A Method For Evaluating Anti-Parkinson’s Activity Of Lantana Camara Leaf Extracts In Wistar Rats

Abstract

The present invention is related tothe anti-Parkinson’s activity of Lantana camara leaves through an experimental model using male Wistar rats. The leaves were extracted via maceration in methanol, resulting in a methanolic extract rich in phytochemicals such as flavonoids and phenolic compounds, known for their neuroprotective properties. Rats were divided into five groups, with varying treatments, including standard medications and different dosages of the extract. Behavioral assessments were conducted using the Rota Rod, Actophotometer, and Y-Maze apparatus to evaluate motor coordination and cognitive functions. Preliminary findings indicated that Lantana camara extract improved both motor performance and cognitive abilities compared to the control groups. These results suggest that Lantana camara may hold potential therapeutic value in the management of Parkinson’s disease, warranting further investigation into its active constituents and mechanisms of action.

Specification

Description:The invention is related to a method for screening the anti-Parkinson's activity of Lantana camara leaves in Wistar rats consists of several systematic and carefully designed steps aimed at evaluating the therapeutic potential of the plant. Figure 1 illustrates various constituents present in Lantana camara. Lantana camara is a flowering plant belonging to the verbena family (Verbenaceae) and is native to the tropical regions of the Americas. Commonly referred to by various names, such as big sage, wild sage, red sage, white sage, tick berry, West Indian lantana, and umbelanterna, this species is well-known for its vibrant flowers and potential medicinal properties. Its diverse nomenclature reflects its widespread presence and use in different cultures.For the experimental work involving Lantana camara, all chemicals and reagents were procured from Sigma Aldrich, ensuring a high standard of quality with laboratory-grade materials. The study utilized several specialized instruments, as detailed in Table 1, which includes essential equipment such as a cooling centrifuge, digital balance, heating mantle, pH meter, rotary evaporator, semi-auto analyzer, rotarod, actophotometer, and Y-maze. These instruments played a critical role in the extraction and analysis processes. Additionally, the reagents used in the experiments, listed in Table 2, encompassed various chemical solutions such as Mayer's reagent, Molisch reagent, Dragendroff's reagent, Tollen's reagent, Benedict's reagent, DTNB, and Fehling's A and B solutions, facilitating a comprehensive assessment of the phytochemical properties of the plant extract.
The process begins with the collection and authentication of Lantana camara leaves to confirm their identity and ensure that the correct species is used in the study. This initial step is crucial, as the efficacy of the extracts may depend on the authenticity and quality of the plant material used. The selection of Lantana camara leaves for this study was based on a thorough literature survey that highlighted their medicinal properties and potential benefits in treating Parkinson's disease. This approach ensures that the research is grounded in existing scientific knowledge, guiding the focus toward plants with proven efficacy in traditional medicine and pharmacological studies.
Following authentication, the leaves undergo a maceration technique using methanol to extract phytochemical compounds. This method (200) as shown in Figure 2, is well-regarded for its efficiency in solubilizing a variety of phytochemicals, including flavonoids, alkaloids, and terpenes, which may contribute to the desired anti-Parkinsonian effects. After extraction, varying doses of the methanolic extract are administered to Wistar rats, allowing researchers to determine a dose-response relationship and identify the optimal dosage for potential therapeutic effects. For the maceration process, approximately 100 g of the powdered leaves were immersed in 400 ml of methanol and soaked for four days. The prolonged soaking period enhances the extraction of bioactive compounds. Following the soaking period, the plant material was removed by filtration through cheesecloth, ensuring that the liquid extract contained the dissolved phytochemicals. The resulting filtrate was then concentrated using a rotary
evaporator, which reduces the solvent volume and increases the concentration of the extract. This extraction method is known to yield a variety of phytochemicals that are responsible for the plant's therapeutic effect.
The final phase of the method involves evaluating the anti-Parkinson's effect through a series of behavioral tests, including the Rota rod, actophotometer, and Y-maze apparatus. The Rota rod test assesses the motor coordination and balance of the rats, while the actophotometer measures their general locomotor activity. The Y-maze apparatus evaluates cognitive functions such as memory and learning. These behavioral assessments provide valuable insights into the efficacy of the Lantana camara extract in alleviating Parkinsonian symptoms and contribute to a deeper understanding of its potential as a natural therapeutic agent.
Experimental Protocol: In this evaluation study, the experimental animals were divided into five distinct groups to assess the effects of the treatments. Group I served as the vehicle control group and was administered 2 mL of normal saline to establish a baseline for comparison. Group II received Haloperidol at a dosage of 1 mg/kg body weight per day for a duration of 21 days, a common antipsychotic used in research to induce Parkinsonian symptoms in animal models.
Groups III and IV were designated as drug-treated groups and were pre-treated for one week with varying doses of the methanolic extract of Lantana camara, specifically at 250 mg/kg and 500 mg/kg body weight per day, administered orally. This pre-treatment aimed to evaluate the potential neuroprotective effects of the plant extract against the behavioral and physiological alterations induced by Haloperidol. Finally, Group V functioned as the standard drug-treated group and received Levodopa at a dosage of 30 mg/kg via intraperitoneal injection after the completion of the 21-day treatment regimen. This setup allowed for a comparative analysis of the efficacy of Lantana camara extracts against a well-established Parkinson's disease treatment.





Table1: Treatment Groups

Groups Treatment Dose Animals required
Group I Vehicle Normal saline(2ml/kg) 6AlbinoWistarrats
Group II Disease
control Haloperidol(1mg/kg,i.p) 6AlbinoWistarrats
Group III Test- I MELC low dose(250mg/kg) +
Haloperidol(1mg/kg,i.p) 6AlbinoWistarrats
Group IV Test- II MELChighdose(500mg/kg)
+Haloperidol (1mg/kg,i.p) 6Albino Wistarrats
Group V Standard Levodopa(30mg/kg. i.p) +
Haloperidol 6Albino Wistarrats

The Rota Rod test was performed to evaluate motor coordination, following the methodology outlined by Ibarrola et al. (2006). In this procedure, the rodents were placed on a Rota Rod apparatus, which consists of a horizontal rod divided into six equal compartments. The rod, with a diameter of 2.5 cm, rotates at a speed of 12 revolutions per minute (rpm). For inclusion in the test, the animals must remain on the rod for a minimum of two minutes across two consecutive trials. The rodents were divided into four distinct groups: a control group, a group receiving Haloperidol at a dose of 1 mg/kg via intraperitoneal injection, and two groups treated with the methanolic extract of Lantana camara (MELC) at doses of 250 mg/kg and 500 mg/kg, respectively.
The Actophotometer test assessed the locomotor activity of the rodents to investigate their mental alertness and wakefulness. This test utilized a digital device equipped with photoelectric cells. Upon placing a rat inside the apparatus, a light beam is directed toward a photoelectric cell, which registers movement when interrupted by the rat's activity. As the rodent moves, the device records the number of locomotor counts, providing insights into the animal's general activity levels.
The Y-Maze spontaneous alternation test was conducted to evaluate the learning and memory capabilities of the rodents. This behavioral test employed a Y-shaped apparatus with three arms arranged at a 120° angle to each other. The rodents were positioned at the junction of the three arms and allowed to explore the maze for five minutes. A rodent exhibiting good memory would preferentially explore the less-frequently visited arm compared to the previously explored ones. Both the number of arm entries and alterations were recorded, from which the percentage of alternation was calculated, offering insights into the cognitive function of the animals in relation to anti-Parkinson's activity.
The preliminary phytochemical screening of Lantana camara leaves indicated the presence of a variety of bioactive compounds. The methanolic extract was found to contain carbohydrates, proteins, amino acids, phenolic compounds, tannins, flavonoids, and steroids. These findings are detailed in Table 2, highlighting the rich phytochemical profile of the plant, which may contribute to its medicinal properties. Such diverse compounds are often linked to various therapeutic effects, reinforcing the potential of Lantana camara in traditional medicine and pharmacology. The study reported the presence of different kind of secondary metabolites like flavonoids, phenolic compounds, triterpenoids, tannins, saponins, amino acids, proteins, and carbohydrates in Methanolic extract of Lantana camara (MELC).
Table2: Results of Phytochemical screening

Name of the Phytochemical MELC
Carbohydrates +
Aminoacids +
Proteins +
Alkaloids +
Cardiacglycosides +
Triterpenoids +
Saponins +
Flavonoids +
Phenolic compounds +
Tannins +
Steroids -
Gums -
+means Positive and-means negative
The Rota Rod test was conducted to assess motor coordination in experimental animals, with each group consisting of six animals (n=6). The results are expressed as Mean ± SEM, and statistical analysis was performed using a one-way ANOVA followed by Dunnett's test. The comparison between the control and vehicle control groups showed a significant difference (ᵃP < 0.001), while comparisons between the experimental groups and the control group yielded non-significant (ns) or significant differences at various levels (*P < 0.05, **P < 0.01, ***P < 0.001).
Motor coordination was evaluated by observing the falling latency of animals on a rotating rod (25 rpm) over a five-minute period. In the control group, the falling latency was 166.5 ± 2.86 seconds. However, in the Haloperidol-treated group, a significant reduction in falling latency was observed (15.00 ± 2.65, p < 0.01), indicating impaired motor coordination. While Lantana camara-treated groups displayed altered falling latencies at varying doses, the changes were not statistically significant compared to the Haloperidol-treated group (p > 0.05). The detailed findings are presented in Table 3.
Table3: Effect of MEL ConRotaRod
S. No Treatment Time taken to fall from
Rotating rod (secs)
1 Normal control 166.5±2.86
2 Disease control 15.00±2.65*
3 Standard 112.66±0.42*
4 MELC250mg/Kg 100.83±0.30*
5 MELC500mg/Kg 108.00±0.68*
The Actophotometer test was conducted to measure locomotor activity in each experimental group, with six animals per group (n=6). Results are presented as Mean ± SEM. Statistical analysis was performed using a one-way ANOVA followed by Dunnett's test. The comparison between the control and vehicle control groups showed a highly significant difference (ᵃP < 0.001), indicating a distinct change in locomotor activity. Comparisons between the experimental groups and the control group also revealed significant changes in activity at various levels: *P < 0.05, **P < 0.01, and ***P < 0.001. However, in some cases, the results were non-significant (ns), indicating no significant difference between the groups for particular conditions.
Table 4: Effect of MELC on Locomotor activity
Groups LC(sec)
Day3 Day5 Day7
Normal control 384±3.60 394±1.15 386.33±1.56
Disease control 186±2.30*** 162.67±2.02*** 145.33±0.23***
Standard 196.33±2.60*** 217.33±0.02*** 246.33±0.77***
MELC250mg/Kg 189.33±1.48*** 194.67±0.91*** 215.67±0.56***
MELC500mg/Kg 192±1.55*** 198±1.12*** 236.67±1.85***
The statistical analysis highlights the varying effects of treatments on locomotor activity, with certain treatments showing significant improvement or reduction compared to the control group. These results help in evaluating the effectiveness of Lantana camara extracts in improving mental alertness and motor function.
In the Y-Maze test, each experimental group comprised six animals (n=6), with results represented as Mean ± SEM. Statistical analysis was performed using a one-way ANOVA followed by Dunnett's test. A highly significant difference between the control and vehicle control groups was indicated by ᵃP < 0.001. Further comparisons between the experimental groups and the control group were assessed at different levels: *P < 0.05, **P < 0.01, and ***P < 0.001, while some results were deemed non-significant (ns).
Notably, animals treated with the higher dose of Methanolic Extract of Lantana camara (MELC) exhibited an increase in spontaneous alternation behavior following a single administration, which suggests an enhancement in short-term memory. This improvement led to a statistically significant increase in spontaneous alternations, as reported in Table 5. These findings underscore the potential cognitive-enhancing effects of MELC in the tested animals.
Table6: Effect of MELC on Y-Maze equipment
S.No Treatment groups Number of Entries
1 Normal control 13.5±2.87
2 Disease group 5.00±0.65*
3 Standard 10.66±0.42*
4 MELC250mg/Kg 8.83±0.30*
5 MELC500mg/Kg 9.00±0.68*
Parkinson's disease is a chronic neurodegenerative condition primarily characterized by the progressive loss of dopamine-producing neurons in the substantia nigra, caudate nucleus, and putamen. This neuronal degeneration results in classic symptoms such as resting tremor, bradykinesia (slowness of movement), shuffling gait, a flexed posture, and muscular rigidity. Although the exact cause of this neurodegeneration remains unclear, oxidative stress has been identified as a significant contributing factor. Oxidative stress, which arises from dopamine metabolism, generates harmful free radicals. Compared to other regions of the brain, the substantia nigra pars compacta is exposed to higher levels of reactive oxygen species (ROS), increasing its vulnerability to oxidative damage.
Studies have shown that patients with Parkinson's disease exhibit heightened oxidative stress in the brain, which exacerbates neuronal damage. Haloperidol, a commonly used neuroleptic drug, is known to induce motor dysfunctions like tardive dyskinesia by blocking postsynaptic striatal dopamine D2 receptors. This blockade, combined with increased ROS production, contributes to haloperidol's neurotoxicity. Medications that mitigate haloperidol-induced motor disorders may offer protective effects against Parkinson's disease-related motor impairments.
Motor coordination in experimental animals was assessed using the Rota Rod performance test. In this test, animals were placed on a rotating rod (25 rpm) for five minutes, and their ability to remain balanced was measured. The control group showed a significantly higher latency to fall (166.5±2.86 seconds) compared to the Haloperidol-treated group, which had a markedly reduced fall latency (15.00±2.65 seconds). Treatment with Lantana camara extract at different doses improved fall latency, though the effect was not statistically significant compared to the Haloperidol-affected group (p > 0.05). However, at higher doses, the extract did significantly enhance motor performance (p < 0.01).In addition to motor coordination, the extract of Lantana camara significantly improved locomotor activity as observed in the actophotometer test, with results showing increasing activity on Day 3 (192±1.55), Day 5 (198±1.12), and Day 7 (236.67±1.85). These results suggest a potential role for Lantana camara in managing motor symptoms associated with Parkinson's disease. Cognitive function, assessed using the Y-Maze test, also improved with Lantana camara treatment. Animals treated with a higher dose of 500 mg/kg showed increased spontaneous alternation (9.00±0.68), indicative of enhanced short-term memory. This significant improvement in spontaneous alternations further supports the cognitive benefits of Lantana camara . These findings suggest that further research may provide deeper insights into the potential use of Lantana camara for treating Parkinson's disease and its associated neurological effects.
, Claims:We claim
1. A method for screening the anti-Parkinson's activity of Lantana camara leaves in Wistar rats, comprising the steps of:
(a) Collecting and authenticating Lantana camara leaves;
(b) Extracting phytochemical compounds from the leaves using a maceration technique with methanol;
(c) Administering varying doses of the methanolic extract to the Wistar rats; and
(d) Evaluating the anti-Parkinson's effect through behavioral tests including Rota rod, actophotometer, and Y-maze apparatus.

2. A composition comprising a methanolic extract of Lantana camara leaves containing phytochemical constituents, including but not limited to, geraniol, lauric acid, linalool, and flavonoids, wherein the composition exhibits neuroprotective properties against Parkinson's disease.

3. A method for evaluating anti-Parkinson's activity in animal models, which includes:
(a) Measuring motor coordination via Rota rod performance;
(b) Assessing locomotor activity through an actophotometer; and
(c) Determining cognitive function using the Y-maze apparatus.

4. A dosage regimen for administering Lantana camara leaf extract to subjects, wherein the effective dosages are determined based on acute toxicity studies and are selected to be 250 mg/kg and 500 mg/kg body weight.
5. A method for assessing the safety profile of Lantana camara leaf extract in Wistar rats, demonstrating that the extract is safe up to 5000 mg/kg, based on an acute toxicity study compliant with OECD guidelines.

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