Amelioration of Antipsychotic Activity of Ethanolic Fruit Extract of Piper longum and its effects on TNF-α Expression in Rat Hippocampus

Spoorthi. N¹, Pinki Verma²*, Hemavathi. N³

¹Research Scholar, Department of Pharmacology, Aditya Bangalore Institute of Pharmacy Education and Research, Rajiv Gandhi University of Health Sciences, Yelahanka, Bengaluru, 560064 Karnataka, India.

²Associate Professor, Department of Pharmacology, Aditya Bangalore Institute of Pharmacy Education and Research, Rajiv Gandhi University of Health Sciences, Yelahanka, Bengaluru, 560064 Karnataka, India.

³Research Scholar, Department of Pharmacology, Aditya Bangalore Institute of Pharmacy Education and Research, Rajiv Gandhi University of Health Sciences, Yelahanka, Bengaluru, 560064 Karnataka, India.

*Corresponding Author E-mail: spoorthi1507@gmail.com, vermapinki05@gmail.com, hemavathi089@gmail.com

ABSTRACT:

Aim: The present research is to evaluate the Antipsychotic activity of ethanolic fruit extract of Piper longum and its effect on TNF-α expression on rat hippocampus. Materials and Methods: The fruit was collected, dried, and pulverized. Extraction was carried out with 98% ethanol by the Soxhlet apparatus. Preliminary phytochemical studies of the extract were investigated in this study. Two doses of the extract (200 and 400mg/kg) were used for this study. Antipsychotic activity was evaluated against psychosis-induced rats using Cook’s pole climbing apparatus, Actophotometer, and Open field model. Further, Immunohistochemistry studies of TNF-α expression and Histopathological studies of the brain and liver were also performed. Result and Discussion: The extract contains phytochemicals, including alkaloids, flavonoids, phenolic compounds, saponins, steroids, tannins, and starch. The extract produced a significant increase in the latency to climb the pole and a decrease in the locomotor activity by Actophotometer and Open field test (crossing, rearing, sniffing & grooming, etc.). Piper longum extract significantly reduced the expression of TNF-α in the hippocampus. Histopathology of the brain stated recovery of the cells in Piper longum (400mg/kg) treated while the liver revealed that the extract is free fromhepatotoxicity. Conclusion: Overall, our findings suggest that ethanolic extract of Piper longum possesses potent antipsychotic activity which might be due to its modulatory action on dopamine neurotransmission.

KEYWORDS: Piper longum, Antipsychotic activity, Immunohistochemistry, TNF-α Expression, Histopathology.

INTRODUCTION:

Psychosis is a neuropsychiatric condition characterized by recurrent episodes that are persistent and have a negative influence on the quality of life of those affected.¹ An estimated 2% of people worldwide will suffer psychosis at some point in their lives, with 80% of these people having it between the ages of 16 and 40.^2,3 Suicide, sadness, anxiety, abuse, drug abuse, cognitive impairment, victimization, poverty, and increased medical problems are more common in people who suffer from psychosis.⁴

Psychosis is a characteristic of numerous psychiatric, neuropsychiatric, neurologic, and neuro developmental disorders.⁵

Neuroinflammation is observed in neurodegenerative diseases such as psychiatric diseases. The formation and enhancement of inflammation within the Central nervous system (CNS) depend heavily on TNF-𝛼. It is typically generated by activated microglia. TNF-𝛼 can also be created by endothelial cells, neurons, and engulfing immune cells. Glial cells can increase their local production of TNF-𝛼 in response to other inflammatory cytokines that are transported to the CNS by invading T cells. TNF-𝛼 modifies the balance of excitatory and inhibitory impulses, this increases the amount of calcium that enters the cells and aids in the neuronal excitotoxic cell death process. TNF-𝛼 is a key player in the breakdown of the blood-brain barrier and the invasion of inflammatory chemicals and cells into the central nervous system (CNS).⁶

Antipsychotic drugs cause many side effects which may depend on a combination of the receptors, but the dopamine pathway is still considered as the primary common target for all antipsychotic drugs.⁷ The main side effect of “typical” antipsychotics was Tardive Dyskinesia, which involves uncontrollable movements of the face, hands, and feet. The other effects of antipsychotic drugs include cardiovascular effects, metabolic effects, sexual dysfunction, sedation, dry mouth, dizziness, akathisia, reduced brain volume, and shortened life span.⁸ Medicinal plants are used for various research purposes. Hence in the present investigation, an attempt has been made to evaluate the antipsychotic activity of ethanolic fruit extract of the Piper longum in animal models of psychosis.

Piper longum (Piperaceae) is commonly known as Long Pepper, is a medicinal plant belonging to the family Piperaceae. Fruits are black cylindrical, Irregular, up to 2-5cm long and 5mm wide, and compact. Fruits are tiny, oval-shaped berries that develop as spikes and are gathered when they are fully developed. These spikes are known as "pippali" when they are dried, but "pippalimula" is the name of the rootradix.⁹

Piper longum fruits have been used as folk and in traditional medicine, including the Ayurvedic system of medicine. In Ayurveda, this plant is mainly used to cure respiratory disorders. Piper longum is used in Ras Ayana therapy which helps prevent COVID-19 symptoms. It is used as a bio enhancer that helps in removing endotoxins from the body.¹⁰ The fruit extract is used to treat chronic bronchitis, asthma, constipation, gonorrhea, paralysis of the tongue, diarrhea, cholera, chronic malaria, viral hepatitis, respiratory infections, stomach ache, bronchitis, diseases of spleen, cough, and tumors. They are also used as antidepressants, antiepileptics, acaricidal, antiulcers, antimicrobials, anti-snake venom, antioxidants, antifertility, neuroprotective, and antifertility.^10,11

MATERIALS AND METHODS:

Collection and authentication of plant material:

The fruits of Piper longum, were chosen for examination and were obtained from the nearby area of our college. Plant material was taxonomically recognized and authenticated by Dr. N M Ganesh Babu Associate Professor, Heading Centre for Herbal Gardens, The University of Trans-Disciplinary Health Sciences and Technology, Bangalore 560064, Karnataka, India.

Preparation of plant extract:

The fruits of Piper longum were cleaned and dried at room temperature. About 50gm of dried powder was extracted with 500ml of 98% of ethanol. The extraction was maintained until the solvent in the thimble became clear. After complete extraction, the extract was collected and the solvent was evaporated. Then it was concentrated to dry residue in the water bath and the extract (dry residue) was weighed. The dried extract was stored carefully to determine the Antipsychotic activity.

The percentage of extract yield was calculated by using the formula:

Percentage of extract yield = (Weight in gm of extract obtained)/(Weight in gm of plant material taken) × 100

Phytochemical Analysis:

The extract was tested for phytochemicals.

Acute toxicity studies¹²

Acute toxicity study of Piper longum has been performed by authors Shah AH, Al-Shareef AH et al., 1998. Ethanolic extracts of Piper longum was orally administered to mice at doses of 500, 1000, and 3000 mg/kg and the animals were observed for 14 days which showed no moral toxicity effect in acute toxicity parameters. In the present study, the dose of extract was fixed to 200, 400mg/kg.

Experimental animals¹³

Wistar albino rat is currently one of the most popular rats used for laboratory research. It is characterized by its wide head, long ears, and having tail length that is always less than its body length. Adult male or female Wistar Albino rats (150-200gm) were used to evaluate the antipsychotic activity. The rats were maintained under standard laboratory conditions in polypropylene cages under a 12-hour light/dark cycle, controlled temperature (24±2°C) and humidity (50-60%), and fed with a commercial pellet diet and water at libitum. Experimental protocols and procedures used in this research was accepted by the Institutional Animal Ethics Committee of Aditya Bangalore Institute of Pharmacy Education and Research, Kogilu Cross, Yelahanka, Bengaluru, India. (Project Proposal No: 76/1611/CPSCEA).

Experimental protocol:

Twenty-four Wistar Albino Rats were separated into four groups of six individuals each was treated orally for 21 days.

Group I: Control – Vehicle (Normal Saline – 0.9% w/v)

Group II: Standard – Haloperidol was administered at a dose of 2mg/kg

Group III: Ethanolic extract of Piper longum fruit was administered at a dose of 200mg/kg Group IV: Ethanolic extract of Piper longum fruit was administered at a dose of 400mg/kg.

Evaluation of Antipsychotic activity:

Cooks pole climbing:

In a chamber with a stainless-steel pole 2.5cm in diameter and a 2.8 kHz speaker mounted on top, a condition stimulus (buzzer sound) was administered for 10 seconds, and then an unconditional stimulus a scrambling shock was applied to the grid floor. Animals were trained to resist the shock that follows the conditional stimulus to avoid the unconditionalreaction.

Training and testing of rats were carried out in the pole climbing apparatus, which contains a floor that acts as a shock source. A wooden pole stands in the center of the roof. The animals were trained in the following manner. When we press the buzzer, a 20-volt shock was sent to the floor grid. To prevent shock, the animal was trained to climb the pole. This was repeated until the animals learned to climb the pole as soon as they heard the buzzer, even if they did not receive the shock. For this study, rats that climbed the pole within 3 seconds of pressing the buzzer were chosen. For control, standard, and treatment groups, the latency period to climb the pole was measured.¹⁴

Actophotometer:

Dews P.B. (1953) described an Actophotometer for monitoring animal locomotor behavior. Animal locomotor activity (horizontal movement) was measured using an Actophotometer equipped with a digital counter, photocell, and light source. Each animal was placed in an Actophotometer for ten minutes, and their basal activity score was measured. Each animal was given the respective drug, and the activity score was determined after 1hour.¹⁵

Open field test:

The Open Field space was constructed of acrylic (60cm x 60cm x 40cm, transparent walls, black floor), and it was divided into sixteen equal-sized squares. The animal's exploratory behavior was assessed using the Open Field. The characteristics were observed for five minutes after adjusting for one minute. The activities observed were: the number of squares crossed (with the four paws) (locomotor activity), and the number of rearing, sniffing and grooming were also measured.¹⁶

Immunohistochemistry Examination:

The fixation fluid-immersed left cerebral hemisphere was dehydrated with gradient alcohol and xylene, then paraffinized and sliced as thin as 5μm with a Leica rotary microtome. Tissue was then placed on coated-object glass followed by rehydrated with xylene and alcohol at concentrations of 96%, 90%, 80%, and 70% before being washed with tap water. The heat-induced epitope retrieval method was used for the next stage, where the slides were placed in a citrate buffer solution and heated at 95°C for 60 minutes. TNF-α 1:700 (cloud clone) antibody was then stained, followed by an overnight incubation at 4°C. The secondary antibody, biotinylated- horseradish peroxidase, was then painted on and incubated for 1hour at room temperature. The slide was then treated with chromogen. In addition, the dehydration process was repeated using strong alcohol and xylene. The next step was to mount and assess the TNF-α expression.¹⁷

Histopathology of the Brain and Liver:

Animals were sacrificed and the organs were collected. Tissues were removed and fixed with 10% formalin, embedded in paraffin, and sectioned at 7μm. Subsequently, sections were deparaffinized with xylene and then stained with hematoxylin and eosin.¹⁸

Statistical Analysis:

^{All results are presented as means ±} ^{standard errors of mean (SEM). The
statistical analysis involved two}groups and one-way analysis of variance (ANOVA) was employed, followed by Dunnett's multiple comparison post-test, to compare the control and various treatment groups using Gram pad prism 10 software. Statistical significance was accepted at *p<0.05, **p<0.01 and***p<0.001.

RESULTS:

Extraction:

The percentage yield of Piper longum extract was 13.56%.

Table 1: Preliminary phytochemical screening of Piper longum

Sl. No.	Phytochemical constituent	Test performed	Observation	Inference
01	Alkaloids	Dragendroff’s Test	Orange brown ppt	Presence
01	Alkaloids	Mayers Test	Creamy white ppt	Presence
02	Flavonoids	Shinoda Test	Pink colour	Presence
02	Flavonoids	Ferric chloride Test	Yellow colour	Presence
03	Proteins	Biuret Test	Pink colour	Absence
04	Carbohydrates	Fehling’s Test	Brick red ppt	Absence
05	Glycosides	Keller Killani Test	Blue-green colour	Absence
06	Saponins	Foam Test	Formation of foam	Presence
07	Tannins/Phenolic compounds	Lead acetate Test	Yellow ppt	Presence
07	Tannins/Phenolic compounds	Ferric chloride Test	Yellow colour	Presence
08	Steroids	Salkowski Test	Greenish yellow fluorescence	Presence
08	Steroids	Liebermann Burchard Test	Deep red colour	Presence
09	Starch	Iodine Test	Blue colour	Presence

Antipsychotic activity:

Effect of EEPL on Pole Climbing Avoidance Test:

Administration of EEPL for twenty-one successive days significantly (p<0.05) inhibited the conditioned avoidance response in rats as indicated by increased time spent on the grid floor of the chamber. However, a high dose of Piper longum significantly (p<0.05) increased the latency period compared to the vehicle-treated group.

Figure 1: Effect of EEPL on Pole Climbing Avoidance in Wistar Albino Rats.

All the values are expressed in mean±S.E.M., where EEPL - Ethanolic Extract of Piper longum, n = 6, One way ANOVA followed by Dunnett’s multiple comparison test, *p<0.05, **p<0.01 and ***p<0.001 compared with the control group.

Effect of EEPL on Actophotometer model:

Administration of EEPL orally for 21 successive days markedly decreased locomotor activity in rats measured using Actophotometer. However, the Piper longum (400mg/kg) treated group was remarkably (p<0.05) effective in decreasing locomotor activity. The effect of EEPL was found to be comparable to that of Haloperidol.

Figure 2: Effect of EEPL on Locomotor Activity Count by Actophotometer in Wistar Albino Rats.

All the values are expressed in mean ± S.E.M., where EEPL - Ethanolic Extract of Piper longum, n = 6, One way ANOVA followed by

Dunnett’s multiple comparison test, *p<0.05, **p<0.01, and ***p<0.001 compared with the control group.

Effect of EEPL on Open Field Test:

The study's findings demonstrate that all stereotypical behaviours, such as crossing, rearing, sniffing, and grooming were considerably less common in the treatment groups than in the control groups. However, the degree of this reduction varied among the treatment groups, and there was no significant decrease with a lower dose of the extract. Piper longum (400 mg/kg) significantly (P<0.01) decreased all the activities compared to the control group. Every neuroleptic at every dosage dramatically reduced the hypermotility induced by a week of pretraining.

Expression of TNF-α in the Hippocampus:

The control group had higher levels of TNF-α expression in the hippocampus, an indication of inflammation followed by neurodegenerative diseases (psychosis, depression and Alzheimer's diseases etc.). In comparison to the control group, EEPL significantly reduced TNF-α expression of in hippocampus region of Wistar albino rat.

Figure 3: TNF-α expressing cells in the rat hippocampus with ×100 and ×400 magnification (a) control (b) Haloperidol 2mg/kg (c)EEPL200mg/kg (d) EEPL 400mg/kg

Histopathological Studies of the Brain:

The group treated with Haloperidol and EEPL (400mg/kg) indicate recovery of the cells, compared to that of vehicle treated group.

Figure 4: Histopathological sections of rat brain (a) control (b) Haloperidol 2mg/kg (c) EEPL 200mg/kg (d) EEPL 400mg/kg

Histopathological Studies of the Liver:

The liver's histological structure was normal, consisting of regular, polygonal hepatocyte cords. There was normal sinusoidal spacing between the cords. The central vein and portal vein were normal whereas the haloperidol-treated group showed enlargement of the sinusoid, foci of necrotic hepatocytes disseminated in the parenchyma because of its uncommon effects on the liver showing hepatotoxicity.

Figure 5: Histopathological sections of rat liver (a) control (b) Haloperidol 2mg/kg (c) EEPL 200mg/kg (d) EEPL 400mg/kg

DISCUSSION:

The antipsychotic effects of ethanolic extract of Piper longum fruit were tested in numerous behavioral animal models of psychosis. Preliminary phytochemical screening of the extract was investigated in this study, showing the presence of alkaloids, flavonoids, phenolic compounds, saponins, steroids, tannins, and starch.

The appropriate and targeted regulation of the multiple Dopamine pathways appears to be necessary to increase the effectiveness of antipsychotic drugs. For instance, it is believed that newer drugs have distinct effects on the frontal cortex and striatum, which accounts for the diminished extrapyramidal symptoms found with them.¹⁹ According to Cook and Catania (1964), the mesocortical dopaminergic neuron circuit is the primary mechanism via which pole climb avoidance conduct impacts cognitive behaviour. It is a method of separating neuroleptics

from sedatives and anxiolytics by avoidance escape. Sedative substances suppress both avoidance and escape responses at roughly the same levels, whereas neuroleptic medications reduce avoidance responses at lower doses than those that impact escape responses.^[20] In this study, an ethanolic extract of Piper longum given for twenty-one successive days considerably decreased the time required by the rats to climb the pole. This revealed the antipsychotic potential of ethanolic extract of Piper longum.

Numerous neurotransmitter systems, including Gamma-aminobutyric acid (GABAergic), opioidergic, and dopaminergic (D2) systems and receptors, control the behaviour of rat during the early stages of life.²¹ Reduced motor effects and/or greater sedation may be the cause of the drug-induced reduction in locomotory activity in experimental animals.²²

TNF-α boosted the absorption of dopamine in PC12 cells through the mitogen-activated protein kinase kinase (MEK) signaling pathway. The increase was antagonized by the anti- TNF-α antibody (Ab) and soluble TNF receptor, suggesting that TNF-α certainly increases dopamine uptake in PC12 cells.^[23] This study showed that EEPL improved clinical psychosis by inhibiting the expression of TNF-α in the hippocampus by which we can state dopamine levels would also decrease. ^24,25

In present histopathological studies of the brain, the control group shows unorganized and congested pyramidal cells with a high reduction of white and and grey matter indicating the disease condition. Whereas the Haloperidol and high dose of extract treated groups, the cells are dilated and spread throughout evenly, they also show white and and grey matter voluminously dilated. According to histopathological examinations of the liver, in the control group there is no difference in the liver, showing CV and PV, round centrally placed nuclei, hepatocytes, endothelial cells, and normal sinusoids. The haloperidol-treated group shows moderate changes i.e., no proper cell arrangement and enlargement of sinusoids. This is because therapy with Haloperidol shows uncommon effects on the liver showing hepatotoxicity. The other groups treated with Piper longum (200 and 400 mg/kg) show normal functioning of the liver.

CONCLUSION:

By the findings of the current study, it can be concluded that the ethanolic fruit extract of Piper longum has exceptional antipsychotic properties exhibited by flavonoids, alkaloids and terpenes using various psychosis models. In this study, the extract has shown promising effects by reducing the positive symptoms of psychosis in Wistar albino rats by lowering the expression of TNF-α in the hippocampus. Histopathological studies of the brain showed recovery in the cell arrangements whereas histopathological studies of the liver revealed that the extract is free from hepatotoxicity. Further, chemical segregation, purification, and identification of its active phytoconstituents could result in the discovery of novel molecular entities for potential development as antipsychotic drugs, which can also be a significant field of future research.

ABBREVIATIONS:

CNS - Central nervous system

EEPL - Ethanolic extract of Piper longum

SEM - Standard errors of mean ANOVA - Analysis of variance

TNF-α - Tumor necrosis factor -alpha sRI - Soluble TNF receptor I

CV - Central vein PV – Portal vein

ACKNOWLEDGEMENT:

The authors of this article would like to express their gratitude to Dr. B. A. Vishwanath, Chairman of the Aditya Group of Institutions, Yelahanka, Bengaluru, for giving me access to use the college's research facilities and for providing me with the materials required.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

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Received on 09.11.2023 Modified on 13.03.2024

Research J. Pharm. and Tech 2024; 17(9):4214-4220.

DOI: 10.52711/0974-360X.2024.00651

Sl. No.	Treated Groups	LocomotorActivityCountsbyOpenField(meanS.E.M)
Sl. No.	Treated Groups	Crossing	Rearing	Sniffing	Grooming
01	Control	58.5±6.64	13.67±1.41	14.83±1.58	6.5±0.76
02	Haloperidol (2mg/kg)	28.67±4.02	5.5±0.56	7±0.89	2.83±0.60
03	EEPL (200mg/kg)	43±2.53	9.5±1.54	10.5±.85	4.17±0.60
04	EEPL (400mg/kg)	33.67±2.40	7.33±0.67	8.67±1.12	3.67±0.33

Spoorthi. N1, Pinki Verma2*, Hemavathi. N3