Investigation of Antidepressant Effects of Ethanolic Extract of Roots of Piper Methysticum G. Forst in Swiss Albino Mice

Depression is a prevalent mental health disorder characterized by persistent sadness, loss of interest in activities, and various emotional and physical issues,(1)Its causes are complex, involving genetic, biological, environmental, and psychological factors, with neurotransmitter imbalances playing a significant role. The World Health Organization identifies depression as a leading cause of disability globally, affecting approximately 280 million individuals. Effective treatments include psychological therapies and medications(2). Plant-based medications are becoming more and more popular and are being researched for a variety of conditions, including neurological conditions like depression(3).medications might be used to create a multi-ingredient herbal combination that would undoubtedly aid in the treatment of anxiety and depression, among other neurodegenerative conditions. A thorough and well-thought-out investigation in this field could lead to significant advances in the field of therapy(2). Piper methysticum, commonly known as kava, is a plant native to the South Pacific islands, traditionally used for its calming effects. The roots contain kavalactones, compounds responsible for its psychoactive properties. Kava holds significant cultural importance in Pacific communities, used in ceremonies to promote relaxation and social bonding. In recent years, kava has gained global attention as a natural remedy for anxiety and insomnia. However, concerns about potential liver toxicity have led to regulatory scrutiny in some countries. Despite these concerns, research continues to explore kava's therapeutic potential, focusing on its anxiolytic and muscle-relaxant properties(4)^. The primary active compounds in kava are kavalactones, including kavain, dihydrokavain, methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin. These interact with the central nervous system, particularly GABA-A receptors, to produce calming effects. Additionally, kava contains flavonoids with antioxidant properties, as well as coumarins, alkaloids, and essential oils contributing to its aroma and mild sedative effects(5). While kavalactones are the main active constituents, the combined effects of these compounds contribute to kava's overall pharmacological profile. However, excessive consumption has been linked to potential liver toxicity, warranting ongoing research into its safety. Traditionally, kava has been integral to Pacific Island cultures, used in ceremonies to promote relaxation, social bonding, and spiritual connection(6). The root is prepared into a beverage consumed during gatherings, symbolizing hospitality and respect. Kava is also used in healing rituals to address ailments such as nervous tension and insomnia. Its significance is deeply embedded in the customs and traditions of these regions, where it is revered as a sacred plant(7).

MATERIAL AND METHOD   

Collection and authentication of plant

On September 11, 2023, the powder extract of piper methysticum G. Forst were collected from the from Herbo Nutra, Noida. Mahesh R. Atale, a botanist at ALARSIN, Andheri, received the sample specimen voucher. The specimen is consistent with the characteristics of " piper methysticum," which is a member of the " Piperaceae" family. The plant is commonly referred to as "Kava kava."

Preparation of extract

The leaves were washed with tap water and shade dried at normal room temperature with the aid of circulating airflow using a fan. The leaves were dried. Air-dried leaves were cut into small pieces and macerated with aqueous ethanol mixture at room temperature for 10 days. The macerate was filtered and the solvent was removed by rotatory evaporator under reduced pressure (8)

Physicochemical analysis of powdered roots

Physicochemical parameters were carried out on powdered roots of Piper methysticum G. Forst for loss on drying, total ash value, acid-insoluble ash value, water-soluble ash value, and determination of extractive values and were investigated as described by the well-established methods.

Preliminary phytochemical screening of the extract

Preliminary chemical tests were carried out on the ethanolic extract of roots of Piper methysticum G. Forst for the determination of the presence of phytoconstituents such as alkaloids, flavonoids, glycosides, saponins, steroids, carbohydrates, and phenolic compounds and were investigated as described by the well-established methods.

Animal model specifications

The animal model specification is shown in table 1.

Swiss Albino Mice (20-35gm) were used for the study. The animals were obtained from Jay Agro, Posari Tal -Karjat, Dist.- Raigad, Karjat-410201. The use of these animals and the study protocols were approved by CPCSEA recognized local ethical committee of Oriental College of Pharmacy under protocol no. OCP/IAEC/2023-2024/05(R) titled “Evaluation of antidepressant activity of Ethanolic extract of roots of piper methysticum G. Forst in Swiss Albino Mice” of thesis entitled “Evaluation of Pharmacological Activity of Herbal Plant Extract”. Mice were kept at the animal house of Oriental College of Pharmacy, Sanpada, Navi Mumbai; in polypropylene cages, at 22 ± 2°C, with 12:12 hrs dark: light cycle. They were provided with commercial mice feed and water given ad libitum.

Table 1: Animal model specifications

Selection of doses

In the literature survey, it was found that the Ethanolic Extract of Piper methytsticum G. Forst safe. LD50 of the ethanolic extract is reported to be 750 mg/kg. The plant is often eaten by animals, which is also an indicator to prove it is less toxic. Thus, for purpose of research study, the doses of EERPM were finalized are 35mg/kg, 75mg/kg(9,10).

Drugs and chemicals

Imipramine (Abbott, Mumbai, India) was used as reference standards for the antidepressant activity.

Experimental Design

Thirty mice will be randomly divided into Five experimental groups. Group-I (normal control) mice will receive normal saline (10 ml/kg, p.o.) daily for 14 days. Group-II (stress control) mice will receive normal saline (10 ml/kg, p.o.) daily for 14 days and will be subjected to restraint stress on 15th day. Group-III (standard control) mice will receive Imipramine (15 mg/kg, p.o.) daily for 14 days. Group-IV and V mice will be treated with Hydroalcoholic Extract of Brassica oleracea var. sabellica (100 mg/kg and 200 mg/kg, p.o.) daily for 14 days. Stress-like behavior was assessed by subjecting the mice to behavioral paradigms such as tail suspension test (TST), 40 min post-restraint stress procedure. Pretest of 10 minutes for forced swim test (FST) was also given to each mouse simultaneously. Oxidative stress parameters such as SOD, CAT, MDA, and extent of LPO were analyzed in restraint stress-induced animals and control group, following forced swim test on the 15th day. The experimental design is shown in table 2.

NOTE: EERPM=Ethanolic Extract of Roots of piper methysticum G. Forst

Acute restraint stress was accomplished by placing mice in an individual plastic rodent restraint device for 12 h. This restrained all physical movements without subjecting the animal to pain. Animals were deprived of food and water during the entire period of exposure to stress. After 12 h, the animals were released from their enclosure and 40 min post-release, the animals were subjected to behavioral tests and then to biochemical estimations. In the normal control group, the mice were kept in the animal cage in the experimental room(11).

Behavioural tests

Numerous rodent behavioral tests are currently used to assess characteristics like sensory-motor function, stress-like behavior, and depression-like behavior. These tests are inherently complex, and their use necessitates careful consideration of a number of factors, particularly test validity, which has a significant impact on the likelihood that preclinical results will be successfully translated to clinical settings. Behavioral research may have been founded by Charles Darwin. In order to better comprehend the central nervous system (CNS) and discover cures for illnesses, behavioral testing has been widely utilized ever since(12).

Tail-suspension test (TST)

Mice will be suspended from the edge of a table 50 cm above the floor, by the adhesive tape placed approximately 1 cm from the tip of the tail. Total duration of immobility will be recorded for next 4 min during a 6 min test. Mice will be considered to be immobile only when they hung passively and will completely motionless. Recording of duration of immobility of animals will be done by observer’s blind to the treatments given to the animals under study. Antidepressant decreases the immobility of mice in this test(12)

Forced Swim Test (FST)

Mice were forced to swim in a cylinder (diameter 40 cm, height 60 cm) containing 30 cm of fresh water maintained at 25°C ± 1°C. Water in the cylinder was changed after each animal to prevent behavioral alteration among animals due to used water. Each animal showed vigorous movement during the initial 2 min period of the test. Duration of immobility will be manually recorded during the next 4 min of total 6 min testing period by the observer. Mice were considered to be immobile when they floated in an upright position, making only small movements to keep their head above the water level. Following swimming session, mice were dried using a cotton towel and returned to home cages after the experiment. A decrease in the duration of immobility is indicative of antidepressant-like effect, whereas an increase of immobility time, when compared with the control group, is associated with depressive-like effects(13).

Biochemical Estimation

All the animals were sacrificed by euthanasia, after behavioral observations. The brains were quickly removed, washed in ice-cold sterile isotonic saline, and weighed. 10% (w/v) tissue homogenates were prepared with phosphate buffer solution (PBS) of (pH 7.4). The supernatant was obtained by centrifugation of the homogenate at 1000 rpm for 20 min at 5 c and used for further biochemical estimation(14).

Determination of Catalase activity:

The supernatant (50 μl) was added to a cuvette containing 2.95 ml of 19 mM/L solution of H2O2 prepared in potassium phosphate buffer. The change in absorbance was monitored at 240 nm wavelength at the 1minute interval for 3 minutes. Presence of catalase decomposes H2O2 leading to a decrease in absorbance.

 Determination of Superoxide Dismutase activity:

The SOD activity in the supernatant was measured by the method of Misra and Fridovich. The supernatant (500 μl) was added to 0.800ml of carbonate buffer (100mM, pH 10.2) and 100 μl of epinephrine (3mM). The change in absorbance of each sample was then recorded at 480 nm in spectrophotometer for 2 min at an interval of 15 sec. Parallel blank and standard were run for determination of SOD activity. The reaction mixtures are diluted 1/10 just before taking the readings in a spectrophotometer.

Determination of Malondialdehyde (MDA) formation:

1 ml of suspension medium was taken from the 10% tissue homogenate. 0.5 ml of 30% TCA will be added to it, followed by 0.5 ml of 0.8% TBA reagent. The tubes were then be covered with aluminium foil and kept in shaking water bath for 30 minutes at 80O C. After 30 minutes tubes were taken out and kept in ice-cold water for 30 minutes. These were then be centrifuged at 3000 rpm for 15 minutes. The absorbance of the supernatant was read at 540 nm at room temperature against an appropriate blank. Blank consists of 1 ml distilled water, 0.5 ml of 30% TCA and 0.5 ml of 0.8% TBA.

Determination of Lipid peroxidation assay:

To 0.2 ml of the test sample, 0.2 ml of SDS, 1.5 ml of acetic acid and 1.5 ml of TBA were added. The mixture was made up to 4 ml with water and then heated in a water bath at 95°C for 60 minutes. After cooling, 1 ml of water and 5 ml of n-butanol/pyridine mixture were added and agitated smartly. After centrifugation at 4000 rpm for 10 minutes, the organic layer was taken and its absorbance was scan at 532 nm. The level of lipid peroxides was expressed as n moles of MDA released/ g wet tissue.

Statistical analysis:

The data obtained from animal experiments were analyzed by Software GraphPad Prism (version 10.2.1). It was expressed as mean ± SEM (standard error of the mean). For statistical analysis, the data were subjected to analysis of variance (ANOVA) followed by Bonferroni's multiple comparisons test. Results were considered to be statistically significant at P ≤0.05. Significance levels were as follows:

 * Indicates p ≤0.5 as significant;

 ** indicates p ≤0.01 as highly significant;

*** indicated p ≤0.001 as very significant.

RESULTS AND DISCUSSION

Physicochemical analysis of powdered roots

The result of Physicochemical analysis of powdered roots of piper methysticum G. Forst are shown in table 3.

Table 3: Results of physiochemical analysis of powdered roots of Piper methysticum G. Forst

Qualitative phytochemical screening

The result of quantitative phytochemical analysis of powdered roots of piper methysticum G. Forst are shown in table 4.

Tail suspension test:

All the two doses of the Ethanolic extract of Roots of piper methysticum showed a dose-dependent decrease in immobility time when compared against stress control as well as against imipramine which was used as a standard. The result of forced swim test is shown in table 5.

Values are the mean ± SEM of n=6 mice/treatment. Significance *p ≤0.5

Figure No 1: Effect of Ethanolic extract of Roots of Piper methysticum on Immobility time of Tail suspension test in Swiss Albino Mice.

All the two doses of the Ethanolic extract of Roots of piper methysticum G. Forst showed a dose-dependent decrease in immobility time when compared against stress control as well as against imipramine which was used as a standard.  The result of forced swim test is shown in table 6.

Table 6: Effect Ethanolic extract of Roots of Piper methysticum G. Forst on Immobility time of forced swim test in Swiss Albino Mice.

Values are the mean ± SEM of n=6 mice/treatment. Significance *p ≤0.5

Fig No 2: Effect of Ethanolic extract of Roots of Piper methysticum G. Forst on Immobility time of forced swim test in Swiss Albino Mice.

Evaluation of CAT activity revealed that stressed mice presented a significant decrease in CAT activity, which was significantly prevented by EERPM (35mg/kg and 75mg/kg) pretreatment when compared to unstressed group as shown in table 7.

Fig No 3: Effect of EERPM pretreatment on ARS induced changes on catalase activity. NC: Normal control; ARS: Acute restraint stress; EERPM: Ethanolic extract of Roots of Piper methysticum G. Forst. Values are expressed as mean ± standard error of mean N=6).

The level of SOD was considerably higher in the mice pretreatment with EERPM at doses of 35 mg/kg and 75 mg/kg p.o. than in the ARS animals. Significant and dose-dependent recovery from ARS-induced decreased SOD levels in the animal as a result of EERPM is shown in Table 7.

Fig No 4: Effect of EERPM pretreatment on ARS induced changes on catalase activity. NC: Normal control; ARS: Acute restraint stress; EERPM: Ethanolic extract of Roots of Piper methysticum G. Forst. Values are expressed as mean ± standard error of mean (N=6).

The results illustrate that ARS significantly increased MDA level in mice brain as compared to normal and treated mice. The results indicated that EERPM (35 mg/kg and 75mg/kg) pretreatment and imipramine significantly abolished the increase in MDA level caused by ARS.

Fig No 5: Effect of EERPM pretreatment on ARS induced changes on MDA activity. NC: Normal control; ARS: Acute restraint stress; EERPM: Ethanolic Extract of Roots of Piper Methysticum G. Forst Values are expressed as mean ± standard error of mean (N=6).

Quantitative measurement of LPO in the whole brain was assessed based on the amount of malondialdehyde (MDA) formed, the statistical analysis revealed that ARS produced a significant increase in MDA level whereas EERPM (35 mg/kg and 75 mg/kg) pretreatment significantly abolished the increase in MDA level compared to stressed animals.

Fig No 6: Effect of EERPM pretreatment on ARS induced changes on MDA activity. NC: Normal control; ARS: Acute restraint stress; EERPM: Ethanolic Extract of Roots of Piper Methysticum G. Forst Values are expressed as mean ± standard error of mean (N=6).