INTEGRATED IN-VITRO & IN-VIVO PHARMACOLOGICAL EVALUATION OF ANTI-PARKINSONIAN ACTIVITY OF COMBINED ETHANOLIC EXTRACTS OF PHYSALIS MINIMA AND PERONEMA CANESCENS

INTEGRATED IN-VITRO & IN-VIVO PHARMACOLOGICAL EVALUATION OF ANTI-PARKINSONIAN ACTIVITY OF COMBINED ETHANOLIC EXTRACTS OF PHYSALIS MINIMA AND PERONEMA CANESCENS

Mehnoor Farheen *, Isra Naaz, Razia Zubaidi, Syeda Qadar Unnisa and Afifa Begum

Department of Pharmacology, Shadan Women’s College of Pharmacy, JNTUH, Hyderabad, Telangana, India.

ABSTRACT: This study investigates the anti-Parkinsonian properties of ethanolic extracts derived from Physalis minima and Peronema canescens. Efficacy was assessed using a combination of in-vitro biochemical assays and in-vivo behavioural screening models in Swiss albino mice. Parkinson’s disease symptoms were induced and subsequently evaluated using the body swing test. Oral administration of the extracts, individually and in combination, noticeably reduced behavioural abnormalities associated with Parkinsonism. Treated groups exhibited significant increases in monoamine neurotransmitters, specifically dopamine (DA), compared to control groups (DA levels approximately 0.35 vs. 0.05). Furthermore, the extracts enhanced the endogenous antioxidant defence by elevating crucial biomarkers, including glutathione (GSH), glutathione peroxidase (GPX), catalase (CAT), and superoxide dismutase (SOD). These biochemical findings were complemented by histological investigations of brain tissue. The results indicate that both Physalis minima and Peronema canescens extracts possess considerable neuroprotective and anti-parkinsonian activity, demonstrating an effect comparable to conventional standard pharmaceutical treatments like Levodopa. The study concludes that these plant extracts warrant further investigation as potential natural therapeutic agents for managing Parkinson's disease symptoms.

Keywords: Parkinson’s disease, Neurodegeneration, Physalis minima, Peronema canescens, Haloperidol, Levodopa

INTRODUCTION: Parkinson's disease (PD) is a chronic and progressive neurodegenerative disorder, currently the second most common worldwide, characterized primarily by the loss of dopaminergic neurons in the brain's Substantia nigra region ¹.

This neuronal degeneration leads to a significant dopamine deficit, resulting in characteristic motor symptoms such as tremors, rigidity, and bradykinesia (slowness of movement) ².

While current pharmacological treatments, notably levodopa, remain highly effective for symptomatic relief, they do not halt disease progression and are often associated with long-term motor complications like dyskinesias. The complex pathophysiology of PD, involving oxidative stress, neuroinflammation, mitochondrial dysfunction, and the accumulation of misfolded alpha-synuclein proteins into Lewy bodies, highlights the urgent need for novel therapeutic agents, particularly those with neuroprotective capabilities ³. Medicinal plants and their derived natural products have emerged as a promising source for developing alternative and adjunctive therapies in PD management due to their multifaceted mechanisms of action, including potent antioxidant and anti-inflammatory properties.

Traditional medicine systems have long utilized botanical compounds to manage various neurological conditions. The bioactive constituents in these plants, such as flavonoids, alkaloids, and phenolic compounds, offer potential avenues to target the underlying pathological pathways of PD, such as scavenging free radicals, inhibiting inflammatory mediators, and promoting neuronal survival. Preclinical studies have identified numerous phytochemicals that demonstrate significant neuroprotective effects in animal and cellular models of PD by modulating key signaling pathways like the Nrf2/ARE antioxidant system and inhibiting apoptosis.

This paper reviews the pharmacological and traditional uses of two such plants: Physalis minima (Pygmy Groundcherry) and Peronema canescens (Sungkai). Physalis minima, a species of the Solanaceae family ⁴, is rich in withanolides and flavonoids and has been reported to possess anti-inflammatory, antioxidant, and acetylcholinesterase inhibitory activities ^{5, 6,} suggesting a potential role in improving cognitive function and reducing neuronal damage. The anti-inflammatory activities of its withanolides, for instance, are noted for acting on NF-κB, STAT3, and HO-1 pathways in cellular models ⁸.

Peronema canescens, belonging to the Lamiaceae family ⁴, contains valuable diterpenoids and phenolic compounds, employed traditionally for malaria, fever, and immune system support ^{4, 9}. Recent studies highlight its potent immunomodulatory, anti-inflammatory, and antidiabetic activities, linking specific compounds to mechanisms like DPP-4 inhibition in diabetic models ^{9, 10, 11}. This combination aims to boosts the therapeutic power, reduce side effects and explore the existing knowledge on the phytochemical profiles, report pharmacological activities, assessing their potential as sources of neuroprotective agents to supplement current therapeutic strategies for Parkinson's disease, while also considering challenges such as bioavailability and the need for rigorous clinical validation.

Aim and Objectives:

Aim: The study is aimed at evaluating the anti-parkinsonian activity of the combination of ethanolic extracts of Physalis minima and Peronema canescens using rats as an experimental model.

MATERIALS AND METHODS:

Collection & Authentication of Plant Material: The leaf powder of Physalis minima and Peronema canescens is obtained and authenticated by the botanist Dr. K. Madhava Chetty, Assistant Professor, Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India.

Chemicals & Materials Required:

Apparatus: Porcelain jars, alluminium foil, muslin cloth, beakers, petri dish.

Chemicals: 99% ethanol & other required chemicals to carry out phytochemical screening tests.

Drugs: Plant test drug, standard drug (Levodopa) & toxicant (Haloperidol).

Dose Preparation: Levodopa 1mg/kg of animal i.e., 0.25mg dissolve in 1ml of normal saline (0.9 w/v) at PH-7 *Haloperidol 1mg/kg i.e., 0.25mg dissolve in 1ml of normal saline (0.9 w/v) at PH-7

Animals Required: Swiss Albino mice weighing around 18-25 g.

Plants Extraction by Maceration Technique: The leaves of Physalis minima and Peronema canescens were dried at room temperature before the extraction procedure was carried out. Then the dried plants were crumbled into powder form and transferred in jars, ethanol was added, stirred regularly for 7 days. The jars are wrapped with aluminium foil near their mouth. After one week, the plant powders are sieved using a muslin cloth. After filtration, evaporation of ethanol gave a thick consistency to the plant mixture. Thus, plant extracts are ready for experimentation.

FIG. 1: EXTRACTION BY MACERATION TECHNIQUE

Phytochemical Screening Tests: Phytochemical evaluation of ethanolic extracts of plants were carried out to check the presence of biologically active constituents for anti-Parkinson's activity ¹².

Sample Size Determination: The sample size for this study was calculated using power analysis (G*Power, version 3.1) with 80% power (α = 0.05) and an expected medium effect size (Cohen’s f = 0.25). Based on this analysis, each group required 6 animals to detect statistically significant differences in lipid parameters among the experimental groups. This resulted in a total of 54 animals across 9 groups for the main study (6 animals per group × 9 groups).

Acute Toxicity Studies: OECD guidelines 423 (Up and Down Procedure) were followed for conducting toxicological information to determine the safe dose. The animals were split into 2 groups of 3animals each, then the biological extract was given orally in increasing doses of about 100, 200, 500, 1000, and 2000 mg/kg of body weight. After 48 hrs animals are looked for death/toxicity signs.

No differences were observed in the body, skin, fur/eyes of the animal after giving plant doses ¹³.

Experimental Animals: Experimental studies conducted at Shadan Women's College of Pharmacy. Swiss albino mice were kept under a good amount of light i.e 12 hrs of light and dark cycles. Animals are provided with a regular pellet diet, water. The whole experiment was carried out according to ethical standards & CPCSEA guidelines IAEC-07/SES-2025/41/106.

Experimental Design:

Inducing Parkinson's disease: Disease is induced in mice by giving a toxic dose of the drug to animals i.p. The toxic control was prepared freshly in normal saline, given at a single dose. The development of Parkinson's is confirmed by animal screening models, and the efficacy of plants is also determined.

The use of 54 Swiss Albino mice was done. It is divided into 9 groups of 6 mice each.

TABLE 1: GROUPING OF ANIMALS

Plant 1: Ethanolic extract of Physalis minima. Plant 2: Ethanolic extract of Peronema canescens. Toxic control: Haloperidol Standard drug: Levodopa.

Experimental Methods:

Gas Chromatography (GC-MS):

Principle: Gas chromatography and mass spectrometry combines two analytical techniques of separating mixtures by GC and identifying components by MS-GC separates compounds within a temperature limit. Modern GC instruments involve use of an oven, and by adjusting the temperature, compounds can be analysed by retention time. MS is paired with GC, undergo ionisation and generate charged fragments. These ions are sorted and even unstable components are also detected.

Procedure: The sample for identification is passed from the GC entry point, where it is vaporised and passed into the chromatography column by gas.

After passing through the column, sample gets separated.

Later, the column passes into heat transfer, where compounds are converted into ions and detected by charge-to-mass ratio.

Applications:

• It is used for food service analysis.
• It's involved in chemical and elemental analysis.
• It is used in environmental assessment.
• It is helpful for refinery purposes.
• It's found in forensic analysis ¹⁴.

In-vivo Screening Methods:

Elevated Body Swing Test:

• In this method, Animals are divided into groups as mentioned earlier.
• Rats in the Test group receive anesthesia with pentobarbital (60mg/kg i.p).
• Haloperidol is injected.
• After 7 days, 14 days, 21 days behavioural testing is carried out.
• The control group animals are placed in a box but without inducing agent.
• After the required time period all the animals are subjected for testing.
• The rat is gently lifted by the base of the tail and held at 2.5cms above the testing surface, swings are recorded.
• Before another swing the animal must be in a vertical position.
• The swings are recorded for 60sec.

FIG. 2: BODY SWING TEST

Evaluation: The total number of swings is recorded. The no. of left side swings and the number of right-side swings are recorded, compared with standard ¹⁶.

Biochemical Analyses of Brain Tissue: Following behavioral assessments on day 22, the animals were sacrificed, and their brains collected for various biochemical evaluations.

Tissue Preparation (Homogenization): Collected brain samples were homogenized in a phosphate buffer. The resulting mixture was subjected to high-speed centrifugation (between 15,000 and 25,000 rpm) for 25 minutes. The clarified supernatant was carefully collected and reserved for subsequent analytical tests.

Measurement of Lipid Peroxidation (MDA Assay): Lipid peroxidation was assessed by quantifying the concentration of malondialdehyde (MDA), a key biomarker of oxidative damage. The tissue homogenate was combined with acetic acid (20% concentration), dodecyl sulfate (8% concentration), and thiobarbituric acid (0.8% concentration). This reaction mixture was heated to 90°C for 60 minutes, subsequently cooled, and then centrifuged at a speed of 1500–2000 rpm for 15 minutes. Absorbance readings were taken using a spectrophotometer set at 532 nm. MDA production levels were calculated and reported in nmol/g of tissue.

Quantification of Reduced Glutathione (GSH): The level of reduced glutathione (GSH) was measured using a standard colorimetric method. A sample of the homogenate (0.1 mL) was mixed with 10% trichloroacetic acid and Ellman’s reagent (dithiobis-nitrobenzoic acid prepared in a sodium citrate and phosphate buffer solution). After mixing, the solution was centrifuged at 2000 rpm for 15 minutes. The absorbance of the resulting supernatant was measured at 412 nm using a UV spectrophotometer. Results were presented as nmol GSH per gram of tissue.

Superoxide Dismutase (SOD) Activity Assay: Superoxide dismutase (SOD) activity was determined based on its capacity to inhibit the reduction of nitroblue tetrazolium (NBT) by superoxide radicals. A reaction mixture containing NBT, phosphate buffer, supernatant, and xanthine oxidase was incubated. Enzyme activity was calculated based on the degree of NBT reduction inhibition, where one unit of SOD activity corresponds to the amount of protein that inhibits NBT reduction by 50%. Results were expressed as units per milligram of protein.

Catalase (CAT) Activity Assay: Catalase activity was assessed by measuring the rate of hydrogen peroxide H₂O₂ degradation. To a cuvette containing phosphate buffer (2 mL) and H₂O₂ (1 mL), 0.5 mL of the brain supernatant was added. The decrease in absorbance was monitored at 240 nm over a 30-second interval using a spectrophotometer. Activity was calculated as mmoles of H2O2oxidized per minute per milligram of protein.

Neurotransmitter Profiling: The concentrations of key neurotransmitters and their metabolites including dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) were determined using commercially available Rat Enzyme-Linked Immunosorbent Assay (ELISA) kits. All assays were conducted strictly according to the manufacturer's provided protocols.

Neuroinflammatory Marker Analysis: The levels of specific pro-inflammatory cytokines, namely Interleukin-6 (IL-6), Interleukin-1 beta (IL-1), and Tumor Necrosis Factor-alpha (TNF-alpha), were measured using respective commercial ELISA kits. Protein was extracted from the brain homogenate, and samples were loaded into antibody-coated ELISA plates. The concentration of each cytokine was quantified following the standard assay procedures ¹⁷. நன்றி

உங்கள்கருத்து Googleளைமேம்படுத்தஉதவும். எங்கள்தனியுரிமைக்கொள்கையைப்பாருங்கள்.

மேலும்கருத்தைப்பகிருங்கள்சிக்கலைப்புகாரளிமூடு

Histopathological Evaluation: Mice were made unconscious and dissected by using chloroform as anaesthetic. For lab examination isolated brains are preserved in formalin solution. Histopathological assessment is done to evaluate parameters.

RESULTS:

Calculation of Percentage Yield:

Physalis minima: Weight of plant ethanolic extract: 130gms; Weight of plant crude extract: 500gms.

Formula used for calculation of % yield is:

Wt of sample / Wt of crude extract × 100

% yield = 130 / 500 × 100 = 26%

Hence, % yield of Physalis minima is 26%.

Peronema canescens: Weight of plant ethanolic extract: 122gms, weight of plant crude extract: 500gms.

Formula used for calculation of % yield is: Wt of sample/Wt of crude extract ×100

% yield = 120 / 500 × 100 = 24.4%. Hence, %yield of Peronema canescens is 24.4%.

TABLE 2: PHYTOCHEMICAL SCREENING RESULTS OF PHYSALIS MINIMA AND PERONEMA CANESCENS

GC-MS Results:

GCMS Graph of Physalis minima:

FIG. 3: PHYTOCHEMICAL TENTATIVELY IDENTIFIED BASED ON RETENTION TIME MATCHING IN ETHANOLIC EXTRACTS OF PHYSALIS MINIMA BY GC-MS

TABLE 3: PHYTOCHEMICALS TENTATIVELY IDENTIFIED BASED ON RETENTION TIME MATCHING IN ETHANOLIC EXTRACTS OF PHYSALIS MINIMA BY GC-MS

GC-MS Graph of Peronema canescens:

FIG. 4: PHYTOCHEMICAL TENTATIVELY IDENTIFIED BASED ON RETENTION TIME MATCHING IN ETHANOLIC EXTRACTS OF PERONEMA CANESCENS BY GC-MS

TABLE 4: PHYTOCHEMICAL TENTATIVELY IDENTIFIED BASED ON RETENTION TIME MATCHING IN ETHANOLIC EXTRACTS OF PERONEMA CANESCENS BY GC-MS

In-vitro Results:

Effects of Plant Extract in Haloperidol Induced Parkinson's disease:

TABLE 5: EFFECTS OF PLANT EXTRACT IN HALOPERIDOL INDUCED PARKINSON’S

The values are expressed as mean ± SEM, (𝑛=6). ***p << 0.001 vs. normal control. **p << 0.001 vs disease control group.

In-vivo Results:

Physiological Parameters:

Body Weight:

TABLE 6: EFFECTS OF ETHANOLIC EXTRACTS OF PHYSALIS MINIMA AND   PERONEMA CANESCENS ON BODY WEIGHT

The above values are expressed in mean ±SEM *P≤0.05,**P≤0.01,***P≤0.001 compared to control group of mice (done by two-way ANNOVA).

FIG. 5: SHOWING EFFECTS OF E.E.P.M & E.E.P.C. ON BODY WEIGHT

Biochemical Estimations in Blood Sample:

TABLE 7: IMMUNOMODULATORS LEVELS IN BLOODS

The data was analysed by 2way ANOVA test & expressed as means ±SEM,*P≤0.01 is compared to non stressed+standard vehicle groups, **P≤0.001 compared to the stressed+control group.

FIG. 6: SHOWING EFFECTS OF E.E.P.M & E.E.P.C AND THEIR COMBINATION IN MICE ABOUT TNF, IL LEVELS

TABLE 8: EFFECTS OF ETHANOLIC EXTRACTS OF PHYSALIS MINIMA AND PERONEMA CANESCENS AND THEIR COMBINATION IN MICE ABOUT DA, GSH, GPX LEVELS

The above data was analysed by two way ANOVA test and expressed as means ±SEM,*P≤0.01 is compared to non stressed+standard vehicle groups, **P≤0.001 compared to the stressed+control group.

FIG. 7: SHOWING EFFECTS OF E.E.P.M & E.E.P.C AND THEIR COMBINATION IN MICE ABOUT DA, GSH, GPX LEVELS

Biochemical Estimations in Anti-Oxidant Activity:

TABLE 9: EFFECTS OF ETHANOLIC EXTRACTS OF PHYSALIS MINIMA AND PERONEMA CANESCENS AND THEIR COMBINATION IN MICE ABOUT ANTI-OXIDANT LEVELS

The above data was analysed by two way ANOVA test and expressed as means ±SEM,*P≤0.01 is compared to non-stressed + standard vehicle groups, **P≤0.001 compared to the stressed+control group.

FIG. 8: SHOWING EFFECTS OF E.E.P.M & E.E.P.C AND THEIR COMBINATION IN MICE ABOUT ANTI-OXIDANT LEVELS

Histopathological Analysis:

FIG. 9: CHANGES IN SUBSTANTIA NIAGARA OF BRAIN CELLS. B 1: Represents Normal control group, B 2: Represents Toxic control (Haloperidol) group, B 3: Represents E.E.P.M (plant 1) group, B 4: Represents E.E.P.C (Plant 2) group, B 5: Represents Standard control (Levodopa) group

The above photograph shows the changes in substantia nigra of the brain viewed at 400× Magnification and pathological sections are stained with Hematoxylin and Eosin (H&E). Histopathology of brain reveals ,(B1) There are no signs for  neurodegeneration in normal control group, (B2) In toxic group neuronal damage, structural damage is observed,(B3) Upon treatment with E.E.P.M slight change in structure & growth occurs,(B4) Treatment with E.E.P.C leads to improvement in form, shape observed,(B5) Standard drug treatment leads to increase in pigmentation, firm structure is seen.

DISCUSSION: The use of various medicinal plants &herbs for the treatment leads to the development of healthcare system. In this study, anti-Parkinsons activity of Physalis minima & Peronema canescens is carried out. Ethanolic extraction of both plants were obtained by the maceration. Albino mice are animal model used, exposed to toxicant to induce PD (Haloperidol). Thus, the botanical extracts are evaluated by screening models, compared with standard drug. The animals are divided into 9 groups containing 6mice in each. The groups involves normal, toxic, standard, low & high doses of plant extract and lastly, combination of both plants. Before doses, ATS (acute toxicity studies) is carried out. After dosing behavioural, biochemical estimations has done by collecting blood samples, dissection & isolation of brain is performed for further results of study.

CONCLUSION: The present research evaluates ethanolic extracts of P. minima and P. canescence for their potential Anti-Parkinsonian effects. The phyto-chemicals tests shows positive response for phenols, flavonoids, Sterols, terpenoids, coumarins for Anti-parkinsons activity as according to previous research. In-vivo assessment using Body Swing Test is demonstrated. The effectiveness of plant & their combinations showed almost close response to standard drug. The behavioural & biochemical estimations reveals increase in the levels of dopamine, IL, GSH, CAT, SOD by plants P. minima & P. cenescens and results are similar to the standard drug. Histopathological assessment of treated mice brains, shows the decrease in loss & degeneration of neurons of substantia Niagara region which shows effectiveness of plants.

Hence, this study can be concluded with an impression that these plants exhibits great potential which can be utilised in future treatment of PD and other such brain related disorder.

ACKNOWLEDGEMENTS: Nil

CONFLICTS OF INTEREST: Nil

REFERENCES:

1. Bloem BR, Okun MS and Klein C: Parkinson's disease. The Lancet 2021; 397(10291): 2284-303.
2. Schapira AH: Etiology of Parkinson’s disease. Neurology 2006; 66(10-4): 10-23.
3. MacMahonCopas AN, McComish SF, Fletcher JM and Caldwell MA: The pathogenesis of Parkinson's disease. Frontiers in Neurology 2021; 12: 666737.
4. Calne DB: Treatment of Parkinson's disease. New England Journal of Medicine 1993; 329(14): 1021-7.
5. Chothani PG & Vaghasiya HU: A phyto-pharmacological overview on Physalis minima Indian Journal of Natural Products and Resources 2012; 3(4): 477-482.
6. Kaur P, Satti NK & Parle M: Anti-inflammatory, analgesic and antipyretic activities of Physalis minima Journal of Medicinal Plants Research 2008; 3(1): 057-063.
7. Alik LMA & Handayani FF: Review of phytochemical and pharmacological activities of Physalis minima. International Journal of Pharmaceutical Research and Applications 2020; 5(5): 415-422.
8. Li RJ: The anti-inflammatory activities of two major withanolides from Physalis minima via acting on NF-κB, STAT3, and HO-1 in LPS-stimulated RAW264. Cells Inflammation 2017; 40(2): 401-413.
9. Rahardhian MRR, Sari PM & Latief M: A review of sungkai (Peronema canescens): traditional usage, phytoconstituent, and pharmacological activities. Intern J of Applied Pharmaceutics 2022; 14(5): 15-23.
10. Latief M, Sari PM & Hidayat M: An in-vogue plant, Peronema canescens Jack: traditional uses and recent bioactivity studies with emphasis on anti-inflammatory, immunomodulatory, and antidiabetic activities. Journal of Future Foods 2024; 4(1): 1-17.
11. Latief M: Bioassay-guided fractionation, compound profiling by LC-MS/MS and molecular docking of Peronema canescens (Sungkai) leaves as a Dipeptidyl peptidase-4 (DPP-4) inhibitor. Journal of Applied Pharmaceutical Science 2024; 14(7): 160-170.
12. Ku Halim KF, Jalani KJ, Mohsin HF and Abdul Wahab I: Phytochemical screening of Peronema canescens International Journal of Pharmaceuticals, Nutraceuticals and Cosmetic Science (IJPNaCS) 2020; 1: 7-15.
13. Akhila JS, Shyamjith D and Alwar MC: Acute toxicity studies and determination of median lethal dose. Current Science 2007; 917-20.
14. Al-Rubaye AF, Hameed IH and Kadhim MJ: A review: uses of gas chromatography-mass spectrometry (GC-MS) technique for analysis of bioactive natural compounds of some plants. International Journal of Toxicological and Pharmacological Research 2017; 9(1): 81-5.
15. Kearns SM, Scheffler B, Goetz AK, Lin DD, Baker HD, Roper SN, Mandel RJ and Steindler DA: A method for a more complete in-vitro Parkinson's model: slice culture bioassay for modeling maintenance and repair of the nigrostriatal circuit. Journal of Neuroscience Methods 2006; 157(1): 1-9.
16. Roghani M, Behzadi G and Baluchnejadmojarad T: Efficacy of elevated body swing test in the early model of Parkinson's disease in rat. Physiology & Behavior 2002; 76(4-5): 507-10.
17. Humpel C: Organotypic brain slice cultures: A review. Neuroscience 2015; 305: 86-98. doi: 10.1016/j.neuroscience.2015.07.086. Epub 2015 Aug 5. PMID: 26254240; PMCID: PMC4699268.
    

    ---

    How to cite this article:

    Farheen M, Naaz I, Zubaidi R, Unnisa SQ and Begum A: Integrated in-vitro & in-vivo pharmacological evaluation of anti-parkinsonian activity of combined ethanolic extracts of Physalis minima and Peronema canescens. Int J Pharm Sci & Res 2026; 17(5): 1504-14. doi: 10.13040/IJPSR.0975-8232.17(5).1504-14.

    ---

    All © 2026 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.