Neuroprotective Evaluation of Green-Synthesized Silver Nanoparticles of Magnolia Champaca and Origanum Majorana in A Haloperidol-Induced Parkinson’s Disease Model in Wistar Rats

Neurodegenerative diseases present serious medical challenges, characterized by the progressive dysfunction of the nervous system. These conditions involve the gradual deterioration of neural structures, affecting key areas such as the brain or the entire nervous network. Although each disease under this category has its own unique symptoms, they all share a common trait: a relentless progression that steadily impairs the nervous system. Their subtle onset often masks the damage occurring within, and by the time symptoms become evident, it is frequently too late for effective medical intervention due to delayed diagnosis. At the heart of neurodegenerative diseases lies the progressive deterioration of neuron structure or function—a process known as neuro-degeneration. This relentless decline affects multiple levels of the brain's neural circuitry, from molecular mechanisms to broader systemic consequences. Unfortunately, the damage to neurons is irreversible, making these diseases currently incurable. Nevertheless, research has highlighted oxidative stress and inflammation as key contributors to the neurodegenerative process. Parkinson’s Disease (PD) is relatively rare before the age of 50, but its prevalence increases significantly with age. Globally, the annual incidence ranges from 5 to over 35 new cases per 100,000 individuals, a variation likely influenced by demographic differences and methodological inconsistencies across studies. A Minnesota-based study using validated diagnoses reported an incidence rate of 21 cases per 100,000 person-years. The incidence of PD rises sharply between the sixth and ninth decades of life. While the overall global prevalence is estimated at approximately 0.3%, it exceeds 3% among individuals aged 80 and older. Mortality rates in PD do not show a marked increase during the first decade after diagnosis, but they double in subsequent years. Advances in healthcare and increased life expectancy have contributed to the growing prevalence of PD over time.

2.1.1 Taxonomy:

• Family: Magnoliaceae
• Genus: Magnolia
• Species: champaca

2.1.2 Botanical Name:

• • Magnolia champaca (L.) Baill. ex Pierre

2.1.3 Synonyms:

• • Michelia champaca L.
  • Champaca michelia (historical usage)

2.1.4 Chemical Constituents of Magnolia champaca:

Different parts of the plant—flowers, bark, leaves, seeds, and roots—contain a variety of bioactive compounds. Key classes include alkaloids, essential oils, lignans, flavonoids, terpenoids, and phenolics.

Major Constituents (by plant part):

Flowers:

• Essential Oil - Linalool, β-Caryophyllene, Methyl eugenol, α-Humulene, Farnesol
• Flavonoids – Quercetin, Kaempferol
• Phenolic compounds

Leaves: Flavonoids, Tannins, Saponins, Alkaloids

Bark: Alkaloids (e.g., magnoflorine), Lignans, Triterpenoids, Sterols, Tannins

Seeds: Fixed oils, Fatty acids (palmitic acid, linoleic acid), Proteins, Saponins

Roots: Alkaloids, Lignans

Fig: 2 Magnolia champaca (L.) Baill. ex  Pierre

2.2 ORIGANUM MAJORANA L:

2.2.1 Synonym:

Origanum majorana has a few botanical synonyms, although it's quite a well-defined species. Some of these may reflect older classifications or variations in taxonomy:

• Majorana hortensis Moench
• Amaracus majorana (L.) Benth.
• Origanum onitesvar. majorana (L.) Nyman
• Marjorana majorana (L.) H.Karst.

2.2.2 Taxonomical Classification:

• Kingdom: Plantae
• Order: Lamiales
• Family: Lamiaceae
• Genus: Origanum
• Species: Origanum majorana

2.2.3 Chemical Constituents:

Essential Oil Components (Main Bioactive Compounds): Terpenoids: Terpinen-4-ol, γ-Terpinene, α-Terpinene, Sabinene, Linalool, Carvacrol, Thymol, Cis-sabinene hydrate

Other Phytochemicals: Flavonoids (e.g., apigenin, luteolin), Phenolic acids (rosmarinic acid, caffeic acid), Tannins, Saponins, Ascorbic acid (Vitamin C)

3.1 Plant Collection:

Fresh Flower of Magnolia champaca (L.) Baill. ex Pierre and Whole Plant of Origanum majorana L were collected from the local area of Thiruvannamalai district. It was washed gently with distilled water in order to remove the dust particles may present on the surface of the flower and whole plant. and it was dried under shade for 5 days straight. The stem barks are crushed into smaller pieces and stored in air tight container for further use.

3.2 Green Synthesis of Nanoparticle:

Take 10mg of silver nitrate in 100ml DI-water & set temperature to 80?-90?. The Fresh Flower of Magnolia champaca (L.)Baill. ex Pierre and Whole Plant of Origanum majorana L extract has been mixed drop wise until the color develops to light brownish orange. The plant Fresh Flower of Magnolia champaca (L.) Baill. ex Pierre and Whole Plant of Origanum majorana L extract has been used as a reducing agent and capping agent. The mechanism involved in the synthesis of AgNPs is reduction. The product has been centrifuged using centrifugal apparatus. AgNPs are stored in an air tight container.

3.3 Evaluation of AGNP’s:

Specific methods must first confirm the formation of AgNPs before they can be used for their intended application. The most basic method to monitor AgNPs production by visually observing the change in the color of the solution from yellow to brown. A spectrophotometer can further confirm the tracking process and detect nanoparticle peaks in the visible area of the UV–vis spectrum at a wavelength between 400 and 450 nm. Other techniques, including SEM can be used to investigate the size, morphology, dispersion, and composition of nanoparticles. Moreover, FTIR spectroscopy can help track biomolecules that influence nanoparticle formation and stability.

3.4 Phytochemical Test:

The extracts of Fresh Flower of Magnolia champaca (L.)Baill. ex Pierre and Whole Plant of Origanum majorana Lwere subjected to the following preliminary phytochemical analysis.

3.5 Experimental Method:

Thirty male Wistar albino rats were randomly divided into five groups (n=5 per group) to evaluate the neuroprotective potential of NP-MCOM, a green-synthesized silver nanoparticle formulation prepared using the flower extract of Magnolia champaca and the whole plant extract of Origanum majorana, in a haloperidol-induced Parkinson’s disease model.

• Group I – Control: Received only normal saline via oral gavage once daily for 21 days.
• Group II – Negative Control: Received haloperidol (1 mg/kg, i.p.) once daily for 21 days, without any treatment.
• Group III – Standard Treatment: Received levodopa (100 mg/kg) and carbidopa (25 mg/kg) via intraperitoneal injection one hour before haloperidol (1 mg/kg, i.p.) for 21 days.
• Group IV – NP- MCOM Low Dose: Treated with NP-MCOM (200 mg/kg, p.o.) one hour prior to haloperidol (1 mg/kg, i.p.) for 21 days.
• Group V – NP- MCOM High Dose: Treated with NP-MCOM (400 mg/kg, p.o.) one hour prior to haloperidol (1 mg/kg, i.p.) for 21 days.

This protocol was designed to investigate the dose-dependent prophylactic efficacy of NP-MCOM (green-synthesized silver nanoparticle formulated by flower extract of Magnolia champaca and the whole plant extract of Origanum majorana in alleviating Parkinsonian symptoms induced by haloperidol in Wistar rats, using standard therapy as a positive control. Certainly, Here's the entire experimental procedure rewritten into a clear, single paragraph format, suitable for inclusion in your Materials and Methods section: Thirty male Wistar albino rats were randomly divided into five groups (n=5 per group) to evaluate the neuroprotective effects of NP-MCOM, a green-synthesized silver nanoparticle formulation derived from the flower extract of Magnolia champaca and the whole plant extract of Origanum majorana. Group I (Control) received normal saline via oral gavage once daily for 21 days. Group II (Negative Control) was administered haloperidol (1 mg/kg, intraperitoneally) once daily for 21 days without any co-treatment. Group III (Standard) received levodopa (100 mg/kg) and carbidopa (25 mg/kg) intraperitoneally, one hour prior to haloperidol (1 mg/kg, i.p.), for 21 days. Group IV and Group V received NP-MCOM orally at doses of 200 mg/kg and 400 mg/kg, respectively, one hour prior to haloperidol administration (1 mg/kg, i.p.) daily for 21 consecutive days. This protocol was designed to assess the dose-dependent prophylactic potential of NP-MCOM in mitigating haloperidol-induced Parkinsonian symptoms in Wistar rats by comparing it with standard dopaminergic therapy.

3.6 Pharmacological Activity:

• Catalepsy Bar Test
• Tardive Dyskinesia (TD) Test
• Open Field Test
• Rotarod
• Wire Hang Test
• Pole Test
• Beam Walking Test
• Assessment Of Serum Superoxide Dimutase
• Assessment of Serum Catalase
• Glutathione
• Histopathological Study

3. RESULTS AND DISCUSSION:
   1. Preparation Of Nanoparticle:

4.3 Evaluation of Nanoparticles:

4.3.1 Particle Size & Zeta Potential:

Table: 2 Evaluation of Nanoparticles

4.3.2 UV Analysis:

Fig: 7 UV Spectrum Analysis

4.3.3 Surface Morphological Analysis:

Fig: 8 SEM Analysis

4.4.1 Effect Of NP-MCOM On Catalepsy Bar Test & Catalepsy Score:

Fig: 10 Effect Of NP-MCOM On Catalepsy Bar Test & Catalepsy Score

Fig: 12 Effect Of NP-MCOM On Wire Hang Test & Pole Test

Table: 7 Effect Of NP-MCOM On Pole Test (T- Total) & Beam Walking Test C

Fig: 13 Effect Of NP-MCOM On Pole Test (T- Total) & Beam Walking Test

Table: 8 Effect Of NP-MCOM On Open Field Test (Central Exploration), Superoxide Dimutase (SOD), Catalase (CAT) & Glutathione (GSH)

Group I (Control Group): The normal control group (Group I) exhibited an intact histological architecture with well-preserved cellular morphology and tissue organization. No signs of inflammation, degeneration, or necrosis were observed, indicating healthy and uncompromised neural tissue integrity. These findings confirm the suitability of the control group as a baseline for comparison in this study. Overall, the histopathology validates that no adverse effects occurred in untreated healthy animals throughout the experimental period.

Group II (Disease Model – Haloperidol Induced Group): The disease control group showed marked neuronal degeneration with disrupted cellular architecture. Mild congestion in blood vessels and slight hemorrhage were evident, indicating vascular impairment. Additionally, neurofibrillary tangles and plaques were observed in the brain tissues, reflecting hallmark pathological features of neurodegeneration induced by haloperidol (1 mg/kg).

Group III (Standard Group – Levodopa (100 Mg/Kg) + Carbidopa (25 Mg/Kg)): The standard treatment group exhibited significant restoration of healthy, active neurons accompanied by improved vascular nourishment and enhanced blood supply to the brain tissue.   Enhanced microvascular integrity contributed to reduced neuroinflammation and facilitated regeneration.

Group IV (NP-MCOM 200 mg/kg): This group showed notable neuronal regeneration with partial restoration of normal neuronal morphology and reduced signs of degeneration. Mild vascular congestion was present but less pronounced than in the disease group, indicating improved microcirculation. Gliosis was also attenuated, suggesting reduced neuroinflammation.

Group V (NP-MCOM 400 mg/kg): Group V exhibited significant histopathological recovery with nearly intact neuronal architecture and minimal degeneration. Vascular congestion and hemorrhagic changes were substantially diminished, reflecting enhanced cerebral perfusion. Marked reduction in gliosis indicated effective suppression of neuroinflammatory responses.