Terminalia chebula Extract Protects OGD-R Induced PC12 Cell Death and Inhibits LPS Induced Microglia Activation

Terminalia chebula, native to Southeast Asia, is a popular medicinal plant in Ayurveda. It has been previously reported to have strong antioxidant and anti-inflammatory efficacy. In this study, we aimed to investigate if fruit extract from T. chebula might protect neuronal cells against ischemia and related diseases by reduction of oxidative damage and inflammation in rat pheochromocytoma cells (PC12) using in vitro oxygen-glucose deprivation followed by reoxygenation (OGD-R) ischemia and hydrogen peroxide (H2O2) induced cell death. Cell survival was evaluated by a 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Free radical scavenging, lipid peroxidation and nitric oxide inhibition were measured by diphenyl-1-picrylhydrazyl (DPPH), thiobarbituric acid (TBA) and Griess reagent, respectively. We found that T. chebula extract: (1) increases the survival of cells subjected to OGD-R by 68%, and H2O2 by 91.4%; (2) scavenges the DPPH free radical by 96% and decreases malondialdehyde (MDA) levels from 237.0 ± 15.2% to 93.7 ± 2.2%; (3) reduces NO production and death rate of microglia cells stimulated by lipopolysaccharide (LPS). These results suggest that T. chebula extract has the potential as a natural herbal medicine, to protect the cells from ischemic damage and the possible mechanism might be the inhibition of oxidative and inflammatory processes.


Introduction
Natural antioxidants, from medicinal or edible plants, have recently received much attention as promising agents for reducing the risk of oxidative stress-induced neurological diseases [1,2]. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the main source of free radicals, and they lead to serious damage causing neurodegeneration in the pathogenesis of neurological disorders such as Alzheimer's disease, Parkinson's disease and strokes [3][4][5]. Overproduction of ROS, during ischemia/reperfusion, causes an imbalance between oxidative and anti-oxidative processes leading to mitochondrial dysfunction, excitotoxicity, lipid peroxidation, and inflammation. Therapeutic efforts aimed to counteract ROS or inhibit their formation, have been shown to be beneficial in various neuro-degenerative diseases, including ischemia [6].
Based on the information regarding antioxidant and anti-inflammatory effects, we hypothesized that fruit of T. chebula extract may have protective effects on neuronal cell death against ischemia and related diseases. In this study, we aimed to investigate the protective effect of T. chebula extract against OGD-R and hydrogen peroxide (H 2 O 2 ) induced cell injury on the rat pheochromocytoma cell line (PC12 cells). The reason for choosing PC12 cells is that, they display phenotypic characteristics of adrenal chromaffin cells and sympathetic neurons and therefore, PC12 cells are a well-known model for neuronal differentiation [17,18]. OGD-R, a well-accepted in vitro ischemia model [19,20], was used to evaluate the protective effect of T. chebula extract. H 2 O 2 induced cell death; an extensively used in vitro oxidative stress model [21,22], was used to evaluate the effect of T. chebula extract in PC12 cells. Furthermore, the activation of the primary microglia is one of the major reasons of cellular inflammation in ischemic condition and lipopolysaccharide (LPS) activated BV2 cells are reported as the suitable alternatives for primary microglia in the culture or animal experiments [23]. Therefore, we investigated the inhibitory effect of T. chebula extract on LPS activated microglia cells (BV2 cell). We also determined the diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, lipid peroxidation and nitric oxide (NO) inhibition activity of T. chebula extract.

HPLC Analysis and Total Phenol Content of T. chebula Extract
The 3D HPLC spectrum of T. chebula extract is shown in Figure 1. The major polyphenols isolated from the T. chebula extract were gallic acid and ellagic acid.

DPPH Radical Scavenging Activity of T. chebula Extract
T. chebula extract showed dose dependent radical scavenging activities with a maximum of 96% inhibition against DPPH radical at 100 μg/mL. The effective concentration of T. chebula extract to scavenge 50% of DPPH free radical (EC 50 ) was found as 5.5 ± 1.1 μg/mL. Ascorbic acid (EC 50 of 4.7 ± 0.01 μg/mL) was used as a positive control. Table 1 showed the content of gallic acid, ellagic acid and total phenols in T. chebula extract.

Effect of T. chebula Extract in Lipid Peroxidation
The TBA colorimetric assay was performed to determine whether T. chebula extract can inhibit the formation of MDA induced by H 2 O 2 . The cells treated with H 2 O 2 significantly increased MDA levels as compared with control cells (set 100%). Pretreatment of T. chebula extract at 0.1 μg/mL and 1 μg/mL significantly reduced MDA level from 237.0 ± 15.2% in the H 2 O 2 treated group to 115.8 ± 5.8% (p < 0.001) and 93.7 ± 2.2% (p < 0.001) respectively ( Figure 3B).

Discussion
In the present study, fruit extract of T. chebula significantly reduced PC12 cell death induced by 4 h of OGD followed by 24 h of reoxygenation and 24 h exposure of H 2 O 2 . T. chebula extract showed the strong scavenging activity against DPPH free radicals and markedly reduced the H 2 O 2 -induced MDA production. T. chebula extract also reduced LPS induced cell death and inhibited the production of NO by BV2 cells in a dose dependent manner.
As OGD-R mimics tissue ischemia, it results in the production of ROS, release from glutamate toxicity, and inflammatory cytokines [25,26]. Generations of the free radicals, such as, superoxide and superoxide-derived oxidants have been invoked as key mediators of tissue injury during ischemia-reperfusion [27]. Cellular injury followed by ischemic reperfusion is a complex pathophysiological event associated with impairment of multiple vascular and cellular responses. Oxidative damage due to the ROS/RNS play the vital role to initiate a wide range of intracellular stress signaling processes that culminate in excessive cytokine and chemokine response, adhesion molecule upregulation, etc. [28]. Once the ROS/RNS are produced within the cells, oxidative cascades of events take place simultaneously, including free radical production, sudden start and aggravation of mitochondrial respiratory chains, excitotoxicity, enzymatic changes, and the stimulation of the inflammatory processes [29]. Among such cascades, oxidative stress and inflammation play the key role for the cellular damage. The compounds having antioxidant, anti-inflammatory, anti-glutamate activity show protective activity against hypoxia induced cell damage [26,[30][31][32].
T. chebula extract significantly protected the PC12 cells against OGD-R. The exposure of PC12 cells to 4 h of OGD followed by 24 h of re-oxygenation resulted decrease in cell viability as compared to control (non-OGD) cells. Incubation of PC12 cells with T. chebula extract, 30 min before and during OGD period, led to significant increase in cell viability. It is reported that, OGD mediates the oxidative stress in the cells by activating the different oxidative cascades such as activation of lipoxygenase, IκB kinase, nuclear factor-κB, and OGD-R is the major source of ROS in ischemia [33,34]. In our study, T. chebula extract markedly scavenged the nitrogen-centered free radical DPPH (by 96%) in a dose dependent manner, similar to that of ascorbic acid, so the potential nature of antioxidant activity of T. chebula extract might be the key factor to sequester the ROS, that are generated in the ischemic cascades, like OGD-R.
The maximum protective effect of T. chebula extract (70%) against H 2 O 2 induced PC12 cell death, was found to be significantly higher than that of the positive control, baicalein (46.3%). H 2 O 2 is not a free radical and has a limited reactivity; however, it is the major precursor of the highly reactive hydroxyl radical. Previous studies have shown a close association between H 2 O 2 and neurodegenerative disease, and it has been suggested that H 2 O 2 levels are increased during pathological conditions, such as ischemia [35,36]. ROS, such as H 2 O 2 , readily damage biological molecules that can eventually lead to apoptotic or necrotic cell death [37]. In our study, interestingly, the extent of cell protection by T. chebula extract after H 2 O 2 induced cell death was higher in less concentration of extract than that in higher concentration ( Figure 3A). The mechanism of cell protection by T. chebula extract, against H 2 O 2 induced cell death is largely due to the antioxidant efficacy of polyphenols present in T. chebula, such as quercetin, gallic acid and ellagic acids. These polyphenols are reported to show pro-oxidant activity at higher concentrations [38,39]. This might explain the lesser protective effect of T. chebula extract at higher concentration (1 μg/mL).
T. chebula extract markedly scavenged the nitrogen-centered free radical DPPH in a dose dependent manner. From HPLC analysis, it was observed that main phenolic compounds in T. chebula are gallic acid and ellagic acid, which are well-known natural antioxidants [40]. Plant polyphenols are multifunctional in the sense that they can act as reducing agents, hydrogen atom donors, and singlet oxygen scavengers [41]. The DPPH radical scavenging activity of T. chebula is mostly related to the hydroxyl group in gallic acid and ellagic acid.
Our results showed that MDA production during H 2 O 2 treatment was significantly inhibited by T. chebula. H 2 O 2 is believed to be the major precursor for highly reactive free radicals, expressed indirectly by the increase in MDA levels, which is one of the most important organic expressions of oxidative stress in various neuronal diseases including ischemia [42]. Oxidative stress induces cellular damage and lipid peroxidation, which may lead to alterations in membranes and produce significant changes in their biophysical function [43]. Previous studies have shown that the radical scavengers and inhibitors of lipid peroxidation can ameliorate ischemic cell damage [5,44]. Therefore, antioxidant effect through the inhibition of lipid peroxidation and free radical scavenging could be the possible protective mechanisms of T. chebula against ischemia and H 2 O 2 induced cell death.
T. chebula extract inhibited the accumulation of NO by 76.2%, at 10 μg/mL. LPS activated BV2 cells release NO in the culture medium which is recognized as a mediator and regulator in pathological reactions, especially in acute inflammatory responses [45]. In addition to ROS, overproduction of NO also plays an important role in various models of inflammation [46][47][48]. The reaction of NO with superoxide anion forms peroxynitrite [49], a potent cytotoxic oxidant eliciting lipid peroxidation and cellular damage [50]. Activated microglia is thought to be involved in neuronal inflammation by overproducing various bioactive molecules such as NO, ROS and pro-inflammatory cytokines [51].
T. chebula extract significantly inhibited NO release by LPS activated BV2 cells, elucidating that it can inhibit the activation of microglia. Therefore, anti-inflammatory effect through the inhibition of NO release could be another possible protective mechanism of T. chebula against ischemia.
Besides these, T. chebula and its active compounds gallic acid and ellagic acids are previously reported to inhibit cytochrome p450 enzyme and protect against mitochondrial dysfunction [52,53]. T. chebula treatment significantly increased the level of Cytochrome C oxidase and other mitochondrial enzymes. Gallic acid down-regulates the protein expression and activity of caspase-3, an essential effector molecule in the course of programmed cell death [54]. T. chebula is also reported to inhibit inducible nitric oxide synthesis by decreasing iNOS protein and iNOS mRNA levels suggesting the possible intercellular mechanism of T. chebula extracts and its active constituents [55]. Taken together, the protective efficacy of the T. chebula extract against OGD-R induced PC12 cell death and the inhibition of activated microglia must be due to the antioxidant and anti-inflammatory potential of T. chebula extract and its polyphenols.

Plant Material and Chemicals
The fruits of T. chebula were collected from Pokhara, Nepal after proper identification by the experts. A voucher specimen (HP203) was deposited in the Herbarium of the Kyung Hee University,

Extraction of Plant Material
T. chebula fruit powder (100 g) was extracted under reflux with 70% methanol (1000 mL). The extract was evaporated with a rotary evaporator under reduced pressure to remove the organic solvent and then lyophilized until dryness. A stock solution of extract was prepared in DMSO and deionized water, respectively. Final DMSO concentration did not exceed 0.1% (v/v) in all treatments.

HPLC Analysis of T. chebula Extract
Methanol extract of T. chebula was analyzed by the HPLC with a 600 pump (Waters, Milford, MA, USA) using 250 mm × 4 mm Hypersil TM Gold C18 column (ThermoElectron, Bellefonte, PA, USA), at a flow rate of 1 mL/min under the following conditions: solvent A (0.5% v/v H 3 PO 4 ) and solvent B (CH 3 CN); a linear gradient from 5% to 50% v/v of solvent B in A for 60 min. The isolated compounds were monitored with photodiode array detector (926, Waters). Content of major compounds was calculated by comparing with the standard compounds.

Free Radical Scavenging Activity and Total Phenol Content in T. chebula Extract
Free radical scavenging activity was measured spectrophotometrically by DPPH free radical scavenging assay [56] using ascorbic acid as a positive control. Phenolic compounds in the T. chebula extracts were determined by the colorimetric assay as described previously [57] with some modifications. Briefly, 1 mL of sample was mixed with an equal volume of Folin and Ciocalteu's phenol reagent. After 3 min, 1 mL of saturated sodium carbonate solution was added to the mixture. After 90 min of reaction, the optical density (OD) was measured on a plate reader (SPECTRA max Plus 384 , CA, USA) at 725 nm. Gallic acid (10-500 mg/L) was used for calculation, and the results were expressed as mg of gallic acid equivalents (GCE) per gram of extract.

Cell Culture
PC12 cells and BV2 cells were obtained from the Medical Department, Kyung Hee University, Seoul, Korea, and maintained at 37 °C in a humidified atmosphere containing 5% CO 2 . PC12 cells were seeded at a density of 1.5 × 10 3 cells/well whereas BV2 cells were seeded at a density of 5 × 10 4 cells/well and cultured at 37 °C in DMEM is supplemented with 10% heat-inactivated FBS, penicillin (1 × 10 5 U/L) and streptomycin (100 mg/L) in a 5% CO 2 incubator.

Oxygen-Glucose Deprivation Followed by re-Oxygenation
Oxygen-glucose deprivation followed by re-oxygenation experiments were carried out 24 h after seeding the cells. For the measurement of cytotoxic effects, PC12 cells were treated with T. chebula extract (at 0.1, 1, 10 μg/mL) and incubated for 28 h. For OGD injury, PC12 cells were first washed with phosphate buffer saline (PBS, pH 7.2) then with glucose-free DMEM. The cultures were then placed in fresh glucose free DMEM and kept in a hypoxic chamber (Forma Science, UK) containing 95% N 2 and 5% CO 2 for 4 h. At the end of the exposure period, glucose solution was added, and the cells were incubated (5% CO 2 at 37 °C) for an additional 24 h. Samples of different concentrations were treated 30 min before and during OGD exposure. The control group was treated with the same amounts of serum free glucose DMEM and incubated. Baicalein, a potent antioxidative and neuroprotective compound [58] was used as a positive control.

Lipid Peroxidation Assay
Malondialdehyde (MDA), the most abundant lipid peroxidation product from PC12 cells, was measured using the TBA colorimetric assay. Briefly, 24 h after the treatment of the cells with H 2 O 2 (200 μM) in the presence or absence of T. chebula extract (0.1 and 1 μg/mL), cultures were washed with ice-cold PBS, pooled in 0.1 mol/L PBS-5% Triton X-100 buffered solution, and incubated for about 1 h at 37 °C. Trichloroacetic acid (350 μL; 20% w/v) was added to 250 μL of cellular lysate and centrifuged (1,000 × g at 4 °C for 10 min). Aliquots (450 μL) of supernatant were mixed with equal volume of 0.5% (w/v) TBA. The mixture was boiled at 100 °C for 30 min. After cooling MDA formation was measured at 520 nm and the results were expressed as a percentage of the control group.

LPS-Induced Microglia Activation and the Measurement of NO
For LPS induced cell death and nitric oxide inhibitory assay of T. chebula extract, BV2 cells were incubated with/without 1 μg/mL of LPS, in the absence or presence of different sample solutions for 24 h. For the measurement of NO produced by activated cells, 90 μL of the supernatant from the BV2 cells was mixed with equal volume of the Griess reagent. The OD was measured at 540 nm. Curcumin, a potent anti-inflammatory compound [59] was used as a positive control.

Cell Viability
Cell viability was evaluated by observing the ability of viable cells to reduce yellow colored MTT to purple colored formazan. MTT was dissolved in DMEM and added to the culture at a final concentration of 0.5 mg/mL. After an additional 2 h of incubation, the media was carefully removed and 100 µL DMSO was added to each well. The OD was measured on a plate reader at 570 nm. Results were expressed as a percentage of the control group.

Statistical Analysis
All results are presented as mean ± S.E.M. Significant differences between experimental groups were determined by using one way ANOVA followed by the Tukey's post hoc test using GraphPad Prism 4 (GraphPad Software Inc., La Jolla, CA, USA). p < 0.05 is considered as statistically significant for the experimental analysis. Each experiment was performed at triplicate.

Conclusions
In conclusion, T. chebula extract showed a protective effect against OGD-R, H 2 O 2 and LPS induced cell death. T. chebula has an antioxidative and anti-inflammatory activity which could be the possible mechanism for the protection of cells against ischemia. Our results suggest that T. chebula fruit could be the useful remedy for ischemia, oxidative stress and activated microglia-induced secondary damage, which occurs in many neurodegenerative diseases. The exact protective mechanism of T. chebula extract against OGD-R is unknown, however, it can be estimated that the protective effect of T. chebula might be due to its antioxidative and anti-inflammatory effects. Further study is necessary to evaluate the protective effect of T. chebula extract on in vivo ischemia.