|Year : 2019 | Volume
| Issue : 61 | Page : 309-316
Neuroprotective potential of methanolic extract of Saraca asoca bark against doxorubicin-induced neurotoxicity
Sri Pragnya Cheruku1, Mallikarjuna Rao Chamallamudi1, Grandhi Venkata Ramalingayya1, Subhankar Biswas1, Karthik Gourishetti1, Krishnadas Nandakumar1, Raviraj Devkar2, Sanchari Basu Mallik1, Madhavan Nampoothiri1, Nitesh Kumar1
1 Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India
2 Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India
|Date of Submission||24-Feb-2018|
|Date of Decision||21-Mar-2018|
|Date of Web Publication||6-Mar-2019|
Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal - 576 104, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Doxorubicin (DOX) is a widely used anthracycline-based anticancer agent, which causes a gradual decline in cognition on long-term usage in breast cancer patients that affect their quality of life negatively. Aim: The present study was designed to assess the neuroprotective potential of Saraca asoca bark against Dox-induced neurotoxicity, considering its previously reported anticlastogenic effect. Materials and Methods: Methanolic extract of bark was prepared by Soxhlet extraction and characterized by high-performance thin-layer chromatography (catechin-8.82%). In vitro studies were performed in IMR-32 neuroblastoma cells using cell viability and neurite growth assay to determine the neuroprotective potential of the extract against DOX-induced neurotoxicity. In vivo neuroprotection study of extract (100 and 200 mg/kg p. o) was performed by assessing episodic memory through novel object recognition test (NORT) in DOX-induced (10 cycles, 2.5 mg/kg, 5 days apart) memory deficit model. In addition, antioxidant markers and acetylcholinesterase activity in hippocampus and frontal cortex were evaluated. Results and Conclusion: The extract showed significant in vitro neuroprotection toward differentiated cells in morphological features and cell viability assessment against DOX (IC50-63.35 and 45.17 μg/ml against 1 μg/ml and 2 μg/ml DOX, respectively, and 0.46 μg/ml for DOX alone in 3-[4,5 dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay). DOX-treated animals showed depletion in episodic memory while extract at 100 mg/kg significantly prevented it. Similar changes were observed in antioxidant markers levels in the brain. DOX treatment elevated acetylcholinesterase activity which was significantly reversed by the treatment with extract. Thus, it can be stated that the neuroprotective and antioxidant potentials of extract possibly underlie the efficacy against the DOX-induced neurotoxicity.
Abbreviations Used: DOX: Doxorubicin; S. asoca: Saraca asoca; DMEM: Dulbecco's Modified Eagle's Medium (DMEM); MTT: 3-[4,5 dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide; FBS: Fetal bovine serum; RA: Retinoic acid; CMC: Carboxymethyl cellulose; SOD: Superoxide dismutase; GSH: Reduced glutathione; HPTLC: High-performance thin-layer chromatography; NORT: Novel object recognition task; ITI: Intertrial interval; MPO: Myeloperoxidase; MDA: Malondialdehyde; RI: Recognition index; DI: Discrimination index.
Keywords: Chemoprotection, doxorubicin, episodic memory, novel object recognition test, Saraca asoca
|How to cite this article:|
Cheruku SP, Chamallamudi MR, Ramalingayya GV, Biswas S, Gourishetti K, Nandakumar K, Devkar R, Mallik SB, Nampoothiri M, Kumar N. Neuroprotective potential of methanolic extract of Saraca asoca bark against doxorubicin-induced neurotoxicity. Phcog Mag 2019;15:309-16
|How to cite this URL:|
Cheruku SP, Chamallamudi MR, Ramalingayya GV, Biswas S, Gourishetti K, Nandakumar K, Devkar R, Mallik SB, Nampoothiri M, Kumar N. Neuroprotective potential of methanolic extract of Saraca asoca bark against doxorubicin-induced neurotoxicity. Phcog Mag [serial online] 2019 [cited 2019 Mar 19];15:309-16. Available from: http://www.phcog.com/text.asp?2019/15/61/309/253494
- Saraca asoca at 100 mg/kg p. o showed effective protection against doxorubicin (DOX)
- Neuroprotection by modulation of cholinergic activity, oxidative, and nitrergic stress
- In vitro protection in IMR-32 against DOX and glutamate-induced toxicity.
| Introduction|| |
Doxorubicin (DOX) is widely used to treat a wide range of malignancies by multiple mechanisms such as interfering with DNA replication causing DNA damage, by inhibiting topoisomerase enzyme and also by free radical generation causing oxidative stress, thereby producing a cytotoxic effect,, In contrast to its therapeutic effect against cancer cells, the normal cells are also affected leading to various toxicities such as cumulative cardiotoxicity, hepatotoxicity, potent myelosuppression, and also cognitive decline., Impaired cognition due to chemotherapy is the biggest challenge for cancer survivors and is commonly known as “chemobrain” or “chemofog” which attributes to the disorganized mental state during or after chemotherapy in patients with cancer. It is manifested by a decrease in learning and memory, executive functioning, and information processing speed.,, Asher and Myersreported that chemobrain is prevalent in about 75% of patients with cancer during treatment while about 15%–35% cancer survivors experienced subtle cognitive decline months and years after treatment impacting their quality of life (QOL). Since the early 1990s, empirical research is being conducted on chemotherapy-induced cognitive impairment caused by antineoplastic agents.,, Over the past decade, authors reported that the patients diagnosed with breast cancer were profoundly affected with cognitive decline during and after chemotherapy.,,, Previous studies suggested that chronic administration of DOX has resulted in a cognitive decline in patients with breast cancer., Preclinically, DOX demonstrated a deteriorating effect in the cognitive domain.
Treatment-related toxicities due to chemotherapy are inevitable. Neurostimulants such as modafinil and methylphenidate, herbal supplements such as Gingko biloba, natural antioxidants such as Vitamin E are regarded as effective interventions against cognitive impairment. Although no sufficient evidence is available on the efficacy of these interventions in combating chemobrain condition.,, Preclinical studies purported that antioxidants have proved to prevent oxidative stress and implied to slow the rate of cognitive decline in aging and disease.
Saraca asoca (SA), commonly known as Saraca indica or Sita-Ashok or Ashoka, is native to India., It is purported to have various biological activities such as aphrodisiac, antibacterial, anti-oxytocic, and anti-menorrhagic activity. Apart from this, the bark of SAwas demonstrated for hemoprotective and anticlastogenic effect against cisplatin, cardioprotective effect against cyclophosphamide-induced toxicity. There are no clear reports till now indicating its neuroprotective effect against antineoplastic agents especially DOX.
DOX has demonstrated to cause cognitive impairment in healthy rodents by the mechanism of neuroinflammation, DNA damage and oxidative stress mediated by central and peripheral activation of inflammatory cytokines,, which makes it paramount to circumvent the toxic effect of chemotherapy by antioxidant supplementation. The bark of SA has been studied extensively for the accumulation of various metabolic components and is rich in catechin components. The present study is designed to standardize the methanolic bark extract of SAby quantifying the catechin components by high-performance thin layer chromatography (HPTLC) compared against a standard (+) catechin hydrate and to investigate the chemoprotective effect of the bark extract against DOX-induced neurotoxicity along with its protective effect against other major organ toxicities.
| Materials and Methods|| |
DOX (Fresenius Kabi Oncology Ltd., Solan, India), Trypsin - ethylenediaminetetraacetic acid (EDTA), dipotassium EDTA, 3-(4,5 dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), fetal bovine serum (FBS), retinoic acid (RA), gallic acid, and methanol were acquired from Himedia chemicals, India. (+) Catechin hydrate (catechin), Dulbecco's Modified Eagle's Medium (DMEM), Ellman's reagent or 5,5'-Dithiobis-(2-nitrobenzoic acid), Acetylthiocholine iodide was procured from Sigma Aldrich, USA. Sterile 96 and 6 well plates, 25 and 75 cm2 culture flasks were procured from Tarsons Product Pvt Ltd., USA.
Preparation of methanolic extract of Saraca asoca bark by Soxhlet extraction method
The bark of SAwas acquired from a local vendor in Udupi, Karnataka and authenticated by a taxonomist. The bark was desiccated at room temperature for 1 week until completely dry and ground in an electric grinder. The powder obtained was macerated in 100% methanol for 24 h followed by extraction at 50°C–55°C using hot percolation method by Soxhlet extraction. After 2 to 3 cycles, the methanolic extract obtained was evaporated to dryness under reduced pressure using rotavapor at 50°C–55°C. This process was repeated 4 to 5 times until all the components are extracted. The obtained extract is concentrated and dried at 37°C. Traces of methanol were evaporated by lyophilization at −48°C. The extract was stored in the desiccator until use.,
Quantitative analysis of catechin
Catechin was estimated in the bark extract of SA by densitometry using HPTLC using CAMAG HPTLC instrumentation system from Switzerland. A solution of (+)-Catechin hydrate (standard, from Sigma Aldrich, USA.) (100 μg/ml) and SA extract (1000 μg/ml) were prepared in methanol. Ten microliter of standard (100 μg/ml) and extract (1 mg/ml) samples were spotted in duplicates on the HPLC plate (size 20 cm × 10 cm, precoated with silica gel GF254) using automated Linomat 5 applicator under nitrogen gas flow with a band of 6 mm and the track distance of 8 mm. The chromatographic conditions were mobile phase: Toluene: Ethyl acetate: Formic acid: methanol (3:6:1.6:0.4), detection wavelength: 254 nm. The chamber (CAMAG Twin Trough) was saturated for 20 min after which, the plate was run up to 8 cm and air dried. CAMAG TLC scanner-3 with slit dimensions 6 mm × 0.45 mm and CATS evaluation software for densitometry was used for the evaluation. The percentage of catechin in the extract was calculated by the software.
In vitro cytoprotection studies
IMR-32 cell line is derived from 13-month old Caucasian male diagnosed with neuroblastoma. The cell line as purchased from NCCS, Pune, maintained and subcultured in T-25 flasks containing proliferation medium supplemented with DMEM, 10% FBS, 1% gentamicin at 37 °C in 5% humidified atmospheric CO2 incubator with 95% air. After 70%–80%, confluency was obtained, the cells were trypsinized by the addition of proliferation medium containing 0.2% Trypsin – 0.02% disodium EDTA and the viable cells counted by trypan blue exclusion were used to study the neuroprotective effect of the compounds.
3-(4,5 dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide cell viability assay in undifferentiated and differentiated IMR-32 cell line
Five-thousand cells/well were seeded in microplate consisting of 50 μl of proliferation medium and incubated overnight. After 24 h, from the stock solutions of catechin hydrate (standard) and SA(50 mg/ml), 50 μl of concentrations ranging from 31.23 to 250 μg/ml was added to the wells, and the cells were pretreated for an hour followed by the addition of 50 μl of DOX (1 and 2 μg/ml). After a 24 h treatment, 50 μl of MTT was added and incubated for 3 h, and then, the medium was discarded. Formed blue formazan crystals observed under the microscope was dissolved in 100 μl of dimethyl sulfoxide, and the absorbance was read at 540 nm. The protective effect of pretreated compounds against DOX toxicity was evaluated. Along with this, cytoprotective effect of standard catechin hydrate, plant extract SA, and DOX at different concentrations were evaluated individually by the same method. IC50 of the percentage cell viability of the compounds were obtained.
Evaluation of neuroprotection against Doxorubicin and Glutamate-induced neurotoxicity in differentiated IMR-32 cell line
Two million cells/well were seeded in 6-well plates supplemented with proliferation medium overnight, after which the media was replaced by differentiation medium containing DMEM, 5% FBS for first 3 days, 15 μM alltrans RA (ATRA), 1% gentamicin, thereby arresting proliferation and promoting neuronal differentiation. Depending on the extent of differentiation, the complete media was replaced by serum-free differentiation media. The cells were used for experimentation after 12 days of differentiation into long neurites extensions observed under an inverted microscope. The cells were supplemented with fresh media every 3 days. Glutamate (0.5 mM) and DOX (1.5 mg/ml) were used to induce neurotoxicity in the neuronal cells and investigated for the neuroprotective effect of SA(60 μg/ml) based on the IC50 values of the compounds obtained in MTT assay. L-glutamate has shown its neurotoxicity at 0.5 mM concentration. The cells were treated for 48 h, after which, the medium was removed, and the cells were washed and replaced with phosphate-buffered saline.
The cells were observed for the effect of compounds on the neurite length which was viewed under an inverted microscope at × 40 objective, and the images were taken. The neurite length defined as a straight-line distance from the tip of the neurite to the junction between the cell body and the neurite base expressed in μm. It was calculated for the treatment groups and evaluated the neuroprotective effect.
In vivo studies
The study was conducted after obtaining Ethical Approval from the Institutional Animal Ethics Committee, Kasturba Medical College, Manipal University (No: IAEC/KMC/80/2014). Six months old healthy male Wistar rats, weighing 200–250 g were procured from the Central Animal Research Facility of Manipal University, Manipal as per CPCSCEA guidelines. The animals could acclimatize for 1 week before the initiation of the experiment and maintained under controlled conditions of temperature and humidity with a 12 h day and night cycle with free access to rat pellet diet and water ad libitum. The animals were randomized according to their body weight.
Doxorubicin-induced memory deficit model
The animals were divided into five treatment groups (n = 6 each), Group I served as vehicle control, administered with 0.25% carboxymethylcellulose (CMC) p. o. and saline intraperitoneallyGroups II, III, and IV were induced with DOX as per protocol. Group II served as disease control and was administered with 0.25% (CMC) p. o. Groups III and IV were pretreated with plant extract 100 and 200 mg/kg p. o.prepared in 0.25% CMC. The animals were pretreated 45 min before the administration of DOX (2.5 mg/kg i.p.). Ten cycles of DOX (Adriamycin) 2.5 mg/kg, freshly prepared in distilled water was administered i.p.every 5th day for 57 days at a dosing volume of 2 ml/kg body weight.
The animals pretreated with SA bark extract 1 week before the first cycle of DOX induction. On day 57, at the last cycle of DOX, the animal behavior toward the novel and familiar objects in an open field arena of size 49 cm × 49 cm × 49 cm was observed at fixed time intervals by novel object recognition task (NORT). The behavior was monitored with the help of a camera (model: QuickCam Pro9000, Logitech International S. A., Lausanne, Switzerland) mounted 160 cm above the observation arenas, two stopwatches, and a timer alarm. The study was carried out between 9 am and 4 pm. The animals could habituate to the observation arena during the habituation phase. The familiarization and choice trials with an intertrial interval (ITI) of 4 h was performed. During the familiarization, trial animals could explore two similar objects (a1 and a2). After an ITI, a choice trial was conducted during which a familiar and a novel object was placed (a3 and b). The exploration time, recognition and discrimination indices (DIs) of an animals' on the familiar and novel objects were calculated. Immediately after the study, animals were anesthetized using ketamine. The brain was surgically removed, and hippocampus (H) and frontal cortex (FC) areas were isolated for further biochemical estimations.
Assessment of oxidative and nitrosative stress markers
Oxidative stress and nitrosative markers which include catalase, superoxide dismutase (SOD), reduced glutathione (GSH) and lipid peroxidation, nitrite levels, and myeloperoxidase (MPO) levels were estimated in the H and frontal cortex.
Estimation of ex vivo and in vivo acetylcholinesterase activity
The brain tissue homogenates were estimated for the protective effect of standard Catechin hydrate and SAbark extract against DOX-induced toxicity by the UV-spectrophotometric method. A volume of 0.02 ml of acetylthiocholine iodide, 0.1 ml of DTNB, 2.6 ml of phosphate buffer (pH 8), and 0.4 ml of tissue supernatant was added directly to the cuvette. The change in the absorbance was recorded at 412 nm at a time interval of the 60 s for 4 min by the UV-kinetic method.
Estimation of ex vivo acetylcholinesterase activity was performed in untreated rat brain, which was isolated and homogenized at specified conditions by ice-cold 0.1 M phosphate buffer. The brain supernatant collected was incubated at different concentrations with standards donepezil and catechin hydrate ranging from 1000 μM to 31.25 μM, and extract SA ranging from 1000 to 31.25 μg/ml for 45 min with the brain tissue supernatant after which acetylcholinesterase activity was estimated. The result was expressed as micromoles of acetylthiocholine iodide hydrolyzed per min per mg of tissue.
Estimation of in vivo acetylcholinesterase activity was performed in the treated tissue samples. The result was expressed as micromoles of acetylthiocholine iodide hydrolyzed per min per mg of protein.
Data were statistically analyzed using Graph Pad Prism 5.0 software. Results of the in vitro and in vivo studies were expressed as mean ± SEM. Statistical comparisons for novel object recognition task results were analyzed by nonparametric test and paired Student's t- test, two-tailed, Kruskal–Wallis test followed by post hoc Dunnett's multiple comparison tests. All the other results were analyzed by one-way ANOVA followed by post hoc Dunnett's Multiple Comparison tests. The DOX-treated group was compared with normal control group. P < 0.05 was considered statistically significant at 95% confidence interval.
| Results|| |
Densitometric evaluation of (+) catechin by high-performance thin-layer chromatography
Methanolic extract of SAbark showed 8.82% of catechin by (HPTLC) [Figure 1].
|Figure 1: Characterization of extract by high-performance thin-layer chromatography. Densitometric evaluation of standard catechin (a), catechin in methanolic extract of Saraca asoca bark (b)|
Click here to view
In vitro studies
Effect of methanolic bark extract ofSAon cell viability in IMR-32 cells:SA bark extract has shown an IC50 of 494 μg/ml on the undifferentiated IMR-32 cell line. Dox showed IC50 value 0.48 μg/ml. Bark extract of SAdefended the cell death. The IC50 of bark extract along with 1 and 2 μg/ml DOX was 63.35 and 45.17 μg/ml, respectively, on the differentiated IMR-32 cell line.
Effect of treatment on DOX and glutamate-induced neurotoxicity in differentiated IMR-32 cells The differentiated cells were treated with bark extract SAalong with DOX (1.5 μg/ml) and glutamate (0.5 mM) and incubated for 24 h after which the cells were observed under an inverted microscope for the morphological alterations. There was significant toxicity in IMR-32 cells after treatment with glutamate and DOX alone. The cells were found degenerated, rounded, and detached. This degeneration caused due to the toxicants were significantly protected by treatment with extract at 60 μg/ml. Microscopically, images showed a very few numbers of cell bodies in DOX alone treatment group and DOX and S. acoca combination treatment group and a wide number of intact cells [Figure 2].
|Figure 2: Effect of the extract against doxorubicin and Glutamate-induced morphological changes on IMR-32. Effect of doxorubicin alone and glutamate alone; doxorubicin + treatment and glutamate + treatment on the length of the neurites. ###P < 0.001 versus normal control; ***P < 0.001 versus doxorubicin control; *P < 0.05 versus glutamate control|
Click here to view
In vivo pharmacology (doxorubicin-induced memory deficit model)
In DOX-induced memory deficit model, chronic DOX treatment for 50 days induced episodic memory deficits which were demonstrated by the significant reduction in the exploration time of the novel object compared to saline and CMC treated groups (P < 0.001) which was also evident by recognition index (RI) and DI (P < 0.01 RI; P < 0.001; DI). Animals pretreated with bark extract at 100 mg/kg (P < 0.001) showed a significant increase exploration time of novel object (P < 0.001) and significant RI (P < 0.05) and DI (P < 0.01) comparable to DOX and saline-treated rats. However, animals treated at a higher dose of bark extract SA200 mg/kg p. o. explored both novel and familiar objects almost equally [Table 1].
|Table 1: Novel object recognition test in doxorubicin-induced memory deficit model|
Click here to view
Effect of treatment on ex vivo and in vivo acetylcholinesteraseactivity
Ex vivo treatment of bark extract of SAdemonstrated potent acetylcholinesterase inhibition potential at an IC50 of 36.91 μg/ml compared to standards donepezil and catechin hydrate with an IC50 of 190.18 μM (79 μg/ml) and 72.85 μM (21.46 μg/ml), respectively [Table 2].
In vivo chronic administration of DOX increased the AchE activity in hippocampal samples (P < 0.001) compared to the vehicle group. Pre-treatment with bark extract significantly prevented the toxic effect of DOX. t. A similar trend was evident in FC samples; however, the result was not significant [Table 2].
Effect of treatment on oxidative stress markers in brain
Chronic DOX administration significantly increased the malondialdehyde (MDA) levels compared to the vehicle control in H (P < 0.01) and FC (P < 0.001) samples which was significantly reversed by bark extract at 100 mg/kg (P < 0.01 [H]; P < 0.001 [FC]) and 200 mg/kg p. o.(P < 0.001, H; P < 0.01, FC). A trend in the decrease in CAT and GSH antioxidant levels due to DOX administration was observed which was improved by treatment with bark extract at doses 100 and 200 mg/kg. SOD levels significantly decreased in FC samples due to DOX. However, there was no effect on SOD levels of H due to DOX and treatment [Table 3].
Effect of treatment on myeloperoxidase and nitrite in brain
There was a notable increase in nitrite and MPO levels by DOX treatment in the H and FC which was significantly reversed by treatment with bark extract at 100 and 200 mg/kg [Table 3].
| Discussion|| |
There are no clinically proven treatments to prevent cognitive decline due to chemotherapy. Antioxidants supplements have known to prevent oxidative stress peripherally and delay the process of degeneration in the brain. SAbark extract is known for its antioxidant and anticancer activity. This may be one such alternative for enhancing the quality of life in the patients undergoing DOX chemotherapy. As in the earlier studies, it has shown the myeloprotective effect against cisplatin and cardioprotective effect against cyclophosphamide in rodents. With this evidence, we further evaluated the extract for its potential anti-chemo brain activity in an established DOX-induced chemobrain model for episodic memory.
Earlier studies have confirmed that the bark of SAis composed of chemical constituents such as catechin, (-)-epicatechin, epiafzelechin-(4β-8)-epicatechin, procyanidin B2, tannins, lignin glycosides, steroids, saponins, leukocyanidin, leukopelargonidin, and β-sitosterol. Previous research shows that flavan-3-ols (catechins), a subclass of flavonoids is one type of phenolic components known to have medium polarity. For the extraction of phenolic components, most widely used solvents are water, methanol, ethanol, and acetone. Among them, methanol is considered as the best suitable solvent for extraction of phenolic compounds, since, it is medium polar in nature, and mediates considerable extraction efficiency of antioxidant compounds. In light of the previous research, methanol was proved to be the suitable solvent for extraction of catechin components from Saraca asoca bark.,, Our study standardized the SAextract based on the presence of catechin by HPTLC analysis. Catechin was detected in the methanolic bark extract comparable to standard (+) catechin hydrate at Rf and peak area of 0.73 and 8.82%. Polyphenolic components, such as catechin, epicatechin, and other polyphenols, are known to be potent natural antioxidants. In an earlier study, we found that catechin ameliorated DOX-induced neurotoxicity and prevents the episodic memory loss. Geetha et al. reported antimutagenic properties of epicatechin. In addition, it showed profound improvement in learning and spatial memory in mice and also improved cognition in older adults. Thus, the potent antioxidant property of the extract strengthens the hypothesis that the extract may be a potent chemoprotective agent against DOX.
The in vitro study was conducted in IMR-32 human neuroblastoma cell line to evaluate the protective effect against DOX-induced neurotoxicity. Previous literature indicates that the IMR-32 neuroblastoma cell line has been studied for neuroprotective evaluation., The cell viability assay conducted on undifferentiated IMR-32 cell line was to estimate the toxicity profile of the bark extract. An IC50 of 494 μg/ml concentration implies that the bark extract may not be cytotoxic in nature at such high concentration; therefore, it may not interfere with antitumor properties of DOX. Cassano et al. and Silvis et al. demonstrated that the cell line showed normal neuronal phenotypic differentiation and formation of neurite extensions when treated with ATRA., Induction with ATRA manifested formation of neurite extensions. SAbark extract at 60 μg/ml along with DOX at 1.5 μg/ml showed comparative retention in neurite length in comparison to the DOX alone treated well. The presence of apoptotic cell bodies in treatment wells, however, less when compared to DOX alone, could be due to DOX treatment. A similar course was observed in studies conducted to evaluate neuroprotective potential against DOX. Comparatively fewer cell bodies and presence of more neurite growth extensions purport the extract's protective effect (P < 0.001). In addition, the treatment showed significant reversal (P < 0.01) against glutamate-induced neurotoxicity. DOX mediates TNF-alpha induced upregulation of the excess release of glutamate from hemichannels leading to glutamate-induced excitotoxicity causing neurodegeneration. The conducted in vitro study confirmed its neuroprotective effect of the extract against glutamate, demonstrating its possible role in preventing glutamate-induced excitotoxicity. Furthermore, this can be proposed for molecular evaluation studies to identify the possible protective mechanism.
An in vivo study was conducted to evaluate the effect of extract in counteracting effect DOX systemic toxicity mainly by cognitive decline mediated by oxidative and nitrergic stress. The study was conducted using 50 days treatment including 10 cycles, given every 5 days at a dose of 2.5 mg/kg. The chronic exposure of DOX for 57 days produced a reduction in body weight, comparable to the vehicle although it was not significant. This was significantly reversed by treatment with the SA(100 mg/kg) treatment.
Episodic memory is mainly associated with the H and FC. Most of the conscious learning is performed by the declarative memory function in the H and FC, which is said to be temporally dated conscious recollection of past experiences and events. The animals' ability to recollect past experiences (episodic memory) is evaluated by NORT in which the animals were allowed to explore two familiar objects in the familiarization trial followed by choice trial after anITI. In the choice trial, one object was replaced with a novel object and the ability to explore and discriminate the objects was evaluated. The animals irrespective of the treatment tend to explore the two similar objects for almost equal time during the familiarization phase. However, the animals which were able to remember the familiar object were able to discriminate the novel object and spent significantly more time exploring a novel object. In DOX study, animals were treated for 57 days, after which the episodic memory was assessed. An ITIof 4 h was given which was standardized in our laboratory. DOX-treated animals showed insignificant difference in exploration time of the novel or familiar object. This signifies that the animal failed to recognize the previously encountered familiar objects. Vehicle-treated animals were able to discriminate the novel object from the familiar one. Treatment with extract at 100 mg/kg p. o. showed significantly more exploration time toward the novel object (P < 0.001) compared to familiar one and reversed the impairment caused due to DOX. This suggests the beneficial effect of SAagainst DOX-induced cognitive impairment. However, the higher dose of extract, DOX + SA200 mg/kg did not reverse the impairment caused due to DOX as the animals failed to recognize the novel object over the familiar object.
Cholinergic system in the brain is mainly responsible for learning and memory function, which contributes to the cognitive ability. Acetylcholinesterase is an enzyme responsible for the degradation of acetylcholine terminating the cholinergic transmission. DOX causes neurodegeneration leading to impairment in the cognitive domain which makes it necessary to evaluate the acetylcholinesterase activity in the brain. Chronic exposure of the toxicant may attribute to the decreased acetylcholine levels in the body due to a decrease in the choline uptake due to neurodegeneration in the H and FC. According to Agamy et al. suggested a possible mechanistic role of DOX-induced oxidative and nitrosative stress which might abnormally increase AchE levels implying an increased degradation of acetylcholine which could be one of the pathways of DOX-induced neurotoxicity. Ex vivo acetylcholinesterase inhibition activity study signified potential degradation of substrate acetylthiocholine iodide when treated with SA, determined by intense yellow-colored formed byproduct, thionitrobenzoic acid. This entails a possible acetylcholinesterase inhibition potential of SA. After chronic exposure of DOX, a similar activity was shown. S. acosa had significantly reduced the AchE activity in the H which could be due to its potent anti-oxidant activity indicating the restoration of cognitive function.
Oxidative stress is one of the major pathways for the neurodegenerative disorders such as Alzheimer's disease. Most of the cancer chemotherapeutic drugs including DOX produce cognitive decline by producing oxidative stress and nitrergic stress. DOX will undergo redox cycling and absorbed into the body not only affects the cancer cells but also the normal cells. This effect in the brain leads to neuroinflammation and mitochondrial dysfunction leading to apoptosis and neurodegeneration. In the present study, oxidative stress markers such as catalase, SOD, GSH, MDA and inflammatory markers, nitrite, and MPO levels were evaluated. DOX increased the oxidative and nitrosative stress which was significantly ameliorated by SA.
| Conclusion|| |
SAbark extract showed an improvement in episodic memory which was impaired by DOX treatment. The neuroprotective and antioxidant potential of the test extract possibly underlies the efficacy to reverse cognitive deficits associated with the DOX-induced chemobrain like condition. SA extract at 100 mg/kg p.o. showed better chemoprotective activity against extract at 200 mg/kg, p.o.
The authors would like to thank Manipal Academy of Higher education, Manipal, for providing infrastructural facility to conduct the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Aluise CD, Sultana R, Tangpong J, Vore M, St. Clair D, Moscow JA, et al.
Chemo brain (chemo fog) as a potential side effect of doxorubicin administration: Role of cytokine-induced, oxidative/nitrosative stress in cognitive dysfunction. Adv Exp Med Biol 2010;678:147-56.
Goodman LS. Goodman and Gilman's the Pharmacological basis of Therapeutics. New York: McGraw-Hill; 1996.
Rang HP. Rang and Dale's Pharmacology. Philadelphia: Churchill Livingstone; 2007. p. 672-4.
Keeney JT. Doxorubicin-induced, TNF-α-mediated Brain Oxidative Stress, Neurochemical Alterations, and Cognitive Decline: Insights Into Mechanisms of Chemotherapy Induced Cognitive Impairment and Its Prevention; 2013.
Fardell JE, Vardy J, Johnston IN, Winocur G. Chemotherapy and cognitive impairment: Treatment options. Clin Pharmacol Ther 2011;90:366-76.
Janelsins MC, Kesler SR, Ahles TA, Morrow GR. Prevalence, mechanisms, and management of cancer-related cognitive impairment. Int Rev Psychiatry 2014;26:102-13.
Asher A, Myers JS. The effect of cancer treatment on cognitive function. Clin Adv Hematol Oncol 2015;13:441-50.
Boykoff N, Moieni M, Subramanian SK. Confronting chemobrain: An in-depth look at survivors' reports of impact on work, social networks, and health care response. J Cancer Surviv 2009;3:223-32.
Carvalho C, Santos RX, Cardoso S, Correia S, Oliveira PJ, Santos MS, et al.
Doxorubicin: The good, the bad and the ugly effect. Curr Med Chem 2009;16:3267-85.
Reid-Arndt SA, Hsieh C, Perry MC. Neuropsychological functioning and quality of life during the first year after completing chemotherapy for breast cancer. Psychooncology 2010;19:535-44.
Ahles TA, Saykin AJ. Candidate mechanisms for chemotherapy-induced cognitive changes. Nat Rev Cancer 2007;7:192-201.
Wagawade JD, Roy S, Hullatti K, Khode NR, Pednekar H, Hegde HV, et al
. Doxorubicin induced cognition impairment in rat model. Asian J Pharm Clin Res 2015;8:1-4
Barton DL, Burger K, Novotny PJ, Fitch TR, Kohli S, Soori G, et al.
The use of Ginkgo biloba
for the prevention of chemotherapy-related cognitive dysfunction in women receiving adjuvant treatment for breast cancer, N00C9. Support Care Cancer 2013;21:1185-92.
Gill M, Rai A, Kinra M, Sumalatha S, Rao CM, Cheruku SP, et al.
Chemically characterised extract of Saraca asoca improves the sexual function in male wistar rats. Andrologia 2018;50:e13037.
Panchawat S, Sisodia S.In vitro
antioxidant activity of Saraca asoca
Roxb. De wilde stem bark extracts from various extraction processes. Asian J Pharm Clin Res 2010;3:23.
Varghese C, Nair S, Panikkar K. Chemoprotection of Saraca asoca
extracts on cisplatin induced toxicity in mice. Amala Res Bull 1992;12:86-91.
Viswanatha Swamy AH, Patel UM, Koti BC, Gadad PC, Patel NL, Thippeswamy AH, et al.
Cardioprotective effect of Saraca indica
against cyclophosphamide induced cardiotoxicity in rats: A biochemical, electrocardiographic and histopathological study. Indian J Pharmacol 2013;45:44-8.
Mittal A, Kadyan P, Gahlaut A, Dabur R. Nontargeted identification of the phenolic and other compounds of Saraca asoca
by high performance liquid chromatography-positive electrospray ionization and quadrupole time-of-flight mass spectrometry. ISRN Pharm 2013;2013:293935.
Gill M, Kinra M, Rai A, Chamallamudi MR, Kumar N. Evaluation of antidepressant activity of methanolic extract of Saraca asoca
bark in a chronic unpredictable mild stress model. Neuroreport 2018;29:134-40.
Yadav NK, Saini KS, Hossain Z, Omer A, Sharma C, Gayen JR, et al. Saraca indica
bark extract shows in vitro
antioxidant, antibreast cancer activity and does not exhibit toxicological effects. Oxid Med Cell Longev 2015;2015:205360.
Rathee P, Rathee S, Rathee D, Rathee D. Quantitative estimation of (+)-Catechin in stem bark of Saraca asoka
Linn using HPTLC. Der Pharm Chem 2010;2:306-14.
Kataria H, Wadhwa R, Kaul SC, Kaur G. Water extract from the leaves of withania somnifera protect RA differentiated C6 and IMR-32 cells against glutamate-induced excitotoxicity. PLoS One 2012;7:e37080.
Cheruku SP, Ramalingayya GV, Chamallamudi MR, Biswas S, Nandakumar K, Nampoothiri M, et al.
Catechin ameliorates doxorubicin-induced neuronal cytotoxicity in in vitro
and episodic memory deficit in in vivo
in wistar rats. Cytotechnology 2018;70:245-59.
Miguel MG, Nunes S, Dandlen SA, Cavaco AM, Antunes MD. Phenols, flavonoids and antioxidant activity of aqueous and methanolic extracts of propolis (Apis mellifera
L.) from Algarve, South Portugal. Food Sci Technol (Campinas) 2014;34:16-23.
Widyawati PS, Budianta TD, Kusuma FA, Wijaya EL. Difference of solvent po-larity to phytochemical content and antioxidant activity of Pluchea indicia
less leaves extracts. Int J Pharmacognosy Phytochem Res 2014;6:850-5.
Andersen OM, Markham KR. Flavonoids: Chemistry, Biochemistry and Applications. United States: CRC Press; 2005.
Geetha T, Garg A, Chopra K, Pal Kaur I. Delineation of antimutagenic activity of catechin, epicatechin and green tea extract. Mutat Res 2004;556:65-74.
van Praag H, Lucero MJ, Yeo GW, Stecker K, Heivand N, Zhao C, et al.
Plant-derived flavanol (-) epicatechin enhances angiogenesis and retention of spatial memory in mice. J Neurosci 2007;27:5869-78.
Brickman AM, Khan UA, Provenzano FA, Yeung LK, Suzuki W, Schroeter H, et al.
Enhancing dentate gyrus function with dietary flavanols improves cognition in older adults. Nat Neurosci 2014;17:1798-803.
Ramalingayya GV, Cheruku SP, Nayak PG, Kishore A, Shenoy R, Rao CM, et al.
Rutin protects against neuronal damage in vitro
and ameliorates doxorubicin-induced memory deficits in vivo
in wistar rats. Drug Des Devel Ther 2017;11:1011-26.
Shivapriya S, Ilango K, Dubey GP. Evaluation of antioxidant and neuroprotective effect of Hippophae rhamnoides
(L.) on oxidative stress induced cytotoxicity in human neural cell line IMR32. Saudi J Biol Sci 2015;22:645-50.
Silvis AM, McCormick ML, Spitz DR, Kiningham KK. Redox balance influences differentiation status of neuroblastoma in the presence of all-trans retinoic acid. Redox Biol 2016;7:88-96.
Cassano S, Agnese S, D'Amato V, Papale M, Garbi C, Castagnola P, et al.
Reactive oxygen species, Ki-ras, and mitochondrial superoxide dismutase cooperate in nerve growth factor-induced differentiation of PC12 cells. J Biol Chem 2010;285:24141-53.
Antunes M, Biala G. The novel object recognition memory: Neurobiology, test procedure, and its modifications. Cogn Process 2012;13:93-110.
El-Agamy SE, Abdel-Aziz AK, Wahdan S, Esmat A, Azab SS. Astaxanthin ameliorates doxorubicin-induced cognitive impairment (Chemobrain) in experimental rat model: Impact on oxidative, inflammatory, and apoptotic machineries. Mol Neurobiol 2018;55:5727-40.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]