Cytotoxic and anti-excitotoxic effects of selected plant and algal extracts using COMET and cell viability assays

Excess glutamate in the central nervous system may be a major cause of neurodegenerative diseases with gradual loss and dysfunction of neurons. Primary or secondary metabolites from medicinal plants and algae show potential for treatment of glutamate-induced excitotoxicity. Three plant extracts were evaluated for impact on glutamate excitotoxicity-induced in primary cultures of retinal ganglion cells (RGC). These cells were treated separately in seven groups: control; Plicosepalus. curviflorus treated; Saussurea lappa treated; Cladophora glomerate treated. Cells were treated independently with 5, 10, 50, or 100 µg/ml of extracts of plant or alga material, respectively, for 2 h. Glutamate-treated cells (48 h with 5, 10, 50, or 100 µM glutamate); and P. curviflorus/glutamate; S. lappa/glutamate; C. glomerata/glutamate [pretreatment with extract for 2 h (50 and 100 µg/ml) before glutamate treatment with 100 µM for 48 h]. Comet and MTT assays were used to assess cell damage and cell viability. The number of viable cells fell significantly after glutamate exposure. Exposure to plant extracts caused no notable effect of viability. All tested plants extracts showed a protective effect against glutamate excitotoxicity-induced RGC death. Use of these extracts for neurological conditions related to excitotoxicity and oxidative stress might prove beneficial.


Results
Phytochemical screening of methanolic extracts of P. curviflorus shoots, S. lappa roots and C. glomerata demonstrated the presence of anthraquinones, coumarins, tannins, flavonoids, terpenoids, alkaloids, cardiac glycosides, phlobatannins, and saponins. The extraction percentage yield of P. curviflorus shoots, S. lappa roots and C. glomerata varied from 2.55 to 9.55 with a descending order of S. lappa > P. curviflorus > C. glomerata ( Table 1), indicating that the methanol extract of S. Lappa contains the highest concentration of extractable phytoconstituents. Table 1 summarizes that total phenolic components in extracts varied widely, ranging from 49.93 ± 1.8 to 122.82 ± 1.2 mg/g expressed as gallic acid equivalents (GAE). S. lappa extract showed the highest concentration of total phenolic contents followed by P. curviflorus extract. The flavonoid content is expressed as rutin equivalents, varied from 14.85 ± 0.5 to 39.52 ± 1.9 mg rutin equivalent/g extract ( Table 1). The root extract of S. lappa exhibited the highest quantity of the highest amount of flavonoid contents.
The percentage of DNA migrating into the comet tail (indicating the presence of breaks) from the COMET assay is presented in Table 2 and Figs. 1 and 2. It can be easily noticed that the three plant extracts induced nonsignificant increase of tail length and tail moment at the highest concentration used (i.e. 100 µg) ( Table 2). In contrast, glutamate induced dose dependent increase of both comet assay variables recording tail length values of 1.57 ± 0.13, 2.03 ± 0.2, 3.36 ± 0.32, and 4.62 ± 0.41 compared to a value of 1.23 ± 0.09 for control (Table 2). Much higher tail moments were also recorded in glutamate-intoxicated RGCs recording values of 2.88 ± 0.41, 4.12 ± 0.44, 10.32 ± 1.49, and 17.96 ± 4.09 compared to a tail moment value of 1.51 ± 0.10 in control cells (Table 2). Percentage changes of both comet variables in the three plants-treated and glutamate-treated cells are presented in Figs. 1 and 2 respectively compared to control-untreated RGCs.
The cytotoxic effects of plant extracts on RGCs proliferation after two-hour incubation was measured by MTT assay (Table 3 and Fig. 3A). Extracts caused a negligible but dose-dependent reduction in cell viability. In contrast, glutamate after 48 h of exposure to concentrations ranging from 5 to 100 µM, caused numbers of viable cells to fall significantly to 76% and 58% at 50 µM and 100 µM, respectively. Inhibition of cell proliferation was most pronounced at 100 µM concentration suggesting dose dependency. Cell viability of the plant extract treated RGCs cells are significantly different compared to glutamate intoxicated cells (Table 3 and Fig. 3B).
Cytoprotective effects were measured determined for cells pretreated with plant extracts (50 and 100 µg/ ml) for 2 h followed by exposure to either 50 µM or 100 µM glutamate. Cell viability is expressed percent of control cells exposed to vehicle only. Control value was taken as 100%. Data are expressed as mean ± SD of three independent experiments (n = 3) (Table 4), significantly different at *p < 0.05, **p < 0.01). Extracts efficiently rescued cell viability of glutamate excitointoxicated RGCs and demonstrate much lower tail length as measure of DNA damage (Table 4). One hundred µg/ml aliquots of extracts of P. curviflorus, S. lappa or C. glomerata showed maximal protective effects of 27.58%, 31.03%, and 22.41%, respectively against excitotoxicity of a 100 µM glutamate (p < 0.01, n = 4).
Antioxidant activity is the ability of the antioxidants to protect the organism system towards the harmful effects of oxidative stress. In the current study, antioxidant capacity of methanol extract of P. curviflorus, S. lappa, and C. glomerata was assessed by DPPH and ABTS scavenging activity. The IC 50 of DPPH and ABTS scavenging potentials of each extract was compared with ascorbic acid (Table 5).

Discussion
The fact that phenolic and flavonoid constituents are major active components in materials of natural product origin, including herbs, medicinal plants, algae, sponges and cyanobacteria is well documented. All the three tested samples showed noticeable variations in the content level of phenolic components studied among the species. The percentage of free phenolics varied considerably, highest was found in S. lappa (12.28%), followed by P. curviflorus (8.93%) and lowest in C. glomerata (4.99%). These free phenolics are considered as the responsible compounds for the cytotoxic, anti-excitotoxic and antioxidant properties under the experimental conditions applied in this study. The major types of phenolic components contributing in these activities mainly include  7 . In traditional medicine, various therapeutic plants and natural products have been utilized to treat neurological disorders. Excitotoxicity is a well-accepted pathological mechanism of neurodegenerative disease 29 caused by excess glutamate, a crucial excitatory neurotransmitter in mammals. Overstimulation of glutamate receptors leads to an overload of intracellular Ca 2+ , generation of free radicals and subsequent neuronal cell death 30 .
In the current study, single-cell gel electrophoresis (comet assay) was effective for evaluation of single-strand breaks of brain DNA post-exposure of RGCs to high levels of glutamate. The percentage of DNA migrating into the comet tail (indicating the presence of breaks) was not significantly elevated at 5 and 10 mM glutamate, but was increased from 1.23 ± 0.0941 (means ± SEM) in healthy untreated cells to 3.36 ± 0.32 and 4.62 ± 0.41 in the presence of 50 mM and 100 mM glutamate, respectively. Tail length and tail moment from the COMET assay are presented in Table 2 and Figs. 1 and 2 to describe DNA migration. Tail moment calculated by Olive et al. 31 is principally useful in describing heterogeneity within a cell population since it identifies variations in DNA distributions within tails. Tail moment, as a derived measure, should be presented together with primary measurements (e.g., tail length and % tail DNA) 32,33 . In the present study, the other three measured Comet variables were submitted as "Supplementary data S1" (Tailed %, untailed % and % tail DNA).
Understanding the events of glutamate excitotoxicy and subsequent neuronal death is of critical importance in identifying novel therapeutic targets. NAMDA receptor overstimulation and other events are anticipated. Glutamate-induced elevated calcium levels over-activate several enzymes, including nitric oxide synthase, proapoptotic enzymes, phospholipases, protein kinases and phosphatases 34 . Some enzymes can also produce positive feedback loops to accelerate progression toward neuronal death through damage to cell membranes, cytoskeleton, and DNA [34][35][36][37] .
In the present study, Table 2 and Fig. 1, present results of measuring cytotoxic effects of plant extracts (P. curviflorus; S. lappa, and C. glomerata) using COMET assay in comparison to control healthy-untrated RGCs. Higher concentrations (100 µg/ml) of extracts exhibit slightly increased tail length and tail moment, but still much lower when compared to the excitotoxic effects of glutamate on RGCs. significant DNA damage was recorded in RGCs post-exposure to 50 and 100 µM glutamate (Table 2 and Fig. 2) presented as longer tail length, and greater tail moment are consistent with multiple previous studies. Collectively, non-significant cytotoxicity was observed in cultured rat cortical and hippocampal neurons exposed to 10-50 μM glutamate for 10 min, remarkable neuronal death was observed at higher concentrations of glutamate, 100 μM or greater 38,39 .
The cytotoxic effects of plant extracts on RGCs proliferation using MTT show a negligible but dose-dependent reduction in cell viability significantly different from glutamate-treated cells but, non-significantly different when compared to control healthy cells (Table 3 and Figs. 3A,B). This is going parallel with their effects on DNA using COMET assay.
The role of flavonoids as major components of the three phytochemical extracts is notable. Flavonoids can protect against glutamate excitotoxicity through inhibiting protein kinase activation as a signaling event. Flavonoids directly bind with multiple protein kinases such as Akt/PKB, Fyn, Janus kinase 1 (JAK1), mitogenactivated protein kinase kinase 1(MEK1), PI3K, mitogen-activated protein kinase kinase 4 (MKK4), Raf1, and chain-associated 70-kDa protein (ZAP-70) kinase. These kinases are specific proteins essential to intracellular signaling cascades associated with glutamate excitotoxicity 40,41 .
Phospholipase A2 is involved in many inflammatory reactions leading to disease progression and is a possible therapeutic target for attenuating glutamate excitotoxicity. Inhibition of this enzyme might address oxidative   www.nature.com/scientificreports/ stress and neuroinflammation that contribute to disease 42,43 Experimental results on PLA2-inhibition showed good inhibitory activity of quercetin as one of the major flavonoids in the three studied plant extracts 44  Significant ameliorative effects of plant extracts presented as lesser tail lengths and tail moments compared to 100 µM glutamate-induced DNA strand breaks (Table 4). This result could reflect the inhibitory action of flavonoids on protein kinases, phospholipases, and pro-apoptotic signaling. These major events are usually overactivated in response to exposure to high concentrations of glutamate. S. lappa shows the most potency followed by P. curviflorus and C. glomerata.
Possible flavonoid-related anti-excitotoxic effects of the three plant extracts for neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, is supported by multiple studies. Different feeding trials with the flavone, apigenin, show neuroprotective effects for memory and learning deficits, and reduction of fibrillar amyloid deposits in a rodent Alzheimer's disease model. Additionally, restoration of cortical extracellular signal-regulated protein kinase 1 (ERK)/cAMP response element-binding protein (CREB)/BDNF pathway was observed. This pathway is involved in learning and memory deficits typically seen in Alzheimer's disease patients 46,47 . Polyphenols are also as major components of P. curviflorus and S. lappa both of which significantly reduced the initial calcium peak in response to high concentrations of glutamate leading to protection from glutamate-induced cell death. Interestingly, the anti-excitotoxic effects reported in the present study can also find support in the study of Yang et al. 48 which indicated an evidence that terpenes of plant origin protects against cerebral ischemic injury by inhibiting excitotoxicity through the modulation of the imbalance between excitatory glutamate against gamma-Aminobutyric acid (GABA) as inhibitory neurotransmitter, which may support the traditional use of terpenes for the treatment of stroke.
The DPPH radical scavenging activity of standard and methanol extracts tested (P. curviflorus, S. lappa, and C. glomerata) is summarized in Table 5. As shown in Table 5, all the three extracts demonstrated remarkable free radical scavenging activity with 84-95% DPPH radicals scavenged. The result obtained revealed that the methanol extract of S. lappa displayed the highest scavenging activity (lowest IC 50 value; 2.98 ± 0.68 μg/mL) followed by P. curviflorus extract (IC 50 ; 7.23 ± 0.05 μg/mL). However, C. glomerata extract showed moderate antioxidant activity (IC 50 ; 45.32 ± 3.82). The excellent radical scavenging activity profile of S. lappa and P. curviflorus extract may be attributed to the presence of high content of hydrogen donating phenolic components in these extracts. Additionally, the hydroxyl group in the structural backbone of phenolics is the responsible contributors of antioxidant activity 49 .
The results of ABTS radical cation scavenging assay showed that the IC 50 values of extracts were ranged 35.16-65.23 μg/mL (Table 5). Amongst the three tested extracts, S. lappa extract showed the highest activity with IC 50 values of 35.15. P. curviflorus and C. glomerata extracts exhibited moderate activity with with IC 50 values of 60.26 μg/mL and 65.26 μg/mL, respectively. Ascorbic acid was used as standard with IC 50 values 24.23 μg/mL. The outcome the study was analyzed that the higher the concentration of phenolic components in the extracts, higher antioxidant capacity values they possess, which is clearly in agreement with the results observed in previous studies 50   www.nature.com/scientificreports/ this location. Plant species as well as algal biomass were identified and authenticated by Dr Mohamed Yousef, a taxonomist in the Pharmacognosy Department, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. A voucher specimen with catalog Nos: (PC-3-2143), (SC-7806) and (CG-5018) were submitted to the herbarium of the same department for P. curviflorus S. lappa, and C. glomerata respectively. All the three samples were air-dried, coarse powdered, and preserved in airtight bags until extracted in the laboratory. This study complies with the local national regulations for the use of cultivated plants in experimental studies.
Extraction of plant samples. Methanol was selected as an organic extractant using the ratio of 1:4 plant material. One thousand grams of powdered P. curviflorus shoots, S. lappa roots and C. glomerata biomass were individually soaked in 3 L of methanol in an airtight glass containers for four days at ambient temperature with shaking at 2 h intervals for maximum extraction of bioactive constituents [51][52][53] . Liquid extracts were separated from solid residues by filtration through Whatman No. 1 filter paper. All the experiments were performed in two replications under similar conditions. The organic solvent of each combined extract was evaporated to dryness in a rotaevaporator under reduced pressure at a temperature of 40 °C to yield dark brown (85.3 g), brown (92.5 g) and dark green (25.5 g) residues for P. curviflorus shoots, S. lappa roots and C. glomerata biomass, respectively. The dried residues were transferred to tightly stoppered glass tubes and stored at 5 °C until further use.

Preparation of retinal cell suspensions. Newborn
Cell treatment. Pure RGCs were divided into seven treatment groups: control (untreated); plant extract treated independently with 5, 10, 50, or 100 µg/ml of P. curviflorus, S. lappa or C. glomerata. Control and treated cells were exposed to extracts for 2 h. Glutamate-treated cells were exposed to glutamate for 48 h using 5, 10, 50, or 100 µM glutamate). Finally, cells pretreated individually with extracts (50, or 100 µg/ml) were exposed to 100 µM glutamate for 48 h.
Comet assay. The method described by Singh et al. 55  www.nature.com/scientificreports/ from the center of the body of the nuclear core) and tail intensity of DNA (TI = % of genomic DNA that migrated during the electrophoresis from the nuclear core to the tail).
Determination of cell viability. MTT test was used to measure cell viability using RGC seeding density of 0.625 × 10 5 (i.e. 62,500 cells/ml) in a 100 µL media. To assess viability, the medium was exchanged for an MTT working solution (5 mg/ml in cell culture medium) and incubated for 4 h at 37 °C. Afterwards, the reaction was terminated by adding 200 µL of DMSO to each well for 15 min. The absorbance was recorded at 490 nm using a micro plate reader. The results are presented as a percentage of control (untreated cells) or glutamate excitotoxicity.
DPPH radical scavenging assay. The antioxidant potential of plant extracts was evaluated by UV specterophotometrically against against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-Azino-Bis-(3-ethylbenzothiazoline-6-Sulfonic Acid) (ABTS) radical. DPPH free radical scavenging activity of each plant extract was performed according to previously described method by Huang et al. 56 , with slight modification. Briefly, 2.5 ml of each sample extract was thoroughly mixed with 0.5 ml of freshly prepared 0.2 ml DPPH in ethanol solution and allowed to stand for 30 min at room temperature. The blank samples were prepared by mixing the same amount of ethanol and DPPH. After that, each reaction mixture was examined for DPPH radical scavenging effect by measuring the absorbance at 517 nm against blank samples on a UV-Vis spectrophotometer. Lower the value of absorbance of reaction mixture higher would be the free radical scavenging activity. The concentration of extract causing the 50% inhibition (IC 50 ) was determined from the graph plot of percentage inhibition versus extract concentration. Ascorbic acid was used as a standard. Following equation was used to calculate the radical scavenging activity: where Ab and Aa is the absorption of the blank sample and the extract, respectively.
ABTS radical scavenging assay. ABTS cation scavenging activity of plant extracts was conducted by obeying a procedure described in previous study 57 . Briefly, ABTS reagent was prepared by mixing equal volumes of freshly prepared stock solutions of ABTS (7 mM) and potassium persulphate (2.4 mM) and were allowed to react in the dark for 12 h at ambient temperature. After 12 h incubation, the resultant dark colored ABTS reagent solution was diluted with ethanol until an absorbance of 0.700 ± 0.005 at 734 nm was attained. 200 μl of each test sample solution was treated with 2 mL of ABTS stock solution. The reaction mixture was vortoxed for 30 min and absorbance was recorded at 734 nm. Similarly, different concentrations (1-100 μg/mL) of ascorbic acid was treated with ABTS solution. The amount of extract required to decrease the absorbance of ABTS by 50% (IC 50 ) was determined graphically. Following equation was applied to calculate the antioxidant activity of each extract: Statistical analysis. Data were analyzed using the Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA). Results are presented as mean ± standard error (SEM). All statistical comparisons among the control, P. curviflorus treated; S. lappa treated; C. glomerate -treated, and glutamate-treated groups used one-way analysis of variance (ANOVA) complemented with Dunnett's test for Multiple Comparisons. Significance was considered p < 0.05. www.nature.com/scientificreports/