The curative and mechanistic acumen of curcuminoids formulations against haloperidol induced Parkinson’s disease animal model

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https://doi.org/10.1007/s11011-022-01122-1 ORIGINAL ARTICLE

The curative and mechanistic acumen of curcuminoids formulations against haloperidol induced Parkinson’s disease animal model

Uzma Saleem¹ · Sundas Khalid¹ · Zunera Chauhdary¹ · Fareeha Anwar² · Muhammad Ajmal Shah³ · Ifat Alsharif⁴ · Ahmad O. Babalghith⁵ · Rana O. Khayat⁶ · Aishah E. Albalawi⁷ · Tourki A. S. Baokbah⁸ · Maryam Farrukh¹ ·

Celia Vargas‑De‑La‑Cruz^9,10 · Pharkphoom Panichayupakaranant¹¹

Received: 17 August 2022 / Accepted: 28 October 2022

Abstract

Parkinson’s disease (PD) is slowly developing neurodegenerative disorder associated with gradual decline in cerebration and laboriousness to perform routine piece of work. PD imposed a social burden on society through higher medical cost and by loss of social productivity in current era. The available treatment options are expensive and associated with serious adverse effect after long term use. Therefore, there is a critical clinical need to develop alternative pharmacotherapies from natural sources to prevent and cure the pathological hall marks of PD with minimal cost. Our study aimed to scrutinize the antiparkinsonian potential of curcuminoids-rich extract and its binary and ternary inclusion complexes. In healthy rats, 1 mg/kg haloperidol daily intraperitoneally, for 3 weeks was used to provoke Parkinsonism like symptoms except control group. Curcuminoids rich extract, binary and ternary inclusion complexes formulations 15–30 mg/kg, L-dopa and carbidopa (100 + 25 mg/kg) were orally administered on each day for 3 weeks. Biochemical, histopathological and RT-qPCR analyses were conducted after neurobehavioral observations. Findings of current study indicated that all curcuminoids formulations markedly mitigated the behavioral abnormalities, recovered the level of antioxidant enzymes, acetylcholinesterase inhibitory activity and neurotransmitters. Histological analysis revealed that curcuminoids supplements stabilized the neuronal loss, pigmentation and Lewy bodies’ formation. The mRNA expressions of neuro-inflammatory and specific PD pathological biomarkers were downregulated by treatment with curcuminoids formulations. Therefore, it is suggested that these curcuminoids rich extract, binary and ternary supplements should be considered as promising therapeutic agents in development of modern anti-Parkinson’s disease medications.

Keywords Curcuminoids · Curcuminoids rich extract · Anti-Parkinson · Animal behavior · Natural product · Curcuma longa

Introduction

The second most prevalent progressive neurodegenerative disorder includes Parkinson’s disease (PD) (Ball et al.

2019), associated with motor (stiffness, tremors, perturba- tions, and slowed movement) and non-motor dysfunctions (mood and sleep disturbances, and cognitive impairment)

(Saleem et al. 2021a). PD influences 10 million people over the world and is growing promptly at the age of 60 and 80 years (Srivastav et al. 2017). As the global age rate rises, by 2030, the overall population is predicted to be increased by factor of two (Wang et al. 2017). α-Synuclein protein accumulation, depletion of dopamine and dopaminergic neurons, neuronal inflammation and axonal migration are all neuropathological features associated with PD (Dexter and Jenner 2013). α-Synuclein protein aggregation and deposition due to oxidative stress, and mitochondrial mutation and dysfunctions mainly con- tributed to neurodegeneration and thus PD (Doke and Lakhdive nd). The obvious underlying cause of this het- erogeneous neuronal syndrome is still unknown, although the risk factors include: genetic changes, oxidative stress,

* Uzma Saleem [email protected]

* Muhammad Ajmal Shah [email protected]

* Pharkphoom Panichayupakaranant [email protected]

Extended author information available on the last page of the article

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mitochondrial malfunction, neuro-inflammatory response and environmental factors (Barreto et al. 2015). However, therapeutic interventions and differential diagnosis has clinically limited success (Lang and Espay 2018). Cur- rently, PD has only two treatment options: pharmacological (MAO (monoamine oxidase)-B inhibitors, L-dopa (levodopa), DAs (Dopamine agonists), COMT (catechol- O-methyltransferase) inhibitors) and nonpharmacologi- cal (surgical therapy and physical therapy) (Marsili et al.

2017). Long-term pharmacotherapy has led to more com- plications like dyskinesia, cognitive impairment (Hamed et al. 2019) and surgery may increase the incidence of morbidity and mortality due to the effect of intracellular hemorrhage (Doke and Lakhdive nd). Therefore, current therapeutic strategies are in progress to develop plant based therapeutic moieties to mitigate the progression of a disease and provision of symptomatic relief with no adverse events. Plant derived compounds like resvera- trol, curcumin, ginsenosides etc. are considerably more

convenient to use and tend to improve the motor capabili- ties and neuronal activities in PD (Saleem et al. 2021a).

Curcuma longa (turmeric, Family: Zingeberaceae) is an herbaceous rhizomatous perennial plant that has been used as a flavor and as medicinal agent in Asia. C. longa is also used as an adjuvant, diuretic, stimulant, carminative, mor- dant and detergent in traditional Chinese medicine (Li et al.

2011). The most beneficial part of the plant used for dietary and therapeutic purposes is root (Shishodia et al. 2005).

Three major curcuminoids of C. longa is curcumin 77% (Cur I), demethoxycurcumin 18% (Cur II) and bisdemethoxycur- cumin 5% (Cur III) (Fig. 1) (Darvesh et al. 2012). These curcuminoids have anticancer, antioxidant, hepatoprotective, anti-fungal, anti-mutagenic, and antiviral features (Aggarwal et al. 2003).

To date, both in vitro and in vivo researches indicated that curcuminoids specially Cur I have the competency to treat PD symptoms in animal models (Jagatha et al. 2008).

The propensity of Cur I mitigated the disease progression by

Fig. 1 Chemical structures of curcuminoids

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binding and inhibiting the assemblage of α-synuclein protein (Singh et al. 2013). The low water solubility, therapeutic effectiveness, and bioavailability of curcuminoids made its preclinical and clinical trials difficult and limited (Yallapu et al. 2012). By using green extraction and enrichment phi- losophy, curcuminoids-rich extract (CRE) containing 88%

curcuminoids content was prepared then its cyclodextrin (CD) inclusion complexes binary (CRE, hydroxypropyl- β-cyclodextrin) and ternary (CRE, hydroxypropyl-β- cyclodextrin and polyvinyl pyrolidine K30) has been prepared to gear up the dissolution rate and water solubility (Gould and Scott 2005; Lateh et al. 2019). The purposed research has been deliberated to elucidate the comparative anti-Parkinson potential of CRE and CD inclusion complexes on the haloperidol-induced Parkinsonian rat model.

Materials and methods

Plant material

Curcumminoids rich extract (CRE) and its cyclodextrin inclusion binary (CRE, hydroxypropyl-β-cyclodextrin) and ternary (CRE, hydroxypropyl-β-cyclodextrin and polyvinyl pyrolidine K30) complexes were gifted from the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat- Yai campus, Songkla, Thailand.

Chemicals and medications

Haloperidol (a searl invention), levodopa and carbidopa (Platinum pharmaceuticals (Pak) Ltd.), traizol (Invitro- gen™), primers (Thermo Fisher Scientific-US), cDNA kit (Thermo Scientific), cyber green (SYBR® Green master mix of Bio-Rad) carboxy methyl cellulose, methanol (Sigma- Aldrich; USA) and all reagents, chemicals and other supplies used were of an analytical grade.

Animals

Albino rats (weight ranging to 100–150 g) were obtained at the animal house of Government College University, Fais- alabad, Pakistan. The animals were well ventilated, lighted and maintained at a temperature (20 °C- 25 °C) with a 12/12 h light and a humidity level of 30% to 60%. Animals were kept in separate cages in groups and they were had unrestricted access to food and water (Saleem et al. 2021b).

The experimental study was performed after the acceptance of ethical review committee (ERC) of Government College University Faisalabad with reference no. GCUF/ERC/102.

Anti‑Parkinson’s activity exploration

Experimental induction of PD

Haloperidol (1 mg/kg) once daily dose was giving intraperitoneally for 21 days to all rats except the normal control group 30 min before the administration of respective treatment.

Experimental design

Healthy ratswere divided into nine distinct categories (n = 5).

Group I: Received vehicle (carboxy methyl cellulose 1%, 10 mL/kg, p.o) were considered as control (C).

Group II: Administered haloperidol (1 mg/kg i.p) were considered as disease control (DC).

Group III: Received L-dopa (100 mg/kg) and carbidopa (25 mg/kg) p.o + haloperidol (1 mg/kg, i.p) were served as standard (SD).

Group IV: Haloperidol (1 mg/kg i.p) + CRE 15 mg/kg p.o were given.

Group V: Haloperidol (1 mg/kg, i.p) + CRE 30 mg/kg p.o were given.

Group VI: Haloperidol (1 mg/kg, i.p) + 15 mg/kg binary inclusion complexes (Bin-15) p.o were given.

Group VII: Haloperidol (1 mg/kg, i.p) + 30 mg/kg binary inclusion complexes (Bin-30) p.o were given.

Group VIII: Haloperidol (1 mg/kg, i.p) + 15 mg/kg ternary inclusion complexes (Ter-15) p.o were given.

Group IX: Haloperidol (1 mg/kg, i.p) + 30 mg/kg ternary inclusion complexes (Ter-30) p.o were given.

Following treatment in groups were given for a period of 21 days. Weight was noted and behavioral indicators were examined at the start and conclusion of the study. On the 22^nd day, rats were humanly sacrificed under light anesthesia with cervical dislocation; brains were secluded, drenched with phosphate buffer, and conserved for histopathological and biochemical tests at -80 °C.

Behavioral studies

Catalepsy Catalepsy is a behavioral incapacity of rodents like rats and mice to correct externally inflicted posture.

Blockage of dopamine receptors in the brain induces catatonia following intraperitoneal administration of neuroleptic drugs (Sanberg et al. 1996). After haloperidol administration (by standard bar test) with 3 cm and 9 cm heightened bar; cataleptic behavior were observed (for 30 to 120 min).

The score were recorded at varying interval based on their position as follow: Score zero (0) = when the rat was move freely about on the table. Score half (0.5) = when the rat was

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moved if pushed on back or touch. Score one (1) = when the rat was incompetent to pull out his front paws posed on bar i.e. 3 cm high (0.5 score was given to each paw). Score two (2) = when the rat was unable to set his forepaws placed on the bar i.e. 9 cm high (1 score was given to each paw) (Elliott et al. 1990).

Hole bard test The rats were noticed by head dipping for anxiety and exploratory actions in the hole board test. The apparatus was made up of Plexiglas material with wall height of 25 by 25 cm and of 30 cm. 16 equally divided holes were made on the floor of the apparatus which were 1.5 m high from the earth surface. Rats were placed one by one on the level of the apparatus and permitted to investigate for eight minutes. The estimated distances travelled in the middle and perimeter of the apparatus as well as enumerated head dips in the holes, were measured. The head dipping was only evaluated when the pupils of both eyes covered by the hole (Tillerson and Miller 2003).

Wire hanging test Neuromuscular strength of the rats fore- arm was measured with wire hanging test. The apparatus used for this test was made up of wooden walls which were adorned with horizontal stainless steel grids. Rats were grasped from their tails and set on the grid to behold up until their forepaws and hind paws clasped the grid and clung on wire/grid downward in an upright posture. Rats were required to hang for up to 120 s on a wire. The hanging duration were recorded for 3 min and repeated it consecu- tively 3 times (Tillerson and Miller 2003; Chitra et al. 2017).

Grip‑walk test The goal of the study was to examine loco- motors abilities, neuromuscular coordination, and a hind limb/forelimb discrepancy in PD rats. Due to higher level of control and precision, this test is typically utilized instead of an open field test. This test was carried out by putting the rat on a wooden stepladder constructed with 45 steps (2 cm distance between each step) inclined at an angle 45°.

A small wooden crate was built on top to give the animals a safe and concealed location. Muscular activity of the rats were detected by spotting the arrival time to the tiny box (Cummings et al. 2007).

Open‑field test This study was aimed to investigate the exploration, anxiety, and movement in animals at the same time. Its apparatus was built of wood paneling measuring 72 × 72 cm on all four sides and was had a height of 36 cm.

To show the rat moving in the device, one of the walls was constructed of Plexiglas. The black lines were strained on the floor to identify the main area from other locations in its vicinity and the red lines were drawn in the central square (Carrey et al. 2000). After performing each experiment on each rat ethanol (70%) were used to clean the device.

The rat was treated delicately and then tracked for 10 min (RajaSankar et al. 2009). Spending time in the middle was recorded, as well as defecation, stretching postures, groom- ing, rearing, and freezing (Brown et al. 1999).

Foot printing test The measurement of foot length and stride width provided numerical facts to examine gait abnormalities. The marking was made on a paper sheet by dipping forepaws in black ink. Animals were walked on paper for 10 min while their stride length was recorded (Klapdor et al.

1997).

Assessment of biochemical parameters

Tissue homogenate preparation

Each animal was humanly killed by giving isoflurane as an anesthetic and the brain was taken off via euthanasia. Brains were cleansed with frozen NS (normal saline) and cooled in a biomedical freezer at -80 °C. The brain tissues were homogenized in a tissue homogenizer; supernatant was collected by centrifuging at 800 rpm for 30 min at 4 °C (Lak- shmi et al. 2015).

Assessment of malondialdehyde (MDA) level

The MDA level signifies the degree of lipid peroxidation.

For this assessment, brain homogenate (200 μL) was com- bined with trichloroacetic acid (TCA) 15%, 0.25 M HCl and TBA (thiobarbituric acid) 0.38% (w/w) in a falcon tube and mixed the solution. Before cooling, the whole mixture was held for 15 min in a water bath and the temperature was kept at 90 °C. After cooling, the solution was centrifuged at 4000 rpm for ten minutes. The underlying top layer was collected and absorbance was measured at 532 nm (Hira et al.

2020). The MDA was computed by the following formula:

where, Y is absorbance, Vt is the total volume of assay mixture (mL), E is coefficient 1.56 × 10⁵, wt is the weight of brain (g) and Vu is the volume of aliquots (mL).

Assessment of catalase (CAT) activity

For this study, the brain homogenate of 50 μL, 1.95 mL of 50 mMphophate buffer (pH 7.4), and hydrogen peroxide (1 mL, 30 mM) was taken. Optical density was measured at 240 nm. CAT was computed by the following formula:

(Han et al. 2022).

MDA= Y×100× Vt E× wt× Vu

Catalases activity= change in absorbance per minute

extinction coefficient×volume of sample×mg of protein

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Extinction co-efficient i.e. 0.071 mmol cm⁻¹, Extinction co-efficient i.e. 0.071 mmol cm⁻¹, volume of sample (mL) and mg of proteins in brain homogenate.

Assessment of superoxide dismutase (SOD)

The activity was carried out preparing 3 mL total mixture by adding 0.1 M potassium phosphate buffer of 7.4 pH (2.8 mL) and pyragallol solution (0.1 mL) to tissue homogenate (100μL). Absorbance was assessed at 325 nm via spectro- photometer (Hira et al. 2019) and correlating with standard curve of SOD (unit/mL).

Assessment of glutathione peroxidase (GPx) level

To estimate glutathione peroxidase level, 0.1 mL of brain homogenate, EDTA (0.2 mL), 0.2 mL of sodium azide, and distilled water were taken in a tube and mixed. The reac- tion was terminated by adding TCA (trichloracetic acid) and centrifuged for 10 min at 2000 rpm to take out the supernatant. The overlying upper layer was joined with 0.5 mL DTNB (5,5´-dithiobisnitrobenzoic acid) and 4 mL disodium hydrogen phosphate. Absorbance was measured at 420 nm. Glutathione peroxidase level was assessed as glutathione oxidized (μmol)/ protein (min/mg).

Assessment of reduced glutathione (GSH) level

In this experiment, 1 mL tissue homogenate was com- bined with 1 mL of trichloracetic acid which was then centrifuged for 30 min at 3000 rpm. The supernatant (2 mL) was mixed with 0.5 mL of DTNB and 4 mL of 0.1 M sodium phosphate buffer (7.4 pH). At 412 nm, the absorbance of the sample and control (including all reagents except for brain tissue homogenate) was recorded.

GSH was computed by the following formula: (Saleem et al. 2019)

where, A is absorbance, DF is dilution factor, BT is brain tissue, and Vu is the volume of aliquots.

Assessment of nitrite level

The nitrite level was estimated by preparing Griess mixture by adding 2.5% phosphoric acid, 0.1% N-1-naphthyl ethylene amine dihydrochloride and 1% sulphanilamide and then it was mixed with tissue homogenate equally.

The resulting assay solution was incubated for 10 min and GSH= A−0.00314

0.0314×df×BT×Vu

after which time the absorbance at 546 nm was measured (Bais et al. 2015).

Assessment of neurotransmitters

For the preparation of aqueous phase, mix HCl-n butanol (5 mL) solution with brain tissue homogenate and centrifuges for ten minutes at 2000 rpm. The outer phase was separated by centrifugation, where it was mixed with 0.3 mL of HCl and 2.5 mL of heptanes solution and shaken vigorously. The resulting mixture was again centrifuges for 10 min at 2000 rpm after which the organic layer was separated from the aqueous layer for the estimation of neurotransmitters (Parambi et al. 2020;

Sanawar et al. 2020).

Assessment of acetylcholinesterase activity

A 0.1 M phosphate buffer solution of pH 8.00 (2.6 mL) was mixed with 2, 4 dithiobisnitrobenzoic acid (100 μL) and acetylthiocholine iodide (20 μL) in a small quantity of brain tissue homogenate (0.4 mL). In this test, the reac- tion of 2, 4 dithiobisniotrobenzoic acid with thiocholine produced a yellow color and absorbance was measured at 412 nm (Lakshmi et al. 2015). AChE activity was computed as follow

Assessment of dopamine and noradrenaline

Aqueous phase of 0.2 mL was taken and EDTA solution of 0.1 mL and HCL (0.4 M) of 0.05 mL was added in it.

Then iodine solution (0.1 M) in 0.1 mL ethanol and Na₂SO₃ solution was added next followed by 0.1 mL acetic acid.

The mixture was warmed up for 6 min at 100 °C and then allowed to come at 25 °C (room temperature). After that, absorbance at 352 and 452 was measured for dopamine as well as for noradrenaline. Blank for dopamine and noradrenaline was prepared by adding Na₂SO₃ prior to the solution of iodine (Hira et al. 2020).

Assessment of serotonin

Serotonin estimation was done by adding aqueous phase (0.2 mL) in O-phthaldialdehyde and subjected to heat at 100 °C for ten minutes. After cooling at room temperature the absorbance of resulting extract was measured at 440 nm. For blank, concentrated HCl 0.25 mL was used only (Ali et al. 2021).

Hydrolysed tissue substrate ( ^mol minute

g )

= ^5.74^×¹⁰⁻

4× absorbance variation per min

tissue concentration (mg mL⁾

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Examination of the histopathological changes in rat brain

Brains was removed and stored in 4% paraformalde- hyde. Paraffin-embedded brain tissues were sliced with a microtome at 5 μm thickness at transverse sections, hema- toxylin and eosin dye was used for staining, and the sample was examined using a 10X light microscope.

Hematological and biochemical analyses

Blood samples were collected after anesthesia, CBC (com- plete blood count), lipid profile, liver and urinary function tests were performed by kit (Elabsciences) according to manufacturer protocol.

PCR amplification in real‑time

To determine the genes expressions that are associated with Parkinson’s disease PCR technique was conducted by using the following primersα-synuclein, IL-1α, IL-1β, TNF-α, AChE (acetylcholinestrase), β-secretrase, and ABPP (β-amyloid precursor protein) (Fig. 2). RNA extracted using TRIzol method was quantified by measuring 260/280 nm absorbance ratio using nanodrop spectro- photometer (Hummon et al. 2007). RNA was then tran- scribed into cDNA using Thermo Scientific cDNA kit.

The level of gene expression was assessed via qRT-PCR by using GADPH as a house-keeping gene. For the experiment, a forward primer (0.5 μL) was used along with corresponding reverse primer (0.5 μL), and cDNA volume of 5 μL plus cyber green volume of 5 μL was placed in microplate wells. Thermal cycler was programmed at

forty cycles of denaturation at 95 °C, annealing at 60 °C and extension at 72 °C by putting microplate in it (Coura et al. 2015). Gel electrophoresis was performed by pipette together 5μL sample with 5μL DNA ladder to visualize bands pattern.

Statistical analysis

The results were presented as mean ± SEM. Two way ANOVA followed by turkey’s post-hoc test was applied using the software Graphpad prism version 6. P < 0.05 was considered as statistically significant.

Results

Behavioral studies

Examine for Catalepsy

Catalepsy from 30 to 180 min was induced by haloperidol in DC group significantly (p < 0.05) in comparison to the C group (Table 1). CRE-15, CRE-30, Bin-15, Bin-30, Ter-15, Ter-30 and SD significantly (p < 0.05) reversed catatonia on comparison with DC group. Bin-15, Bin-30, Ter-15 and Ter- 30 indicated that they are more neuroprotective potential than CRE-15 and CRE-30.

Hole board test

It was investigated that head dip count decreased significantly (P < 0.05) in DC, CRE-15, and CRE-30 group in comparison to the C, SD, Bin-15, Bin-30, Ter-15, and Ter-30 groups (Fig. 3). Locomotor and exploratory behavior was impaired by haloperidol in DC group.

Fig. 2 List of primers with sequence and accession number

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Wire hanging test

Wire hanging test was used to explore the latency or fall-off time which reduced significantly (P < 0.05) in the DC group in comparison to the C group (Fig. 4) and fall-off time was

improved significantly (P < 0.05) in dose dependent manner in CRE-15, CRE-30, Bin-15, Bin-30, Ter-15, Ter-30 and SD group when compared to the DC group. In this study Bin-15, Bin-30, Ter-15 and Ter-30 strengthen neuromuscular coordination by improving the hang time than CRE-15 and CRE-30 group.

Grip‑walk test

Grip-walk test by ladder climbing was designed to investigate the impaired motor function. DC group significantly (p < 0.05) consumes more time to climb ladder then the C group (Fig. 5). Foot slips decreased significantly (p < 0.05) in Bin-15, Bin-30, Ter-15, Ter-30 and SD groups on comparison with CRE-15, CRE-30 and DC groups.

Open‑field test

The purpose of this test was to investigate anxiety and locomotion such as frequency of rearing and number of

Table 1 Effect of CRE and its cyclodextrin inclusion complexes (binary and ternary) on catalepsy in the haloperidol-induced PD model

###p < 0.05 compared to C group and ^***p < 0.05 compared to DC group Groups Dose (mg/kg) Scores of catalepsy

30 min 60 min 90 min 120 min 150 min 180 min

Control (C) 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

Disease control (DC) 1 0.71 ± 0.02^### 1.13 ± 0.06^### 1.2 ± 0.029^### 1.56 ± 0.013^### 1.76 ± 0.023^### 1.84 ± 0.021^###

Standard (SD)

(L Dopa/Carbidopa) 100/25 0.36 ± 0.027^*** 0.41 ± 0.021^*** 0.84 ± 0.023^*** 0.79 ± 0.021^*** 0.50 ± 0.032^*** 0.36 ± 0.073^***

CRE-15 15 0.51 ± 0.021^*** 0.67 ± 0.029^*** 0.99 ± 0.021^*** 1.02 ± 0.026^*** 1.01 ± 0.021^*** 0.58 ± 0.029^***

CRE-30 30 0.43 ± 0.026^*** 0.56 ± 0.023^*** 0.94 ± 0.026^*** 0.95 ± 0.029^*** 0.92 ± 0.021^*** 0.74 ± 0.026^***

Bin-15 15 0.53 ± 0.032^** 0.55 ± 0.029^*** 0.98 ± 0.026^*** 0.90 ± 0.023^*** 0.70 ± 0.02^*** 0.61 ± 0.021^***

Bin-30 30 0.44 ± 0.023^*** 0.507 ± 0.03^*** 0.91 ± 0.021^*** 0.81 ± 0.023^*** 0.58 ± 0.02^*** 0.54 ± 0.035^***

Ter-15 15 0.53 ± 0.032^** 0.41 ± 0.021^*** 0.61 ± 0.021^*** 0.33 ± 0.024^*** 0.43 ± 0.026^*** 0.2 ± 0.029^***

Ter-30 30 0.38 ± 0.035^*** 0.34 ± 0.017^*** 0.34 ± 0.023^*** 0.24 ± 0.017^*** 0.28 ± 0.023^*** 0.05 ± 0.023^***

0 5 10 15

20 Control (C)

Disease control (DC) Standard (SD) CRE-15 CRE-30 Bin-15 Bin-30 Ter-15 Ter-30

*** ***

###

*** *** *** *** ***

Effect on hole board test

Groups

setunim/tnuoCgnippiD-daeH

Fig. 3 Effect of CRE and its CD inclusion complexes (binary and ternary) on hole borad test in the haloperidol-induced PD model.

###p < 0.05 compared to C group and ^***p < 0.05 compared to DC group

0 20 40 60

80 Control (C)

Disease Control (DC) Standard (SD) CRE-15 CRE-30 Bin-15 Bin-30 Ter-15 Ter-30 Groups

###

***

*** ***

*** *** *** ***

Effect on wire hanging test

sdnoces/emitffo-llaF

Fig. 4 Effect of CRE and its CD inclusion complexes (binary and ternary) on wire hanging test in the haloperidol-induced PD model.

Effect on grip-walk test

0 20 40 60 80

100 Contro (C)l

###

***

*** *** ***

Groups

)sdnoceS(emiT

Effect on grip-walk test

0 20 40 60 80

100 Contro (C)l

###

***

*** *** ***

Groups

)sdnoceS(emiT

Fig. 5 Effect of CRE and its CD inclusion complexes (binary and ternary) on grip-walk test in the haloperidol-induced PD model.

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lines crossed. It was determined that locomotor activity was significantly (p < 0.05) declined in DC group on comparison with C group (Fig. 6). SD, CRE-15, CRE-30, Bin- 15, Bin-30, Ter-15 and Ter-30 improved the locomotion and exploration of rats significantly (p < 0.05) then the DC group. Binary and ternary inclusion complexes of CRE at the dose of 15 and 30 mg/kg showed significantly higher locomotor activity and exploration as compared with CRE- 15 and CRE-30.

Foot printing test

This test was performed to examine the stride length, it was noted that the stride length was decreased significantly (p < 0.05) in DC group on comparison with C group because the dopamine level was depleted in the substantia nigra by the administration of haloperidol at the dose of 1 mg/kg (Fig. 7). Rats treated with Bin-15, Bin-30, Ter-15, Ter-30 and SD groups demonstrated

significantly (p < 0.05) improved stride length and reversed akinesia and motor dysfunction when compared with DC, CRE-15 and CRE-30 groups.

Assessment of biochemical parameters

The level of biochemical oxidative stress markers like CAT, SOD, GPx and GSH reduced significantly (p < 0.05). MDA and nitrite level markedly increased (p < 0.05) in the DC group on the administration of haloperidol. Bin-15, Bin-30, Ter-15, Ter-30 and SD groups restored the declined level of CAT, SOD, GPx and GSH, and the raised level of MDA and nitrite on compared to DC, CRE-15 and CRE-30 groups (Fig. 8). Following regression equation was obtained by standard curve of BSA to determine mg of protein:

The calibration curve of SOD was generated via use of various concentrations i.e., 10 μL to 100 μL and the following equation was employed to determine the SOD level:

Following regression equation was derived from the curve of sodium nitrite:

Assessment of neurotransmitters

Acetylcholine, dopamine, noradrenaline and serotonin are the main neurotransmitters associated with cognitive abilities and the basal ganglia regulating motor coordination. The level of acetylcholine, dopamine, serotonin and noradrenaline were reduced significantly (p < 0.05) in the DC group in contrast to C group Y =0.00007571x +0.0000476

Y =0.0095x +0.1939

Y =0.003432x +0.0366

Fig. 6 Effect of CRE and its CD inclusion complexes (binary and ternary) on open-field test in the haloperidol-induced PD model. ^###p < 0.05 compared to C group and ^***p < 0.05 compared to DC group

Effect on footprinting test

0 5 10 15

20 Control (C)

###

***

*** *** ***

* *

Groups

)mc(htgneledirtS

Fig. 7 Effect of CRE and its CD inclusion complexes (binary and ternary) on foot printing test in the haloperidol-induced PD model.

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(Fig. 9). Treatment with CRE-15, CRE-30, Bin-15, Bin- 30, Ter-15, Ter-30 and SD groups elevated the level of neurotransmitters significantly (p < 0.05) on comparison with DC group. Bin-15, Bin-30, Ter-15 and Ter-30 groups have more neuroprotective propensity then the CRE-15 and CRE-30 groups.

Examination of histopathological changes in rat brain

Histopathological analysis revealed the normal neuronal architecture of C group. Neurofibrillary tangles, lewy body inclusion and depigmentation were observed in the DC group

Fig. 8 Effect of CRE and its cyclodextrin inclusion complexes (binary and ternary compounds) on biochemical stress markers in the haloperidol-induced PD model. ^###p < 0.05 compared to C group and ^***p < 0.05 compared to DC group

Fig. 9 Effect of CRE and its cyclodextrin inclusion complexes (binary and ternary compounds) on neurotransmitters level in the haloperidol- induced PD model. ^###p < 0.05 compared to C group and ^***p < 0.05 compared to DC group

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(Figs. 10, 11). Treatment with CRE-15, CRE-30 and SD moderately improved the neurofibrillary tangles and depigmentation, while Bin-15, Bin-30 Ter-15 and Ter-30 markedly improved the neuronal architecture and neuronal loss.

Hematological and biochemical analyses

Tables 2, 3, 4 represented the result of blood chemistry, lipid profile and liver and renal function which showed

minor alteration in their parameters but within the normal physiological range.

PCR amplification in real‑time

The mRNA expression of interleukin-1 beta (IL-1β), alpha- synuclein (α-synuclein), beta-amyloid precursor protein (ABPP), tumor necrosis factor alpha (TNF-α), acetyl cho- linesterase (AChE), interleukin 1 alpha (IL-1α) and beta

Fig. 10 Histopathological examination of rat brain tissues in haloperidol induced PD model using a 10X light microscope. (A) Control group (B) Disease control group (C) Standard group (D) CRE-15 (E) CRE-30 (F) Bin-15 (G) Bin-30 (H) Ter-15 (I) Ter-30. Abbreviations:

DP, Depigmentation; NFT, Neurofibrillary tangles; LB, Lewy bodies;

CRE-15, Low dose curcuminoids-rich extract; CRE-30, High dose curcuminoids-rich extract; Bin-15, Low dose binary inclusion complexes; Bin-30, High dose binary inclusion complexes; Ter-15, Low dose ternary inclusion complexes; Ter-30, High dose ternary inclusion complexes

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secretase (β-secretase) were markedly upregulated (p < 0.05) by haloperidol in DC group compared to C group. Treat- ment with CRE-15, CRE-30, Bin-15, Bin-30, Ter-15, Ter-30 and SD were significantly downregulated the raised level of mRNA expression compared to DC group. Bin-15, Bin-30, Ter-15 and Ter-30 appreciably down regulated the mRNA expressions of pathological biomarkers than CRE-15 and CRE-30.

Discussion

In this study, anti-Parkinson’s activity of CRE and its cyclodextrin inclusion complexes (binary and ternary) were investigated in PD rat model induced by haloperidol. Dyskinesia and rigidity are the main symptoms of PD. The dyskinesia are caused by dysfunction in the basal ganglia that is performed a variety of motor-related functions, including smooth movements like walking (Skinner et al. 2019).

According to the study results, the cataleptic scores were awarded in the standard bar apparatus. The rats’ attained higher scores showed greater constraint and dexterity to hold the horizontal bar without slipping off (Deacon 2013). In the standard bar apparatus, binary and ternary compound showed improved cataleptic scores compared to CRE and DC groups, indicating a notable movement in motor excit- ability, these findings are consistent to previous work, in which Tribulus terrestris methanol extract improved neuromuscular coordination and recover catalepsy (Chauhdary

et al. 2019; Saleem et al. 2017). The hole-board test was performed to evaluate the non-motor symptoms of PD, namely depressive and exploratory behavior in rats. The anxiousness of rats was reflected through the way they dipped their heads and interpretation of these findings showed the depression and less exploratory behavior in DC group (Aruna et al.

2017). The head dipping counts in DC group were lowered when compared to C group, where the Bin and Ter groups showed a significantly higher number of head poking then CRE and DC groups.

To determine the motor coordination and muscular strength, ladder climbing and hang test were performed.

Additionally, walking patterns were analyzed via foot printing test. DC group showed slow movement while climbing a ladder and more repetitive behavior (hanging) similar to effect of Tribulus terrestris extract and Cucurbita pepo extract (Saleem et al. 2021b). Bin and Ter groups showed the improvements of locomotor and neuromuscular strength in ladder climbing and hanging task. Abnormally slow limb movements and gait linked to brain dysfunction were observed in foot printing test in DC group. Reduction in stride length is significantly related to liberalized cell degeneration within the substantia nigra of rat brain (Nascimento- Ferreira et al. 2013). Improved pattern of foot prints were observed in Bin and ter groups than CRE and DC groups.

Results from the open-field test in DC group showed stress-induced hypolocomottion that harmonious to previous findings like vinpocetine in paraquat induced Parkin- son’s disease mice model (Ishola et al. 2018). Anxiety and

Fig. 11 Effect of CRE and its cyclodextrin inclusion complexes (binary and ternary compounds) on mRNA expression of various genes in the haloperidol-induced PD model. ^###p < 0.05 compared to C group and ^***p < 0.05 compared to DC group

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motor dysfunction induced by haloperidol were recovered by administration of Bin and Ter compounds. Ter groups showed appreciably improvements in behavioral tasks compared to other treatment groups.

The dopaminergic neuronal degeneration increased the pro- duction of reactive oxygen species (ROS) and shrinkage of safeguarding mechanism of brain, perpetuating the oxidative stress. Antioxidants compounds are helpful to overcome the oxidative stress and mitigate the stress induced pathogenesis (Surendran and Rajasankar 2010). GSH is one of the most well-known endogenous antioxidant living cells maintained the homeostasis of oxygen and oxidation level. It plays a vital role in defense mechanism and to get rid from damaging superoxide molecules, hydroxyl radicals and damaging free oxygen radicals. It reacts by releasing hydrogen peroxide (H₂O₂) that converted into oxygen (O₂) and water (H₂O) (Saleem et al.

2021a). The declined level of glutathione is a precipitating factor in the mitochondrial damage, which is a worsening con- dition in Parkinson’s patients. The catalases enzymes naturally exist in our body to protect against oxidative stress and breaks down H₂O₂ into O₂ and H₂O. MDA (malondialdehyde) is an index lipid per-oxidation. Its raised level has been related to mitochondrial dysfunction and oxidative stress.

Current findings revealed that antioxidant enzymes depletion CAT, SOD, GPx and GSH as well as increased nitrite and MDA level are linked with oxidative stress induced by haloperidol in DC group. These results are similar to the previous findings in the literature that showed a strong association between PD progression and oxidative stress (Pal and Ghosh 2018).

Parkinson’s disease causes an imbalance of important neurotransmitters in the brain such as dopamine, serotonin, acetylcholinestrase (AChE) and noradrenaline. These neurotransmitters are disturbed throughout the cerebral cortex but they also exist in other parts of the brain including the basal ganglia and limbic structures. It is evident from literature that the level of dopamine and noradrenaline level markedly decreased in locus coeruleus and involved in motor as well as non-motor deficits. Previous studies have showed that when administered intraperitoneally, haloperidol produced apoptotic effects on dopaminergic neurons in the substantia nigra. Moreover, levels of dopamine, serotonine AChE and noradrenaline were also significantly reduced (Abdel-Salam et al. 2012). Of note, our study also found that DC group led to decrease in dopamine, serotonin, AChE and noradrenaline concentration. Recent studies have showed that Bin and Ter compounds allows to improve the neurotransmitter level and therefore regaining motor functions as well as non-motor functions in PD.

Histopathological findings revealed that Bin and Ter treated groups recovered the neurofibrillary tangles and pigmentation induced by haloperidol when compared with CRE and DC groups which was in agreement with previous studies (Bhangale and Acharya 2016).

Table 2 Effect of CRE and its cyclodextrin inclusion complexes (binary and ternary compounds) on blood chemistry in the haloperidol-induced PD model ###p < 0.05 compared to C group and ***p < 0.05 compared to DC group ParametersControl (C)Disease control (DC)Standard (SD)CRE-15CRE-30Bin-15Bin-30Ter-15Ter-30 Haemoglobin (g/dl)12.06 ± 0.09310.98 ± 0.073 ###8.98 ± 0.159***12.03 ± 0.044***11.23 ± 0.1 ns10.85 ± 0.058 ns11.53 ± 0.060*10.38 ± 0.088*12.28 ± 0.088*** P.C.V (%)38.01 ± 0.04439.53 ± 0.06###38.2 ± 0.104***38.53 ± 0.117***37.63 ± 0.109***38.53 ± 0.06***42.13 ± 0.073***40.41 ± 0.22**40.33 ± 0.169** Total RBCs (10¹²

/L)4.98 ± 0.2897.11 ± 0.088###4.9 ± 0.076***5.91 ± 0.203***5.46 ± 0.117***5.15 ± 0.115***5.28 ± 0.088***4.99 ± 0.087***5.08 ± 0.088*** M.C.V (fl)84 ± 2.0855.73 ± 0.13###83 ± 2.08***68.66 ± 2.028***77.33 ± 1.764***79.33 ± 2.333***85.66 ± 1.202***89.71 ± 0.415***92.25 ± 0.946*** M.C.H (Pg)28.31 ± 0.15919.68 ± 0.289###28.33 ± 0.363***26.31 ± 0.492***25.41 ± 0.464***25.06 ± 0.54***22.49 ± 0.134*28.21 ± 0.788***28.88 ± 1.067*** M.C.H.C (g/dl)34.51 ± 0.26226.33 ± 0.363###35.93 ± 0.101***31.08 ± 0.22***31.73 ± 0.242***35.51 ± 0.259***34.37 ± 0.102***35.57 ± 0.085***35.9 ± 0.076*** WBCs (K/uL)12.41 ± 0.2494.51 ± 0.176###10.33 ± 0.22***12.03 ± 0.478***9.91 ± 0.082***11.16 ± 0.215***8.88 ± 0.448***11.46 ± 0.289***10.46 ± 0.318*** Neutrophils (%)66.66 ± 0.88234.33 ± 2.33###64.66 ± 0.88***44 ± 0.577***47.33 ± 0.882***52.33 ± 1.453***58 ± 0.577***58 ± 1.155***63.33 ± 1.453*** Lymphocytes (%)37.33 ± 1.45378 ± 1.155###42.66 ± 1.202***43 ± 0.577***56.33 ± 0.882***26.66 ± 2.404***32 ± 2.082***43.66 ± 1.856***47.66 ± 882*** Monocytes (%)5.47 ± 0.2491.33 ± 0.220###7.16 ± 0.441***3.26 ± 0.344**3.95 ± 0.535***4.31 ± 0.348***5.13 ± 0.073***5.02 ± 0.123***5.85 ± 0.217*** Eosinophils (%)5.47 ± 0.2491.33 ± 0.220###7.16 ± 0.441***3.26 ± 0.344**3.95 ± 0.535***4.31 ± 0.348***5.13 ± 0.073***5.02 ± 0.123***5.85 ± 0.217*** Platelets (K/UI)254.66 ± 2.404430.66 ± 12.73###229.33 ± 2.963***430 ± 5 ns389 ± 3.464*286.66 ± 4.41***293.33 ± 4.41***292.33 ± 5.364***357.33 ± 16.149*** E.S.R (mm/1st Hr.)13.66 ± 0.8827.33 ± 0.882###13 ± 0.577**11.61 ± 0.448*12.66 ± 0.882**14 ± 0.520***14.91 ± 0.583***12.75 ± 1.443**14.61 ± 0.311***

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Table 3 Effect of CRE and its cyclodextrin inclusion complexes (binary and ternary compounds) on lipid profile in the haloperidol-induced PD model ###p < 0.05 compared to C group and ***p < 0.05 compared to DC group

ParametersControl (C)Disease control (DC)Standard (SD)CRE-15CRE-30Bin-15Bin-30Ter-15Ter-30 Cholestrol (mg/dL)150.33 ± 5.175131.78 ± 0.897###145.35 ± 0.058***140.66 ± 0.22*143.41 ± 0.3**145.58 ± 0.176***147.54 ± 0.292***148.96 ± 0.531***152.48 ± 0.289*** Triglyceride (mg/dL)80.23 ± 0.1338.75 ± 0.382###91.75 ± 0.629***81.33 ± 0.22***66.61 ± 0.311***87.4 ± 0.284***66.46 ± 0.274***83.5 ± 0.276***60.81 ± 0.606*** HDL (mg/dL)55.58 ± 0.335.33 ± 0.22###41 ± 0.577**42.58 ± 0.87***40.36 ± 0.252**41.75 ± 0.629***44.66 ± 1.202***42.66 ± 1.202***47.58 ± 0.821*** LDL (mg/dL)99.25 ± 0.62950.33 ± 0.22###93.25 ± 0.901***55.33 ± 0.882*56.33 ± 0.882**66.91 ± 0.583***75 ± 1.155***77.58 ± 0.961***83.66 ± 0.882*** Table 4 Effect of CRE and its cyclodextrin inclusion complexes (binary and ternary compounds) on liver and urinary function tests in the haloperidol-induced PD model ###p < 0.05 compared to C group and ***p < 0.05 compared to DC group ParametersControl (C)Disease control (DC)Standard (SD)CRE-15CRE-30Bin-15Bin-30Ter-15Ter-30 Bilirubin (total) (mg/dL)0.86 ± 0.0850.15 ± 0.029###0.68 ± 0.044***0.99 ± 0.006***0.64 ± 0.023***0.58 ± 0.045***0.54 ± 0.023***0.84 ± 0.026***0.65 ± 0.029*** Alkaline phos- phatase (μ/L)179 ± 0.57792 ± 1.155###184.33 ± 1.202***132 ± 1.155***145.33 ± 1.453***158.33 ± 0.882***165.33 ± 1.453***135 ± 0.577***167.66 ± 1.453*** SGPT (ALT) (μ/L)33.33 ± 1.20214.33 ± 1.453###38.33 ± 1.202***27.66 ± 1.453***21.66 ± 1.202*26 ± 1.155***37.66 ± 0.882***30.33 ± 1.453***35.66 ± 1.453*** Blood urea (mg/ dL)46.66 ± 0.88216.66 ± 0.882###43.33 ± 1.202***38.33 ± 0.882***32.33 ± 1.453***44.66 ± 1.202***39.66 ± 1.453***38.33 ± 1.202***46.66 ± 0.882*** Serum creatinine (mg/dL)0.93 ± 0.0440.61 ± 0.006###0.96 ± 0.006***0.78 ± 0.009***0.91 ± 0.012***0.73 ± 0.018*0.84 ± 0.023***0.92 ± 0.015***0.74 ± 0.026**

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manuscript. ROK, TASB, MF, IA, AOA, AEA, CVL and PP revised and edited the manuscript.

Data availability Data will be provided upon request.

Declarations

Ethical statement Animals studies were conducted according to all national and international ethical guidelines required for animal use and care.

Consent to participate All authors declared willingness to participate.

Publication consent It is declared by all authors to publish.

Conflict of interest All authors declared no conflict of interest.

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α-synuclein misfolding has been found as the primary hallmark for PD. When too much α-synuclein is expressed, release of dopamine markedly decreased. The present study illustrated that α-synuclein protein have a greater expression in the DC group which is consistent with earlier research (Nemani et al. 2010). Proinflammatory cytokines including IL-1β (interleukin-1 β), IL-1α (interleukin-1 α) and TNF-α (tumor necrosis factor-α) have been linked to neurobiologi- cal consequences of neuro-inflammation, which can lead to symptoms as debilitating as those seen in PD (Leal et al.

2013). Elevated levels of IL-1β, IL-1α and TNF-α was evident in DC group, which was in agreement with previous research (Koprich et al. 2008). Acetylcholinestrase (AChE) mediates the synaptic termination of a variety of choliner- gic neuromascular signals by hydrolyzing acetylcholine. In earlier studies it was evident that increased expression of a neuronal isoform of the AChE induces apoptosis within numerous brain tissues samples (Jiang and Zhang 2008).

In Dc group, the mRNA expressions of AChE was higher and its activity was enhanced in the brain, which was col- laborated with previous findings in which MPTP induced PD in mice model showing the overexpression of syn- apse-anchored synaptic AChE (AChE-S) variant. Bin and Ter groups ameliorated α-synuclein, IL-1β, AChE, IL-1α and TNF-α expression compared to CRE and DC groups.

Ter group shows more appreciable results because of the enhanced water solubility of curcuminoids.

Therefore, we have found that ternary inclusion complexes exhibited neuroprotective effects in behavioral and biochemical change induced by haloperidol and suggest that these curcuminoids formulations may act through antioxidant and anti-inflammatory actions. These will be considered as an excellent therapeutic compounds for developing modern neuroprotective drugs.

Conclusion

Based on the behavioral, biochemical, histopathological and RT-PCR estimation; it is concluded that ternary inclusion complexes has the potential to alleviate the Parkinson’s disease hallmarks. These formulations improved the level of first line antioxidant enzymes, neurotransmitters and downregulated the mRNA expression of pathological biomarkers α-synuclein, AChE, IL-1β, IL-1α and TNF-α. Hence, ternary inclusion complexes should be explored as a potential resource to function as neuroprotective agents in development of modern neuroprotective medicines.

Author contributions US, MAS and PP designed the research concept.

SK, ZC and FA performed the experiment and collected the results.

IA, AOA, TASB and AEA analyzed the results and prepared the