Prasansuklab and Tencomnao BMC Complementary and Alternative Medicine (2018) 18:278 https://doi.org/10.1186/s12906-018-2340-4 RESEARCHARTICLE Open Access Acanthus ebracteatus leaf extract provides neuronal cell protection against oxidative stress injury induced by glutamate Anchalee Prasansuklab1 and Tewin Tencomnao2* Abstract Background: Acanthus ebracteatus (AE), an herb native to Asia, has been recognized in traditional folk medicine not only for its antioxidant properties and various pharmacological activities but also as an ingredient of longevity formulas. However, its anti-neurodegenerative potential is not yet clearly known. This work aimed to evaluate the protective effect of AE leaf extract against glutamate-induced oxidative damage in mouse hippocampal HT22 cells, a neurodegenerative model system due to a reduction in glutathione levels and an increase in reactive oxygen species (ROS). Methods: Cell viability, apoptosis, and ROS assays were performed to assess the protective effect of AE leaf extract against glutamate-induced oxidative toxicity in HT22 cells. The antioxidant capacity of AE was evaluated using in vitro radical scavenging assays. The subcellular localization of apoptosis-inducing factor (AIF) and the mRNA and protein levels of genes associated with the nuclear factor erythroid 2–related factor 2 (Nrf2) antioxidant system were determined to elucidate the mechanisms underlying the neuroprotective effect of AE leaf extract. Results: We demonstrated that AE leaf extract is capable of attenuating the intracellular ROS generation and HT22 cell death induced by glutamate in a concentration-dependent manner. Co-treatment of glutamate with the extract significantly reduced apoptotic cell death via inhibition of AIF nuclear translocation. The increases in Nrf2 levels in the nucleus and gene expression levels of antioxidant-related downstream genes under Nrf2 control were found to be significant in cells treated with the extract. Conclusions: The results suggested that AE leaf extract possesses neuroprotective activity against glutamate-induced oxidative injury and may have therapeutic potential for the treatment of neurodegenerative diseases associated with oxidative stress. Keywords: Acanthus ebracteatus, HT22 cells, Glutamate toxicity, Oxidative stress, Oxytosis, Neuroprotection, Antioxidant, Nrf2/ARE pathway Background important role in cellular injury in various tissues, par- Oxidative stress is classically described as an imbalance ticularly the central nervous system (CNS), which is of redox homeostasis, resulting from the overproduction highly sensitive to oxidative damage due to its large de- of free radicals relative to the innate ability of cells to pendence on oxygen consumption [3, 4]. In fact, oxida- scavenge them. This detrimental event causes damage to tive stress is associated with aging and is a common cellular components and alterations in cellular function pathological feature of age-related neurodegenerative that ultimately contribute to cell death [1, 2]. Reactive diseases, such as Alzheimer’s disease (AD), Parkinson’s oxygen species (ROS) are the most common type of free disease (PD), and amyotrophic lateral sclerosis (ALS), in radicals produced in the human body and play an which ROS accumulation is implicated in the mechan- ism of neuronal loss [5–7]. Currently, many researchers * Correspondence: [email protected] believe that compounds or drugs possessing powerful 2Age-Related Inflammation and Degeneration Research Unit, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University antioxidant activity could be effective in treating such Bangkok 10330, Thailand ROS-related diseases [8–10]. Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Prasansuklab and Tencomnao BMC Complementary and Alternative Medicine (2018) 18:278 Page 2 of 15 Glutamate, the principal excitatory neurotransmitter in Methods the brain, has been suggested as a critical trigger of neur- Plant material and preparation of the extracts onal cell death in several CNS disorders and neurodegener- The plant material used in this study is the leaves of A. ative diseases [11]. In addition to its involvement in many ebracteatus collected from the Princess Maha Chakri Sir- aspects of normal brain functions, glutamate can also act indhorn Herbal Garden (Rayong Province, Thailand). The as a neurotoxin when it is present in excessively high con- plant was authenticated by Professor Dr. Thaweesakdi centrations in the brain extracellular space, causing cellular Boonkerd and deposited with voucher specimen number damage in the context of neurodegeneration. One of the A013422(BCU) at the herbarium of Kasin Suvatabhandhu main mechanisms underlying the neurotoxic effects of glu- (Department of Botany, Faculty of Science, Chulalongkorn tamate is an oxidative stress-induced programmed cell University, Thailand). The extraction was carried out twice death pathway called oxytosis [12]. In this cell death para- using hexane and absolute ethanol as extracting solvents. digm, glutamate at pathological levels induces inhibition of Briefly, the leaves were dried in a ventilated incubator at a cystine uptake via the cystine/glutamate antiporter (system temperature of at 40 °C and ground into a fine powder. − Xc ), leading to impaired production of the endogenous Then, the extracts were prepared by macerating 35 g of antioxidant glutathione (GSH) and thereby enhancing ac- the dried leaf powder in 350 mL of each solvent for 48 h cumulation of ROS as well as oxidative stress. Subse- under agitation at room temperature (RT), followed by fil- quently, the elevated ROS level disrupts mitochondrial tration. The residue powder was re-extracted by a similar membrane integrity and results in the release of process, and all filtrates were subsequently combined be- apoptosis-inducing factor (AIF), which eventually triggers fore removing the solvent by vacuum evaporation. The neuronal death via a caspase-independent pathway [13, yield of hexane extract (AEH) and ethanolic extract (AEE) 14]. Therefore, suppression of glutamate-induced oxidative of A. ebracteatus leaves was found to be 2.14% and 7.98% stress-mediated neuronal cell death may have the potential (w/w), respectively. Each resulting extract was dissolved in to provide a beneficial therapeutic approach for the treat- dimethyl sulfoxide (DMSO) as a stock solution of 100 mg/ ment of neurodegenerative diseases. mL, stored at − 20 °C, and protected from light until fur- At the present time, there is growing worldwide use of ther analysis. herbal medicines for preventive and therapeutic applications basedonhistoricalknowledge.Inconnectionwiththeafore- Determination of total flavonoid content mentioned mechanism of glutamate-induced oxidative tox- The total flavonoid content was determined using the icity, medicinal plants or naturally derived compounds with aluminum chloride colorimetric method modified for a antioxidant and antiapoptotic effects are currently being microplate format as described previously [33]. In brief, researched as neuroprotective agents [15–19]. Acanthus 50 μL of the extract sample (1 mg/mL) was made up to ebracteatus Vahl. (AE), commonly known as “Sea Holly”,isa 200 μL with 95% ethanol, and mixed well with 10 μLof medicinal mangrove plant in the family Acanthaceae and is 10% (v/v) AlCl3 solution and 10 μL of 1 M NaOAc solu- widely distributed in Southeast Asia, including China, India, tion. After the reaction was allowed to stand for 40 min in and Australia [20, 21]. All parts of this plant have been used the dark, the absorbance of the reaction mixture was mea- historically for a variety of medicinal purposes, such as hair sured at 415 nm using a microplate reader (Perkin-Elmer). root nourishment, reduction of cough and fever, expulsion of Quercetin (Sigma-Aldrich) was used as a standard to con- kidney stones, relief of rheumatoid arthritis pain and inflam- struct the calibration curve for quantification, and the mation, and treatment of hypertension, cancer, skin diseases content of total flavonoids was reported as mg of quer- such as rash, chronic wounds and snakebites [22–26]. Inter- cetin equivalent (QE) per g of dry weight extract. estingly,AEisalsousedasanimportant ingredient in trad- itional Thai longevity and neurotonic remedies for Determination of total phenolic content improving brain and body functions [23, 27]. Moreover, pre- The total phenolic content was determined using the vious chemical investigations on this plant revealed the pres- Folin-Ciocalteu method adapted for analysis with a micro- ence of some bioactive components possessing considerable plate reader, as previously described [33]. Briefly, 50 μLof antioxidant activity, neuromodulatory function or the extract sample (1 mg/mL) was mixed thoroughly with memory-improving effects [28–32]. However, currently, 50 μL of 10-fold diluted Folin-Ciocalteu’sphenolreagent