson disease in rats. These tests include effects on rats body weight, and catalepsy score using standard procedures.
Effects of plant extract on oxidative parameters of tissue homogenate
To investigate the protective effects of methanolic plant extract on various parameters of tissue homogenate was investigated using standard protocols. The effects of plant extract on the reduction of lipid peroxidation, standard method of Jollow et al. (1973) while glutathione content was determined using procedure of Iqbal et al. (1996). Protocols of Chance and Maehly (1955) was used to determine Catalase activity (CAT), method of Kakkar et al. (1984) was followed for Superoxide Dismutase (SOD) and procedures of Joseph et al. (2007) was used to determined Glutathione Peroxidase (GPx).
Statistical analysis
Data were expressed as mean and Standard Error (SE). Statistical analysis of parametric data for IC50 was carried out using graph prism pad software. Experimental results were further analyzed for Pearson correlation coefficient between TPCs, flavonoids and different anti-oxidant assays and tested for significance by Student’s t-test (p<0.05). SPSS ver. 14.0 (Chicago, IL, USA), Microsoft Excel 2007 (Roselle, IL, USA) and origin were used for the statistical and graphical evaluations.
RESULTS
Qualitative phytoconstituent analysis
Various bioactive groups of phytoconstituents were determined using different fractions of plant extracts. The findings of the present study revealed that methanol, butanol, ethyl acetate and chloroform contain flavonoids, alkaloids, tannins, terpenoids and saponins however N-hexane fraction was deficient in terpenoids, and tannins while terpenoids and saponins were absent in aqueous fraction.
Determination of phenolic compounds and total flavonoids constituents
Phenolic and flavonoids compounds play a crucial role in the activity of various plant extracts. The findings of the present study showed that comparatively higher amount of total phenolic compounds in methanolic plant extract (215.12±1.92 mg GAE/g) followed by butanolic (123.67±2.07 mg GAE/g), chloroform (88.80±1.05 mg GAE/g), ethyl acetate (56.78±0.98 mg GAE/g), n-Hexane (37.14±0.92 mg GAE/g) and aqueous (19.73±0.75 mg GAE/g) fractions respectively. Similarly quantitative phytoconstituent analysis revealed that maximum concentrations of flavonoids were present in methanol fraction of plant extract as compare to other fractions (Table 1).
| Sample | Total phenolic components as mg gallic acid equivalent (GAE mg/g extract) | Total flavonoids as mg rutin equivalent (mg/g extract) |
|---|---|---|
| Methanol | 215.12±1.92e | 47.89±1.78f |
| Butanol | 123.67±2.07d | 32.02±2.01d |
| Chloroform | 88.80±1.05c | 19.12±1.38c |
| Ethyl acetate | 56.78±0.98b | 10.13±0.98b |
| n-Hexane | 27.14±0.92a | 7.53±0.45b |
| Aqueous | 19.73±0.75a | 3.07±0.12a |
Effect of various fraction on scavenging of various free radicals
Various free radicals were used to investigate the efficacy of various plant fractions.
DPPH free radical scavenging efficacy
The effect of various fractions of plant extracts and ascorbic acid used as standards was checked on scavenging of DPPH free radical capability. The results revealed that methanolic fraction (85.24±3.21) show maximum efficacy as compare to other fractions like butanol (73.89±2.54), chloroform (65.43±2.78), ethyl acetate (55.13±1.90), N-hexane (45.21±1.42) and aqueous fraction (36.11±1.76) as presented in Figure 1A. Ascorbic acid (94.53±2.53) used as standard positive control higher efficacy in scavenging of DPPH free radicals comparatively of all plant fractions.
ABTS free radical scavenging assay
The ABTS free radical scavenging efficacy of various fractions of plant extracts and ascorbic acid was carried out. The findings shows that ascorbic acid (92.4±4.29) has maximum inhibition of ABTS free radicals scavenging as compare to various plant fraction viz; methanol (79.21±3.21), butanol (68.43±2.78), chloroform (59.45±2.76), ethyl acetate (53.54±1.89), N-hexane (32.76±1.66) and aqueous fraction (22.14±0.92) (Figure 1B).
Hydrogen peroxide (H2O2) scavenging ability
Various fractions of plant extracts were characterized for the assessment of free radical efficacy of H2O2. The data revealed that H2O2 free radicals were significantly inhibited by methanol fraction as compare to other fraction however, showed less inhibition as compare to ascorbic acid used as standard antioxidant positive control as shown in Figure 2A.
Superoxide radical scavenging activity
Superoxide free radical scavenging activity of various fraction of the plant and ascorbic acid was determined in the current study. Reports obtained presented that ascorbic used as standard antioxidant show higher percent inhibition of superoxide radical followed by various plant fraction in order of ascorbic acid <Methanol fraction <butanol fraction <chloroform fraction <ethyl acetate fraction <N-hexane fraction <aqueous fraction respectively (Figure 2B).
Total Antioxidant Activity Assessment of Various fractions
Total antioxidant activity of various fractions and ascorbic used a standard was calculated as presented in Figure 3. The data showed that various fraction have the potency to remove free radical however revealed less efficacy as compare to standard antioxidant ascorbic acid. The comparative analysis among the various fractions of plant showed that methanol extract has higher potency as compare to other fractions.
Determination of hydroxyl radical inhibition of various fractions
Various fractions of plant extracts and ascorbic acid revealed were characterized using hydroxyl radicals. The findings revealed that methanol fraction (72.51±3.21) among the various fractions of plant extract showed higher hydroxyl free radical scavenging potential as compare to other fractions viz; followed by butanol (61.67±2.68), chloroform (50.31±2.11), ethyl acetate (39.21±1.89), N-hexane (23.98±0.89) and aqueous fraction (14.32±0.23) respectively as shown in Figure 4.
In vivo anti-Parkinson activity
Effects of Torilis nodosa on Body Weight
The body weight of trial rats plays a crucial part in the anti-parkinson effects of plant extracts. The rats’ body weight was aimed to evaluate their overall healthiness. Induction of rats with 1.5 mg/kg body weight rotenone triggered substantial decrease in body weight of rats. Co-treatment with 150 mg/kg and 300 mg/kg body weight plant extracts and 5 mg/kg body weight stalevo significantly improved the abnormality caused by rotenone depends on the amount of dose as shown in Table 2.
| Groups | After 7 days | After 14 days | After 21 days |
|---|---|---|---|
| Control | 190.78±2.78 | 198.55±2.42 | 214.24±2.52b |
| 1.5 mg/kg Rotenone | 188.23±3.11 | 178.24±4.27 | 170.11±1.43a |
| 1.5 mg/kg Rotenone +5 mg/kg stalevo | 193.38±3.34 | 196.91±2.04 | 212.03±4.13b |
| 1.5 mg/kg Rotenone +150 mg/kg T. nodosa | 191.12±4.73 | 198.23±3.92 | 208.27±2.98b |
| 1.5 mg/kg Rotenone +300 mg/kg T. nodosa | 190.53±3.04 | 193.75±2.77 | 213.65±3.57b |
Catalepsy score
Catalepsy score plays a key role in behavioral study of Parkinson activity. In the study bar test was used to study the effects of various fractions of plant extract. Table 3 revealed that administration of 1.5 mg/kg b.w., rotenone group significantly increased cataleptic score as compared to the normal control group. Rats treated with 150 and 300 mg/kg body weight of plant extracts as well as 5 mg/kg body weight stalevo, showed significantly (p<0.01) decrease against catalepsy by decreasing cataleptic score (Table 3).
| Groups | After 3rd day | After 6th day | After 9th day | After 12th day | After 12th day | After 12th day | After 12th day |
|---|---|---|---|---|---|---|---|
| Control | 2.12±0.06 | 4.68±0.19 | 7.12±0.26 | 8.43±0.12 | 9.92±0.98 | 10.24±0.87 | 12.87±1.75 |
| 1.5 mg/kg Rotenone | 5.21±0.09** | 10.12±0.31** | 13.33±0.33** | 16.11±0.32** | 18.23±1.14** | 21.28±1.76** | 25.21±1.98** |
| 1.5 mg/kg Rotenone +5 mg/kg stalevo | 3.11±0.21++ | 5.25±0.28++ | 7.23±0.71++ | 9.12±0.27++ | 10.67±0.58++ | 11.72±1.23++ | 13.74±0.89++ |
| 1.5 mg/kg Rotenone +150 mg/kg T. nodosa | 4.42±0.12+ | 7.62±0.27+ | 8.56±0.58++ | 10.14±0.57++ | 12.54±1.02++ | 14.64±1.24++ | 16.26±2.01++ |
| 1.5 mg/kg Rotenone +300 mg/kg T. nodosa | 2.67±0.09++ | 4.35±0.13++ | 7.11±0.08++ | 8.52±0.67++ | 9.24±1.21++ | 11.39±0.78++ | 13.10±1.02++ |
Oxidative Biomarkers Test
Rat brain samples were examined for oxidative stress indicators such as antioxidant enzyme activities, TBARS and GSH contents. Rats treated with 5 mg/kg body weight rotenone caused significant increase (p<0.01) in TBARs and reduced the level of GSH contents. Co-treatment of rats treated with 150 mg/kg and 300 mg/kg body weight Torilis nodosa showed significant improvement (p<0.01) by reversing the abnormal change caused by rotenone in the activities of antioxidant enzymes, level of TBARs and GSH as compared to the rotenone-treated group rats. Similarly, markedly decreased (p<0.01) TBARs levels and increased GSH levels were reported in rats treated with rotenone as compared to normal non treated control rats (Table 4). Activities of antioxidant enzymes revealed the efficiency of body defense system. Administration of 1.5 mg/kg body weight rotenone caused significant reduction (p<0.01) in the activities of antioxidant enzyme such as Superoxide Dismutase (SOD), Catalase (CAT), and Glutathione Peroxidase (GPx). The administration of Torilis nodosa (150 mg/kg and 300 mg/kg) body weight significantly increased (p<0.01) the activities of SOD, CAT and GPx. Similar observations were recorded by the treatment of 5 mg/kg body weight stalevo (Table 4).
| Groups | TBARS nM /min/mg protein) | GSH (M /g tissue) | SOD (U/mg protein) | CAT(U/min) | GPx (nM /min/mg protein) |
|---|---|---|---|---|---|
| Control | 23.5±1.15++ | 33.5±1.3++ | 27.13±0.87++ | 26.70±0.82++ | 34.14±1.45++ |
| 1.5 mg/kg Rotenone | 56.7±2.23** | 16.5±1.1** | 10.45±0.25** | 12.01±0.91** | 14.76±0.94** |
| 1.5 mg/kg Rotenone +5 mg/kg stalevo | 22.8±1.45++ | 32.5±1.7++ | 26.78±1.17++ | 24.97±0.78++ | 33.57±1.17++ |
| 1.5 mg/kg Rotenone +150 mg/kg T. nodosa | 32.6±1.51++ | 23.6±2.1+ | 19.58±0.91+ | 22.23±0.88++ | 26.65±1.22++ |
| 1.5 mg/kg Rotenone +300 mg/kg T. nodosa | 26.5±1.56++ | 31.2±1.6++ | 24.93±1.02++ | 25.29±0.95++ | 30.71±1.16++ |
Liver Profile Test
In our study, the liver profile test was also taken, but the liver profile test was not directly related to Parkinson's disease. Administration of 1.5 mg/kg body weight of rotenone caused significant changes (p<0.01) in the liver profile of rats. Treatment of 150 mg/kg and 300 mg/kg body weight Torilis nodosa improved the abnormalities in SGPT, serum albumin, total protein, and total bilirubin as compared to rotenone treated rats. Similar effects were reported in rats treated with 5 mg/kg body weight stalevo.
Kidney function and profile play a key role in the health of any individual. Administration of 1.5 mg/kg body weight rotenone caused significant elevation (p<0.01) in serum creatinine and blood urea level. Co-treatment of 150 mg/kg and 300 mg/kg body weight Torilis nodosa significantly reversed (p<0.01) the serum creatinine and blood urea dose dependently. Similar reports were obtained from the rats treated with 5 mg/kg body weight stalevo (Table 5).
| Groups | SGPT(ALT) (U/L) | Serum Albumin (µmol/L) | Total Proteins (g/dL) | T. Bilirubin (mg/dL) | Serum Creatinine (mg/dL) | Blood Urea (mg/dL) |
|---|---|---|---|---|---|---|
| Control | 25.45±1.87++ | 7.32±0.76++ | 9.56±0.56++ | 2.33±0.45++ | 0.89±0.45++ | 23.9±0.93++ |
| 1.5 mg/kg Rotenone | 55.35±2.05** | 2.95±0.34** | 4.17±0.71** | 8.21±0.57** | 1.72±0.16** | 43.5±0.57** |
| 1.5 mg/kg Rotenone +5 mg/kg stalevo | 28.23±0.98++ | 7.78±0.19++ | 9.98±0.99++ | 2.73±0.67++ | 0.90±0.34++ | 26.4±0.55++ |
| 1.5 mg/kg Rotenone +150 mg/kg T. nodosa | 38.01±1.98+ | 4.98±0.28+ | 6.12±0.77+ | 6.12±0.78+ | 1.21±0.25+ | 37.7±0.71+ |
| 1.5 mg/kg Rotenone +300 mg/kg T. nodosa | 29.67±0.95++ | 7.12±0.17++ | 9.43±0.58++ | 3.81±0.56++ | 0.94±0.38++ | 27.9±1.52++ |
DISCUSSION
Parkinson disease is a neurodegenerative dysfunction that effected huge population worldwide. In market several therapeutic allopathic drugs are available but have side effects. Therefore, scientists are busy in the development of new beneficial drugs from medicinal plants which has less or no side effects and have easy access (Ferreira and Massano, 2017; Garabadu and Agrawal, 2020). In the present study Torilis nodosa plant was analyzed for its phytochemical composition, antioxidant capacity and antiparkinsonian activity. Phytochemical analysis of different fractions showed that alkaloids, tannins, glycosides, saponins and flavonoids are present in the crude methanol extract of the plant. Other phytochemicals, including steroids, terpenoids, and saponins are also found in other fractions of plant extract. Previous studies have shown that plant extracts contain many phytochemicals, including steroids, terpenoids, flavonoids, saponins and alkaloids. These phytochemicals are important in the prevention and treatment of infectious diseases and act as free radical scavengers (Farombi, 2003).
Quantitative evaluation showed that the methanol fraction of Torilis nodosa plant extract composed of maximum quantity of phenol and flavonoids contents as compare to other fractions. Similar data were reported in other studies (Khan et al., 2012).
In this study, various fractions of Torilis nodosa exhibited antioxidant activity measured by DPPH, ABTS, H2O2 and other scavenging methods. Methanol has the highest value in DPPH; in ABTS, butanol showed the highest percentage of inhibitory activity, but in H2O2, ethyl acetate showed the highest percentage of activity. Previous studies have shown that antioxidant behavior, one of the most frequently observed functions of bioactive substances which reduces the effects of oxidative stress (Madihaand Haider, 2019).
In the present study due to higher activity of methanol fraction, it was preceded for in vivo anti-Parkinson activities. In the Parkinson activity the methanol fraction of Torilis nodosa markedly improved the abnormalities in the body weight of rats treated with rotenone. Similarly, it was reported that rats administered with both safflower flavonoid extract and madopar showed significant improvement as compared to rats in the rotenone group which are in consistent with our findings (Rodriguez, 2008; Nour et al., 2013).
Behavioral marker plays a crucial role in the assessment of Parkinson activities. In the recent study the rotenone-treated group rats showed lowest time on rotarod as compared with the normal control group rats. Torilis nodosa plants showed marked improvement in rod spinning and sucrose feeding tests. Similar findings were obtained in the study conducted before and after the addition of quercetin in rotenone-induced Parkinson's disease (Procaccio et al., 2024).
The impact of the therapy on memory performance was investigated using the Morris water maze and footprint test. Data of the current study revealed that rotenone injection considerably increased (p<0.01) escape latency and dramatically decreased (p<0.01) time spent in and crossings over the target quadrant in comparison to the control rats. Torilis nodosa (150 g/kg and 300 mg/kg body weight) has been shown to have a significant effect in the improvement of this alteration caused in rats. Our results are consistent with previous studies showing that rotenonen treated rats exhibited higher tonicity scores and beam walking latencies compared to control rats which were restored (Seppi et al., 2019).
Injection of 1.5 mg/kg body weight caused significant differences in front and hind limb step length and front limb base width compared to normal controls. Torilis nodosa treatment had a positive effect on the rotenone-induced footprint test in mice. The results of this study are consistent with those of Madiha et al. (2019) showed decreased walking and reduced forward movement in rotenone-treated rats.
The antioxidant defense system plays an important role in eliminating harmful chemicals entering the brain. In this study, rotenone-induced parkinsonian effects also caused oxidation, ultimately reducing the activity of antioxidant enzymes such as SOD and CAT. Various treatments of Torilis nodosa and stalevo showed significant improvement in antioxidant enzyme activity. Similar findings are found in previous reports (Shen et al., 2016; Liu et al., 2020).
Lipid peroxidation and glutathione content are very important at the cellular level. In this study, rotenone treatment caused an increase in lipid peroxidation TBARs contents and a decrease in GSH levels. Obeso et al. (2008) obtained similar observations when evaluating the effect of Tymol on brain homogenates from patients with Parkinson's disease.
The liver is an important organ, and its interior is very sensitive to all kinds of toxins. Rotenone-induced liver injury in mice eventually increased serum ALT, total proteins and total bilirubin levels. These findings suggests that rotenone causes damage to hepatocytes. These damages can be repaired by treatment with multiple doses of Torilis nodosa. Our results are consistent with those of Liu et al. (2020) reported that quercetin protects the liver from damage by increasing liver function biomarkers. The kidney is the powerhouse of the body and plays an important role in maintaining homeostasis and detoxification and elimination of toxic metabolites from cells. Toxic metabolites of rotenone cause significant changes in renal serum creatinine and blood urea, indicating that rotenone causes nephrotoxicity. Torilis nodosa and stalevo treatment reversed the anemia. Ramos (2007) and Talarowska et al. (2016) reported same findings during their study in rats. These findings revealed that Torilis nodosa has the potency to be used for the treatment of Parkison disease. Further research on the exact mechanism of action of Torilis nodosa and isolation of its bioactive component is recommended.
CONCLUSION
From the current data it was revealed that the methanol extract of Torilis nodosa significantly improved antioxidant enzymes, behavioral, learning and memory parameters due to the presence of bioactive metabolites. Further research work is needed to investigate the mechanism of action of these bioactive constituents present in the plant extract.