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Neuroprotective effects of Hibiscus Sabdariffa against hydrogen peroxide-induced toxicity
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Neuroprotective effects of Hibiscus Sabdariffa against hydrogen peroxideinduced toxicity Aiman Shalgumb, Manoj Govindarajulua, Mohammed Majrashia,c, Sindhu Ramesha, Willard E. Collierb, Gerald Griffinb, Rajesh Amina, Chastity Bradfordb, Timothy Moorea, ⁎ Muralikrishnan Dhanasekarana, a
Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States Department of Biology, Tuskegee University, Tuskegee, AL, United States c Department of Pharmacology, Faculty of Medicine, University of Jeddah, Jeddah, Saudi Arabia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hibiscus sabdariffa Antioxidant Apoptosis Endogenous neurotoxin Mitochondrial dysfunction Neuroprotection
The World Health Organization and the National Institute of Mental Health (United States of America) states that neurodegenerative diseases leads to significant loss of regular activity of the patients, their family and the caretakers leading to a huge economic loss. Current treatments provide modest and temporary symptomatic relief, without altering the underlying mechanisms associated with the onset and the progression of the neurodegenerative diseases. Strong scientific evidence points the involvement of oxidative stress in the pathogenesis of neurodegenerative diseases. Thus, the current therapeutic efforts have been directed to find beneficial agents that could reduce the oxidative damage and promote a functional recovery of neurons in degenerative disorders. Hydrogen peroxide is an endogenous neurotoxin which can initiate and propagate (promote) neurodegeneration. Hibiscus sabdariffa (roselle) exhibits multiple pharmacological activities. Hence in this study, we evaluated the neuroprotective effects and the possible mechanisms of action of Hibiscus sabdariffa (roselle) against the hydrogen peroxide-induced neurotoxicity. Hibiscus sabdariffa exhibited antioxidant and antiapoptotic effects and significantly attenuated the neurotoxicity of hydrogen peroxide. Hibiscus sabdariffa exhibits neuroprotective effects and can be an effective and novel alternative approach to reduce the risk of various neurodegenerative disorders.
1. Introduction Neurotoxins (endogenous or exogenous) are substances that act primarily on the cells and tissues in the central and peripheral nervous system. Endogenous neurotoxins can induce cell death by acting on receptors (pre or post synaptic), enzymes (associated with the synthesis and metabolism of neurotransmitters & hormones, second messenger system), pumps, channels, storage vesicles or the nucleic acid (Breedlove et al., 2007; Popoff and Poulain, 2010). Furthermore endogenous neurotoxins can alter the neurotransmissions, induce energy dysfunction, generate free radicals & pro-inflammatory cytokines, initiate programmed cell death, affect the transcription factors which can target the cytoskeletal disruption, cellular functions and behavior leading to the death of the neuron (Popoff and Poulain, 2010). Current literature has clearly revealed that these endogenous neurotoxic substances can significantly enhance the risk of trauma, ischemic-
reperfusion injury, and various neurodegenerative diseases (Parkinson’s disease, Alzheimer’s disease, Huntington's disease, multiple sclerosis and amyotrophic lateral sclerosis) (Opazo et al., 2002; Uttara et al., 2009). The major endogenous neurotoxins are derivatives of isoquinoline, beta-carboline, 3,4-dihydroxyphenylacetaldehyde (DOPAL), Quinolinic acid, and hydrogen peroxide (Moser, 1997). In addition to the endogenous neurotoxins, emerging diffusible small-molecule messengers (reactive oxygen species and hydrogen peroxide) generated independently or in the presence of neurotoxins has also been associated with neurodegeneration (Bao et al., 2009). Presently, no remedies are available to cure the progressive damage and death of neurons. Hydrogen peroxide is an ubiquitous neuronal signaling substance in the central nervous system that affects the transcription factors, phosphatases, kinases, and ion channels (Kishida and Klann, 2007; Morgan and Veal, 2007). Hydrogen peroxide is generated from the powerhouse
⁎ Corresponding author at: Department of Drug Discovery and Development, 4306 Walker building, Harrison School of Pharmacy, Auburn University, Auburn, AL, 36849, United States. E-mail address: [email protected] (M. Dhanasekaran).
https://doi.org/10.1016/j.hermed.2018.100253 Received 7 June 2017; Received in revised form 24 November 2018; Accepted 14 December 2018 2210-8033/ © 2018 Elsevier GmbH. All rights reserved.
Please cite this article as: Shalgum, A., Journal of Herbal Medicine, https://doi.org/10.1016/j.hermed.2018.100253
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2. Materials and methods
of the cell-mitochondria (during mitochondrial respiration), neurotransmitters metabolism by monoamine oxidase and NADPH oxidase. Mitochondrial respiration and oxidative metabolism produces superoxide anion by the reduction of oxygen followed by subsequent conversion to hydrogen peroxide by superoxide dismutase or spontaneous dismutation (Boveris and Chance, 1973). Deamination of monoaminergic neurotransmitter by monoamine oxidase occurs through a two-electron reduction of oxygen to hydrogen peroxide. Tetrahydrobiopterin (autoxidation) in combination with tyrosine hydroxylase generates hydrogen peroxide and reactive oxygen species (Haavik et al., 1997). NADPH oxidase helps in the formation of hydrogen peroxide by catalyzing the electron reduction of oxygen. Physiologically, oxidative metabolism requires divalent metals. Interestingly, accumulation of divalent metals occurs progressively with ageing in some regions of the brain which are associated with motor and cognitive dysfunction. In Alzheimer's disease and Parkinson's disease, changes in local metal homoeostasis result in altered cellular distribution and accumulation, ultimately inducing neurotoxicity (Crichton and Ward, 2013). Divalent metals in the presence of hydrogen peroxide can generate reactive oxygen species and pro-inflammatory mediators resulting in oxidative stress, mitochondrial dysfunction, inflammation and apoptosis which can considerably promote neurodegeneration. Thus, hydrogen peroxide accumulation can significantly increase the risk of various neurodegenerative diseases. Hibiscus sabdariffa (roselle) is an annual herb that belongs to Malvaceae family and Hibiscus genus. They are widely distributed in tropical and subtropical regions (West Africa, India, and China). Plants from Hibiscus family contain neuroprotective nutrients such as acids (citric acid, malic acid, tartaric acid and allo-hydroxycitric acid), alkaloids, carotenoids, coenzyme Q-10, emodins, flavonoids, glycosides, lactone, polyphenols, saponins, steroids, tanins and triterpenoids (Foyet et al., 2011; Hritcu et al., 2011). Neuroprotective nutrients present in the botanicals are known to attenuate reactive oxygen species, chelate metals and reduce apoptosis, enhance mitochondrial functions and reduce the risk of neurodegenerative diseases (Dhanasekaran et al., 2009, 2008, 2007; Lohani et al., 2013). Polyphenols and coenzyme Q-10 exhibit resilient potential to attenuate the etiology of several neurological disorders because they address their complex pathophysiology by modulating multiple targets (receptors, enzymes, pumps, channels, etc.) at the same time. Hibiscus rosa sinensis is the well-established botanical in Hibiscus family. This is because numerous pharmacological and toxicological studies have been performed with Hibiscus rosa sinensis compared to Hibiscus sabdariffa. Hibiscus sabdariffa has shown to exhibit antimicrobial, anti-inflammatory, cardioprotective, hepatoprotective, nephroprotective, and anticancer activity. Hibiscus sabdariffa also affects the endocrine and exocrine secretions in the body and induce vasodilation resulting in increased sexual and reproductive functions (Carvajal-Zarrabal et al., 2012; Da-Costa-Rocha et al., 2014; Guardiola and Mach, 2014). However, there are no studies regarding the neuroprotective effects of Hibiscus sabdariffa against endogenous neurotoxins. Our previous study with various botanicals such as Bacopa monniera (Dhanasekaran et al., 2007), Mucuna pruriens (Dhanasekaran et al., 2008), Centella asiatica (Dhanasekaran et al., 2009), Scutellaria lateriflora (Lohani et al., 2013) have shown to exhibit antioxidant and neuroprotective effects. Epidemiological studies link vascular and endocrine disorders like diabetes mellitus, hypertension, and stroke with neurodegenerative disorders. Vascular risk factors are common comorbidities of nigral, hippocampal and cortical neurodegeneration. Abnormalities in metal and hydrogen peroxide homeostasis with additional genetic factors play critical roles in the metal-catalyzed oxidative oligomerization which may lead to possible protein aggregation and neurodegenerations. Hence, in this study we evaluated the neuroprotective effects against an endogenous neurotoxin (hydrogen peroxide) and established the neuroprotective mechanisms of Hibiscus sabdariffa extract.
Materials-Antibodies, Chemicals, and reagents: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA (0.25%), penicillin/streptomycin mixture, hydrogen peroxide (H2O2), 3-(4,5-dimethyl thiazol-2-yl), 2,5- diphenyltetrazoliumbromide (MTT), thiobarbituric acid, butylated hydroxytoluene (BHT), 2,7- dichlorodihydrofluorescein diacetate, dimethyl sulfoxide (DMSO). thiazolyl blue tetrazolium bromide (MTT), O-phthaldialdehyde (OPT), Trisbuffered saline tablet, phosphate buffered saline tablet, reduced glutathione (GSH) were purchased from Sigma Aldrich (St. Louis, MO). Cell lysis buffer, secondary anti-rabbit antibody conjugated with fluorophore DyLight 550, Bcl-2, caspase-3 and β-actin primary antibodies were purchased from Cell Signaling Technologies (Cell Signaling Technology, Inc., Danvers, MA). Thermo Scientific Pierce 660 nm Protein Assay reagent kit was purchased (Pierce, Rockford, IL) for protein quantification. 2.1. Extraction procedure The Hibiscus sabdariffa seeds were obtained from a well-established commercial source, Strictly Medicinal, Williams, OR, USA. Strictly Medicinal Company identified it based on their herbarium. Seeds are maintained from year to year in Department of Biology, Tuskegee University and are provided to other researchers free of charge on request. A voucher specimen has been deposited in the Freeman Herbarium (AUA) at Auburn University (accession #77678). Hibiscus sabdariffa was grown organically and sepals were harvested at Tuskegee University. Dried Hibiscus sabdariffa sepals (5 g) were weighed and placed into a large test tube of ethanol (40 ml) and the test tube was capped. The tube was placed in a shaker bath set to 50°-55 °C with a speed of 80 rpm and allowed to extract for 1 h. The contents of the test tube were filtered to remove the extracted sepals. These crude extracts were filtered again, using 0.2 μm filters, into weighed amber vials. The ethanol was evaporated using a rotary evaporator. The extract was evaporated to remove all extraction solvent. A stock solution of 10 mg/ ml of Hibiscus sabdariffa extract was prepared in sterile water and stored in dark at 4 °C. For the experimental purpose, the extract was then diluted in water to make solutions of varying concentrations for the cytotoxicity assay and for the evaluation of the neuroprotective effects. Initial cell viability experiments were carried out with different concentration of Hibiscus sabdariffa extract (25, 50, 100, 150, 200, 250, 500 μg/ml). The dose of 25, 50, 100 μg did not show any cytotoxicity. Hence, this dose was used to establish the neuroprotective effects of Hibiscus sabdariffa extract. Quantification of neuroprotective nutrients (alkaloid, protein, polyphenols, sulfhydryl and coenzyme Q-10) in Hibiscus sabdariffa extract: Alkaloid, protein, polyphenols, sulfhydryl and coenzyme Q-10 were measured using our previously published procedure (Lohani et al., 2013). 2.2. Cell line acclimatization SH-SY5Y cells were purchased from ATCC (Manassas, VA, USA). The cells were cultured in DMEM containing Fetal Bovine Serum (10%), Penicillin-Streptomycin Solution (1%), L-glutamine (4 mM), sodium bicarbonate (1.5 g/L) and glucose (4.5 g/L). Cells were incubated at 37 °C and supplemented with 5% CO2. Cells were initially propagated in the 75 cm2 flasks. 2.3. Treatment procedure and cytotoxicity assay by MTT SH-SY5Y cells were harvested from the flasks by trypsinization after reaching 70–85% confluence and plated into 96 well plates at a density of 1 × 105 cells/ml. To investigate the neuroprotective effects, SH-SY5Y cells were incubated with or without Hibiscus Sabdariffa extract (50 and 2
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100 μg) for 24 h. After the pre-treatment, SH-SY5Y cells were exposed to the endogenous neurotoxin hydrogen peroxide (32 μM) or saline for another 8 h. Saline treated cells served as the control. For positive control studies, Vitamin C (1 and 5 μM) substituted the Hibiscus Sabdariffa extract. Subsequently, MTT (2 mg/ml) was added to each well, followed by 4 h of incubation at 37 °C in a dark environment to allow the formation of purple formazan dye. The medium was removed and DMSO was added to solubilize the insoluble formazan crystals. The absorbance was measured using a microtiter plate reader (Synergy HT, Bio-Tek Instruments Inc., Winooski, VT, USA) at 570 nm.
2.10. Apoptosis induction assays by western blot Standardized and established western blot procedure was used to study the expression of caspase-3 and Bcl-2. B –actin was used as a loading control. Band intensity was calculated by densitometric analysis using AlphaView software, normalized to β-actin and reported as percentage change from the control (Zheng et al., 2014). 2.11. Statistical analysis Statistical analysis was performed using Prism-IV software (La Jolla, CA, USA). The results obtained from the present study were expressed as means ± SEM. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons test (P < 0.05 was considered to be statistically significant).
2.4. Establishing the antioxidant mechanisms associated with the neuroprotective Hibiscus Sabdariffa extract SH-SY5Y cells were harvested after the treatment with Hibiscus Sabdariffa extract (50 and 100 μg) and / or hydrogen peroxide (32 μM). Content of reactive oxygen species, lipid peroxides & glutathione, and the activity of superoxide dismutase & catalase were measured to establish the antioxidant effects of the Hibiscus Sabdariffa extract.
3. Results Endogenous neurotoxin, hydrogen peroxide (32 μM) was used in the current study for investigating the neuroprotective effects of Hibiscus Sabdariffa extract in the SH-SY5Y neuroblastoma cells. Hydrogen peroxide (32 μM) significantly decreased the neuronal viability as seen by the MTT assay and microscopy as compared to the control (*P < 0.05, n= 12, Fig. 1a). Hibiscus Sabdariffa extract (50 and 100 μg) significantly exhibited neuroprotection against hydrogen peroxide-induced neurotoxicity (*P < 0.05, n= 10, Fig. 1a). Vitamin C (1 and 5μM) also protected the neuroblastoma cells against the endogenous neurotoxin (*P < 0.05, n= 10, Fig. 1b). Based on the above findings, the next step was to establish the neuroprotective mechanisms of Hibiscus Sabdariffa extract using the above model. Hydrogen peroxide (32 μM) significantly induced oxidative stress (increase in reactive oxygen species & lipid peroxide content, decrease in glutathione content & catalase activity), mitochondrial dysfunction (decreased mitochondrial Complex-I activity & mitochondrial membrane potential) and induced apoptosis (increase in caspase-3 and decrease in Bcl-2 expression, *P < 0.05, n= 6, Fig. 2a–e, 3 a and b, 4 a and b). Due to the presence of neuroprotective nutrients, Hibiscus Sabdariffa extract has the potential to scavenge the reactive oxygen species and inhibit lipid peroxidation. With regard to the antioxidant activity, Hibiscus Sabdariffa extract significantly scavenged the reactive oxygen species induced by hydrogen peroxide (aP < 0.05, n= 6, Fig. 2a and b). Increased generation of reactive oxygen species by hydrogen peroxide mainly affects the lipids and causes peroxidation, which decreases the functions of the neuronal lipid membranes resulting in neurotoxicity. Since Hibiscus Sabdariffa extract significantly scavenged the reactive oxygen species, it significantly blocked the lipid peroxide formation as compared to the hydrogen peroxide (aP < 0.05, n= 6, Fig. 2c). Increased generation of reactive oxygen species is attributed mainly due to increase in the pro-oxidants content or decrease in the antioxidant levels. Furthermore, the reactive oxygen species and the hydrogen peroxide’s proxidant effects are reduced by the presence of antioxidants. Glutathione is a potent antioxidant which significantly scavenges the free radicals generated in the body. Superoxide dismutase and catalase play a vital role in decreasing the oxidative stress induced by various proxidants. Hydrogen peroxide induces oxidative stress resulting in neuronal death. To exert its neurotoxic effect by inducing oxidative stress, hydrogen peroxide significantly depleted glutathione and inhibited the catalase activity in the SH-SY5Y neuroblastoma cells (*P < 0.05, n= 6, Fig. 2d and e). Hibiscus Sabdariffa extract significantly blocked the depletion of glutathione induced by hydrogen peroxide and significantly increased the catalase activity (aP < 0.05, n= 6, Fig. 2d and e). Oxidative stress can significantly affect the functions of the mitochondria leading to decreased production of energy and resulting in decreased neuronal viability. Hydrogen peroxide inhibited the mitochondrial Complex-I activity and significantly reduced the mitochondrial membrane potential (*P < 0.05, n= 6,
2.5. Quantifying total reactive oxygen species (ROS) Our previously established spectrofluorometric procedure (using 2`, 7-dichlorofluorescin diacetate) was used to quantify the reactive oxygen species (Zheng et al., 2014). Results were expressed as percentage change from the control. Mitochondrial Reactive Oxygen Species Quantification: This was measured using mitochondrial specific dihydrorhodamine (DHR) probe (Biotium, no. 10055, (Mouli et al., 2015). Fluorescence intensity was detected using a fluorescence microscope and the results were expressed as percentage change from the control. 2.6. Lipid peroxidation Thiobarbituric acid based colorimetric method was used to measure the lipid peroxide content. Results were expressed as percentage change from the control (Zheng et al., 2014). 2.7. Catalase activity (CAT) Spectrophotometric procedure using hydrogen peroxide as the substrate was used to assess the catalase activity. A standard curve of hydrogen peroxide was obtained and the results were expressed as percentage change from the control (Zheng et al., 2014). 2.8. Glutathione content Established fluorimetric procedure was used to measure the glutathione content. A standard curve of glutathione was obtained and the results were expressed as percentage change from the control (Zheng et al., 2014). Effects of Hibiscus Sabdariffa extract on the mitochondrial function: Mitochondrial Complex-I activity: Mitochondrial Complex-I activity is assessed based on the NADH oxidation, which was measured spectrophotometrically. A standard curve of NADH was obtained and the results were expressed as percentage change from the control (Zheng et al., 2014). 2.9. Mitochondrial membrane potential assay The cells were stained and the signals were imaged using a fluorescence microscope. The mitochondrial membrane potential was measured utilizing tetramethylrhodamine ethyl ester (TMRE; Biotium, no.70016; 2). TMRE florescent intensity was accomplished by BioTek Synergy HT plate reader (BioTek, VT, USA). The results were expressed as percentage change from the control (Mouli et al., 2015). 3
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Fig. 1. Effect of Hibiscus sabdariffa on proliferation of neuroblastoma cell line (SH-SY5Y). Cell proliferation was measured calorimetrically by MTT assay. Results are expressed as mean ± SEM normalized to the % of control. a. Hibiscus sabdariffa showed significant neuroprotective effect at both doses (50 & 100 μg) against hydrogen peroxide-induced toxicity (aP < 0.05, n = 10). b. Vitamin C was used as a positive control. It also exhibited neuroprotection against endogenous neurotoxin (aP < 0.05, n = 10). Results are expressed as mean ± SEM normalized to the % of control.
4. Discussion
Fig. 3a and b). Coenzyme Q-10 is a mitochondrial energy enhancer and has exhibited neuroprotection against endogenous and exogenous neurotoxins (Somayajulu-Niţu et al., 2009). Hibiscus Sabdariffa extract possess coenzyme Q-10 and significantly blocked the mitochondrial toxicity induced by hydrogen peroxide (aP < 0.05, n= 6, Fig. 3a and b). Increase in pro-apoptotic factors and decreased antiapoptotic factors have been associated with neurodegeneration. In our study, hydrogen peroxide significantly increased the expression of caspase-3 (proapoptotic) and decreased the expression of Bcl-2-anti-apoptotic factor (*P < 0.05, n= 4, Fig. 4a and b). Increased Bcl-2 and decreased caspase-3 expression was mainly attributed to the anti-apoptotic effects of Hibiscus Sabdariffa extract against hydrogen peroxide-induced neurotoxic insults (aP < 0.05, n= 4, Fig. 4a and b).
Oxidative stress, mitochondrial dysfunction and neuronal death are typical features of neurodegenerative diseases (Karpinska and Gromadzka, 2013). Therefore, extensive research is underway for identifying novel synthetic and natural drugs/agents exhibiting potent neuroprotective effects and with minimal adverse effects and hypersensitivity reactions to treat various neurological disorders. Numerous studies have demonstrated that various botanicals contain cytoprotective agents which can significantly contribute their therapeutic efficacy. Ilex latifolia (Aquifoliaceae) exhibits antioxidant, anti-inflammatory and anti-ischemic effects in the memory-impaired rodent brain (Kim et al., 2015, 2012). The neuroprotective mechanisms of Ilex latifolia involved the prevention of glutathione depletion and lipid peroxidation, decreased expression of phosphorylated tau (p-tau) and 4
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Fig. 2. Effect of Hibiscus sabdariffa on the oxidative stress induced by hydrogen peroxide: Hibiscus sabdariffa exhibited antioxidant activity as seen by scavenging reactive oxygen species, reducing lipid peroxidation, blocking glutathione depletion and affecting catalase activity (*P < 0.05 as compared to control, aP < 0.05 as compared to Hydrogen peroxide treatment, n = 8).
inflammatory mediators (Hwang, 2013; Sriram et al., 1997). In addition to the endogenous toxins accumulation and exposure to exogenous toxins, protein aggregation is an explicit shared biochemical aspect of many neurodegenerative diseases. Amyloid-beta (Aβ) and tau protein in Alzheimer’s disease and α-synuclein in Parkinson’s disease are implicated in generating hydrogen peroxide. Cholinergic and dopaminergic neurodegeneration in Alzheimer’s disease and Parkinson’s disease respectively is attributed to the direct production of hydrogen peroxide during extracellular or intracellular protein aggregation (Winklhofer and Haass, 2010). Endogenous neurotoxin, hydrogen peroxide induces oxidative stress by generating reacting oxygen species and inhibiting antioxidant enzymes (Finkel and Holbrook, 2000). With regard to mitochondrial functions, hydrogen peroxide inhibited Complex-I activity and affected
pro-apoptotic proteins (Fan et al., 2014). Chromogranin A-derived peptide catestatin has shown to possess potent antihypertensive and neuroprotective effects by affecting GABAergic neurotransmission (Choi et al., 2015). Thus, the contemporary scientific literature validates the use of drugs that can inhibit oxidative stress, mitochondrial dysfunction, excitotoxicity, apoptosis and inflammation as a possible valid and a novel therapeutic strategy for reducing the risk and progression of the neurodegenerative disorders (Table 1). Accrual of endogenous and/or exogenous toxins in the central nervous system has shown to elicit a cascade of neurochemical and biochemical lethal modifications that results in neuronal damage. Catecholamines in the presence of endogenous and/or exogenous toxins, metals and enzymes generate potent pro-oxidants (superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen), pro5
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Fig. 3. Effect of Hibiscus sabdariffa on the mitochondrial dysfunction induced by hydrogen peroxide: Hibiscus sabdariffa enhanced the mitochondrial functions as seen by the effect on Complex-I activity and mitochondrial membrane potential. (*P < 0.05 as compared to control, aP < 0.05 as compared to Hydrogen peroxide treatment, n = 8).
et al., 2013; Shults et al., 2002). Similar to coenzyme Q-10, NADH also shown to exhibit protective effects (Kidd, 2005; Wakade and Chong, 2014). Various other studies also have validated the bioactive properties of Hibiscus sabdariffa that may play an essential role in its prophylactic effect against chronic diseases (Khaghani et al., 2011; Sindi et al., 2014). The nutrients can scavenge reactive oxygen and free radicals, inhibit the activity of xanthine oxidase, decrease the peroxidation of lipids and boost the antioxidant activities, decrease inflammation and increase the mitochondrial functions (Da-Costa-Rocha et al., 2014). Hibiscus sabdariffa extract exhibited neuroprotection by its antioxidant action, blocking mitochondrial dysfunction and apoptosis induced by hydrogen peroxide. Based on our current findings, Hibiscus sabdariffa can be a novel botanical therapy for reducing the endogenous toxins mediated neuronal insult. Clinical trials with Hibiscus sabdariffa has shown to have very few adverse effects (Hajifaraji et al., 2018). Clinically, botanicals have recently gained global popularity due to their credible multi-target actions and minimal side effects. The multitarget actions of botanicals provide a wide-range of therapeutic avenue for treating various disorders. Hence, with regard to the global health care, Hibiscus sabdariffa extract may be used to reduce the risk for neurodegenerative diseases. Hibiscus sabdariffa extract exerts multitarget effects and thus therapeutically can be used in the prevention and treatment of Alzheimer’s, Parkinson’s and Huntington’s disorders.
mitochondrial membrane potential leading to neurotoxicity (Winklhofer and Haass, 2010). Hydrogen peroxide also induced apoptosis by increasing the pro-apoptotic protein expression and decreasing the anti-apoptotic expression. In our current study, hydrogen peroxide induces oxidative stress, mitochondrial dysfunction and apoptosis leading to the neuronal death. Currently there are very few neuroprotective studies with Hibiscus sabdariffa. However, various plants form Hibiscus family has exhibited neuroprotective effects in animal models of movement and memory related neurodegenerative diseases. Hibiscus sabdariffa has exhibited nootropic action and memory enhancement effects (Joshi and Parle, 2006; Nade et al., 2011). In our current study, we have established the presence of neuroprotective micronutrients such as polyphenols, sulfhydryl content, NADH and coenzyme Q 10, in addition to the alkaloid and protein. In our previous studies, we have associated the presence of the above nutrients to the neuroprotective effects of various botanicals such as Mucuna pruriens (Dhanasekaran et al., 2008), Trichopus xeylanicus (Tharakan et al., 2005), and Bacopa monniera (Dhanasekaran et al., 2007). With regard to polyphenols, there are other studies that have confirmed the protective effects of polyphenols (Qi et al., 2017; Talarek et al., 2017). Coenzyme Q-10 is an endogenous antioxidant that significantly improves the activity of Complex I and II in the mitochondrial electron transport chain and increases the production of ATP. Several preclinical and clinical studies have established the neuroprotective effects of coenzyme Q-10 in Parkinson’s, Alzheimer’s and Huntington’s disease (Chandra et al., 2014; Huntington Study Group Pre2CARE Investigators et al., 2010; Shetty 6
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Fig. 4. Effect of Hibiscus sabdariffa on apoptosis induced by hydrogen peroxide: Hibiscus sabdariffa decreased apoptosis. (*P < 0.05 as compared to control, a P < 0.05 as compared to Hydrogen peroxide treatment, n = 8).
References
Table 1 Polyphenol, Sulfhydryl, protein, alkaloid, and Coenzyme Q10 content in the ethanolic extract of Hibiscus Sabdariffa. Parameters
Content
Total Polyphenols Sulfhydryl content Total Protein Total Alkaloid NADH Coenzyme Q10
11.35 + 0.06 μg/mg 0.93 + 0.03 μg/mg 0.27 ± 0.02 μg/mg 84 ± 0.19 μg/mg 1.8 + 0.1 ng/mg 9.6 + 0.7 ng/mg
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Conflict of interest As a corresponding author, I have received the input from all the authors and there are no conflicts of interest. On behalf of all the authors, I (corresponding author), I assure that there are no conflict of interest.
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Neuroprotective effects of Hibiscus Sabdariffa against hydrogen peroxideinduced toxicity Aiman Shalgumb, Manoj Govindarajulua, Mohammed Majrashia,c, Sindhu Ramesha, Willard E. Collierb, Gerald Griffinb, Rajesh Amina, Chastity Bradfordb, Timothy Moorea, ⁎ Muralikrishnan Dhanasekarana, a
Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL, United States Department of Biology, Tuskegee University, Tuskegee, AL, United States c Department of Pharmacology, Faculty of Medicine, University of Jeddah, Jeddah, Saudi Arabia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hibiscus sabdariffa Antioxidant Apoptosis Endogenous neurotoxin Mitochondrial dysfunction Neuroprotection
The World Health Organization and the National Institute of Mental Health (United States of America) states that neurodegenerative diseases leads to significant loss of regular activity of the patients, their family and the caretakers leading to a huge economic loss. Current treatments provide modest and temporary symptomatic relief, without altering the underlying mechanisms associated with the onset and the progression of the neurodegenerative diseases. Strong scientific evidence points the involvement of oxidative stress in the pathogenesis of neurodegenerative diseases. Thus, the current therapeutic efforts have been directed to find beneficial agents that could reduce the oxidative damage and promote a functional recovery of neurons in degenerative disorders. Hydrogen peroxide is an endogenous neurotoxin which can initiate and propagate (promote) neurodegeneration. Hibiscus sabdariffa (roselle) exhibits multiple pharmacological activities. Hence in this study, we evaluated the neuroprotective effects and the possible mechanisms of action of Hibiscus sabdariffa (roselle) against the hydrogen peroxide-induced neurotoxicity. Hibiscus sabdariffa exhibited antioxidant and antiapoptotic effects and significantly attenuated the neurotoxicity of hydrogen peroxide. Hibiscus sabdariffa exhibits neuroprotective effects and can be an effective and novel alternative approach to reduce the risk of various neurodegenerative disorders.
1. Introduction Neurotoxins (endogenous or exogenous) are substances that act primarily on the cells and tissues in the central and peripheral nervous system. Endogenous neurotoxins can induce cell death by acting on receptors (pre or post synaptic), enzymes (associated with the synthesis and metabolism of neurotransmitters & hormones, second messenger system), pumps, channels, storage vesicles or the nucleic acid (Breedlove et al., 2007; Popoff and Poulain, 2010). Furthermore endogenous neurotoxins can alter the neurotransmissions, induce energy dysfunction, generate free radicals & pro-inflammatory cytokines, initiate programmed cell death, affect the transcription factors which can target the cytoskeletal disruption, cellular functions and behavior leading to the death of the neuron (Popoff and Poulain, 2010). Current literature has clearly revealed that these endogenous neurotoxic substances can significantly enhance the risk of trauma, ischemic-
reperfusion injury, and various neurodegenerative diseases (Parkinson’s disease, Alzheimer’s disease, Huntington's disease, multiple sclerosis and amyotrophic lateral sclerosis) (Opazo et al., 2002; Uttara et al., 2009). The major endogenous neurotoxins are derivatives of isoquinoline, beta-carboline, 3,4-dihydroxyphenylacetaldehyde (DOPAL), Quinolinic acid, and hydrogen peroxide (Moser, 1997). In addition to the endogenous neurotoxins, emerging diffusible small-molecule messengers (reactive oxygen species and hydrogen peroxide) generated independently or in the presence of neurotoxins has also been associated with neurodegeneration (Bao et al., 2009). Presently, no remedies are available to cure the progressive damage and death of neurons. Hydrogen peroxide is an ubiquitous neuronal signaling substance in the central nervous system that affects the transcription factors, phosphatases, kinases, and ion channels (Kishida and Klann, 2007; Morgan and Veal, 2007). Hydrogen peroxide is generated from the powerhouse
⁎ Corresponding author at: Department of Drug Discovery and Development, 4306 Walker building, Harrison School of Pharmacy, Auburn University, Auburn, AL, 36849, United States. E-mail address: [email protected] (M. Dhanasekaran).
https://doi.org/10.1016/j.hermed.2018.100253 Received 7 June 2017; Received in revised form 24 November 2018; Accepted 14 December 2018 2210-8033/ © 2018 Elsevier GmbH. All rights reserved.
Please cite this article as: Shalgum, A., Journal of Herbal Medicine, https://doi.org/10.1016/j.hermed.2018.100253
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2. Materials and methods
of the cell-mitochondria (during mitochondrial respiration), neurotransmitters metabolism by monoamine oxidase and NADPH oxidase. Mitochondrial respiration and oxidative metabolism produces superoxide anion by the reduction of oxygen followed by subsequent conversion to hydrogen peroxide by superoxide dismutase or spontaneous dismutation (Boveris and Chance, 1973). Deamination of monoaminergic neurotransmitter by monoamine oxidase occurs through a two-electron reduction of oxygen to hydrogen peroxide. Tetrahydrobiopterin (autoxidation) in combination with tyrosine hydroxylase generates hydrogen peroxide and reactive oxygen species (Haavik et al., 1997). NADPH oxidase helps in the formation of hydrogen peroxide by catalyzing the electron reduction of oxygen. Physiologically, oxidative metabolism requires divalent metals. Interestingly, accumulation of divalent metals occurs progressively with ageing in some regions of the brain which are associated with motor and cognitive dysfunction. In Alzheimer's disease and Parkinson's disease, changes in local metal homoeostasis result in altered cellular distribution and accumulation, ultimately inducing neurotoxicity (Crichton and Ward, 2013). Divalent metals in the presence of hydrogen peroxide can generate reactive oxygen species and pro-inflammatory mediators resulting in oxidative stress, mitochondrial dysfunction, inflammation and apoptosis which can considerably promote neurodegeneration. Thus, hydrogen peroxide accumulation can significantly increase the risk of various neurodegenerative diseases. Hibiscus sabdariffa (roselle) is an annual herb that belongs to Malvaceae family and Hibiscus genus. They are widely distributed in tropical and subtropical regions (West Africa, India, and China). Plants from Hibiscus family contain neuroprotective nutrients such as acids (citric acid, malic acid, tartaric acid and allo-hydroxycitric acid), alkaloids, carotenoids, coenzyme Q-10, emodins, flavonoids, glycosides, lactone, polyphenols, saponins, steroids, tanins and triterpenoids (Foyet et al., 2011; Hritcu et al., 2011). Neuroprotective nutrients present in the botanicals are known to attenuate reactive oxygen species, chelate metals and reduce apoptosis, enhance mitochondrial functions and reduce the risk of neurodegenerative diseases (Dhanasekaran et al., 2009, 2008, 2007; Lohani et al., 2013). Polyphenols and coenzyme Q-10 exhibit resilient potential to attenuate the etiology of several neurological disorders because they address their complex pathophysiology by modulating multiple targets (receptors, enzymes, pumps, channels, etc.) at the same time. Hibiscus rosa sinensis is the well-established botanical in Hibiscus family. This is because numerous pharmacological and toxicological studies have been performed with Hibiscus rosa sinensis compared to Hibiscus sabdariffa. Hibiscus sabdariffa has shown to exhibit antimicrobial, anti-inflammatory, cardioprotective, hepatoprotective, nephroprotective, and anticancer activity. Hibiscus sabdariffa also affects the endocrine and exocrine secretions in the body and induce vasodilation resulting in increased sexual and reproductive functions (Carvajal-Zarrabal et al., 2012; Da-Costa-Rocha et al., 2014; Guardiola and Mach, 2014). However, there are no studies regarding the neuroprotective effects of Hibiscus sabdariffa against endogenous neurotoxins. Our previous study with various botanicals such as Bacopa monniera (Dhanasekaran et al., 2007), Mucuna pruriens (Dhanasekaran et al., 2008), Centella asiatica (Dhanasekaran et al., 2009), Scutellaria lateriflora (Lohani et al., 2013) have shown to exhibit antioxidant and neuroprotective effects. Epidemiological studies link vascular and endocrine disorders like diabetes mellitus, hypertension, and stroke with neurodegenerative disorders. Vascular risk factors are common comorbidities of nigral, hippocampal and cortical neurodegeneration. Abnormalities in metal and hydrogen peroxide homeostasis with additional genetic factors play critical roles in the metal-catalyzed oxidative oligomerization which may lead to possible protein aggregation and neurodegenerations. Hence, in this study we evaluated the neuroprotective effects against an endogenous neurotoxin (hydrogen peroxide) and established the neuroprotective mechanisms of Hibiscus sabdariffa extract.
Materials-Antibodies, Chemicals, and reagents: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA (0.25%), penicillin/streptomycin mixture, hydrogen peroxide (H2O2), 3-(4,5-dimethyl thiazol-2-yl), 2,5- diphenyltetrazoliumbromide (MTT), thiobarbituric acid, butylated hydroxytoluene (BHT), 2,7- dichlorodihydrofluorescein diacetate, dimethyl sulfoxide (DMSO). thiazolyl blue tetrazolium bromide (MTT), O-phthaldialdehyde (OPT), Trisbuffered saline tablet, phosphate buffered saline tablet, reduced glutathione (GSH) were purchased from Sigma Aldrich (St. Louis, MO). Cell lysis buffer, secondary anti-rabbit antibody conjugated with fluorophore DyLight 550, Bcl-2, caspase-3 and β-actin primary antibodies were purchased from Cell Signaling Technologies (Cell Signaling Technology, Inc., Danvers, MA). Thermo Scientific Pierce 660 nm Protein Assay reagent kit was purchased (Pierce, Rockford, IL) for protein quantification. 2.1. Extraction procedure The Hibiscus sabdariffa seeds were obtained from a well-established commercial source, Strictly Medicinal, Williams, OR, USA. Strictly Medicinal Company identified it based on their herbarium. Seeds are maintained from year to year in Department of Biology, Tuskegee University and are provided to other researchers free of charge on request. A voucher specimen has been deposited in the Freeman Herbarium (AUA) at Auburn University (accession #77678). Hibiscus sabdariffa was grown organically and sepals were harvested at Tuskegee University. Dried Hibiscus sabdariffa sepals (5 g) were weighed and placed into a large test tube of ethanol (40 ml) and the test tube was capped. The tube was placed in a shaker bath set to 50°-55 °C with a speed of 80 rpm and allowed to extract for 1 h. The contents of the test tube were filtered to remove the extracted sepals. These crude extracts were filtered again, using 0.2 μm filters, into weighed amber vials. The ethanol was evaporated using a rotary evaporator. The extract was evaporated to remove all extraction solvent. A stock solution of 10 mg/ ml of Hibiscus sabdariffa extract was prepared in sterile water and stored in dark at 4 °C. For the experimental purpose, the extract was then diluted in water to make solutions of varying concentrations for the cytotoxicity assay and for the evaluation of the neuroprotective effects. Initial cell viability experiments were carried out with different concentration of Hibiscus sabdariffa extract (25, 50, 100, 150, 200, 250, 500 μg/ml). The dose of 25, 50, 100 μg did not show any cytotoxicity. Hence, this dose was used to establish the neuroprotective effects of Hibiscus sabdariffa extract. Quantification of neuroprotective nutrients (alkaloid, protein, polyphenols, sulfhydryl and coenzyme Q-10) in Hibiscus sabdariffa extract: Alkaloid, protein, polyphenols, sulfhydryl and coenzyme Q-10 were measured using our previously published procedure (Lohani et al., 2013). 2.2. Cell line acclimatization SH-SY5Y cells were purchased from ATCC (Manassas, VA, USA). The cells were cultured in DMEM containing Fetal Bovine Serum (10%), Penicillin-Streptomycin Solution (1%), L-glutamine (4 mM), sodium bicarbonate (1.5 g/L) and glucose (4.5 g/L). Cells were incubated at 37 °C and supplemented with 5% CO2. Cells were initially propagated in the 75 cm2 flasks. 2.3. Treatment procedure and cytotoxicity assay by MTT SH-SY5Y cells were harvested from the flasks by trypsinization after reaching 70–85% confluence and plated into 96 well plates at a density of 1 × 105 cells/ml. To investigate the neuroprotective effects, SH-SY5Y cells were incubated with or without Hibiscus Sabdariffa extract (50 and 2
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100 μg) for 24 h. After the pre-treatment, SH-SY5Y cells were exposed to the endogenous neurotoxin hydrogen peroxide (32 μM) or saline for another 8 h. Saline treated cells served as the control. For positive control studies, Vitamin C (1 and 5 μM) substituted the Hibiscus Sabdariffa extract. Subsequently, MTT (2 mg/ml) was added to each well, followed by 4 h of incubation at 37 °C in a dark environment to allow the formation of purple formazan dye. The medium was removed and DMSO was added to solubilize the insoluble formazan crystals. The absorbance was measured using a microtiter plate reader (Synergy HT, Bio-Tek Instruments Inc., Winooski, VT, USA) at 570 nm.
2.10. Apoptosis induction assays by western blot Standardized and established western blot procedure was used to study the expression of caspase-3 and Bcl-2. B –actin was used as a loading control. Band intensity was calculated by densitometric analysis using AlphaView software, normalized to β-actin and reported as percentage change from the control (Zheng et al., 2014). 2.11. Statistical analysis Statistical analysis was performed using Prism-IV software (La Jolla, CA, USA). The results obtained from the present study were expressed as means ± SEM. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons test (P < 0.05 was considered to be statistically significant).
2.4. Establishing the antioxidant mechanisms associated with the neuroprotective Hibiscus Sabdariffa extract SH-SY5Y cells were harvested after the treatment with Hibiscus Sabdariffa extract (50 and 100 μg) and / or hydrogen peroxide (32 μM). Content of reactive oxygen species, lipid peroxides & glutathione, and the activity of superoxide dismutase & catalase were measured to establish the antioxidant effects of the Hibiscus Sabdariffa extract.
3. Results Endogenous neurotoxin, hydrogen peroxide (32 μM) was used in the current study for investigating the neuroprotective effects of Hibiscus Sabdariffa extract in the SH-SY5Y neuroblastoma cells. Hydrogen peroxide (32 μM) significantly decreased the neuronal viability as seen by the MTT assay and microscopy as compared to the control (*P < 0.05, n= 12, Fig. 1a). Hibiscus Sabdariffa extract (50 and 100 μg) significantly exhibited neuroprotection against hydrogen peroxide-induced neurotoxicity (*P < 0.05, n= 10, Fig. 1a). Vitamin C (1 and 5μM) also protected the neuroblastoma cells against the endogenous neurotoxin (*P < 0.05, n= 10, Fig. 1b). Based on the above findings, the next step was to establish the neuroprotective mechanisms of Hibiscus Sabdariffa extract using the above model. Hydrogen peroxide (32 μM) significantly induced oxidative stress (increase in reactive oxygen species & lipid peroxide content, decrease in glutathione content & catalase activity), mitochondrial dysfunction (decreased mitochondrial Complex-I activity & mitochondrial membrane potential) and induced apoptosis (increase in caspase-3 and decrease in Bcl-2 expression, *P < 0.05, n= 6, Fig. 2a–e, 3 a and b, 4 a and b). Due to the presence of neuroprotective nutrients, Hibiscus Sabdariffa extract has the potential to scavenge the reactive oxygen species and inhibit lipid peroxidation. With regard to the antioxidant activity, Hibiscus Sabdariffa extract significantly scavenged the reactive oxygen species induced by hydrogen peroxide (aP < 0.05, n= 6, Fig. 2a and b). Increased generation of reactive oxygen species by hydrogen peroxide mainly affects the lipids and causes peroxidation, which decreases the functions of the neuronal lipid membranes resulting in neurotoxicity. Since Hibiscus Sabdariffa extract significantly scavenged the reactive oxygen species, it significantly blocked the lipid peroxide formation as compared to the hydrogen peroxide (aP < 0.05, n= 6, Fig. 2c). Increased generation of reactive oxygen species is attributed mainly due to increase in the pro-oxidants content or decrease in the antioxidant levels. Furthermore, the reactive oxygen species and the hydrogen peroxide’s proxidant effects are reduced by the presence of antioxidants. Glutathione is a potent antioxidant which significantly scavenges the free radicals generated in the body. Superoxide dismutase and catalase play a vital role in decreasing the oxidative stress induced by various proxidants. Hydrogen peroxide induces oxidative stress resulting in neuronal death. To exert its neurotoxic effect by inducing oxidative stress, hydrogen peroxide significantly depleted glutathione and inhibited the catalase activity in the SH-SY5Y neuroblastoma cells (*P < 0.05, n= 6, Fig. 2d and e). Hibiscus Sabdariffa extract significantly blocked the depletion of glutathione induced by hydrogen peroxide and significantly increased the catalase activity (aP < 0.05, n= 6, Fig. 2d and e). Oxidative stress can significantly affect the functions of the mitochondria leading to decreased production of energy and resulting in decreased neuronal viability. Hydrogen peroxide inhibited the mitochondrial Complex-I activity and significantly reduced the mitochondrial membrane potential (*P < 0.05, n= 6,
2.5. Quantifying total reactive oxygen species (ROS) Our previously established spectrofluorometric procedure (using 2`, 7-dichlorofluorescin diacetate) was used to quantify the reactive oxygen species (Zheng et al., 2014). Results were expressed as percentage change from the control. Mitochondrial Reactive Oxygen Species Quantification: This was measured using mitochondrial specific dihydrorhodamine (DHR) probe (Biotium, no. 10055, (Mouli et al., 2015). Fluorescence intensity was detected using a fluorescence microscope and the results were expressed as percentage change from the control. 2.6. Lipid peroxidation Thiobarbituric acid based colorimetric method was used to measure the lipid peroxide content. Results were expressed as percentage change from the control (Zheng et al., 2014). 2.7. Catalase activity (CAT) Spectrophotometric procedure using hydrogen peroxide as the substrate was used to assess the catalase activity. A standard curve of hydrogen peroxide was obtained and the results were expressed as percentage change from the control (Zheng et al., 2014). 2.8. Glutathione content Established fluorimetric procedure was used to measure the glutathione content. A standard curve of glutathione was obtained and the results were expressed as percentage change from the control (Zheng et al., 2014). Effects of Hibiscus Sabdariffa extract on the mitochondrial function: Mitochondrial Complex-I activity: Mitochondrial Complex-I activity is assessed based on the NADH oxidation, which was measured spectrophotometrically. A standard curve of NADH was obtained and the results were expressed as percentage change from the control (Zheng et al., 2014). 2.9. Mitochondrial membrane potential assay The cells were stained and the signals were imaged using a fluorescence microscope. The mitochondrial membrane potential was measured utilizing tetramethylrhodamine ethyl ester (TMRE; Biotium, no.70016; 2). TMRE florescent intensity was accomplished by BioTek Synergy HT plate reader (BioTek, VT, USA). The results were expressed as percentage change from the control (Mouli et al., 2015). 3
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Fig. 1. Effect of Hibiscus sabdariffa on proliferation of neuroblastoma cell line (SH-SY5Y). Cell proliferation was measured calorimetrically by MTT assay. Results are expressed as mean ± SEM normalized to the % of control. a. Hibiscus sabdariffa showed significant neuroprotective effect at both doses (50 & 100 μg) against hydrogen peroxide-induced toxicity (aP < 0.05, n = 10). b. Vitamin C was used as a positive control. It also exhibited neuroprotection against endogenous neurotoxin (aP < 0.05, n = 10). Results are expressed as mean ± SEM normalized to the % of control.
4. Discussion
Fig. 3a and b). Coenzyme Q-10 is a mitochondrial energy enhancer and has exhibited neuroprotection against endogenous and exogenous neurotoxins (Somayajulu-Niţu et al., 2009). Hibiscus Sabdariffa extract possess coenzyme Q-10 and significantly blocked the mitochondrial toxicity induced by hydrogen peroxide (aP < 0.05, n= 6, Fig. 3a and b). Increase in pro-apoptotic factors and decreased antiapoptotic factors have been associated with neurodegeneration. In our study, hydrogen peroxide significantly increased the expression of caspase-3 (proapoptotic) and decreased the expression of Bcl-2-anti-apoptotic factor (*P < 0.05, n= 4, Fig. 4a and b). Increased Bcl-2 and decreased caspase-3 expression was mainly attributed to the anti-apoptotic effects of Hibiscus Sabdariffa extract against hydrogen peroxide-induced neurotoxic insults (aP < 0.05, n= 4, Fig. 4a and b).
Oxidative stress, mitochondrial dysfunction and neuronal death are typical features of neurodegenerative diseases (Karpinska and Gromadzka, 2013). Therefore, extensive research is underway for identifying novel synthetic and natural drugs/agents exhibiting potent neuroprotective effects and with minimal adverse effects and hypersensitivity reactions to treat various neurological disorders. Numerous studies have demonstrated that various botanicals contain cytoprotective agents which can significantly contribute their therapeutic efficacy. Ilex latifolia (Aquifoliaceae) exhibits antioxidant, anti-inflammatory and anti-ischemic effects in the memory-impaired rodent brain (Kim et al., 2015, 2012). The neuroprotective mechanisms of Ilex latifolia involved the prevention of glutathione depletion and lipid peroxidation, decreased expression of phosphorylated tau (p-tau) and 4
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Fig. 2. Effect of Hibiscus sabdariffa on the oxidative stress induced by hydrogen peroxide: Hibiscus sabdariffa exhibited antioxidant activity as seen by scavenging reactive oxygen species, reducing lipid peroxidation, blocking glutathione depletion and affecting catalase activity (*P < 0.05 as compared to control, aP < 0.05 as compared to Hydrogen peroxide treatment, n = 8).
inflammatory mediators (Hwang, 2013; Sriram et al., 1997). In addition to the endogenous toxins accumulation and exposure to exogenous toxins, protein aggregation is an explicit shared biochemical aspect of many neurodegenerative diseases. Amyloid-beta (Aβ) and tau protein in Alzheimer’s disease and α-synuclein in Parkinson’s disease are implicated in generating hydrogen peroxide. Cholinergic and dopaminergic neurodegeneration in Alzheimer’s disease and Parkinson’s disease respectively is attributed to the direct production of hydrogen peroxide during extracellular or intracellular protein aggregation (Winklhofer and Haass, 2010). Endogenous neurotoxin, hydrogen peroxide induces oxidative stress by generating reacting oxygen species and inhibiting antioxidant enzymes (Finkel and Holbrook, 2000). With regard to mitochondrial functions, hydrogen peroxide inhibited Complex-I activity and affected
pro-apoptotic proteins (Fan et al., 2014). Chromogranin A-derived peptide catestatin has shown to possess potent antihypertensive and neuroprotective effects by affecting GABAergic neurotransmission (Choi et al., 2015). Thus, the contemporary scientific literature validates the use of drugs that can inhibit oxidative stress, mitochondrial dysfunction, excitotoxicity, apoptosis and inflammation as a possible valid and a novel therapeutic strategy for reducing the risk and progression of the neurodegenerative disorders (Table 1). Accrual of endogenous and/or exogenous toxins in the central nervous system has shown to elicit a cascade of neurochemical and biochemical lethal modifications that results in neuronal damage. Catecholamines in the presence of endogenous and/or exogenous toxins, metals and enzymes generate potent pro-oxidants (superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen), pro5
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Fig. 3. Effect of Hibiscus sabdariffa on the mitochondrial dysfunction induced by hydrogen peroxide: Hibiscus sabdariffa enhanced the mitochondrial functions as seen by the effect on Complex-I activity and mitochondrial membrane potential. (*P < 0.05 as compared to control, aP < 0.05 as compared to Hydrogen peroxide treatment, n = 8).
et al., 2013; Shults et al., 2002). Similar to coenzyme Q-10, NADH also shown to exhibit protective effects (Kidd, 2005; Wakade and Chong, 2014). Various other studies also have validated the bioactive properties of Hibiscus sabdariffa that may play an essential role in its prophylactic effect against chronic diseases (Khaghani et al., 2011; Sindi et al., 2014). The nutrients can scavenge reactive oxygen and free radicals, inhibit the activity of xanthine oxidase, decrease the peroxidation of lipids and boost the antioxidant activities, decrease inflammation and increase the mitochondrial functions (Da-Costa-Rocha et al., 2014). Hibiscus sabdariffa extract exhibited neuroprotection by its antioxidant action, blocking mitochondrial dysfunction and apoptosis induced by hydrogen peroxide. Based on our current findings, Hibiscus sabdariffa can be a novel botanical therapy for reducing the endogenous toxins mediated neuronal insult. Clinical trials with Hibiscus sabdariffa has shown to have very few adverse effects (Hajifaraji et al., 2018). Clinically, botanicals have recently gained global popularity due to their credible multi-target actions and minimal side effects. The multitarget actions of botanicals provide a wide-range of therapeutic avenue for treating various disorders. Hence, with regard to the global health care, Hibiscus sabdariffa extract may be used to reduce the risk for neurodegenerative diseases. Hibiscus sabdariffa extract exerts multitarget effects and thus therapeutically can be used in the prevention and treatment of Alzheimer’s, Parkinson’s and Huntington’s disorders.
mitochondrial membrane potential leading to neurotoxicity (Winklhofer and Haass, 2010). Hydrogen peroxide also induced apoptosis by increasing the pro-apoptotic protein expression and decreasing the anti-apoptotic expression. In our current study, hydrogen peroxide induces oxidative stress, mitochondrial dysfunction and apoptosis leading to the neuronal death. Currently there are very few neuroprotective studies with Hibiscus sabdariffa. However, various plants form Hibiscus family has exhibited neuroprotective effects in animal models of movement and memory related neurodegenerative diseases. Hibiscus sabdariffa has exhibited nootropic action and memory enhancement effects (Joshi and Parle, 2006; Nade et al., 2011). In our current study, we have established the presence of neuroprotective micronutrients such as polyphenols, sulfhydryl content, NADH and coenzyme Q 10, in addition to the alkaloid and protein. In our previous studies, we have associated the presence of the above nutrients to the neuroprotective effects of various botanicals such as Mucuna pruriens (Dhanasekaran et al., 2008), Trichopus xeylanicus (Tharakan et al., 2005), and Bacopa monniera (Dhanasekaran et al., 2007). With regard to polyphenols, there are other studies that have confirmed the protective effects of polyphenols (Qi et al., 2017; Talarek et al., 2017). Coenzyme Q-10 is an endogenous antioxidant that significantly improves the activity of Complex I and II in the mitochondrial electron transport chain and increases the production of ATP. Several preclinical and clinical studies have established the neuroprotective effects of coenzyme Q-10 in Parkinson’s, Alzheimer’s and Huntington’s disease (Chandra et al., 2014; Huntington Study Group Pre2CARE Investigators et al., 2010; Shetty 6
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Fig. 4. Effect of Hibiscus sabdariffa on apoptosis induced by hydrogen peroxide: Hibiscus sabdariffa decreased apoptosis. (*P < 0.05 as compared to control, a P < 0.05 as compared to Hydrogen peroxide treatment, n = 8).
References
Table 1 Polyphenol, Sulfhydryl, protein, alkaloid, and Coenzyme Q10 content in the ethanolic extract of Hibiscus Sabdariffa. Parameters
Content
Total Polyphenols Sulfhydryl content Total Protein Total Alkaloid NADH Coenzyme Q10
11.35 + 0.06 μg/mg 0.93 + 0.03 μg/mg 0.27 ± 0.02 μg/mg 84 ± 0.19 μg/mg 1.8 + 0.1 ng/mg 9.6 + 0.7 ng/mg
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Conflict of interest As a corresponding author, I have received the input from all the authors and there are no conflicts of interest. On behalf of all the authors, I (corresponding author), I assure that there are no conflict of interest.
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