Evaluation of antioxidant and neuroprotective activities of Cassia fistula (L.) using the Caenorhabditis elegans model

Plant material and extraction

C. fistula leaves and fine stems were obtained during the spring (March and April) 2016 from Orman Botanical Garden, Giza, Egypt. Authenticity was confirmed by Professor Mohamed El Gebaly, Professor of Taxonomy at the National Institute of Research, Egypt.

Freshly harvested leaves and stems were homogenized and extracted using 70% v/v (methanol/water). The extract was then filtered through cotton wool followed by passing on charcoal to get rid of chlorophyll, then vacuum concentration at 37 °C using rotary evaporator was done (Nair, 1995). The concentrated filtrate was finally freeze-dried and the final product was stored at −20 °C for future utilization.

Chemicals

Purified EGCG from green tea (purity ≥ 95%), 2,7-dichlorofluorescein diacetate (DCF-DA), 2,2-diphenyl-1-picrylhydrazyl (DPPH^•), butylated hydroxyl anisole (BHA), bis (neocuproine) copper (II) chelate (CUPRAC reagent), vitamin C (Vit C) and juglone (5-hydroxy-1,4-naphthalenedione) were purchased from Sigma-Aldrich, Germany. Sodium azide was bought from AppliChem GmbH, Darmstadt, Germany; Folin-Ciocalteu reagent from Merck, Darmstadt, Germany.

HPLC/PDA/ESI-MSⁿ qualitative chemical profiling

LC-MS/MS analysis was done on a Finnigan LCQ-Duo ion trap mass spectrometer with an ESI source (Thermo Quest, Austin, TX, USA) coupled to a Finnigan Surveyor HPLC system (MS pump plus, autosampler, and PDA detector plus) with an EC 150/3 NUCLEODUR 100-3 C18ec column (Macherey-Nagel, Düren, Germany).

A gradient of water and acetonitrile (ACN), without acid, was applied from 5% to 60% ACN in 45 min at 30 °C. Flow rate was 0.5 ml/min. Injection volume was about 20 µl. MS was operated in the negative mode with a capillary voltage of −10 V, source temperature of 220 °C, and high purity nitrogen as a sheath and auxiliary gas at a flow rate of 80 and 40 (arbitrary units), respectively. Ions were detected in a mass range of 50–2,000 m/z. Collision energy of 35% was used in MS/MS for fragmentation. Data acquisitions and analyses were done using Xcalibur™ 2.0.7 software (Thermo Fisher Scientific, Waltham, MA, USA).

Folin-Ciocalteu assay for quantification of total phenolic content

The assay was performed in a 96-well microplate as described by (Zhang et al., 2006). Briefly, 100 µl of Folin-Ciocalteu reagent were added to 20 µl of the extract. The microplate was kept afterwards for 5 min at room temperature then 80 µl of sodium carbonate solution (7.5%) were added. The reaction was allowed to occur for 1 h in the dark at room temperature and then absorbance was recorded using microplate reader at 750 nm. Gallic acid was used as a standard to establish a calibration curve. The assay was repeated three times. Phenolic content is presented as µg of gallic acid equivalents (GAE)/mg of CF extract.

CUPRAC method to assay total antioxidant capacity

The assay was carried out according to the method described by Apak et al. (2004). In a test tube, BHA standard or CF, 0.3 ml, were added to 0.8 ml purified water to reach a volume of 1.1 ml. To the previous mixture, 1 ml (10 mM) copper chloride, 1 ml (7.5 mM) neocuproine alcoholic solution and 1 ml of (1 M) ammonium acetate buffer were added to reach a final volume of 4.1 ml. Test tubes were closed and kept for 30 min, and then absorbance was recorded at 450 nm against a reagent blank using JASCO V-630 double beam spectrophotometer equipped with quartz cuvettes.

The assay was repeated three times. A calibration curve was drawn for BHA standard using a series of different concentrations (50, 100, 200 and 400 µg/ml) where absorbance was plotted versus concentration to get the 50% effective concentration of the standard antioxidant used, EC₅₀, in µg/ml. Another calibration curve was plotted for CF using the same series of concentrations (50, 100, 200 and 400 µg/ml). EC₅₀ was obtained for CF from the curve using the equation y = ax + b where y is substituted by 50% as follows: ${\displaystyle {\mathrm{EC}}_{50}=\frac{0.5-\mathrm{b}}{\mathrm{a}}}$

DPPH assay

To assay the power of CF to scavenge radicals, DPPH free radical assay was used according to Blois method (Blois, 1958). The assay was done in a 96-well microplate. 100 µl of 200 µM DPPH were added to 100 µl of different concentrations of CF, Vit C or EGCG. Afterwards, incubation was done at room temperature in the dark for 30 min and absorbance was recorded at 517 nm using a microplate reader. The assay was repeated three times. The ability of CF and standard antioxidants to scavenge DPPH radicals was calculated as follows: ${\displaystyle \text{DPPH scavenging activity (\%)}=\frac{A_0-A_1}{A_0}\times 100}$ where A₀ is the control absorbance, and A₁ is the absorbance of CF, Vitamin C or EGCG. EC₅₀ was calculated as µg/ml using sigmoid non-linear regression.

Caenorhabditis elegans strains and maintenance

C. elegans strains used in this study are: Wild type N2, TJ356 (zIs356[daf-16p::daf-16a/b::GFP + rol-6]), CF1553 (mu1s84[pAD76(sod-3::GFP)]), TJ375 (gpIs1[hsp-16.2::GFP]), CL2166 (dvIs19[pAF15(gst-4::GFP::NLS)]), AM141 (rmls133[unc-54p::Q40::YFP]), CL4176 (dvIs27[myo-3p::A-Beta (1–42)::let-851 3′UTR) + rol-6(su1006)]) X and CL802 [smg-1(cc546) I; rol-6(su1006) II]. Nematodes were all cultured in nematode growth medium (NGM) containing E. coli OP50 as food source and kept at 20 °C incubator, except for CL4176 and CL802 which were kept at 16 °C. All strains and E. coli OP50 were obtained from Caenorhabditis Genetics center (CGC), University of Minnesota, U.S.A.

Age synchronization of worms was achieved by isolating eggs from gravid hermaphrodites using 5 M NaOH, 5% NaOCl (1:3) and sterile water, vortexing 10 min for lysis, followed by centrifugation at 1,200 rpm for 1 min. The supernatant was removed till 0.1 ml then sterile water was added till 5 ml and centrifugation was repeated at 1,200 rpm for 1 min. Finally, water was removed and eggs were kept in M9 buffer for hatching. Larvae were then kept after hatching in S-medium containing E. coli OP50 (OD₆₀₀ = 1.0) (Stiernagle, 1999). Different treatments were applied according to each experiment.

Survival assay under juglone–induced oxidative stress

Synchronized N2 worms (L1 larvae stage) were kept in S-medium at 20 °C using E. coli OP50 as food source. They were divided into six groups of 80 worms each, on the day after hatching, and treated differently. First group was left untreated (untreated control group). Second group was treated with 2.1% methanol (solvent control group). This group served to exclude any toxicity of the solvent used for dissolving the extracts on worms. Third group was treated with EGCG 50 µg/ml dissolved in water, used as a positive control for its known antioxidant activity (Abbas & Wink, 2009; Abbas & Wink, 2010). Groups four, five and six were treated with 100, 200 and 300 µg/ml CF dissolved in methanol (maximum 2.1%).

After 48 h of treatment, the pro-oxidant juglone was added at a lethal concentration (80 µM) for 24 h. Alive and dead worms were counted, considering the dead ones as those who did not move upon touching them gently and repetitively with a platinum wire. Results from three repetitions were calculated as mean ± SEM of live worms (in %). Comparison was done using one-way ANOVA then Bonferroni’s (post-hoc) method using Graphpad Prism version 5.01 (Abbas & Wink, 2009).

Intracellular ROS levels

Age synchronized N2 worms (L1 stage) grown in S-medium with E. coli OP50 at 20 °C were divided into six groups, on the day after hatching. First group was left untreated; second one received 2.1% methanol and the third group was treated with 50 µg/ml EGCG. The other three groups were treated with 100, 200 and 300 µg/ml CF respectively. All groups were left for 48 h then washed with M9 buffer. Afterwards, 1 ml 50 µM of H₂DCF-DA was added and samples were incubated for one hour away from light at 20 °C. Then worms were washed with M9 buffer to remove extra dye and mounted on a glass slide, with the addition of 10 mM sodium azide for paralysis induction.

Analysis was done by fluorescence microscope BIOREVO BZ-9000 with mercury lamp (Keyence Deutschland GmbH, Neu-Isenburg, Germany) at λ_ex 480/20 nm, λem 510/38 nm. 30 worms, at least, were photographed from each group using 10X objective lens and constant exposure time. ImageJ software version 1.48 (National Institutes of Health, Bethesda, MD, USA) was then used to measure densitometrically the relative fluorescence of the full body. Results of three independent assays are expressed as mean fluorescence intensity and compared using one-way ANOVA then Bonferroni’s (post-hoc) method (Peixoto et al., 2016b).

DAF-16 distribution

L1 age synchronized TJ356 transgenic worms, expressing DAF-16::GFP fusion protein, were grown in S-medium supplied with E. coli OP50 at 20 °C. On the day after hatching, they were divided into six groups and were treated as described previously. After 24 h, worms were mounted on a glass slide using 10 mM sodium azide for paralysis. Thirty different worms, at least, were visualized via 20X objective lens at constant exposure time. DAF 16::GFP fusion protein was located either in the nucleus or cytoplasm, whereas some worms showed intermediate distribution between the nucleus and cytoplasm. Analysis was done for three different runs as previously described. Worms were sorted and counted according to the location of the DAF-16::GFP to cytoplasmic, intermediate or nuclear (Peixoto et al., 2016a).

SOD-3 expression

Synchronized CF1553 transgenic worms (L1 stage), expressing SOD-3::GFP as a reporter fusion protein, were grown in S-medium inoculated with E. coli OP50 at 20 °C. They were divided into six groups, on day one after hatching, and treated as previously described, except for the EGCG group that was treated with 100 µg/ml. After 72 h of treatment, worms were mounted on a glass slide with 10 mM sodium azide and images of 30 worms were taken at constant exposure time by 10X objective lens. Analysis of three runs was performed by measuring the mean fluorescence intensity of the posterior intestine and results were obtained as described before (Peixoto et al., 2016a).

HSP-16.2 expression under juglone induced oxidative stress

TJ375 transgenic L1 worms expressing HSP-16.2::GFP reporter gene were divided into six groups, treated as previously mentioned on day one after hatching, and kept at 20 °C. After 72 h of treatment, the worms were exposed to a non-lethal dose of 20 µM juglone for 24 h (diluted immediately prior to the assay in M9 buffer from a 10 mM stock solution prepared in 99% ethanol) (Abbas & Wink, 2009). Worms were mounted on a glass slide with 10 mM sodium azide and images of at least 20 worms were taken at constant exposure time by 20X objective lens. Analysis of three runs was performed by measuring the mean fluorescence intensity of pharynx and results were obtained as described before.

GST-4 expression

L1 larvae of synchronized CL2166 worms expressing GST-4::GFP reporter gene were kept in S-medium with E. coli OP50 at 20 °C. They were divided into six groups and treated as previously mentioned. Worms were kept for 48 h at 20 °C. Afterwards, worms were paralyzed with 10 mM sodium azide on a glass slide and 30 worms were imaged using 10X objective lens at constant time of exposure. Mean fluorescence intensity of the whole body was measured. The experiment was repeated three times and analysis was done as previously mentioned.

PolyQ40 aggregates quantitative determination

L1, age synchronized transgenic AM141 worms expressing polyQ40::YFP as a reporter gene were cultured at 20 °C in S-medium containing E. coli OP50. All worms were divided into six groups and were treated for 72 h as mentioned before. Worms were then paralyzed on a glass slide using 10 mM sodium azide. Images were captured for at least 30 worms using 10X objective lens at constant exposure time. PolyQ40 aggregates, present in the muscle cells of the worms, were counted manually by visualizing the worms of three different assays. The results were expressed as mean ± SEM and compared using one-way ANOVA followed by Bonferroni’s (post-hoc) method.

Paralysis assay

CL4176, temperature-inducible nematodes expressing Aβ transgene in their muscle cells were used in this assay to evaluate paralysis as a symptom of Aβ toxicity. CL802 nematodes, lacking Aβ transgene, were used as a control strain for testing effects independent from Aβ formation. CL4176 population was divided into 5 groups, 50–70 worms each. First group was left untreated (untreated control). Second group, solvent control was treated with 2.1% MeOH. This group was done to exclude any toxicity or paralysis delaying effect of the solvent used to dissolve the extracts. EGCG group 100 µg/ml was used as a positive control and the last two groups were given CF (300 and 500 µg/ml respectively). The assay was performed as described by Dostal & Link (2010), where treatments were added to separate NGM plates and left in 16 °C incubator for 24 h. Afterwards, E. coli OP50 was added in the middle of the plate and plates were left again for 24 h at 16 °C. Then bleaching was done, to obtain age-synchronized population, and 50–70 eggs were put in the middle of the plate. All plates were kept for 48 h at 16 °C then an automatic upshift was done to 25 °C to induce Aβ transgene expression.

Paralysis was assayed 22 h after the upshift and L4 stage worms were observed every 1–2 h for 12 h. Paralyzed worms were the ones who did not respond to a gentle touch with a platinum wire. Three independent assays were carried out and the data is expressed as mean ± SEM. Analysis was done using Kaplan–Meier survival analysis to get the time where half worms were paralyzed (PT₅₀) and survival analysis was done to obtain the graph using a log-rank test in Graphpad prism, version 7.04. The results were compared using one-way ANOVA followed by Bonferroni’s method (post-hoc).

LC/MS metabolites profiling

The bioactive extract of C. fistula was analyzed by HPLC, in combination with a mass spectrometry detector. ESI/LC/MS analysis was performed using cone voltage providing useful additional fragmentation data. A representative UV chromatogram is shown in Fig. 1. Secondary metabolites were tentatively identified, based on authentic standards when available, or on retention data as reported in Table 1, by ESI mass spectra and in comparison with the literature.

Quinic acid derivatives were detected as galloyl quinic acid, represented by peak Cf1 which has [M–H]⁻ of m/z 343.04. Furthermore, quinic acid as in peak Cf2 with its deprotonated molecule were detected as [M–H–152]⁻ at m/z 191.02. This was due to elimination of a galloyl group. The corresponding fragmentation at m/z 169 was also detected, which explains the elimination of quinic acid and the formation of deprotonated gallic acid (Wyrepkowski et al., 2014).

Several glycosides were identified as major peaks within the bioactive extract, as peak Cf9 which was identified as kaempferol 3-O-[6″-O-(rhamnosyl) glucoside] 7-O-rhamnoside as [M–H]⁻ at m/z 739 ([K + 2Rha + Glu–H]⁻ ). Another peak appeared at m/z 284, representing kaempferol aglycone (Schatz, Lapierr & Ralph, 2003). Furthermore, peak Cf10 was identified as kaempferol-O-coumaroyl glucoside with m/z 593.3 as the base peak. The main fragment appeared at m/z 284. A difference of 308 indicates the loss of hydrated coumaroyl glucoside moiety (Brito et al., 2014). Kaempferol-related glycosides were previously reported within the same species (Bahorun, Neergheen & Aruoma, 2005).

In addition, various glycosides were identified, such as eriodictyol-7-O-neohesperidoside represented by peak Cf12, as [M–H]⁻ ion at m/z 594.9. Fragments appeared at m/z 449 and m/z 287, and (iso) rhamnetin-3-O-glucoside-7-O-rhamnoside represented by Cf14, which produced [M–H]⁻ at m/z 623.08. In the MS–MS spectrum, two predominant fragments at m/z 315, due to deprotonated aglycone [M–H–308]⁻ and 314, due to [M–H–308]^−• appeared. This indicates that a hexose (162) and a deoxyhexose (146) are linked at the same position of the aglycone. Due to the most abundant fragment at [M–H–308]^−•, the substituent position is suggested to be a 3-OH group. As a result, the aglycone is identified as (iso) rhamnetin, reported previously in C. fistula (Bahorun, Neergheen & Aruoma, 2005).

Cf5 was identified as acetyl-dicaffeoylglycerol producing an ion at m/z 457.13. Further fragments were observed at m/z 161 and 135. Furthermore, acetyl-p-coumaroyl-caffeoylglycerol, represented by peak Cf4, exhibited a deprotonated molecular ion [M–H]⁻ at m/z 441.

Cf11 was identified as sennoside B with deprotonated ion [M–H]⁻ at m/z 861.17 and the fragmentation pattern showed m/z 699 due to the loss of one glucose molecule [M–H–glu]⁻ and m/z 537 due to the loss of 2 glucose molecules [M–H–glu–glu]⁻ (Zhao et al., 2012). It is worth noting that sennoside B was also compared to an authentic standard to prove its identity. Peak Cf13 was identified as rhein-O-glucoside. Their MS/MS spectrum yielded m/z 283 as the base peak, which could further fragment into m/z 257 and m/z 239. These cleavages were similar to those of rhein. Therefore, the corresponding compound was tentatively identified as the known rhein-O-glucoside (Ye et al., 2007). Peak Cf31 was tentatively identified as emodin-O-glucoside; its MS/MS spectrum showed ions at m/z 269 and m/z 268. The MS3 spectrum of m/z 268 gave a fragment at m/z 224, indicating the loss of CO₂ [M–H–CO₂]⁻, and demonstrating the emodin as aglycone, which has been previously detected as a major constituent in C. fistula (Lee, Lee & Kuo, 2001; Ye et al., 2007).

Total phenolic content

In order to generally quantify the amount of phenolics in the extract, the Folin-Ciocalteu assay was carried out. It showed a total phenolic content of 93.79 ± 4.13 µg of GAE/mg of extract, a value that is close to those obtained from different type of extracts of the aerial parts from different Cassia species (Mehta et al., 2017).

CUPRAC method for total antioxidant capacity

Cupric ion reducing antioxidant capacity assay (CUPRAC), is one of the assays based on electron transfer like DPPH, FRAP and Folin-Ciocalteu. It measures the ability of the antioxidant to reduce the oxidant, leading to a color development that could be measured spectrophotometrically. Polyphenolic compounds have distinctively reactive Ar-OH groups, which are able to be oxidized to their quinone analogue.

Consequently, a reduction to the chromogenic CUPRAC reagent, bis (neocuproine) copper (II) chelate to bis (neocuproine) copper (I) chelate takes place leading to development of yellow-orange color having a maximum absorption at 450 nm. Protons liberated from the reaction are buffered by ammonium acetate (Apak et al., 2004). A strong antioxidant capacity is represented by low EC₅₀ value. BHA standard was used as a positive control in this study. CF showed a relatively close EC₅₀value (EC₅₀ = 170 ± 0.55 µg/ml) to BHA (Table 2).

DPPH assay

CF showed a moderate antioxidant activity in DPPH assay (EC₅₀ of 52.6 ± 0.86 µg/ml), compared to the positive controls Vit C and EGCG (Table 2).

Survival assay

Juglone, a yellow pigmented pro-oxidant quinone found in Juglans regia, is used at a high concentration to induce death of the nematode (Inbaraj & Chignell, 2004). Pre-treatment of worms with CFs enhanced survival rates. CF 300 µg/ml showed the highest percentage (68 ± 5.14%) when compared to the solvent control, MeOH, showing only 34 ± 2.53% survival (p-value <0.001), (Fig. 2). These results are close to the EGCG group, positive control known for its powerful antioxidant activity, which showed a 73 ± 6.73% survival rate (Abbas & Wink, 2009).

Effect of CF on intracellular ROS levels

To further test the antioxidant effect of CF in vivo, intracellular ROS levels were evaluated using wild type N2 worms. H₂DCFDA, a membrane permeable compound, is a widely known marker for detecting the production of ROS inside the cell. After entering the cell, the compound is deacetylated by the intracellular esterases. The presence of ROS leads to the oxidation of the compound forming the highly fluorescent 2′,7′-dichlorofluorescin, where the intensity of the fluorescence correlates with the levels of ROS (Eruslanov & Kusmartsev, 2010).

Results show a significant concentration dependent decrease in the fluorescent intensity of the extract treated group, up to 73% decrease in CF 300 µg/ml compared to MeOH group. These results are in accordance with the EGCG group, which showed 77% decrease in fluorescence compared to the untreated control (p-value <0.001) (Fig. 3).

Effect of CF on DAF-16 localization

To detect whether the antioxidant effect of CF is based only on direct ROS scavenging activity or if other mechanisms are involved, DAF-16 assay was carried out. The DAF-16 transcription factor of C. elegans is considered as a homologue to the fork head transcription factor (FOXO), found in humans. Normally, DAF-16 is localized in the cytosol in its inactive phosphorylated form. Certain kinds of triggers, like stress and other environmental factors can induce its dephosphorylation and subsequent nuclear localization.

Upon nuclear localization, DAF-16 activates several genes that are responsible for lifespan extension and stress response (Mukhopadhyay, Oh & Tissenbaum, 2006). TJ356 transgenic worms are used in this assay in which the treatment with CF 300 µg/ml clearly showed high nuclear localization of DAF-16::GFP by 63 ± 1.73%, compared to MeOH group with 8 ± 8% nuclear localization. On the other side, EGCG treated group showed 81 ± 6.24% nuclear localization (p-value <0.001). The previous results highly imply that the in vivo antioxidant effect of CF is mediated through DAF-16/FOXO pathway (Fig. 4).

Effect of CF on SOD-3 expression

DAF-16 activation subsequently results in activating other stress response genes like SOD-3, which encodes mitochondrial Mn-SOD. The SOD-3 enzyme protects worms from ROS via scavenging O${}_2^{.-}$ radical (Honda & Honda, 1999). Antioxidant compounds show a high capability to increase the expression of SOD-3 (Zhou, Pincus & Slack, 2011). EGCG group showed 83% increase in fluorescence intensity compared to the untreated control.

CF-treated worms show a higher expression of the SOD-3::GFP compared to the negative control. CF 300 µg/ml showed 67% increase in fluorescence intensity compared to MeOH group (p-value <0.001) (Fig. 5).

Effect of CF on HSP expression

To further support the antioxidant effect of the extract, quantification of heat shock protein (HSP) expression in TJ375 mutant worms was investigated. HSP-16.2 is used to test stress levels in worms, as it is induced by both oxidative stress and heat (Strayer et al., 2003). The lowest concentration tested of the extract, CF 100 µg/ml, showed a lower expression of HSP-16.2, induced by juglone (34.34 ± 5.59%) when compared to solvent control + juglone treated group. EGCG showed a reduction in the fluorescence intensity with a value of 47.45 ± 5.67%, compared to juglone treated group (p-value <0.001) (Fig. 6).

Effect of CF on GST-4 expression

GST-4 is one of the glutathione S-transferases contributing to Phase II detoxification process (Choe, Przybysz & Strange, 2009). This group of enzymes is involved in the response towards oxidative stress. They are regulated by the SKN-1 transcription factor, a homologue to the human NRF2, which targets downstream genes (Kahn et al., 2008). Results show a marked decrease in the expression of GST-4 in worms treated by both EGCG and CF, compared to the negative control groups. CF showed 65% decrease in fluorescence intensity compared to MeOH group. These results are in accordance with EGCG treated group that showed 69% decrease in fluorescence intensity compared to the negative control (p-value <0.001) (Fig. 7).

Effect of CF on PolyQ40 aggregation

PolyQ40 aggregation is linked to Huntington’s disease and other neurodegenerative diseases (Williams & Paulson, 2008). Results in this study show that treatment with 300 µg/ml CF is able to decrease the number of polyQ40 aggregates by 80 ± 0.64% compared to MeOH group. This result matches with the positive control, EGCG treated group thaat also showed 80 ± 0.83% decrease in aggregate formation compared to the control group (p-value <0.001). This data demonstrates the strong influence of CF in attenuating protein aggregation (Fig. 8).

Effect of CF in Aβ induced paralysis

To further support the neuroprotective effect of CF, a paralysis assay was carried out. The CL4176 strain was used to test the effect of the plant extract on Aβ accumulation and toxicity, that result in worm paralysis. Results are expressed as a delay in PT₅₀, time where 50% of the worms are paralyzed, compared to the control group. Three hundred µg/ml CF showed a 4 h delay in paralysis time while CF 500 µg/ml showed a 5 h delay, a result that is close to that of the positive control, 100 µg/ml EGCG, used (p-value <0.0001) (Table 3). CL802 control strain, showed no paralysis at all, regardless of treatment.