Evaluation of Antidepressant Activity of Ethanolic Extract of Cissus quadrangularis on Swiss Albino Mice

 

Mr. Salman Kapadia1, Dr. (Mrs.) Vanita Kanase2*

1Department of Pharmacology, Oriental College of Pharmacy, Sector 2, Behind Sanpada Railway Station, Sanpada West, Navi Mumbai, Maharashtra 400705.

2HOD Pharmacology, Department of Pharmacology, Oriental College of Pharmacy, Sector 2, Behind Sanpada Railway Station, Sanpada West, Navi Mumbai, Maharashtra 400705.

*Corresponding Author E-mail: vanita.kanase@gmail.com

 

ABSTRACT:

Background: Cissus quadrangularis has been used for cure of various ailments that includes bone cracks, frail bones (osteoporosis), scurvy, malignant growth, annoyed stomach, hemorrhoids, peptic ulcer sickness, excruciating menstrual periods, asthma, jungle fever (malaria), and mental distress. Specially it is used for bone healing properties. According to this background, the aim of the study was to evaluate the antidepressant-like effect of the ethanolic extract of Cissus quadrangularis (EECQ) in different behavioral models such as forced swimming test (FST) and tail suspension test (TST) on mice after two weeks treatment. Methods: Mice were divided into six groups (n=6/group): control group (normal saline), stress control (normal saline), standard group where Imipramine (15mg/kg) was used as standard drug and three test groups where three doses of ethanolic extract of C. quadrangularis (EECQ) (100, 250, and 350mg/kg) was used for two weeks treatment. All the medication and test samples were managed by means of gavage through oral course. To evaluate the antidepressant like impact of EECQ forced swim test (FST) and tail suspension test (TST) have been done in mice. Results: The outcomes demonstrated that a solid and dose-dependent antidepressant effects in various mice models. The main findings of the EECQ significantly decreased the duration of immobility times in the forced swimming test (p<0.5). Likewise, the extract significantly decreased the immobility time in the tail suspension test (p<0.5) when compared against stress control as well as against imipramine which was used as a standard. Conclusion: The present results clearly demonstrate that the ethanolic extract of C. quadrangularis possesses antidepressant-like activity in the animal behavioral models. Cissus quadrangularis plant can be used for the treatment of neurological disorders and may be recommended as a supplement for the antidepressant activity.

 

KEYWORDS: Cissus quadrangularis, Imipramine, Antidepressant-like effect, Forced swimming, Tail suspension, Biochemical analysis.

 

 


INTRODUCTION:

Depression is an extremely common psychiatric condition. It is a type of serious mood affective disorder which refers to a pathological change in mood state; depression varies from mild to severe depression accompanied by hallucinations and delusions.1 It is recognized to be symptomatically, psychologically and biologically heterogeneous.2,3

 

According to WHO, depression is expected to become the second leading cause of disease related disability by the year 2020, following heart disease. Currently available treatment of depression is often associated with several undesirable side effects and it is effective only in a certain portion of the patients.4 A search for novel pharmacotherapy from medicinal plants for psychiatric illnesses has progressed significantly in the past decade. A large number of herbal preparations for antidepressant activity have been evaluated in a variety of animal models.5 Now, Depression has been found to be the fourth leading cause of overall disease burden and the leading cause of nonfatal disease burden worldwide6.

Cissus quadrangularis is the most well-known species having a place with the family Vitaceae, usually known as Hadjod in Hindi or bone setter because of its bone break healing property.7 It is a bulky cactus flora like, jointed climber, conveyed all through more summary regions of India; additionally developed in nurseries. It is local to India, Bangladesh and Sri Lanka. It is likewise found in Africa and Southeast Asia. It is being imported to Brazil and the southern United States. Cissus quadrangularis arrives at a peak of 1.5 m and has quadrangular-segmented branches with internodes of around 8 to 10 cm long and 1.2 to 1.5 cm wide.8

 

It was found to have Antiosteoporotic activity9, Antioxidant and Antimicrobial activity10, Anticonvulsant and sedative activity11, Neuropharmacological effect12, CNS Activity13, Anthelmintic activity14, Antidiabetic activity15, Antistress activity16, Antinociceptive activity.17

 

MATERIALS AND METHODS:

Collection and Preparation of extract:

The twigs of Cissus quadrangularis L. were collected from Medicinal garden of Oriental College of Pharmacy, Navi Mumbai in July 2019. Sample specimen voucher was submitted to Dr. Bindoo Gopalkrishnan asst. Professor Dept. of Botany, at Mithibai College of Arts, Chauhan Institute of Science and Amruthben Jivanlal College of Commerce and Economics. The plant stems were washed with tap water and shade dried at normal room temperature with the aid of circulating airflow using a fan. The stem was dried and coarse powder was made of the stem in a mixture and it was stored in a container.

 

Preparation of extract:

The powdered plant of Cissus quadrangularis L. was extracted with ethanol in the Soxhlet apparatus. The content of the round bottom flask were emptied in the Petri plate, and the solvent was allowed to evaporate. The extracts were evaporated to obtain the dry powder of extract. This crude dry extract were stored in a suitable container and kept in the refrigerator (0-4)°C until use.

 

Qualitative phytochemical screening:

Preliminary chemical tests were carried out on ethanolic extract of Cissus quadrangularis L. for determination of the presence of phytoconstituents like alkaloids, flavonoids, cardiac glycosides, saponins, vitamic C, steroids, triterpenoids, tannins and phenolic compounds was investigated as described by the well- established methods.

 

Experimental animal:

Swiss Albino Mice (20-35gm) were used for the study. The animals were obtained from Bombay veterinary college, Parel, Mumbai, Maharashtra 400012. 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/2019-20/03 of thesis entitled “Evaluation of Pharmacological activity of selected Medicinal Plant”. 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.

 

Experimental design:

Thirty-six mice will be randomly divided into six experimental groups. Group-I (normal control) mice will receive normal saline (1.0mL/kg, p.o.) daily for 14 days; Group-II (stress control) mice will receive normal saline (1.0mL/kg, p.o.) daily for 14 days and will be subjected to restraint stress on 15th day. Group III (Standard) mice will receive Imipramine (15mg/kg, p.o.) daily for 14 days. Group-IV, V and VI mice will be treated with EECQ (100mg/kg, 250mg/kg and 350mg/kg, p.o.) daily for 14 days subjected to ARS on 15th day.

 

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 mice simultaneously. Then 23.5 hours later, the relevant samples were administered and the main test performed 30 minutes later. 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.

 

Acute oral toxicity:

In the literature survey, it was found that the ethanolic extract of Cissus quadrangularis was safe. LD50 of the ethanolic extract is reported to be 5000mg/kg.18 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 EECQ were finalized to 100mg/kg, 250mg/kg and 350mg/kg.

 

Behavioural Tests:

Procedure for acute restraint stress:

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.19

Tail-suspension test (TST):

Mice will be suspended from the edge of a table 50cm above the floor, by the adhesive tape placed approximately 1cm 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 observers blind to the treatments given to the animals under study. Antidepressant activity decreases the immobility of mice in this test.20

 

Forced Swim Test (FST):

On day 14, all the mice were allowed to swim individually for 10 min for adaptation. Then 23.5 hours later, the relevant samples were administered and the main test performed 30 minutes later i.e. on day 15. Mice were forced to swim in a cylinder (diameter 40 cm, height 60cm) containing 30cm 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 span of immobility is characteristic of antidepressant-like effect, while an expansion of immobility time, when contrasted with the control group, is related with depressive-like effects.21

 

Biochemical estimation22:

Catalase activity:

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

 

Sodium oxide Dismutase activity:

The SOD activity in the supernatant was measured by the method of Misra and Fridovich. The supernatant (500μl) was added to 0.800ml of carbonate cushion (100mM, pH 10.2) and 100μl of epinephrine (3mM). The change in absorbance of each sample was then recorded at 480nm 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:

From the 10% tissue homogenate 1ml suspension medium was taken. 0.5ml of 30% TCA will be added to it, followed by 0.5ml of 0.8% TBA reagent. The tubes were then be covered with aluminum 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 540nm at room temperature against an appropriate blank. Blank consists of 1ml distilled water, 0.5ml of 30% TCA and 0.5ml of 0.8% TBA.

 

Determination of Lipid peroxidation assay:

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

 

Statistical Analysis:

The data obtained from animal experiments were analyzed by Software Graph Pad Prism (version 8.3.0). 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 Dunnett’s t-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:

Antidepression Evaluation:

Tail suspension test:

All three doses of the Ethanolic extract of stems of Cissus quadrangularis showed a dose dependent decrease in immobility time when compared against stress control as well as against imipramine which was used as a standard.

 

Table 1: Effect of Ethanolic extract of Cissus quadrangularis plant on Immobility time of Tail suspension test in Swiss Albino Mice.

Sr. No.

Treatment

Duration of Immobility (sec)

1.

Normal

47.33 ± 1.202

2.

ARS

78.50 ± 2.527

3.

Imipramine (15mg/kg p.o.)+ARS

64.00 ± 2.817

4.

EECQ (100mg/kg p.o.)+ARS

68.17 ± 2.810

5.

EECQ(250mg/kg p.o.)+ARS

68.00 ± 2.646

6.

EECQ(350mg/kg p.o.)+ARS

66.83 ± 2.548

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

 

Figure 1: Effect of Ethanolic extract of Cissus quadrangularis on Immobility time of Tail suspension test in Swiss Albino Mice.

 

Forced swim test:

All the three doses of the Ethanolic extract of Cissus quadrangularis showed a dose-dependent decrease in immobility time when compared against stress control as well as against imipramine which was used as a standard.

 

Catalase:

Evaluation of CAT activity revealed that stressed mice presented a significant decrease in CAT activity, which was significantly prevented by EECQ (100 mg/kg, 250mg/kg and 350 mg/kg) pretreatment when compared to unstressed group as shown in table 3.

Table 2: Effect of Ethanolic Extract of Cissus quadrangularis Stems on Immobility Time of forced Swim Test in Swiss Albino Mice.

Sr. No.

Treatment

Duration of Immobility (sec)

1.

Normal

28.17 ± 1.302

2.

ARS

59.67 ± 2.906

3.

Imipramine (15mg/kg p.o.)+ARS

45.00 ± 2.828

4.

EECQ (100mg/kg p.o.)+ARS

49.17 ± 2.315

5.

EECQ(250mg/kg p.o.)+ARS

47.67 ± 2.499

6.

EECQ(350mg/kg p.o.)+ARS

47.50 ± 2.513

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

 

Figure 2: Effect of Ethanolic extract of Cissus quadrangularis on Immobility time of forced swim test in Swiss Albino Mice.

 


 

Biochemical Estimations:

Table 3: Effect of Oxidative Stress Markers in Brain.

Sr. No.

Treatment

Catalase

SOD

MDA

LPO

1.

Normal Control

16.13 ± 0.230

1.098 ± 0.045

0.193 ± 0.012

0.141 ± 0.003

2.

ARS

12.18 ± 0.177

0.633 ± 0.026

0.316 ± 0.007

0.275 ± 0.004

3.

Imipramine (15mg/kg)+ARS

13.48 ± 0.415

0.816 ± 0.014

0.270 ± 0.008

0.192 ± 0.007

4.

EECQ (100mg/kg)+ARS

13.05 ± 0.111

0.780 ± 0.043

0.298 ± 0.004

0.261 ± 0.005

5.

EECQ (250mg/kg)+ARS

13.22 ± 0.833

0.798 ± 0.044

0.291 ± 0.007

0.249 ± 0.004

6.

EECQ (350mg/kg)+ARS

13.29 ± 0.138

0.806 ± 0.042

0.275 ± 0.017

0.249 ± 0.005

 


 

Figure 3: Effect of EECQ pretreatment on ARS induced changes on catalase activity. NC: Normal control; ARS: Acute restraint stress; EECQ: Ethanolic extract of Cissus quadrangularis. Values are expressed as mean ± standard error of mean (N=6). ***P<0.01, *P<0.05. A versus NC group and b versus ARS group.

Superoxide Dismutase Level:

In the mice pretreated with EECQ 100mg/kg, 250mg/kg and 350mg/kg p.o. the level of SOD was significantly increased (p<.001) as compared to ARS mice. Table 3 shows significant and dose dependent recovery on ARS induced reduced level of SOD in the animal due to EECQ.

 

Malondialdehyde (MDA) formation:

The results depicted in [Figure 5] illustrate that ARS significantly increased MDA level in mice brain as compared to unstressed mice. The results indicated that EECQ (100mg/kg, 250mg/kg and 350mg/kg) pretreatment and imipramine significantly abolished the increase in MDA level caused by ARS.

 

Figure 4: Effect of EECQ pretreatment on ARS induced changes on SOD activity. NC: Normal control; ARS: Acute restraint stress; EECQ: Ethanolic extract of Cissus quadrangularis. Values are expressed as mean ± standard error of mean (N=6). ***P<0.01, *P<0.05. A versus NC group and b versus ARS group.

 

Figure 5: Effect of EECQ pretreatment on ARS induced changes on MDA activity. NC: Normal control; ARS: Acute restraint stress; EECQ: Ethanolic extract of Cissus quadrangularis. Values are expressed as mean ± standard error of mean (N=6). ***P<0.01, *P<0.05. A versus NC group and b versus ARS group.

 

Lipid Peroxidation:

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 EECQ (100mg/kg, 250mg/kg and 350mg/kg) pretreatment significantly abolished the increase in MDA level compared to stressed animals.

 

Figure 6: Effect of EECQ pretreatment on ARS induced changes on LPO activity. NC: Normal control; ARS: Acute restraint stress; EECQ: Ethanolic extract of Cissus quadrangularis. Values are expressed as mean ± standard error of mean (N=6). ***P<0.01, *P<0.05. A versus NC group and b versus ARS group.

 

DISCUSSION:

It was observed from the results that Cissus quadrangularis showed a significant dose- dependent decrease in duration of immobility time in TST and FST when compared with the control group in a dose dependent manner. In addition to behavioral tests, EECQ also normalized oxidative stress markers such as Catalase, SOD, MDA, and LPO in a dose-dependent manner.

 

CONCLUSION:

It can be concluded from the study that the ethanolic extract of Cissus quadrangularis stem possess significant antidepressant property, which is probably due to flavonoids which play an active role in providing Antidepressant-like effect. Cissus quadrangularis plant can be used for the treatment of neurological disorders and may be recommended as a supplement for the antidepressant activity.

 

ACKNOWLEDGMENT:

We are thankful for their guidance and help to our Principal Dr. (Mrs.) Sudha Rathod, Dr. (Mrs.) Vanita G. Kanase, as well as to the Department of Pharmacology, Oriental College of Pharmacy, Navi Mumbai.

 

AUTHORS’ CONTRIBUTIONS:

Dr. (Mrs.) Vanita Kanase guided with designing the study, making of protocol, and managed the work done. Salman Kapadia performed the literature searches, models, phytochemical screening, and completed the manuscript writing.

 

CONFLICTS OF INTEREST:

We announce we do not have conflicting interests.

 

AUTHORS’ FUNDING:

We thank Oriental College of Pharmacy for funding the project.

 

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Received on 06.06.2020           Modified on 16.08.2020

Accepted on 19.10.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(8):4197-4202.

DOI: 10.52711/0974-360X.2021.00727