|Year : 2016 | Volume
| Issue : 1 | Page : 112
Antidepressant activity of enicostemma littorale blume in shp2 (protein tyrosine phosphatase)-inhibited animal model of depression
VA Doss, Dharaniyambigai Kuberapandian
Department of Biochemistry, PSG College of Arts and Science, Coimbatore, Tamil Nadu, India
|Date of Submission||04-May-2015|
|Date of Acceptance||09-Sep-2016|
|Date of Web Publication||27-Sep-2016|
V A Doss
Department of Biochemistry, PSG College of Arts and Science, Coimbatore, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: The objective of this study is to develop a new animal model based on signaling pathways to understand the pathophysiology, therapy of depression, and to investigate the antidepressant activity of Enicostemma littorale which is not yet established.
Methods: Animal models of depression were raised by physical methods and administration of methyl isobutyl ketone (100 mg/kg b.w., i.p.,) and a protein tyrosine phosphatase inhibitor, sodium orthovanadate (30 mg/kg b.w., i.p.,) to young Wistar rats. E. littorale aqueous extract (100 mg/kg b.w., oral) was administered. Forced swimming test (FST), biochemical, and histopathological parameters were performed with reference to fluoxetine (20 mg/kg b.w., oral) treatment.
Results: High-performance thin-layer chromatography confirmed the presence of swertiamarin, a unique glycoside present in the Gentianaceae family. FST indicated high rates of immobility in depressed groups and low rates in plant extract-administered group with reference to fluoxetine. Biochemical assays indicated significantly (P < 0.05) increased levels of total protein, superoxide dismutase, triglycerides, and total serum cholesterol, whereas significant reduction (P < 0.05) of glutathione peroxidase, catalase, and lipid peroxidation in plant extract-administered groups in comparison to the depressed groups. Histopathological analysis indicated disorganized neuronal architecture during depression whereas rejuvenation of neuronal patterns was observed during treatment with plant extract and fluoxetine.
Conclusions: This study shows that sodium orthovanadate induces depression in animals and also establishes the antidepressant activity of E. littorale.
Keywords: Depression, Enicostemma littorale Blume, sodium orthovanadate
|How to cite this article:|
Doss V A, Kuberapandian D. Antidepressant activity of enicostemma littorale blume in shp2 (protein tyrosine phosphatase)-inhibited animal model of depression. Int J Prev Med 2016;7:112
|How to cite this URL:|
Doss V A, Kuberapandian D. Antidepressant activity of enicostemma littorale blume in shp2 (protein tyrosine phosphatase)-inhibited animal model of depression. Int J Prev Med [serial online] 2016 [cited 2021 Oct 21];7:112. Available from: https://www.ijpvmjournal.net/text.asp?2016/7/1/112/191187
| Introduction|| |
Depression is a complex and heterogeneous disorder which involves several neurotransmitters and neurohormonal pathways that play crucial roles in the pathophysiology of depression whose mechanisms are not well understood.  The absence or low levels of brain-derived neurotrophic factor (BDNF) or altered serotonin signaling, stress pathways, and other genetic or epigenetic factors influence the sequential activation of BDNF-mitogen-activated protein kinase (MAPK) pathway thereby resulting in depression. , The knowledge about the downstream targets of the MAPK signaling and their interactions in the regulation of anxiety and depression in the brain is obscure  [Figure 1]. Enicostemma littorale Blume (Indian Gentian) possess antioxidant, hepatoprotective, antinociceptive, anti-inflammatory, and antilipidemic activities. ,
|Figure 1: Molecular signaling events of brain-derived neurotrophic factor-mitogen-activated protein kinase pathway and depression|
Click here to view
| Methods|| |
All the chemicals were of analytical grade purchased from Hi-Media Laboratories Pvt., Limited, (Mumbai, Maharashtra, India). Sodium orthovanadate was purchased from Sigma-Aldrich, (Coimbatore, Tamil Nadu, India). Methyl isobutyl ketone (MIBK) was purchased from Qualigens Pvt. Limited, (Mumbai, Maharashtra, India). The standard drug fluoxetine was purchased commercially as fluon capsules at PSG Healthcare Pharmacy, Coimbatore, India. Swertiamarin standard was purchased from Aktin Chemicals, China.
Plant material and extraction
E. littorale plant on the whole was collected from the local herbal shops in Coimbatore district, Tamil Nadu, India, and was authenticated by the Botanical Survey of India, Southern Regional Centre, Coimbatore (No. BSI/SRC/5/23/2010-11/Tech-2051). The vegetative and reproductive parts of E. littorale (1000 g) were shade dried, powdered in a mixer grinder, and stored in airtight containers. The dried plant powder was mixed with various solvents (1:3 ratio), namely water, ethanol, acetone, chloroform, and petroleum ether and filtered, and the phytochemical analysis , was performed to identify the efficient solvent for the study. The aqueous extract which showed the presence of more phytochemicals was prepared in large scale. 
High-performance thin-layer chromatography of Enicostemma littorale
Two microliter plant extract and standard solutions were loaded as 5 mm band length in a 3×10 silica gel 60F 254 TLC plate using Hamilton syringe and CAMAG Linomat 5 instrument. TLC was performed using the mobile phase consisting of ethyl acetate, methanol, and water in the ratio 7.7:1.2:0.5, respectively. The plate was taken out and developed when the mobile phase was at 90 mm of height, and the developed plate was photo-documented using CAMAG Reprostar 3 in which the images were exposed and captured at visible light, ultraviolet (UV) 254 nm, and UV 366 nm. Then, the plate was scanned at UV 254 nm using CAMAG TLC Scanner 3 and then was derivatized using p-anisaldehyde-sulfuric acid and dried at 100°C in a hot air oven and photo-documented in visible light and UV (366 nm).
Young male Albino rats of Wistar strain (30 ± 20 g) were procured from Chennai, Tamil Nadu, India. The ethical clearance for handling of these experimental animals was obtained from the Institutional Animal Ethics Committee (IAEC) of PSG Institute of Medical Sciences and Research, Coimbatore, that acts under the guidance of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India (CPCSEA/No: 158/1999/CPCSEA). The animals were acclimatized under standard laboratory conditions for 3 days with controlled temperature (29° ± 5°C), humidity (55% ± 5%), and 12 h light/dark cycles and maintained under the same conditions throughout the experimental period.
The experimental animals were divided into seven groups with three rats each including the normal healthy animals as Group I. Rats that were depressed using MIBK (100 mg/kg b.w., i.p., 5 days) and physical methods served as untreated depression control (Group II). The MIBK-induced depressed rates were treated with the standard drug, fluoxetine (20 mg/kg b.w., oral, 7 days) (Group III). The MIBK-induced depressed rats were treated with the aqueous extract of E. littorale (100 mg/kg b.w., oral, 7 days) (Group IV). Simultaneously, a group of animals were administered with sodium orthovanadate (protein tyrosine phosphatase [PTP] inhibitor (LD 50 is 330mg/kg b.w., rat) (30 mg/kg b.w., i.p., 5 days)  and subjected to physical methods for developing depression (Group V). The PTP inhibitor-induced depressed rats were treated with fluoxetine (20 mg/kg b.w., oral, 7 days) (Group VI). The PTP inhibitor-induced depressive rats were treated with aqueous extract of E. littorale (100 mg/kg b.w., oral, 7 days) (Group VII). Previous studies ,, on medicinal activities of E. littorale indicate various dosages of the plant extracts based on which the dosage was fixed in this study.
Induction of depression
Depression was induced by physical and chemical methods in the young albino Wistar rats. Animals were starved for 24 h due to food deprivation and were injected with MIBK  (100 mg/kg, i.p.) with a 1:20 dilution to induce depression. Sodium orthovanadate (PTP inhibitor) (30 mg/kg, i.p.) was prepared in saline. After such chemical treatments, the rats were subjected to food deprivation for 24 h and were kept in a rotatory shaker (300 rpm for 10 min) which can further increase the susceptibility to depression. The status of depression was diagnosed by the forced swimming test (FST) on the 7 th day after induction of depression and followed by treatments on the 3 rd and 7 th .  FST was performed thrice for each test animal in each group and their mean was calculated for data analysis. Data are expressed as mean ± standard deviation.
Collection of serum and tissues
The animals were anesthetized by chloroform and sacrificed by cervical dislocation at the end of the treatment period. Cardiac puncture method was employed for collecting the blood, and the clotted blood was centrifuged at 5000 rpm for 10 min to obtain the serum. The brain was excised from the skull, thoroughly washed in ice-cold saline, and used for biochemical and histopathological analysis.
The collected brain tissues were used for the estimation of protein,  enzymic, and nonenzymic antioxidants, namely superoxide dismutase (SOD),  CAT,  glutathione peroxidase (GPx),  and lipid peroxidation (LPO).  The collected serum was used for the estimations of triglycerides  and serum cholesterol. 
The histopathological analysis was performed with the excised brain tissue to evaluate the neuronal plasticity. 
Data are expressed as mean ± standard error of the mean. Statistical analysis was performed using standard t-tests, and the P < 0.05 was considered statistically significant.
| Results|| |
The preliminary phytochemical analysis of the aqueous extracts of E. littorale revealed the presence of alkaloids, flavonoids, saponins, carbohydrates, proteins, phenols, glycosides, and tannins. High-performance thin-layer chromatography (HPTLC) profiling of glycosides [Figure 2] after derivatization showed a brownish zone at visible light mode which confirmed the presence of glycoside, swertiamarin in the sample, and the standard used.
|Figure 2: High-performance thin-layer chromatography of swertiamarin after derivatization. In the figure, SWN indicates swertiamarin and A indicates plant extract|
Click here to view
FST shows the status of depressive condition in the experimental animal groups [Figure 3]. In the normal control, the immobility time is very less compared to other groups as they are normally active. During the course of study in a week time, the rate of immobility can be found to be lesser than that of the 1 st day of the experiment due to their acclimatization. Depression was induced in Groups II to IV with MIBK (indicated as C) and Groups V to VII by sodium orthovanadate, a PTP inhibitor (indicated as I), respectively, followed by treatment with the standard antidepressant, fluoxetine, and the plant extract. The graph indicates the impact of PTP inhibitor in inducing depression with the increased rate of immobility compared to the normal group. As illustrated in the graph [Figure 3], it can be observed that the status of depression reduced on treatment and a marked reduction can be observed with the treatment of plant extract in comparison with fluoxetine-administered group.
|Figure 3: Immobility periods of experimental animals by forced swimming test to determine the status of depression|
Click here to view
In the biochemical analysis, the level of total protein content was found to be low in depression induced by MIBK (50 ± 3.86 μg) and PTP inhibitor (54 ± 4.18 μg) as compared with the normal control group (100 ± 3.85 μg). On comparison with the depression control, the groups administered with fluoxetine (Group III - 74 ± 4.15 μg; Group V - 60 ± 4.04 μg) and plant extract showed increased total protein levels (Group IV - 100 ± 4.41 μg; Group VII - 70 ± 4.41 μg) [Table 1]. The levels of SOD were found to have least activity against superoxide radicals in depression-induced groups (Group II - 0.06 ± 0.01 [U/g]; Group V - 0.12 ± 0.39 [U/g]) and was found to increase on treatment with fluoxetine (Group III - 0.68 ± 0.14 [U/g]; Group VI - 0.18 ± 0.04 [U/g]) and plant extract (Group IV - 0.62 ± 0.14 [U/g]; Group VII - 0.68 ± 0.12 [U/g]) [Table 2]. The activity of catalase (CAT) was found to be higher in depression groups induced by MIBK (4.0 ± 3.50 IU) and by the PTP inhibitor (7.8 ± 4.20 IU). The CAT activities were high on administration with fluoxetine (Group III - 5.4 ± 3.54 IU; Group VI - 9.6 ± 4.33 IU) and plant extract (Group IV - 8.2 ± 4.0 IU; Group VII - 7.8 ± 4.14 IU) [Table 2]. Thus, the study shows higher levels of GPx in both depression-induced groups (Group II - 400 ± 4.17 IU; Group V - 400 ± 3.54 IU) and depression-treated groups (Group III - 400 ± 3.96 IU; Group IV - 200 ± 3.87 IU; Group VI - 400 ± 4.26 IU; and Group VII - 200 ± 4.06 IU) compared to the normal control (200 ± 4.0 IU) [Table 2].
|Table 2: Estimation of superoxide dismutase, catalase, and glutathione peroxidase |
Click here to view
In the estimation of LPO in brain tissues, the formation of malondialdehyde was lower in groups with depression induced by both MIBK (0.12 ± 0.03 IU) and inhibitor (0.25 ± 0.04 IU). The values turned higher for the drug (Group III - 0.38 ± 0.04 IU; Group VI - 0.32 ± 0.04 IU) and plant extract-administered groups (Group IV - 0.38 ± 0.03 IU; Group VII - 0.57 ± 0.04 IU) when compared with the depression controls. Serum lipid profile analysis showed higher triglyceride levels (Group II - 128 ± 4.07 mg/dl; Group V -122.6 ± 4.19 mg/dl) and low levels of serum total cholesterol (Group II - 125 ± 4.28 μg; Group V - 110 ± 4.28 μg) in depression groups whose levels become lower on treatment with fluoxetine (Group III - 122 ± 4.40 mg/dl; Group VI - 82.6 ± 4.47 mg/dl triglycerides and in cases of cholesterol Group III - 20 ± 4.28 μg; Group VI - 85 ± 4.35 μg) and plant extract (Group IV - 125.3 ± 3.79 mg/dl; Group VII - 122 ± 3.72 mg/dl triglycerides and in cases of cholesterol Group IV - 185 ± 4.48 μg; Group VII - 65 ± 4.28 μg) [Table 3].
|Table 3: Estimation of lipid peroxidation, triglycerides, and serum total cholesterol |
Click here to view
The histopathological examination of the hippocampus in depression groups showed less prominent, disorganized neuronal, and glial cells compared to the normal rats, whereas the treatment with fluoxetine and plant extract resulted in the rejuvenation of normal cellular organization (Data not shown).
| Discussion|| |
HPTLC has shown the presence for swertiamarin which is hypothesized to possess antidepressant and anticholinergic activity due to the presence of a secoiridoid glycoside called swertiamarin which is unique to the Gentianaceae family.  Swertiamarin can be further subjected to structure elucidation and docking studies to reveal the interactions of the glycoside with its receptors to understand its role as an antidepressant which could further enter into clinical trials.  In the present study, the activity of SOD and CAT was less during depression and raises to normal level on treatment with E. littorale. GPx on contrary to previous reports is high during depression and reduces to normal during treatment with E. littorale which can be surmised that in depressed state, genetic polymorphism of GPx with increased proline alleles (Pro197Leu GPx) may be responsible for the abnormal high GPx activity. Enzyme expression studies are required to show whether abnormal antioxidant enzyme expression pattern prevails in depressive conditions.  Moreover, the increased activity can be probably due to increased production of ROS which usually occurs due to the activities of SOD and CAT. 
Another noted outcome in this present study is the reduced level of LPO during depression. Sodium orthovanadate besides as a PTP inhibitor, when administered in lower and optimum doses, was used in protecting cells against direct action of LPO on brain acetylcholinesterase thereby preventing the major complications in diabetes, namely cholinergic neural dysfunction.  Studies reveal that the serum high-density lipoprotein and total cholesterol levels are lower during major depression , as indicated in the present study. Hypertriglyceridemia is another metabolic cause for depression  which is revealed in this study with increased serum triglyceride levels during depression. Another metabolic factor focused in depression is the altered cholesterol transport to the liver from tissues. Hence, the lipid metabolism is involved in depression which warrants further investigations. Evaluation of lipid profile is essential for establishing the relationship between depression and the cardiovascular disease for early diagnosis and prevention at its first phase. ,
Depression is more than a mental disorder causing important alterations of the oxidative stress affecting the whole organism. Currently, the ongoing researches mostly target the analysis of the significant association that depression has with cardiovascular diseases, cancer and why people suffering from depression die at a younger age. As a systemic disease, depression studies look forward for successful animal models to understand the pathophysiology and to study the novel antidepressant strategies. ,
| Conclusions|| |
The present study has put forth a new insight for developing animal models of depression using PTP inhibitor (sodium orthovanadate), the importance of SHP2 inhibitors in identifying downstream targets of cell signaling and the natural or synthetic compounds that interact with them for developing novel therapeutics for depression. Ultimately, this study has also established the antidepressant potential of E. littorale which could be further studied to identify the pharmacologically active antidepressant compound.
Thanks to Dr. S. Ramalingam, Principal, PSG Institute of Medical Sciences and Research and PSG Institutional Animal Ethics Committee, Coimbatore, India, for the project fund (184/2013/PSGIMSR) and providing animal ethical clearance (158/1999/CPCSEA). Sincere thanks to the Botanical Survey of India, for the plant authentication. Thanks to Dalmia Biotech and Research, Coimbatore, for the HPTLC facility. Thankful to Aktin Chemicals, China, and The Indian Customs for the purchase of swertiamarin. Thanks to Ponmani Chemicals Pvt. Ltd., Coimbatore, for the purchase of sodium orthovanadate from Sigma-Aldrich, USA. Thanks to Dr. Arjunan, Lakshmi Pathologies, Coimbatore, for the histopathology facility.
Financial support and sponsorship
The project was funded by PSG Institute of Medical Sciences and Research under the intra-institutional funding scheme (Proposal No: 184/2013/PSGIMSR).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Vaidya VA, Duman RS. Depression - Emerging insights from neurobiology. Br Med Bull 2001;57:61-79.
Li H, Zhang L, Huang Q. Differential expression of mitogen-activated protein kinase signaling pathway in the hippocampus of rats exposed to chronic unpredictable stress. Behav Brain Res 2009;205:32-7.
Easton JB, Royer AR, Middlemas DS. The protein tyrosine phosphatase, Shp2, is required for the complete activation of the RAS/MAPK pathway by brain-derived neurotrophic factor. J Neurochem 2006;97:834-45.
Wefers B, Hitz C, Hölter SM, Trümbach D, Hansen J, Weber P, et al.
MAPK signaling determines anxiety in the juvenile mouse brain but depression-like behavior in adults. PLoS One 2012;7:e35035.
Ramesh B, Dharaniyambigai K. Phytochemical screening and in-vitro
antioxidant activities of aqueous extract of Enicostemma littorale
Blume. Int Res J Biochem Bioinformatics 2012;2:200-4.
Jaishree V, Badami S, Rupesh Kumar M, Tamizhmani T. Antinociceptive activity of swertiamarin isolated from Enicostemma axillare
. Phytomedicine 2009;16:227-32.
Vaidya H, Rajani M, Sudarsanam V, Padh H, Goyal R. Swertiamarin: A lead from Enicostemma littorale
Blume. for anti-hyperlipidaemic effect. Eur J Pharmacol 2009;617:108-12.
Sani AA, Mohammed I, Koita HA. Phytochemical screening of the leaves of Lophira lanceolata
(Ochananceae). Life Sci J 2007;4:75.
Burmeister BT, Taglieri DM, Wang L, Carnegie GK. Src homology 2 domain-containing phosphatase 2 (Shp2) is a component of the A-kinase-anchoring protein (AKAP)-Lbc complex and is inhibited by protein kinase A (PKA) under pathological hypertrophic conditions in the heart. J Biol Chem 2012;287:40535-46.
Okokon EJ, Nwafor AP, Abia OG, Bankhede KH. Antipyretic and antimalarial activities of crude leaf extract and fractions of Enicostemma littorale
. Asian Pac J Trop Dis 2012;2:442-7.
Roy S, Niranjan C, Jyothi T, Shankrayya M, Vishawanath K, Prabhu K, et al.
Antiulcer and anti-inflammatory activity of aerial parts Enicostemma littorale
Blume. J Young Pharm 2010;2:369-73.
Gopal R, Gnanamani A, Udayakumar R, Sadulla S. Enicostemma littorale
Blume - A potential hypolipidemic plant. Nat Prod Radiance 2004;3:401-5.
Umadevi P, Murugan S, Suganthi JS, Subakanmani S. Evaluation of antidepressant like activity of Cucurbita pepo
seed extracts in rats. Int J Curr Pharm Res 2011;3:108-13.
Nirmal J, Babu CS, Harisudhan T, Ramanathan M. Evaluation of behavioural and antioxidant activity of Cytisus scoparius
link in rats exposed to chronic unpredictable mild stress. BioMed Cent 2008;8:1-8.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.
Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 1984;21:130-2.
Sinha AK. Colorimetric assay of catalase. Anal Biochem 1972;47:389-94.
Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: Biochemical role as a component of glutathione peroxidase. Science 1973;179:588-90.
Niehaus WG Jr., Samuelsson B. Formation of malonaldehyde from phospholipid arachidonate during microsomal lipid peroxidation. Eur J Biochem 1968;6:126-30.
Trinder P. Enzymatic calorimetric determination of triglycerides by GOP-PAP method. Ann Clin Biochem 1969;6:24-7.
Zak B. Cholesterol methodologies: A review. Clin Chem 1977;23:1201-14.
Nathiya VC, Vanisree AJ. Investigations on light induced stress model and on the role of Phyllanthus amarus
in attenuation of stress related depression with focus on 5HT2Am-RNA expression. Ann Neurosci 2010;17:167-75.
Vishwakarma SL, Bagul MS, Rajani M, Goyal RK. A sensitive HPTLC method for estimation of Swertiamarin in Enicostemma littorale
Blume, Swertia chirata
(Wall) Clarke, and in formulations containing E. littorale
. J Planar Chromatogr 2004;17:128-31.
Dharaniyambigai K, Doss VA. Swertiamarin: A novel lead to antidepressants. Anc Sci Life 2013;32 Suppl 2:75.
Cumurcu BE, Ozyurt H, Ates O, Gul IG, Demir S, Karlidag R. Analysis of manganese superoxide dismutase (MnSOD: Ala-9Val) and glutathione peroxidase (GSH-Px: Pro 197 Leu) gene polymorphisms in mood disorders. Bosn J Basic Med Sci 2013;13:109-13.
Galecki P, Szemraj J, Bienkiewicz M, Florkowski A, Galecka E. Lipid peroxidation and antioxidant protection in patients during acute depressive episodes and in remission after fluoxetine treatment. Pharmacol Rep 2009;61:436-47.
Ghareeb DA, Hussen HM. Vanadium improves brain acetylcholinesterase activity on early stage alloxan-diabetic rats. Neurosci Lett 2008;436:44-7.
Maes M, Smith R, Christophe A, Vandoolaeghe E, Van Gastel A, Neels H, et al.
Lower serum high-density lipoprotein cholesterol (HDL-C) in major depression and in depressed men with serious suicidal attempts: Relationship with immune-inflammatory markers. Acta Psychiatr Scand 1997;95:212-21.
Rabe-Jablonska J, Poprawska I. Levels of serum total cholesterol and LDL-cholesterol in patients with major depression in acute period and remission. Med Sci Monit 2000;6:539-47.
Glueck CJ, Tieger M, Kunkel R, Tracy T, Speirs J, Streicher P, et al.
Improvement in symptoms of depression and in an index of life stressors accompany treatment of severe hypertriglyceridemia. Biol Psychiatry 1993;34:240-52.
Roohafza H, Sadeghi M, Afshar H, Mousavi G, Shirani S. Lipid profile in patients with major depressive disorder and generalized anxiety disorder. ARYA J 2005;1:15-8.
Martinac M, Karlovic D, Vrkic N, Marcinko D, Barzina N, Babic D. Serum lipids in a depressive disorder with regard to depression type. Biochem Med 2007;17:94-101.
University of Granada. Depression is More than a Mental Disorder: It Affects the Whole Organism. Science Daily; 2016.
Czeh B, Fuchs E, Wiborg O, Simon M. Animal models of major depression and their clinical implications. Prog Neuropsychopharmacol Biol Psychiatry 2016;64:293-310.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]