The protective effect of Moringa oleifera plant extract against glutamate-induced DNA damage and reduced cell viability in a primary retinal ganglion cell line

Plant material

The seeds of the Moringa oleifera were purchased from the local market of the Riyadh, Saudi Arabia. Sample material was identified and authenticated by Prof. Dr. Mohamed Yousef of the Pharmacognosy Department, College of Pharmacy at King Saud University, Saudi Arabia. A voucher specimen (MO-5302) has been kept in the herbarium of the same department. Seeds were cleaned of dirt, washed and dried at room temperature. The dried seeds were finely powdered by using domestic blender, collected in plastic bags and placed in cold place at 4 °C, prior to use.

Extraction of plant samples

The powdered seeds (1 kg) of M. oleifera were soaked in methanol (3 L) with continuous shaking at 2 h intervals for 78 h at room temperature to obtain maximum extraction of bioactive constituents (Do et al., 2014; Dhawan & Gupta, 2017). The obtained extract was filtered through Whatman No. 1 filter paper and centrifuged to remove the suspended material. The extraction process was repeated thrice under the similar conditions. All the organic extracts were pooled and freed from organic solvent on rotaevaporator at 40 °C under reduced pressure to yield a dark brown residue (69.8 g). The dried residues were transferred to glass tubes, tightly stoppered, and stored at 5 °C until further use.

Phytochemical screening M.olifera seed extract

Chemical tests were performed for the phytochemical analysis of compounds present in the extract. These tests include Borntrager’s test for anthraquinones, Shinoda test for phenols, Ferric chloride test for flavonoids, Libermann–Burchard test for steroids–triterpenoids, lead acetate test for tannins, Dragendorff’s test for alkaloids, Rosenthaler test for saponins, and Keller–Killiani test for cardiac glycoside (Brain & Turner, 1975; Harborne, 1998; Samejo et al., 2013).

Quantitative analysis of M.olifera seed extract

Spectrophotometric assay was used to perform the phytochemical analysis of methanolic extract of M.olifera seed by obeying Onwukeame et al. method with little modifications (Onwukeame, Ikuegbvweha & Asonye, 2007). The total phenolic composition in M.olifera extract was calculated by plotting a graph of gallic acid standard as previously mentioned (Luximon-Ramma et al., 2002). Briefly, 0.120 mL of seed extract was mixed with 0.5 mL of distilled water, followed by the slow addition of 0.12 mL of Folin–Ciocalteu reagent and incubated at ambient temperature for 5 min. Afterwards, 7% of sodium bicarbonate solution (1.20 mL) was added to the reaction mixture and volume made up two mL with distilled water and incubated for 1 h at ambient temperature. After 1 h incubation, the absorbance was recorded at 760 nm. The results were calculated as µg of gallic acid equivalents/mg of the seed extract (GAEs). However, the measurement of flavonoid content in the seed extract of M.olifera was performed by using a quercetin reference curve as previously described by Ghosh et al. (Ghosh et al., 2012). Briefly, one mL of seed extract of M.olifera was mixed with one mL of 2% aluminum chloride and incubated for 30 min at room temperature. After 30 min incubation, the absorbance was measured at 368 nm. The results were calculated as µg of quercetin equivalents (QEs)/mg of seed extract. Flavonols content in the seed extract was estimated by using rutin as a reference. The method is based on the complex formation displaying absorption at 440 nm (Grubesic et al., 2005). The results were determined as µg of rutin equivalents/mg of seed extract. The content of tannin in M.olifera seed extract was determined by applying Folin-Denis procedure (Oyaizu, 1986). The method is based on the formation of a precipitate upon treatment with casein showing absorption at 720 nm. The results were calculated as µg of tannic acid equivalents/mg of seed extract.

Antioxidant activity of methanol extract of M.oleifera

Two diverse radical scavenging assays, DPPH (1, 1-diphenyl- 2-picrylhydrazyl) and ABTS (2, 20-azino-bis [3-ethylbenzothiazoline-6-sulphonic acid]) free radical scavenging were applied to examine the antioxidant potential of methanol seed extract of M.oleifera.

DPPH free radical scavenging activity

The DPPH free radical scavenging activity of the seed extract of M.oleifera and butylated hydroxytoluene (BHT) (Sigma-Aldrich, St. Louis, Missouri, USA) as the reference standard, was determined by obeying standard procedure (Mensor et al., 2001). Briefly, 0.1 mM DPPH reagent (Sigma-Aldrich, St. Louis, Missouri, USA) in methanol and three different concentrations (25, 50, 100 µgmL⁻¹) of M.oleifera extract or BHT in methanol were prepared prior to the experiment. The reaction mixture was initiated by adding 50 µL of DPPH solution and varied concentration of M.oleifera extract and BHT to each well of 96-well microplate. The whole reaction mixture was subjected to continuous shaking using orbital shaker for 30 min under dark conditions. After 1 h incubation, the absorbance of the reaction mixture was measured at 593 nm using a UV-vis spectrophotometer (Multiskan GO; Thermo Scientific, Waltham, MA, USA), with methanol as a blank. All the measurements were performed in triplicates. The scavenging abilities of M.oleifera seed extract and BHT on DPPH free radicals was calculated by applying the following equation:

Where Cr and Tr is the absorbance of control and the test samples, respectively

ABTS free radical scavenging activity

A standard method described by Re et al. (1999) was followed to evaluate the ABTS free radical scavenging potential of the seed extract of M.oleifera and BHT taken as a reference standard. Briefly, two stock solutions of 7.4 mM ABTS and 2.6 mM potassium persulfate (Sigma-Aldrich, St. Louis, Missouri, USA) were prepared in methanol individually. To prepare the working solution of ABTS, equivalent amounts of stock solutions were mixed and kept undisturbed for 16 h under dark conditions at room temperature. In a 96-well microplate, 135 µL of the ABTS working solution and different concentration (25, 50, 100 µgmL⁻¹) of M.oleifera extract and BHT to each well. The reaction was mixed and allowed to stand in dark for 2 h at ambient temperature. The UV–Vis spectrophotometer was used to record the absorbance of the reaction mixture at 743 nm, with methanol as a control. All the measurements were carried out in triplicates. The scavenging abilities of M.oleifera seed extract and BHT on DPPH free radicals was calculated by applying the following Eq. (1).

Isolation and purification of RGCs

The Retinal cells were obtained with the help of expert in Animal Reproduction Research Institute (ARRI)-Kafr Nassar, Al Haram, Giza Governorate, Egypt. Six four-day- old Sprague-Dawley rats were used to isolate RGCs. The rats were kept in standard housing conditions with 12 h light/dark cycle in a temperature-controlled room with free access to food and water. Rats were euthanized by using CO₂ inhalation followed by cervical dislocation. Eyes were dissected out, and excess muscle and connective tissue were removed in ice cold phosphate-buffered saline (PBS). RGCs were purified and stored in isotonic buffer solution  containing inorganic salts, glucose, papain, and DNase I for half an hour. Papain activity was stopped using ovomucoid solution. A 40 µm mesh filter was used to obtain the single-cell suspensions using a filtering process. Negative and positive cell selection was performed using rabbit anti-rat macrophage/Thy-1 antibody-coated Petri dishes. Panning plates were incubated overnight at 4 °C and rinsed 3 times with balanced salt solution . Poly-D-Lysine solution was added and cells were incubated overnight at room temperature. Dried cells were incubated with Mouse laminin for 120 min at 37 °C. Finally RGCs were seeded on PDL- and laminin-coated coverslips in RGC growth medium at 37 °C in a 5% CO₂ incubator. Half quantity of medium was replaced on the third day. The protocol of this work was approved by ethical committee, King Saud University with approval number EC Ref No.: 4/67/352670.

Cell treatment

Pure RGCs were divided into three groups depending upon the treatment: control (without treatment); M. oleifera treatment (5, 10, 50, or 100 µg/ml of seed extract); glutamate treatment (5, 10, 50, or 100 µM glutamate for 48 h). Finally, cells individually pretreated 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. (1988) was uperformed for the comet assay. Cells were treated with test material for 24 h in Petri dishes. Cells were trypsinized (0.1% for 4 min), suspended, and centrifuged for 10 min at 800 rpm. Afterward, 600 µl of 0.8% low-melting agarose was added to the cell suspension and relocated to pre-coated agarose slides. The coated slides were dipped in lysis buffer (0.045 M TBE, pH 8.4, having 2.5% SDS) for 20 min. The slides were placed on a gel electrophoresis and covered with ice-cold alkaline solution (300 mM NaOH and 1mM Na2 EDTA, pH 13) in the dark at 0 °C for 20 min, before the electrophoretic run. The electrophoresis settings were 2 V/cm for 20 min and 100 milliampere (mA). Ethidium bromide (20  µg/ml at 4 °C) was used for staining. DNA fragment migration patterns of 100 cells for each dose level were evaluated with a fluorescence microscope. DNA damage was measured as tail length (TL = distance of DNA migration 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

The MTT test was used to measure cell viability. Absorbance was recorded at 490 nm using a microplate reader. The results are presented as a percentage of control (untreated cells) or as a percentage of glutamate excitotoxicity.

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 groups used one-way analysis of variance complemented with Dunnett’s test for multiple comparisons. Significance was considered as p < 0.05.

Phytochemical description of the extracts

The phytochemicals present in the crude extract of M. oleifera seeds are listed in Table 1. The methanol seed extract of M.olifera showed the presence of a variety of bioactive constituents, including phenolics, flavonoids, flavonols, tannins, and steroids. These bioactive components were qualitatively screened by phytochemical analysis and might be responsible for the neuroprotective effects. The content of secondary metabolites such as phenols, flavonoids, flavonols and tannins in the methanol seed extract of M.olifera was summarized in Table 2. The estimation of phenolics, flavonoid, flavonol and tannin content in the M.olifera seed extract was determined by using gallic acid, quercetin, rutin and tannic acid respectively, as standards. The obtained results revealed that the seed extract of M.olifera contains total phenolic, flavonoid, flavonol and tannin content in the range of 78.59 ± 0.98 µg mL⁻¹, 112.23 ± 1.1 µg mL⁻¹, 4.98 ± 0.56 µg mL⁻¹ and 23.73 ± 0.65 µg mL⁻¹, respectively.

Antioxidant activity of methanol extract of M.oleifera

The antioxidant potential M.oleifera seed extract was investigated according to assays performed in various radical scavenging studies, i.e., the DPPH free radical scavenging and ABTS free radical scavenging assays. DPPH and ABTS^•+ cation radicals are reactive against most of the antioxidants and their decolorization indicates the abilities of antioxidants to donate hydrogen atoms or electrons to inactivate the radical species. The IC₅₀ values of the extract for both DPPH radical scavenging and ABTS^•+ cation radical scavenging are presented in Table 3. The obtained results revealed that the M.oleifera seed extract showed similar activity trend towards both the assays. M.oleifera seed extract displayed the lowest IC₅₀ values for both DPPH (18.68 ± 5.54) and ABTS^•+ (21.25 ± 2.22) radical scavenging assays and hence exhibited the highest radical scavenging potential. There was no significant difference (p ≥ 0.05) in the scavenging effect of DPPH and ABTS. The strong DPPH and ABTS^•+ radical scavenging effects of seed extract of M.oleifera may be credited to the presence high phenolic and flavonoid contents in extract.

Comet assay

Protective effect of M. oleifera seed extract against glutamate-induced DNA damage in a primary RGC was evalvated by results of Comet assay. Assessment of the genotoxic activity of glutamate and antigenotoxic activities M. oleifera seed extract were estimated by the parameter related to percentage of tailed DNA in treated cells as compared to control. Table 4 presents the comet assay measured variables of cells in all groups. Glutamate was found to be significantly toxic in a dose-dependent manner for RGCs as compared to control untreated RGCs. Glutamate-induced DNA damage in RGCs is shown in Figs. 1 and 2. We noticed negligible damage with M. oleifera seed extract as compared with glutamate (Figs. 1–2).

Cell viability

Protective effect of M. oleifera seed extract against glutamate-induced neurotoxicity in a primary RGC was done by calculate cell viability and proliferation through MTT assay. In this assay metabolic reduction of the soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) salt to insoluble colored formazan product by mitochondria dehydrogenase of live RGC cells was measured spectrophotometrically at 480 nm. The amount of formazan dye formed in cells treated with glutamate and M. oleifera seed extract was directly correlates to the number of viable cells. Results of cytotoxic effects on RGC proliferation after treatment are presented in Table 5 and Fig. 3. M. oleifera seed extract showed negligible toxicity compared to glutamate and showed a dose-dependent reduction in cell viability. The inhibition of cell proliferation was most obvious at the 100 µM concentration, which suggests dose-dependency. Cell viability of the seed extract-treated RGCs was significantly different compared to glutamate intoxicated cells. Cyto-protective effects of the seed extract were evaluated by pretreating cells with the extract before exposing the RGCs to glutamate. These results are shown in Table 6. RGCs were pretreated with 50 and 100 µg/ml of the seed extract for 2 h before exposing the cells to 50 µM and 100 µM of glutamate. We observed a remarkable increase in cell viability (Table 6 and Fig. 4).