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Neuroprotective behaviour of Phyllanthus emblica (L) on human neural cell lineage (PC12) against glutamate-induced cytotoxicity
Gene Reports 17 (2019) 100545
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Neuroprotective behaviour of Phyllanthus emblica (L) on human neural cell lineage (PC12) against glutamate-induced cytotoxicity
T
⁎
S. Rajalakshmia, S. Vijayakumara, , P.K. Praseethab a b
Computational Phytochemistry Lab, PG and Research Department of Botany and Microbiology, A. V. V. M Sri Pushpam College (Autonomous), Poondi, Tamil Nadu, India Department of Nanotechnology, Noorul Islam Centre for Higher Education, Kumaracoil, Kanyakumari district, Tamil Nadu 629180, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Phyllanthus emblica Neuroprotective Anti-oxidant Glutamate PC 12 cell line
Phyllanthus emblica (L) is an edible, nutrient-rich Indian gooseberry with wide distribution in sub-tropical and tropical belts of India, Indonesia, China, and Malaysia, belonging to the family of Phyllanthaceae and is well known for its traditional medicinal properties. The current study aims to investigate the neuroprotective and antioxidant effects of P. emblica. Radical scavenging action was evaluated by DPPH method, and its hydroxyl moiety scavenging and lipid peroxidation activity were also investigated. Its role in neuroprotection was analyzed on the human neural cell lines (PC12) against glutamate induced cellular inhibition. The cell line study was performed by measuring the viability of cells through MTT assay, and through monitoring LDH activity, GSH and ROS levels. In the current study, results confirmed that P. emblica extract had excellent antioxidant activity. DPPH and hydroxyl radical scavenging assays revealed IC50 levels of 73.21 μg/mL and 0.426 mg/mL, respectively. Significant lipid peroxidation activity (73.21 μg/mL) was also observed. The neuroprotective effect of P. emblica on cell lines was confirmed by cell viability assay after through glutamate-induced cell cytotoxicity.
1. Introduction In the present society, people lead stressful lives, due to lack of physical movement, restlessness of the mind and anxiety. These factors are closely related to the progress of many diseases, including neurodegenerative diseases (Vijayakumar et al., 2016). A large number of deaths have occurred worldwide during the 21st century due to neurodegenerative diseases, such as cognitive dysfunction, neuropsychiatric, and various nervous disorders such as Huntington's, Parkinson's and Alzheimer's disease (Essa et al., 2014). Parkinson's disease falls among the second most neural disease, which completely reduces the quality of life (Jankovic, 2008). Parkinson's disease is characterized by different symptoms like rigidity, bradykinesia, postural instability or facial dyskinesia, muscular stiffness and tremors (Magrinelli et al., 2016). Few medicinal plant extracts have been considered as potential sources of bioactive compounds with neuroprotective effects (Shivapriya et al., 2015). P. emblica (L) is a plant belonging to the Phyllanthaceae family and well known for its medicinal value. It is popular all around the world due to its nutraceutical properties. Many studies have reported that P. emblica has antimicrobial (Ahmad et al.,
1998), antioxidant (Chatterjee et al., 2011; Sumitra et al., 2009) antiinflammatory (Golechha et al., 2011), analgesic and antipyretic (Mythilypriya et al., 2007; Perianayagam et al., 2004) adaptogenic (Baliga et al., 2012) hepatoprotective (Gulati et al., 1995) antitumor (Suresh and Vasudevan, 1994), and anti-ulcerogenic (Sairam et al., 2002) properties. Additionally, the literature suggests P. emblica as a potent antioxidant. Based on this information, through this study we could evaluate the neuroprotective and antioxidant functions of P. emblica using the human PC12 Parkinson's cell line. 2. Materials and methods 2.1. Chemicals The microbial culture media, chemicals and reagents required for the study were purchased from Standard Chemical companies like Sigma Aldrich, HiMedia Laboratories and E. Merck respectively. 2.2. Sample collection The fruits of P. emblica were collected during the months of March,
Abbreviations: FBS, fetal bovine serum; PBS, phosphate buffered saline; DMEM, Dulbecco's modified Eagle's medium; MFEPE, methanolic fruit extract of Phyllanthus emblica; MTT, 3,-4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide; LDH, lactate dehydrogenase; GSH, glutathione; ROS, reactive oxygen species ⁎ Corresponding author. E-mail address: [email protected] (S. Vijayakumar). https://doi.org/10.1016/j.genrep.2019.100545 Received 13 September 2019; Received in revised form 1 October 2019; Accepted 22 October 2019 Available online 23 October 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
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500 nm.
and April 2016, from the Botanical Garden of A.V.V. M Sri Pushpam College, Poondi, Thanjavur district. The plant material was authenticated as being from P. emblica by Rapinot Herbarium, St. Joseph's College, Trichirappalli, India. The plant voucher specimen (PHC1126) was deposited to Pushpam Herbarium Cabinet (PHC), Department of Botany and Microbiology, A.V.V. M Sri Pushpam College, Tamil Nadu, India.
2.7. In-vitro assay and drug treatment Cell cultures were obtained from the National Centre for Cell Sciences (NCCS), Pune, India. The cells were further sub-cultured in DMEM with an added concentration of 10% FBS, penicillin at 100 IU/ mL, streptomycin at 100 mg/mL and amphotericin b at 5 mg/mL. The PC12 cells were cultured for 24 h in 96 well microplates. Then, the culture medium was discarded and the cells were subjected to various concentrations (250, 500, and 1000 μg/mL) of the fruit extract. After that, 100 μL of the fruit extract was diluted in DMEM and added to each well at a concentration of 1 μg/mL. Finally, the plates were incubated for 2 h in humidified conditions with 5% CO2 at 37 °C.
2.3. Preparation of plant extract Fresh fruits collected were washed, cleaned, and shade dried before the synthesis. The dried fruits were powdered using a blender. Then, 100 g of dried fruit powder was mixed well with 1000 mL of 95% methanol, and the resulting mixture was incubated at 25 °C for 72 h. The extract was subjected to filtration through Whatman No. 1 filter paper, and vacuum concentrated with a rotatory evaporator at 40 °C. Then, extracts were stored for further experiments.
2.8. MTT assay The cultured cells in a monolayer fashion was trypsinized, then the cell count was made up to 1.0 × 105 cells/mL with 10% FBS. FBS containing medium was used to determine cell viability via MTT assays as described by Salameh et al. (2010). Absorbance was recorded with a micro-plate reader (540 nm wavelengths). The various concentrations of the test drug required to inhibit the cell growth by 50% (CTC50) were read from the obtained graphs for dose-response from each cell line, and the percentage of inhibition in growth was calculated using the given equation:
2.4. Measurement of free radical-scavenging activity using DPPH assays The antioxidant action of plant extract was determined according to the methodology of Payet et al. (2005). The assay was performed using by a methanolic solution (0.004%). Two hundred microliters of the solvent were transferred to a microplate of 96 wells, with an addition of 20 μL of P. emblica fruit extract, solvent, or control substance. The experimentation mixture was vigorously shaken and further incubated at 30 °C for 1 h. The absorbance was measured at 517 nm using in a microplate reader. The inhibition percentage was calculated as below,
Percentage of Growth inhibition 100 − Mean OD of individual test group = mean OD of the control group.
Inhibition% = 100 − 100 (AS ÷ A O) where
2.9. LDH assay
AO represents the absorbance of blank solution, AS represents the absorbance of the sample (517 nm).
Inhibition% = 1 − (AS–AW/AO) × 100
The cell lines were plated at a concentration of (1.5–2) × 104 cells/ well in 96-well plates, and were kept aside for attachment 24 h before treatment. A single row of wells contained only the medium required for background normalization. Varied concentrations of Methanolic fruit extract of P. emblica (MFEPE) were added successively for 8 h after inducing the toxicity with glutamate for 24 h. The lactate dehydrogenase (LDH) supernatant was gathered for analysis in a cell-free medium. The pellets were lysed in 0.5 mL with a lysis buffer (0.5% Triton X-100) and 0.1 M potassium phosphate buffer at pH 7.0 to release the intracellular LDH to the extracellular medium. The medium was measured to determine the LDH activity which is represented as percentage of total cellular action. The absorbance was measured at 340 nm via a model Digiscan Microplate reader, and the following formula was used to calculate for the percentage of LDH released:
where,
Percentage of LDH released
2.5. Measurement of hydroxyl radical scavenging activity Hydroxyl radical scavenging activity was measured by adopting the methodology of Klein et al. (1981) with minor modifications. Two milliliters of 6 mM ferrous sulfate of different concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mg/mL) were added to 2 mL of the P. emblica fruit extract. The mixture was kept undisturbed for 10 min at room temperature. Then, 2 mL of 6 mM sodium salicylate was added, followed by incubation at 37 °C for 30 min. After incubation, the absorbance was measured at 510 nm. The inhibition percentage was calculated as below,
= (Experimental LDH activity)/(Maximum LDH activity) × 100.
AS represents the absorbance of the sample with sodium salicylate, AW represents the absorbance of the sample without sodium salicylate, AO represents the absorbance of blank solution,
2.10. GSH measurement Cells were inoculated at a density of 4 × 104 cells/cm2 in poly-Dlysine-coated (0.05 mg/mL) flasks of volume 75 cm2 (5 mL/flask) for measuring GSH. After 24 h, different concentrations of P. emblica methanolic fruit extracts were added to the flasks which were then incubated at 37 °C. After the completion of incubation, the cells were harvested from the cultures and homogenized with serine borate buffer (100 mM Tris-HCl, 10 mM boric acid, 5 mm L-serine, 1 mM DETAPAC, pH 7.4). Twenty micromolars of the diluted cell homogenate were mixed with 230 μM of serine borate buffer followed by 750 μM of N-(1pyrenyl) meleimide (NPM) (1 mM in acetonitrile). The solutions were maintained at 35 ± 2 °C for 5 min. The reaction was halted by adding 5 μL of 2 M HCl. The samples were further filtered through a 0.2 μm
2.6. Measurement of lipid peroxidation activity Lipid peroxidase inhibition was performed using a ferric thiocyanate assay (Kikuzaki and Nakatani, 1993). The assay was inhibited by adding 4 mL of ethyl alcohol, 2.51% linoleic acid mixed in absolute ethanol (4.1 mL), 0.05 M phosphate buffer maintained at pH 7.0 (8 mL), in distilled water (3. 9 mL) to a vial with a screw cap. The vial was kept in an oven at 40 °C in the dark. Then, 0.1 mL of the resulting solution was mixed with 9.7 mL of 75% ethanol and 0.1 mL of 30% ammonium thiocyanate. After 3 min, 0.1 mL of 0.02 M ferrous chloride solution was added to 3.5% hydrochloric acid. The absorbance was observed at 2
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filter (Acrodisc) and then injected into the HPLC column. At the cellular level, GSH was determined using the standard procedure described by Park et al. (2003).
(Fig. 1C). In 1923, the terminology-Siddha medicine was framed by the British. Based on the Siddha system, Indian gooseberry possesses an assortment of medicinal properties. Classic manuals (Purananuru) mention the anti-ageing effect of P. emblica. Many studies have reported that Indian gooseberry has excellent antioxidant effect owing to its high flavonoid, polyphenols and tannin contents. Due to the increasing interest in the relationship between antioxidant and diseases, DPPH, hydroxyl radical scavenging assays and lipid peroxidation assay is a crucial factor for assessing antioxidant activity. The results of this study showed that P. emblica extracts possesses excellent radical scavenging ability. These results correlate with those of Akowuah et al. (2005), who reported that P. emblica extract has significant antioxidant activity, using DPPH and FRAP assays.
2.11. Intracellular ROS measurement The dye 2, 7-dichlorofluorescein diacetate (DCFDA), which is oxidized to dichlorofluorescein (DCF) by hydroperoxides, was used to measure the relative levels of cellular peroxides (Surronen et al., 2000). After 24 h cells were treated with various concentrations of MFEPE and the cells were exposed to 10 μg of glutamate for 6 h. At the end of treatment 10 μg of DCFDA was added to the culture with incubation conditions at 30 min at 37 °C. The cells were dissociated for ROS by washing twice with PBS and subsequently scraped from the culture dish with a pipette tip to 200 μL of 1% Triton X-100 in PBS. The cell suspensions at 50 μL were measured by a fluorescence microplate reader at 485 nm (excitation wavelength) and 530 nm (emission wavelength). One hundred microliters of ethyl alcohol (without-DA) was added to control cells to correct auto-fluorescence. Equal volumes of 1% Triton X-100 buffer added with DCFH2-DA free of cells were maintained as a blank control. Data are denoted as percentages of fluorescence intensity for control in units/mg protein.
3.2. MFEPE protected the PC12 cell line from glutamate-induced cytotoxicity Cell viability was measured under different concentrations of MFEPE in toxicity induced PC12 cell lines using MTT assays. As represented in Fig. 2, cells exposed to 10 μg glutamate lost the typical morphology of their neuritis compared to the control. To elucidate whether MFEPE (methanolic fruit extract of P. emblica) can save the cells from glutamate born toxicity in PC12 cells, the survival of the cells was analyzed by optical microscopy and studied using MTT assays. Cytotoxicity activity of MFEPE was carried out against PC12 cell lines at varied concentrations to confirm the IC50 (50% growth inhibition) by MTT assay. The highest cytotoxicity of this extract against PC12 cell was found in 1000 and 500 μg/mL concentrations with 74.15% and 65.03% of cell growth inhibition (Fig. 3). The MFEPE saves PC12 cells from glutamate based lethality by morphological deteriorating and the bulb arrangement on neuritis. PC-12 is a neural cell line which is derived from rodent - pheochromocytoma and is prevalently used for many neurological studies (Shahlei et al., 2014). In this study, the glutamate-induced oxidative toxicity of PC12 cells was determined. It was characterized based on varied potentially and lethal changes inclusive of intracellular LDH activity, GSH concentrations, ROS levels, leading to a reduction in PC12 cell viability.
3. Results and discussion 3.1. MFEPE had good antioxidant activity In this study, free radical and hydroxyl radical-scavenging and lipid peroxidation were measured to evaluate the antioxidant activities of P. emblica fruit extract. The results of the radical scavenging activity showed good antioxidant activity in a concentration-dependent manner. The result proved that MFEPE showed a concentration dependent radical scavenging activity. Fig. 1A shows the IC50 value for the DPPH assay which was observed as 73.21 μg/mL. Hydroxyl radical scavenging activity was the highest (85.16%) at 1.4 μg/mL, with an IC50 value of 0.426 mg/mL (Fig. 1B). The result of lipid peroxidation activity shows that the extract has equivalent activity to ascorbic acids
Fig. 1. Free radical scavenging activity of P. emblica: (A) DPPH assay, (B) OH radical scavenging activity, (C) lipid peroxidation activity. 3
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Fig. 2. Viability of PC12 cells in various concentration of induced glutamate toxicity and and treatment with different concentrations of methanolic fruit extract of Phyllanthus emblica (MFEPE).
3.3. MFEPE inhibited LDH release in glutamate-induced PC12 cells
3.4. MFEPE increased GSH level in glutamate-induced PC12 cells
To quantitatively assess the defence provided by MFEPE to PC12 cell lines, the cells were subjected to 10 μg/mL glutamate concentrations in the presence of MFEPE for 24 h, and the amount of LDH released after the experiment was recorded with an LDH assay (Fig. 4). The results revealed that the different concentrations of MFEPE entirely protected the cells from damage, even with the occurrence of 10 μg/mL glutamate. Similar to our results, Tan and Kim (2016) also showed that glutamate induces cytotoxicity in the PC12 cell line.
The intracellular levels of GSH have important roles in catalysis, the digestive system, and transport. It has a protective action for cells against free radicals, peroxides and other harmful substances (Merlino et al., 2014). GSH additionally assumes an essential role in the kidneys and joints as a vehicle required for the re-absorption of amino acids. In this study, MFEPE treatment increased the GSH content of cells compared to the glutamate-induced PC12 cells. (Fig. 5A). The GSH content in PC12 cells (control) was 8.32 ± 0.20 mol/1 × 105. MFEPE increased the PC12 cell GSH level, contrasted with the level in glutamateinduced cells. The results revealed that treatment with MFEPE 4
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Hydrogen peroxide, a cell porating agent, is formed from the dismutation of the superoxide anion and is viewed as an antecedent to exceptionally responsive oxidants (Pereira and Oliveira, 2000). ROS play a crucial role in cell death paving way to neural senescence. The present study indicated the positive impacts of MFEPE while demonstrating that ROS triggered cell death in PC12 cells. The exhaustion of intracellular GSH might be due to glutamate-instigated accumulation of ROS inside the cell. A rise in ROS accumulation is observed in glutamate-treated cells. ROS include hydrogen peroxide and hydroxyls radicals which damage natural atoms, and can eventually prompt apoptotic or necrotic cell death. As reported in previous studies, glutamateinduced toxicity and hydrogen peroxide prompted oxidative activity which is very harmful in PC12 cells (Drake et al., 2002; Halliwell and Gutteridge, 1999). Hostanska et al. (2003) treated PC12 cells with extract from the medicinal plant o Hypericum perforatum and analyzed ROS function.
Fig. 3. Growth inhibition of MFEPE against PC12 cell line by MTT assay.
increased intracellular GSH level, reducing glutamate-induced toxicity. Intra-cellular levels of GSH are controlled by the Xc and ASC frameworks. The Xc framework transports intracellular cysteine in return for glutamate. The ASC framework is a Na+-subordinate non-partisan amino corrosive transporter that interrupts cysteine transport intracellularly (Choi, 1998). Glutamate toxicity has been described to be an excitatory activity through the actuation of glutamate receptors (Penugonda et al., 2005; Sapakal et al., 2008). Penugonda et al. (2005) reported that N-acetylcysteine amide (NACA) appeared as a sulfhydryl group given for GSH amalgamation in GSH blend captured cells (Penugonda et al., 2005; Sapakal et al., 2008). The decrease in levels of GSH which rises the resistance of neurons to the harmful impacts of neurotoxins, and is related with mitochondrial damage. Lack of GSH has been connected to different neurodegenerative ailments (Bannai and Tateishi, 1986; Offen et al., 2004).
4. Conclusion The current study concludes that the P. emblica fruit extract possess excellent antioxidant action and radical-scavenging activity. It is for the first time the neuro-protective effects of P. emblica have been reported in the human neural cell line PC12. Further investigation into the effect of the plant extract is essential to reveal the molecular mechanisms and gene regulation underlying the effects of MFEPE. Declaration of competing interest The authors declare that there are no conflicts of interest. Acknowledgement The authors are grateful to the external funding provided by DSTSERB (SB/YS/LS-109/2014) for this project. The authors are thankful to DST-FIST (SR/FST/ College-222/2014) for Instrumental facilities (Lyophilizer) provided to carry out this research. We also express our gratitude to the management of A.V.V.M. Sri Pushpam College (Autonomous), Poondi, for providing the necessary infrastructure and
3.5. MFEPE decreased ROS in glutamate-induced PC12 cells DCFDA assay helped in measuring the accumulation of ROS in glutamate-induced toxicity cells (PC12). The ROS values are found to decrease which is confirmed from Fig. 5B. The results showed that the cytotoxicity of glutamate could be decreased by oxidative levels.
Fig. 4. Protective effect of methanolic fruit extract of Phyllanthus emblica (MFEPE) against glutamate cytotoxicity. PC12 cells were plated and grown 24 h in a culture medium and then treated with different concentrations of MFEPE. After 24 h percentage of LDH released was determined using an LDH assay. Results are expressed as the means ± SEM of three replicates and represent the percentage (%) of LDH leaked into the external medium. Results showed that the percentage of LDH released differed significantly (p < 0.001) compared to the control (absence of glutamate).
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Fig. 5. Effect of MFEPE on glutamate induced treated PC12 cells released (A) percentage of GSH, (B) percentage of ROS. Statistically different values of p < 0.01, were determined compared to control conditions in the absence of glutamate at the same incubation period.
support to complete this work.
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Neuroprotective behaviour of Phyllanthus emblica (L) on human neural cell lineage (PC12) against glutamate-induced cytotoxicity
T
⁎
S. Rajalakshmia, S. Vijayakumara, , P.K. Praseethab a b
Computational Phytochemistry Lab, PG and Research Department of Botany and Microbiology, A. V. V. M Sri Pushpam College (Autonomous), Poondi, Tamil Nadu, India Department of Nanotechnology, Noorul Islam Centre for Higher Education, Kumaracoil, Kanyakumari district, Tamil Nadu 629180, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Phyllanthus emblica Neuroprotective Anti-oxidant Glutamate PC 12 cell line
Phyllanthus emblica (L) is an edible, nutrient-rich Indian gooseberry with wide distribution in sub-tropical and tropical belts of India, Indonesia, China, and Malaysia, belonging to the family of Phyllanthaceae and is well known for its traditional medicinal properties. The current study aims to investigate the neuroprotective and antioxidant effects of P. emblica. Radical scavenging action was evaluated by DPPH method, and its hydroxyl moiety scavenging and lipid peroxidation activity were also investigated. Its role in neuroprotection was analyzed on the human neural cell lines (PC12) against glutamate induced cellular inhibition. The cell line study was performed by measuring the viability of cells through MTT assay, and through monitoring LDH activity, GSH and ROS levels. In the current study, results confirmed that P. emblica extract had excellent antioxidant activity. DPPH and hydroxyl radical scavenging assays revealed IC50 levels of 73.21 μg/mL and 0.426 mg/mL, respectively. Significant lipid peroxidation activity (73.21 μg/mL) was also observed. The neuroprotective effect of P. emblica on cell lines was confirmed by cell viability assay after through glutamate-induced cell cytotoxicity.
1. Introduction In the present society, people lead stressful lives, due to lack of physical movement, restlessness of the mind and anxiety. These factors are closely related to the progress of many diseases, including neurodegenerative diseases (Vijayakumar et al., 2016). A large number of deaths have occurred worldwide during the 21st century due to neurodegenerative diseases, such as cognitive dysfunction, neuropsychiatric, and various nervous disorders such as Huntington's, Parkinson's and Alzheimer's disease (Essa et al., 2014). Parkinson's disease falls among the second most neural disease, which completely reduces the quality of life (Jankovic, 2008). Parkinson's disease is characterized by different symptoms like rigidity, bradykinesia, postural instability or facial dyskinesia, muscular stiffness and tremors (Magrinelli et al., 2016). Few medicinal plant extracts have been considered as potential sources of bioactive compounds with neuroprotective effects (Shivapriya et al., 2015). P. emblica (L) is a plant belonging to the Phyllanthaceae family and well known for its medicinal value. It is popular all around the world due to its nutraceutical properties. Many studies have reported that P. emblica has antimicrobial (Ahmad et al.,
1998), antioxidant (Chatterjee et al., 2011; Sumitra et al., 2009) antiinflammatory (Golechha et al., 2011), analgesic and antipyretic (Mythilypriya et al., 2007; Perianayagam et al., 2004) adaptogenic (Baliga et al., 2012) hepatoprotective (Gulati et al., 1995) antitumor (Suresh and Vasudevan, 1994), and anti-ulcerogenic (Sairam et al., 2002) properties. Additionally, the literature suggests P. emblica as a potent antioxidant. Based on this information, through this study we could evaluate the neuroprotective and antioxidant functions of P. emblica using the human PC12 Parkinson's cell line. 2. Materials and methods 2.1. Chemicals The microbial culture media, chemicals and reagents required for the study were purchased from Standard Chemical companies like Sigma Aldrich, HiMedia Laboratories and E. Merck respectively. 2.2. Sample collection The fruits of P. emblica were collected during the months of March,
Abbreviations: FBS, fetal bovine serum; PBS, phosphate buffered saline; DMEM, Dulbecco's modified Eagle's medium; MFEPE, methanolic fruit extract of Phyllanthus emblica; MTT, 3,-4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide; LDH, lactate dehydrogenase; GSH, glutathione; ROS, reactive oxygen species ⁎ Corresponding author. E-mail address: [email protected] (S. Vijayakumar). https://doi.org/10.1016/j.genrep.2019.100545 Received 13 September 2019; Received in revised form 1 October 2019; Accepted 22 October 2019 Available online 23 October 2019 2452-0144/ © 2019 Elsevier Inc. All rights reserved.
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500 nm.
and April 2016, from the Botanical Garden of A.V.V. M Sri Pushpam College, Poondi, Thanjavur district. The plant material was authenticated as being from P. emblica by Rapinot Herbarium, St. Joseph's College, Trichirappalli, India. The plant voucher specimen (PHC1126) was deposited to Pushpam Herbarium Cabinet (PHC), Department of Botany and Microbiology, A.V.V. M Sri Pushpam College, Tamil Nadu, India.
2.7. In-vitro assay and drug treatment Cell cultures were obtained from the National Centre for Cell Sciences (NCCS), Pune, India. The cells were further sub-cultured in DMEM with an added concentration of 10% FBS, penicillin at 100 IU/ mL, streptomycin at 100 mg/mL and amphotericin b at 5 mg/mL. The PC12 cells were cultured for 24 h in 96 well microplates. Then, the culture medium was discarded and the cells were subjected to various concentrations (250, 500, and 1000 μg/mL) of the fruit extract. After that, 100 μL of the fruit extract was diluted in DMEM and added to each well at a concentration of 1 μg/mL. Finally, the plates were incubated for 2 h in humidified conditions with 5% CO2 at 37 °C.
2.3. Preparation of plant extract Fresh fruits collected were washed, cleaned, and shade dried before the synthesis. The dried fruits were powdered using a blender. Then, 100 g of dried fruit powder was mixed well with 1000 mL of 95% methanol, and the resulting mixture was incubated at 25 °C for 72 h. The extract was subjected to filtration through Whatman No. 1 filter paper, and vacuum concentrated with a rotatory evaporator at 40 °C. Then, extracts were stored for further experiments.
2.8. MTT assay The cultured cells in a monolayer fashion was trypsinized, then the cell count was made up to 1.0 × 105 cells/mL with 10% FBS. FBS containing medium was used to determine cell viability via MTT assays as described by Salameh et al. (2010). Absorbance was recorded with a micro-plate reader (540 nm wavelengths). The various concentrations of the test drug required to inhibit the cell growth by 50% (CTC50) were read from the obtained graphs for dose-response from each cell line, and the percentage of inhibition in growth was calculated using the given equation:
2.4. Measurement of free radical-scavenging activity using DPPH assays The antioxidant action of plant extract was determined according to the methodology of Payet et al. (2005). The assay was performed using by a methanolic solution (0.004%). Two hundred microliters of the solvent were transferred to a microplate of 96 wells, with an addition of 20 μL of P. emblica fruit extract, solvent, or control substance. The experimentation mixture was vigorously shaken and further incubated at 30 °C for 1 h. The absorbance was measured at 517 nm using in a microplate reader. The inhibition percentage was calculated as below,
Percentage of Growth inhibition 100 − Mean OD of individual test group = mean OD of the control group.
Inhibition% = 100 − 100 (AS ÷ A O) where
2.9. LDH assay
AO represents the absorbance of blank solution, AS represents the absorbance of the sample (517 nm).
Inhibition% = 1 − (AS–AW/AO) × 100
The cell lines were plated at a concentration of (1.5–2) × 104 cells/ well in 96-well plates, and were kept aside for attachment 24 h before treatment. A single row of wells contained only the medium required for background normalization. Varied concentrations of Methanolic fruit extract of P. emblica (MFEPE) were added successively for 8 h after inducing the toxicity with glutamate for 24 h. The lactate dehydrogenase (LDH) supernatant was gathered for analysis in a cell-free medium. The pellets were lysed in 0.5 mL with a lysis buffer (0.5% Triton X-100) and 0.1 M potassium phosphate buffer at pH 7.0 to release the intracellular LDH to the extracellular medium. The medium was measured to determine the LDH activity which is represented as percentage of total cellular action. The absorbance was measured at 340 nm via a model Digiscan Microplate reader, and the following formula was used to calculate for the percentage of LDH released:
where,
Percentage of LDH released
2.5. Measurement of hydroxyl radical scavenging activity Hydroxyl radical scavenging activity was measured by adopting the methodology of Klein et al. (1981) with minor modifications. Two milliliters of 6 mM ferrous sulfate of different concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mg/mL) were added to 2 mL of the P. emblica fruit extract. The mixture was kept undisturbed for 10 min at room temperature. Then, 2 mL of 6 mM sodium salicylate was added, followed by incubation at 37 °C for 30 min. After incubation, the absorbance was measured at 510 nm. The inhibition percentage was calculated as below,
= (Experimental LDH activity)/(Maximum LDH activity) × 100.
AS represents the absorbance of the sample with sodium salicylate, AW represents the absorbance of the sample without sodium salicylate, AO represents the absorbance of blank solution,
2.10. GSH measurement Cells were inoculated at a density of 4 × 104 cells/cm2 in poly-Dlysine-coated (0.05 mg/mL) flasks of volume 75 cm2 (5 mL/flask) for measuring GSH. After 24 h, different concentrations of P. emblica methanolic fruit extracts were added to the flasks which were then incubated at 37 °C. After the completion of incubation, the cells were harvested from the cultures and homogenized with serine borate buffer (100 mM Tris-HCl, 10 mM boric acid, 5 mm L-serine, 1 mM DETAPAC, pH 7.4). Twenty micromolars of the diluted cell homogenate were mixed with 230 μM of serine borate buffer followed by 750 μM of N-(1pyrenyl) meleimide (NPM) (1 mM in acetonitrile). The solutions were maintained at 35 ± 2 °C for 5 min. The reaction was halted by adding 5 μL of 2 M HCl. The samples were further filtered through a 0.2 μm
2.6. Measurement of lipid peroxidation activity Lipid peroxidase inhibition was performed using a ferric thiocyanate assay (Kikuzaki and Nakatani, 1993). The assay was inhibited by adding 4 mL of ethyl alcohol, 2.51% linoleic acid mixed in absolute ethanol (4.1 mL), 0.05 M phosphate buffer maintained at pH 7.0 (8 mL), in distilled water (3. 9 mL) to a vial with a screw cap. The vial was kept in an oven at 40 °C in the dark. Then, 0.1 mL of the resulting solution was mixed with 9.7 mL of 75% ethanol and 0.1 mL of 30% ammonium thiocyanate. After 3 min, 0.1 mL of 0.02 M ferrous chloride solution was added to 3.5% hydrochloric acid. The absorbance was observed at 2
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filter (Acrodisc) and then injected into the HPLC column. At the cellular level, GSH was determined using the standard procedure described by Park et al. (2003).
(Fig. 1C). In 1923, the terminology-Siddha medicine was framed by the British. Based on the Siddha system, Indian gooseberry possesses an assortment of medicinal properties. Classic manuals (Purananuru) mention the anti-ageing effect of P. emblica. Many studies have reported that Indian gooseberry has excellent antioxidant effect owing to its high flavonoid, polyphenols and tannin contents. Due to the increasing interest in the relationship between antioxidant and diseases, DPPH, hydroxyl radical scavenging assays and lipid peroxidation assay is a crucial factor for assessing antioxidant activity. The results of this study showed that P. emblica extracts possesses excellent radical scavenging ability. These results correlate with those of Akowuah et al. (2005), who reported that P. emblica extract has significant antioxidant activity, using DPPH and FRAP assays.
2.11. Intracellular ROS measurement The dye 2, 7-dichlorofluorescein diacetate (DCFDA), which is oxidized to dichlorofluorescein (DCF) by hydroperoxides, was used to measure the relative levels of cellular peroxides (Surronen et al., 2000). After 24 h cells were treated with various concentrations of MFEPE and the cells were exposed to 10 μg of glutamate for 6 h. At the end of treatment 10 μg of DCFDA was added to the culture with incubation conditions at 30 min at 37 °C. The cells were dissociated for ROS by washing twice with PBS and subsequently scraped from the culture dish with a pipette tip to 200 μL of 1% Triton X-100 in PBS. The cell suspensions at 50 μL were measured by a fluorescence microplate reader at 485 nm (excitation wavelength) and 530 nm (emission wavelength). One hundred microliters of ethyl alcohol (without-DA) was added to control cells to correct auto-fluorescence. Equal volumes of 1% Triton X-100 buffer added with DCFH2-DA free of cells were maintained as a blank control. Data are denoted as percentages of fluorescence intensity for control in units/mg protein.
3.2. MFEPE protected the PC12 cell line from glutamate-induced cytotoxicity Cell viability was measured under different concentrations of MFEPE in toxicity induced PC12 cell lines using MTT assays. As represented in Fig. 2, cells exposed to 10 μg glutamate lost the typical morphology of their neuritis compared to the control. To elucidate whether MFEPE (methanolic fruit extract of P. emblica) can save the cells from glutamate born toxicity in PC12 cells, the survival of the cells was analyzed by optical microscopy and studied using MTT assays. Cytotoxicity activity of MFEPE was carried out against PC12 cell lines at varied concentrations to confirm the IC50 (50% growth inhibition) by MTT assay. The highest cytotoxicity of this extract against PC12 cell was found in 1000 and 500 μg/mL concentrations with 74.15% and 65.03% of cell growth inhibition (Fig. 3). The MFEPE saves PC12 cells from glutamate based lethality by morphological deteriorating and the bulb arrangement on neuritis. PC-12 is a neural cell line which is derived from rodent - pheochromocytoma and is prevalently used for many neurological studies (Shahlei et al., 2014). In this study, the glutamate-induced oxidative toxicity of PC12 cells was determined. It was characterized based on varied potentially and lethal changes inclusive of intracellular LDH activity, GSH concentrations, ROS levels, leading to a reduction in PC12 cell viability.
3. Results and discussion 3.1. MFEPE had good antioxidant activity In this study, free radical and hydroxyl radical-scavenging and lipid peroxidation were measured to evaluate the antioxidant activities of P. emblica fruit extract. The results of the radical scavenging activity showed good antioxidant activity in a concentration-dependent manner. The result proved that MFEPE showed a concentration dependent radical scavenging activity. Fig. 1A shows the IC50 value for the DPPH assay which was observed as 73.21 μg/mL. Hydroxyl radical scavenging activity was the highest (85.16%) at 1.4 μg/mL, with an IC50 value of 0.426 mg/mL (Fig. 1B). The result of lipid peroxidation activity shows that the extract has equivalent activity to ascorbic acids
Fig. 1. Free radical scavenging activity of P. emblica: (A) DPPH assay, (B) OH radical scavenging activity, (C) lipid peroxidation activity. 3
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Fig. 2. Viability of PC12 cells in various concentration of induced glutamate toxicity and and treatment with different concentrations of methanolic fruit extract of Phyllanthus emblica (MFEPE).
3.3. MFEPE inhibited LDH release in glutamate-induced PC12 cells
3.4. MFEPE increased GSH level in glutamate-induced PC12 cells
To quantitatively assess the defence provided by MFEPE to PC12 cell lines, the cells were subjected to 10 μg/mL glutamate concentrations in the presence of MFEPE for 24 h, and the amount of LDH released after the experiment was recorded with an LDH assay (Fig. 4). The results revealed that the different concentrations of MFEPE entirely protected the cells from damage, even with the occurrence of 10 μg/mL glutamate. Similar to our results, Tan and Kim (2016) also showed that glutamate induces cytotoxicity in the PC12 cell line.
The intracellular levels of GSH have important roles in catalysis, the digestive system, and transport. It has a protective action for cells against free radicals, peroxides and other harmful substances (Merlino et al., 2014). GSH additionally assumes an essential role in the kidneys and joints as a vehicle required for the re-absorption of amino acids. In this study, MFEPE treatment increased the GSH content of cells compared to the glutamate-induced PC12 cells. (Fig. 5A). The GSH content in PC12 cells (control) was 8.32 ± 0.20 mol/1 × 105. MFEPE increased the PC12 cell GSH level, contrasted with the level in glutamateinduced cells. The results revealed that treatment with MFEPE 4
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Hydrogen peroxide, a cell porating agent, is formed from the dismutation of the superoxide anion and is viewed as an antecedent to exceptionally responsive oxidants (Pereira and Oliveira, 2000). ROS play a crucial role in cell death paving way to neural senescence. The present study indicated the positive impacts of MFEPE while demonstrating that ROS triggered cell death in PC12 cells. The exhaustion of intracellular GSH might be due to glutamate-instigated accumulation of ROS inside the cell. A rise in ROS accumulation is observed in glutamate-treated cells. ROS include hydrogen peroxide and hydroxyls radicals which damage natural atoms, and can eventually prompt apoptotic or necrotic cell death. As reported in previous studies, glutamateinduced toxicity and hydrogen peroxide prompted oxidative activity which is very harmful in PC12 cells (Drake et al., 2002; Halliwell and Gutteridge, 1999). Hostanska et al. (2003) treated PC12 cells with extract from the medicinal plant o Hypericum perforatum and analyzed ROS function.
Fig. 3. Growth inhibition of MFEPE against PC12 cell line by MTT assay.
increased intracellular GSH level, reducing glutamate-induced toxicity. Intra-cellular levels of GSH are controlled by the Xc and ASC frameworks. The Xc framework transports intracellular cysteine in return for glutamate. The ASC framework is a Na+-subordinate non-partisan amino corrosive transporter that interrupts cysteine transport intracellularly (Choi, 1998). Glutamate toxicity has been described to be an excitatory activity through the actuation of glutamate receptors (Penugonda et al., 2005; Sapakal et al., 2008). Penugonda et al. (2005) reported that N-acetylcysteine amide (NACA) appeared as a sulfhydryl group given for GSH amalgamation in GSH blend captured cells (Penugonda et al., 2005; Sapakal et al., 2008). The decrease in levels of GSH which rises the resistance of neurons to the harmful impacts of neurotoxins, and is related with mitochondrial damage. Lack of GSH has been connected to different neurodegenerative ailments (Bannai and Tateishi, 1986; Offen et al., 2004).
4. Conclusion The current study concludes that the P. emblica fruit extract possess excellent antioxidant action and radical-scavenging activity. It is for the first time the neuro-protective effects of P. emblica have been reported in the human neural cell line PC12. Further investigation into the effect of the plant extract is essential to reveal the molecular mechanisms and gene regulation underlying the effects of MFEPE. Declaration of competing interest The authors declare that there are no conflicts of interest. Acknowledgement The authors are grateful to the external funding provided by DSTSERB (SB/YS/LS-109/2014) for this project. The authors are thankful to DST-FIST (SR/FST/ College-222/2014) for Instrumental facilities (Lyophilizer) provided to carry out this research. We also express our gratitude to the management of A.V.V.M. Sri Pushpam College (Autonomous), Poondi, for providing the necessary infrastructure and
3.5. MFEPE decreased ROS in glutamate-induced PC12 cells DCFDA assay helped in measuring the accumulation of ROS in glutamate-induced toxicity cells (PC12). The ROS values are found to decrease which is confirmed from Fig. 5B. The results showed that the cytotoxicity of glutamate could be decreased by oxidative levels.
Fig. 4. Protective effect of methanolic fruit extract of Phyllanthus emblica (MFEPE) against glutamate cytotoxicity. PC12 cells were plated and grown 24 h in a culture medium and then treated with different concentrations of MFEPE. After 24 h percentage of LDH released was determined using an LDH assay. Results are expressed as the means ± SEM of three replicates and represent the percentage (%) of LDH leaked into the external medium. Results showed that the percentage of LDH released differed significantly (p < 0.001) compared to the control (absence of glutamate).
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Fig. 5. Effect of MFEPE on glutamate induced treated PC12 cells released (A) percentage of GSH, (B) percentage of ROS. Statistically different values of p < 0.01, were determined compared to control conditions in the absence of glutamate at the same incubation period.
support to complete this work.
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