Abstract: Diseases caused due to ‘oxidative stress’ initiated by Reactive Oxygen Species (ROS) is growing at an alarming rate. Several medicinal herbs explored till date were reported to possess numerous pharmacological properties used for combating different diseases. The present study was undertaken to investigate the inhibitory potential of Terminalia chebula by H2O2 induced oxidative stress in THP-1 cell line and also the presence of the marker compounds in T. chebula was confirmed by High Performance Liquid Chromatography (HPLC). Intracellular quantification of ROS was done using a flow cytometer. The percentage of gallic acid and ellagic acid in the extract was performed by HPLC were found to be 4.84% w/w and 2.56% w/w, respectively. Pretreatment of cells with T. chebula extract significantly reduced the intracellular ROS production in a dose-dependent manner. The present study illustrates the effective role of T. chebula in reducing the ROS production which is a causative factor for several disorders. The results describe the possibility and potentiality of natural sources to inhibit the pathogenesis of different diseases caused due to oxidative stress.
INTRODUCTION
During the last few years, new and promising advances have been made by scientists to explore various traditional medicines. The western medicines can cause adverse effects and has been known to develop intolerance in patients towards its pharmacotherapy ADDIN{ ADDIN EN.CITE.DATA (Sham et al., 2014). Therefore herbal medicines are preferred over synthetic ones due to their natural occurrence, presumed safety, nutritional and therapeutic potential. Several herbal medicines as well as purified natural products provide a rich resource for development of many novel drugs (Lin et al., 2014). One among the traditional folk medicines Terminalia chebula, belonging to the family Combretaceae has been used extensively in traditional as well as the modern practice against diabetes, cancer and bacterial infections (Lee et al., 2015). The plant has also been known to possess several pharmacological properties such as antioxidant, anti-inflammatory, antimutagenic, cardioprotective and wound healing activity (Bag et al., 2013). Though different parts of this plant have been explored for various pharmacological properties (Maddinedi et al., 2015; Kathirvel and Sujatha, 2012), the fruits have been known to exhibit several compounds such as hydrolysable tannins, anthraquinone, flavanol and terpenoids and reported for several therapeutic properties like anti-diabetic, anti-mutagenic confirmed by several in vitro and in vivo studies (Rekha et al., 2014). It also demonstrated to be a promising source of antioxidants (Mathew and Subramanian, 2014). The present study utilizes the dried fruit extract of T. chebula as it was observed to have a good efficacy in radical scavenging activity (Saha and Verma, 2014).
The diseases caused due to “oxidative stress’ which is initiated by Reactive Oxygen Species (ROS), produced as a byproduct of the electron transport in mitochondria is growing at an alarming rate (Saha and Verma, 2014). The ROS is considered to be an important target in the development of several anti-cancer drugs. Harmful free radicals like hydroxyl and superoxide anions are being constantly produced as a result of the metabolic reactions occurring inside the living system. The inhaled oxygen may be converted to ROS that can bind to cellular structures and is involved in a number of pathophysiological processes such as aging, inflammation and atherosclerosis (Singhal et al., 2014). Excessive accumulation of ROS can lead to protein oxidation, lipid peroxidation, enzyme inactivation and oxidative DNA damage (Lv et al., 2013). Keeping in view the use of T. chebula, as an effective remedy for oxidative stress associated diseases, the present study was undertaken to evaluate the inhibitory potential of T. chebula fruit extract by hydrogen peroxide (H2O2) induced ROS generation in THP-1 cell line.
MATERIALS AND METHODS
Plant identification and extract preparation: Terminalia chebula was collected from the natural habitat of Salem district, Tamil Nadu, India, in December 2012. The samples were identified and authentication was done by a plant anatomist. A Voucher specimen (ISISM/RES/B0014) has been preserved at the department of the Interdisciplinary Institute of Indian System of Medicine (IIISM), SRM University for future reference. The plant materials were shade dried at room temperature, powdered and stored in air tight containers. Thirty grams of the powdered plant material were soaked in a hydro-alcoholic mixture at room temperature with continuous shaking and the extraction was repeated three times. After completion of the extraction process, the solvent was removed using a rotary vacuum evaporator (Rotovac R-210, Buchi, Germany). The extract was stored in an air-tight container for further use.
Identification and quantification of active compounds by HPLC: Terminalia chebula extract was dissolved in HPLC grade MeOH (1 mg mL–1) and subjected to Reverse Phase High Performance Liquid Chromatography (RP-HPLC) for the qualitative and quantitative analysis of gallic acid and ellagic acid content. The HPLC system consisted of Shimadzu LC 2020 (Shimadzu, Japan) equipped with solvent delivery system, column oven, Photo Diode Array detector (PDA) and Lab solution software was used for all data analysis. The chromatographic separation was performed on Phenomenex-Luna (Torrance, CA, USA) C18 column (i.d. 250×4.6 mm, 5 μm) and the column oven were set at ambient temperature. The isocratic mobile phase consisted of methanol: Acetonitrile: Water (0.05% formic acid, 40:15:45) with a flow rate of 0.8 mL min–1 and the samples were injected using Hamilton Micro liter syringe (Switzerland) into a 20 μL injection loop.
Cell line and treatment: Human monocytic (THP-1) cell line were purchased from National Center for Cell Sciences, Pune, India. The cells were cultured under standard conditions in the roswell park memorial institute (RPMI-1640) supplemented with 10% Fetal bovine serum (FBS), 100 U mL–1 of penicillin and 100 U mL–1 of streptomycin, HiMedia, India in a humidified 5% CO2 at 37°C.
Cytotoxicity assay: The cytotoxic effect of T. chebula on THP-1 cells was assessed by measuring the activity of mitochondrial dehydrogenase as described previously (Wang et al., 2010). Briefly cells (5×103 cells/well) were incubated with various concentrations (3.9-500 μg mL–1) of T. chebula, the culture media was removed and the cells were incubated with 5 mg mL–1 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyltetrazolium bromide, Sigma Aldrich, India) (MTT) in fresh medium at 37°C for an additional 4 h. After this period, the supernatant was removed and 100 μL dimethyl sulfoxide, Sigma Aldrich, India, (DMSO) was added to each well to dissolve the formazan crystals. The plates were read on a microplate reader using MultiSkan GO, Thermo Scientific, USA, at a test wavelength of 540 nm with a reference wavelength of 650 nm.
Quantification of intracellular ROS in H2O2 induced THP-1 cells: The ROS generation was measured after staining the cells with oxidation sensitive probe 2-7-diacetyl dichlorofluorescein, Invitrogen, USA, (DCFH-DA) using flow cytometry as described previously with minor modifications (Krejsa and Schieven, 2000; Vasanth et al., 2014). THP-1 cells (2×105 cells/well) were cultured in 12-well plate treated with indicated concentrations in the presence or absence of T. chebula fruits extract (62.5, 125 and 250 μg mL–1) and incubated at 37°C with 5% CO2 for 24 h. The cells in 12-well plates were washed twice with Hank’s Balanced Salt Solution (HBSS) and incubated in 1 mL working solution of 20 μM DCFH-DA at 37°C in the dark for 30 min. Then the cells were harvested, washed with ice-cold Phosphate Buffered Saline (PBS). Cells were treated with 10 mM H2O2 to induce intracellular ROS. The fluorescence emission of DCF were analyzed in FL-1 channel on Fluorescence Activated Cell Sorting (FACS) (JazzBecton Dickinson, USA) using excitation and emission wavelengths at 488 and 525 nm, respectively. The Mean Fluorescence Intensity (MFI) of 104 cells acquired were quantified.
Statistical analysis: Data are expressed as Mean±SD. The statistical significance of the differences between the mean of various experimental groups was determined by one-way ANOVA followed by Bonferroni test using prism program (Graph-Pad Software Inc., USA). The level of the statistical significance was set at p<0.05.
RESULTS
HPLC analysis: The HPLC have been utilized to standardize many medicinal plant extracts. Standardization of plant based products is more challenging than synthetic drugs. In the present study, T. chebula fruit extract was subjected to HPLC analysis to find out the presence of biomarkers which could be due its synergistic pharmacological activities. The HPLC chromatogram of reference standards like gallic acid and ellagic acid were detected at 280 and 254 nm, respectively is showed Fig. 1. The presence of gallic acid and ellagic acid, was confirmed by comparing chromatographic peaks in hydro-alcoholic extract of T. chebula fruits with the Retention Time (Rt) 3.5 and 5.79 min, respectively. Compounds are reputed for their health promoting properties due to their high antioxidant capacity. The percentage of gallic acid in the T. chebula extract is 4.84% w/w and that of ellagic acid is 2.56% w/w.
| Fig. 1(a-d): | HPLC chromatogram representing (a, b) Hydroalcoholic extract of Terminalia chebula fruits at 254 and 280 nm, respectively inset, (c) Standard ellagic acid and (d) Standard gallic acid |
| Fig. 2: | Effect of Terminalia chebula fruit extract on cellular viability of THP-1 cells was examined by the MTT assay |
| Fig. 3(a-e): | Effect of Terminalia chebula fruit extract on the H2O2 induced ROS production in THP-1 cells. Cells were stained with DCFH-DA and analyzed using FACSCalibur flow cytometer, (a) Control, (b) H2O2, (c) H202+62.5, (d) H2O2+125 and (e) H2O2+250 µg mL–1 |
Effect of Terminalia chebula on cell viability: The MTT assay was performed to determine the safe concentration range of T. chebula in THP-1 cell. As shown in Fig. 2, the HA extract of T. chebula did not exhibit cytotoxicity to THP-1 cells at doses of 3.9-500 μg mL–1. However, the extract inhibited cell viability at the highest dose that was tested (500 μg mL–1). Thus, doses of T. chebula at the concentration that gave cell viability of 80% or more (62.5, 125 and 250 μg mL–1 were used for treatment in the further experiments. These results indicate that the T. chebula did not induce cell death.
Effect of intracellular ROS in H2O2 induced THP-1 cells: Flow cytometric analysis of THP-1 cells treated with different concentrations (62.5, 125 and 250 μg mL–1) of T. chebula stained with DCFH-DA, an indirect method for estimating the intracellular ROS, a specific fluorescent probe for measuring hydrogen peroxide and hydroxyl radicals revealed an increase in DCFH-DA positive cell population indicating generation of reactive oxygen species. As shown in Fig. 3, FACS analysis revealed that exposure to H2O2, markedly increased intracellular ROS levels in THP-1 cells, compared with the control group. Whereas, pretreatment of cells with T. chebula extract significantly reduced the intracellular ROS production in a dose-dependent manner.
DISCUSSION
The present study was undertaken to evaluate the inhibitory potential of T. chebula by hydrogen peroxide induced ROS generation in THP-1 cell line. It is a human leukemia monocytic cell line considered as a suitable, reliable model to study the functions and responses of monocytes and macrophages (Chanput et al., 2014). Herbal drugs have been used since traditional times in the treatment of various diseases. Antioxidants as external supplements are recommended to protect cells from the deleterious effects of oxidative stress conditions. In search of novel and natural antioxidants, various herbal extracts have been explored and researched in the preparation of Ayurvedic formulations. A robust investigation was done on 75 plants for their in vivo anti-inflammatory properties (Kam et al., 2014). Continuous research on the molecular mechanism of the diseases caused due to oxidative stress has resulted in identification of important targets necessary for drug development and thus allowed the investigation of various plant extracts in different cell based assays. One amongst the most popularly used plants since ancient times is T. chebula known for several decades for its various pharmacological properties (Bag et al., 2013). The fruits of this plant have been known to exhibit several medicinal properties such as anti-diarrheic, styptic, anti-bilious and anti-dysenteric activities as been reported by Cheng et al. (2003). The different pharmacological properties of compounds isolated from the fruits of this plant have been reported (Chen et al., 2011). For example chebulagic acid showed anti-proliferative and pro-apoptotic activities in several tumor cell lines. Arjunic acid isolated from this plant was also known as a potent free radical scavenger.
The role of ROS has been implicated in the pathogenesis of many chronic diseases like cancer. It has been known to affect the mitochondrial membrane potential and membrane permeability that can stimulate a number of mitochondria related events. Excessive accumulation of ROS can lead to oxidative DNA damage, protein oxidation and enzyme inactivation. This proves its usefulness in the development of several anti-cancer drugs (Lv et al., 2013). An imbalance of the ROS generation can aggravate inflammation. In the past few years the participation of ROS in the pathogenesis of several diseases has been given several importance. ROS such as hydrogen peroxide, superoxide anions and hydroxyl radicals can cause damage to tissues by activation of the NF-κB pathway and thus initiate as well as perpetuate the inflammatory cascade (Gusman et al., 2015).
Two important biomarker compounds were identified from this plant using HPLC. Gallic acid, a phenolic compound present in T. chebula has been reported for several health benefits such as anti-cancer, anti-inflammatory and cardioprotective properties (Kam et al., 2014). It demonstrated potent antioxidant properties which neutralizes ROS generation and oxidative stress and known to inhibit the tumor growth of liver and colon (Subramanian et al., 2014). Another biomarker, ellagic acid has gained much importance in the recent years due to its multiple pharmacological activities and molecular targets. It has shown several antibacterial, anticarcinogenic and antiatherogenic activities (Garcia-Nino and Zazueta, 2015). Several studies have enumerated the role of ellagic acid in effective lowering of plasma lipids, reduced oxidative stress, inhibition of apoptosis, scavengers of oxygen and hydroxyl radicals as well as inhibition of lipid peroxidation both in vitro and in vivo. It has been known to inhibit the cytokine induced ROS generation and expression of adhesion molecules as well as prevention of oxidized low Density Lipoprotein (LDL) induced apoptosis in the endothelial cells (Lee et al., 2010). The role of T. chebula in inhibiting ROS production may be attributed due to the symbiotic effect of these two important marker compounds present in the extract.
CONCLUSION
Terminalia chebula suppressed the production of ROS, known to participate in the causation of several diseases. It exhibited a high potential against markers involved in oxidative stress and also may act as inhibitor of free radicals mediated oxidative damages as primary antioxidant. The HA of T. chebula extract displayed efficient activities due to presence of high quantitities of phenolic compounds as identified by chromatographic techniques. Further the bioactive constituents present in the extract should be isolated and characterized for determination of the in vivo protective effects.
ACKNOWLEDGMENT
Authors are thankful to SRM University, for providing all necessary facilities.