INTRODUCTION:

Parkinson’s disease is a complicated, progressive neurodegenerative illness first described by James Parkinson in 1817 in his publication “Essay on the Shaking Palsy.” Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra paras’ compacta (SN) and deficiency of dopamine in the striatal region, which is located in the midbrain and is linked with Lewy bodies, which are cytoplasmic inclusions containing insoluble alpha-synuclein clumps¹.

The loss of nigral dopaminergic cells causes rigidity, akinesia, tremor, postural problems and bradykinesia, which are all hallmark motor signs of Parkinson’s disease². Along with these some non-motor manifestations associated with Parkinson’s disease are sleep impairment, olfactory deficits and neuropsychiatric disorders (depression, hallucinations)³.

Psidium guajava is a fruit bearing plant commonly known as guava, which belongs to the family Myrtaceae. Psidium guajava has been traditionally used for the management of various diseases, but only a few studies have tested its uses⁴. Psidium guajava contains many chemical constituents which seems effective as anti- oxidant, allergic conditions, microbial infections, anti-tussive, anti- diabetic, anti-inflammatory, cytotoxic, anti-spasmodic etc. The guava tree's leaves and bark have long been used for medicinal purposes⁵. Guava decoction is used to treat diarrhoea, dysentery, vomiting, Sore throats, and to regulate menstrual cycles. African tribes utilise leaf decoction to treat mouth sores, bleeding gums, vaginal discharges, and to tighten and tone up vaginal walls after childbirth⁶.

Literature survey indicates no scientific data available on anti-parkinsonism activity of Psidium guajava in view of this, the present research aimed to investigate anti-parkinsonism effect of Psidium guajava leaves extract in experimental animal model.

MATERIALS AND METHODS:

Extract fabrication:

The Psidium guajava leaves were dried in the shade at room temperature, ground into a coarse powder, and extracted with ethanol using Soxhlet's extraction process. As a result, the extract was concentrated using a rotary flash evaporator. The extract's total product was 13.2%. For further research, the crude medication was placed in a sealed container and frozen at a temperature of less than 10˚C.

Experimental animals:

In the current research, we used the Wister albino rats (150-200gm) of either sex. Animals were housed in polypropylene cages maintained under standard lab conditions (12/12 h light/dark cycle; 25±1˚C, 35to 40% relative humidity). Animals were maintained under hygienic conditions. Prior to the start of the work, the study protocol was authorised by the Institutional Animal Ethical Committee (IAEC) on 14/08/2021. bldeacop/IAEC/2021/01 (IAEC) permission

Evaluation of anti-parkinsonism activity:

The animals were divided into 6 groups:

Group I: Normal control, were received vehicle

Group II: Control, were received haloperidol (1mg/kg b.w. i.p) Group III: Standard (levodopa 10mg/kg p.o.)

Group IV: EEPGL 125mg/kg p.o. Group V: EEPGL 250 mg/kg p.o. Group VI: EEPGL 500mg/kg p.o.

The animal’s behavioral parameters, such as locomotor activity(Actophotometer)⁷, hang test⁸, and catalepsy⁹ were assessed on the first and seventh days of treatment.

Estimation of biochemical parameters:

Oxidative parameters in brain tissue homogenate for the estimation of superoxide dismutase, Malondialdehyde (MDA) level, Reduced glutathione level^10,11,12.

Histopathological examinations:

The brains were taken from all groups and treated with 10% formalin before being embedded in paraffin wax and sliced into 5m longitudinal sections. For histological examination, the sections were stained with haematoxylin and eosin dye.

Statistical analysis:

All the data were expressed in mean ± SEM. The significance level of mean between control and treated animals for different parameters was determined by one way ANOVA followed by Dunnett’s Multiple Comparison Test. P value <0.05 was considered as statistically significant.

Preliminary phytochemical screening:

The outcomes acquired from the phytochemical examination on ethanolic extract of Psidium guajava leaves executed the presence of phenolic compoun ds, flavonoids, tannins, alkaloids, glycosides, saponins, triterpenoids and sterols.

Acute toxicity study:

The present work, there was no death of the animals (0/3 mice died) at dose of 2000mg/kg by the test extract of Psidium guajava leaves although when dose was repeated using three new mice. Therefore, 2500mg/kg was taken as LD50 cut off value marked as per fixed dose method of OECD guideline number 423. The screening doses opted for the estimation of anti-Parkinson’s activities of the test extract was:125mg/kg - 1/20th dose of LD50 cut off value, 2500mg/kg b.w. 250 mg/kg - 1/10th dose of LD50 cut off value, 2500mg/kg b.w. 500mg/kg - 1/5th dose of LD50 cut off value, 2500 mg/kg b.w.

Evaluation of anti-Parkinsonism activity of test extract:

Haloperidol induced model

Table 1: Effect of EEPGL on locomotor activity using Actophotometer (number of counts/5 min)

Treatment Groups	Actophotometer Apparatus (number of counts/5min)
Treatment Groups	1^st day	7^th day
Normal control	129.2±8.48	119.5±12.03
Control	58.7±2.96^a	34.7±4.80^a
Standard (Levodopa)	137.3±4.18***	119.2±10.12***
EEPGL 125 mg/kg	52.7±4.40^ns	51.8±3.98*
EEPGL 250 mg/kg	55.3±5.38^ns	78.8±3.72***
EEPGL 500 mg/kg	57.7±7.88^ns	82.7±6.90***

All the values are expressed as mean±SEM, n=6, ^ap<0.001, as compared to Normal control and ^nsNonsignificant, *p<0.05, ***p<0.001 as compared to Control group.

Table 2: Effect of EEPGL on Hanging test

Treatment Groups	latency of fall in the hang test
Treatment Groups	1^st day	7^th day
Normal control	51.3±3.98	50.5±3.02
Control	21.8±2.52^a	22.4±2.68^a
Standard (Levodopa)	39.7±4.21***	49.3±3.56***
EEPGL 125 mg/kg	25.2±2.65^ns	29.0±2.28*
EEPGL 250 mg/kg	26.8±3.86^ns	37.5±2.69**
EEPGL 500 mg/kg	29.5±3.33*	48.7±5.06***

All the values are expressed as mean±SEM, n=6, ^ap<0.001, as compared to Normal control and ^nsNonsignificant, *p<0.05, **p<0.01, ***p<0.001 as compared to control group.

Table 3: Effect of EEPGL on Catalepsy Induced by Haloperidol (number of seconds/3 min)

Treatment Groups	Catalepsy (number of seconds/3 min)
Treatment Groups	1^st day	7^th day
Normal control	9.6± 4.45	7.8± 5.09
Control	89.2±9.19^a	97.9 ± 11.07^a
Standard (Levodopa)	9.7 ± 2.43***	8.3 ± 2.36***
EEPGL 125 mg/kg	85.5 ± 5.26^ns	82.4± 5.49**
EEPGL 250 mg/kg	80.1±3.45*	61.8±5.25***
EEPGL 500 mg/kg	73.7± 3.80**	23.2 ± 5.91***

All the values are expressed as mean±SEM, n=6, ^ap<0.001, as compared to Normal control and

^nsNonsignificant, *p<0.05, **p<0.01, ***p<0.001 as compared to control group.

Table 4: Effect of EEPGL on the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) in the brain of Haloperidol treated animals

Groups mg/kg	SOD (nmoles/mg of protein)	GSH(nmoles/mg of protein)	MDA(nmoles/mg of protein)
Normal control	312.4 ± 6.42	13.3 ±0.07	8.3± 0.09
Control	188.3 ± 5.10^a	1.2 ± 0.02^a	38.1 ± 0.16^a
Standard	316.2 ± 4.16***	15.9 ± 0.60***	9.4± 0.28***
EEPGL (125mg/kg)	228.2 ± 3.67*	3.1 ± 0.02*	31.6 ± 0.51*
EEPGL (250mg/kg)	265.7 ± 3.59**	6.5 ± 0.13**	21.5± 0.28**
EEPGL (500mg/kg)	287.8 ± 2.88 ***	13.1± 0.15***	15.6 ± 0.20***

The values are expressed as mean±SEM, n=6, ^ap<0.001, as compared to normal control group and ^nsNonsignificant, *p<0.05, **p<0.01, ***p<0.001 as compared to control group.

Effect of the EEPGL on histopathological studies:

In control group, the animals (Image-B) showed severe changes in degeneration and pyknosis in hippocampal neurons and gliosis with increased infiltration of neutrophils. And moderate changes observed in congestion and hippocampal oedema with increased neuronal degeneration in cerebral cortex, when compared normal control group (Image-A). In treated groups, the animals (Image-D, E and F) showed decreased infiltration of neutrophils and infraction size, reduced intracellular space, increased density of cells and regained normal architecture and moderate necrosis was observed in striatum region of brain as compared to control group. High dose of EEPGL (500mg/kg - Image F) showed significant improvement in striatum region of brain which can be comparable with standard (levodopa) group (Image-C).

DISCUSSION:

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disorder. PD is a neurodegenerative disorder of the basal ganglia characterized by a complex condition of behavioral abnormalities, including tremor, rigidity, and bradykinesia. The most significant PD symptoms are these motor symptoms since they have been emphasized as those that define the clinical condition of an affected person. Its prevalence is anticipated to rise in the ensuing decades as the population ages. The pathogenesis of PD is suggested by evidence from earlier research to entail oxidative damage¹³and mitochondrial dysfunction. Drugs that can alter cellular energy metabolism and/or exert antioxidative actions may therefore be helpful in altering the pathophysiology of PD^14,15,16.

Effect of EEPGL in haloperidol induced PD in rat. Photograph of brain section of different treatment groups stained with Haemotoxylin and Eosin. Image; A: Normal control, B: Control group, C: Standard D: EEPGL (125mg/kg) E: EEPGL (250mg/kg) F: EEPGL (500mg/kg).

Infiltration of neutrophils

Altered architecture and moderate necrosis

Figure 1: Effect of EEPGL on histopathological studies in Haloperidol Induced animal model.

Literature study reveals that despite the invention of levodopa revolutionized the treatment of Parkinsonism; maximum Parkinsonism disease patients suffer massive motor disability after 5–10 years of disease and dementia that do not adequately reply to DA replacement therapies¹⁵. Thus, there is a neuroprotective drug that can be given early inside the course of the Parkinsonism to slow down its progress or arrest or reverse the pathogenesis of PD^17,18,19.

In the current investigation, haloperidol treated animals (7 days) exhibited extreme cataleptic responses along with decreased locomotor and motor coordination^20,21. Biochemical estimations also confirmed that, there is a reduction in levels of glutathione, superoxide dismutase and malondialdehyde compared to the normal control group. The exact mechanism for this biochemical alteration by haloperidol was not clear. Reports suggest, the enzymatic degradation by way of MAOs changed into related to the production of hydrogen peroxide, which changed into readily transformed to the hydroxyl radical in the presence of iron²². Thus, it can initiate an unfavorable LPO cascade, However, increased dopamine (DA) turnover leads to hydrogen peroxide production, which is not directly responsible for oxidative stress degeneration²³. The unbalanced production of free radicals may have been exacerbated by the auto-oxidation of DA, which resulted in the production of superoxide radicals. Other mechanisms, however, may also be involved. Literature study indicates that, haloperidol inhibits the activity of certain detoxifying enzymes, as a result leaving the cell unprotected, in particular if the basal enzyme activity become less or if the free radical searching mechanisms were less effective^24,25.

The capability of the anti-psychotic drugs to clinically induce the extrapyramidal syndrome appears to correlate nicely with their inhibitory impact on the complex-I inhibition. Whatever could have been the mechanism of the unbalanced production of the reactive oxygen species and the oxidative stress by way of haloperidol, EEPGL was found to be effective in decreasing the reactive oxygen species as compared to the control group. This antioxidant activity of EEPGL reduced the cataleptic duration of the animals and also improved the locomotor activity²⁶.

The EEPGL 500 mg/kg has demonstrated almost regular locomotor activity and motor coordination with less cataleptic behavior when compared with the control animals. The animals received EEPGL 250 mg/kg exhibited some cataleptic behavior when as compared to animals administered EEPGL 500 mg/kg.

A photoactometer test can be used to examine hypokinesia, one of the main symptoms of Parkinsonism. This has a mechanism in place to monitor an animal's movement. In this model, widespread development of locomotor activity was found by way of measuring exploration and the voluntary locomotion within an enclosed area. In our study, control animals established reduced locomotor activity suggest PD. whereas, in ethanolic extract of Psidium guajava²^7,28 leaves treated group, this behavioral parameter was improved suggesting ant-PD property^29,30.

Several researchers have been investigated the anti-Parkinson’s activity of the various plant extracts but no such reports available on EEPGL in the literature and this is the 1^st research finding showing the enhancement of locomotor and motor coordination by ethanolic extract of Psidium guajava leaves.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank BLDEAs SSM College of Pharmacy and Research Centre for their kind support during research work and all other lab studies.

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Received on 04.03.2024 Revised on 22.07.2024

Accepted on 26.10.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1568-1572.

DOI: 10.52711/0974-360X.2025.00224

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Rani Bhandarkavathe, Hugar Shivakumar, Nanjappaiah H. M., Virupanagouda P. Patil*, Maharani Bhandarkavathe