Dietary administration of cumin-derived cuminaldehyde induce neuroprotective and learning and memory enhancement effects to aging mice

Evaluation of the cytotoxicity of cuminaldehyde on human neurotypic SH-SY5Y cells

First of all, we assessed the effect of CA on SH-SY5Y cells viability using MTT assay. Cells were treated with 2.5, 5, 10, 25, or 50 μM of CA for 72 h (Figure 2). The exposure of the cells to CA for 72 h showed no significant changes in the percentage of the cell viability compared to the non-treated cells (control group) (P > 0.05). Therefore, CA showed no cytotoxicity on SH-SY5Y cells at varying concentrations.

Figure 2. Effect of CA on SH-SY5Y cells viability. SH-SY5Y cells were seeded onto 96-well plate at a density of 2×10⁵ cells/well. After overnight incubation, cells were treated with 5, 50 or 100 μM of CA for 72 h and the cell viability was evaluated using MTT assay. The experiment was repeated thrice. The bars signify relative viability as compared to the control. Data is represented as mean ± SD.

Evaluation of the neuroprotective effect of cuminaldehyde against dexamethasone-induced neurotoxicity on SH-SY5Y cells

SH-SY5Y cells were treated with several concentrations (5, 25, 50, 200, or 500 μM) of dexamethasone (Dexa), to determine the concentration that would be toxic for cells. Dexa-treated cells showed the significantly decrease in the cell viability in a dose-dependent manner, with a decrease of cell viability up to 55% at 500 μM comparing with the non-treated cells (P < 0.001) (Figure 3A). Therefore, the concentration 500 μM of Dexa was used to induce cell cytotoxicity in the further experiments. Then to estimate the effect of CA neurons, SH-SY5Y cells were treated with CA and/or co-treated with Dexa for 72h. The results showed that cell incubated with CA of 10, 25, or 50 μM reversed the Dexa-induced neuronal cell death (Figure 3B), and the cell viability was considerably increased by more than 20% as compared to only Dexa treated cells (P < 0.01).

Figure 3. Effect on cell viability on SH-SY5Y cells after treatment with (A) dexamethasone (Dexa) and (B) Dexa co-treated with CA. SH-SY5Y cells were treated with CA or 500 μM Dexa for 72 h. Each bar represents the mean ± SD. ***: p < 0.001 as compared to the control; ##: p < 0.01; ###: p < 0.001 as compared to Dexa treatment.

Effects of sub-chromic administration of cuminaldehyde on body weight and brain weight of C57BL/6J mice

CA (25 mg/kg) was administered orally daily for 30 days to C57BL/6J mice, and the effect on body weight was studied. The results obtained showed that the body weight of CA treated mice did not differ from the young water-treated mice throughout the experiment (P > 0.05) (Figure 4A), no mortality and no changes in behavioral activity were recorded.

Figure 4. (A) Effects of CA (25 mg/kg) on body weight during the 30 days experimental period. All mice were fed a normal diet. The body weight was measured every day. Each point presents the values from that group for that day. The values are represented as mean ± standard deviation. (B) Brain weight after 30 days of treatment with CA. The brain was dissected out, washed with ice-cold PBS and weighed. All the values are expressed as mean ± SD. NC: negative control (aged water-treated group); PC: positive control (young water-treated group); CA: Cuminaldehyde treated group.

At the end of probe test of MWM, all mice were sacrificed by cervical dislocation and the brain was dissected and weighed. The results showed that CA-treated mice brain weight (Figure 4B) did not differ from those of young-water-treated mice (positive control) or aged-water-treated mice (negative control). CA did not produce any significant effect on the mice body and brain weights after daily administration for 30 days. This suggests that CA may not have any toxic effect in C57BL/6J mice.

Effect of cuminaldehyde on spatial learning and memory in C57BL/6J mice

To analyze effect CA on the ability of spatial learning and memory in aging C57BL/6J mice, the morris water maze (MWM) test was performed (Figure 5). The time (in seconds) taken by the animal to reach the platform indicates how animal can learn and memorize the platform location. In this experiment, we observed the decrease of escape latency compared to water-treated aging C57BL/6J mice indicating an improvement of spatial learning and memory ability. The escape latency time of both, aged C57BL/6J mice administered with CA (n = 8) and young water-treated C57BL/6J mice (positive control) was significantly decreased by 35% and 31% respectively (P < 0.05) as compared to the water-treated aging C57BL/6J mice (negative control) after 4 days of training session (Figures 4A, 5A). After 7 days of training, the time taken by aged-CA-treated mice (CA group) and young water-treated mice (positive control) significantly decreased by 20% compared to aged water-treated C57BL/6J mice (negative control). On the other hand, there was no significant difference between the escape latency time of aged-CA-treated mice (CA group) and young water-treated mice (positive control).

Figure 5. Effects of CA (25 mg/kg) on escape latency time during the morris water maze trial sessions, on (A) the time spent in the quadrant and (B) crossing time, (C) time taken during probe trial sessions on C57BL/6J mice. Mice were administered orally with water or CA (25 mg/kg/day) for 30 days, 60 min prior to trial sessions. The training trial and probe trial sessions were performed as described in the Materials and Methods. All the values are expressed as mean ± SD. NC: negative control (aged water-treated group); PC: positive control (young water-treated group); CA: Cuminaldehyde treated group. * P < 0.05; ** P < 0.01 as compared to the negative control.

After 7 days of training, a probe test was performed, in which the platform was removed and the time spent in the quadrant (Figure 5B) and time taken to cross the quadrant (Figure 5C) were evaluated. The results showed that aged-CA-treated mice showed a significant increase by 10% in the time spent in the quadrant (P < 0.01) and the crossing time (P < 0.01) as compared to the aged water-treated mice (negative control).

Effect of cuminaldehyde on locomotor activity in C57BL/6J mice

In the probe trial (day 8), we also measured immobility time of mice for evaluation of their motivation. Our results showed that there was a significant increase by 16% (P < 0.05) in the time spent swimming by aged-CA-treated group compared to the aged-water-treated group (negative control), however; no significance was observed between the aged-water-treated mice (negative control) and the young water-treated group (positive control) (P > 0.05) (Figure 6).

Figure 6. Effects of CA on swimming time during probe test of morris water maze trial sessions on C57BL/6J mice. Mice were administered orally with water or CA (25 mg/kg/day) for 30 days 60 min prior to trial sessions. Data is represented as means ± SEM (* P < 0.05 as compared to negative control).

Effect of cuminaldehyde on gene expression in C57BL/6J mice

To determine the molecular mechanism underlying the enhancement of spatial learning and memory of CA, we analyzed changes in gene expression of four genes (Bdnf, IL-6, Icam, and ApoE) in the mice brain using RT-PCR (Figure 6). Compared to the aged water-treated mice (negative control), a dose of 25 mg/kg/day of CA treatment in C57BL/6J mice, for 30 days, up-regulated the expression of Bdnf by 58% (Figure 7A), Icam by 34% (Figure 7B), and ApoE by 58% (Figure 7C), while it down-regulated the expression of IL-6 by 15% (P < 0.05) (Figure 7D).

Figure 7. Effect of CA on mRNA expressions of Bdnf (A), Icam (B), ApoE (C) and IL-6 (D) in C57BL/6J mice brain. The mRNA expression of all genes were normalized to GAPDH mRNA expression and expressed with respect to the negative control (aged water-treated group). C57BL/6J mice were administrated with CA for 30 days. The control group was administered with distilled water. Each bar represents the mean ± SD. * P < 0.05, ** P < 0.01 as compared to negative control group. NC: negative control (aged water-treated group), PC: positive control (young water-treated group), CA: Cuminaldehyde treated group.

Effect of cuminaldehyde on neurotransmitters concentration in C57BL/6J mice brain

To evaluate the effect of CA on the levels of dopamine, adrenaline, and noradrenaline in the brain of C57BL/6J mice, their concentrations in the brain were measured using ELISA technique. The results showed no significant changes in the concentrations of dopamine (Figure 8A) and adrenaline (Figure 8B) in CA-treated mice brain compared to aged water-treated mice (negative control); whereas there was a significant increase (13%, P < 0.05,) in the concentration of noradrenaline (Figure 8C).

Figure 8. Effect of CA on catecholamine concentrations: Dopamine (A), Adrenaline (B), and Noradrenalin (C) in C57BL/6J mice brain. C57BL/6J mice were administrated with CA for 30 days. The control group was administered with distilled water. Each bar represents the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 as compared to negative control group. NC: negative control (aged water-treated group), PC: positive control (young water-treated group), CA: Cuminaldehyde treated group.

Effect of cuminaldehyde on the levels of TNF-α and IL-6 in C57BL/6J mice brain

To evaluate the effect of CA on the levels of TNF-α and IL-6 in the brain of C57BL/6J mice, their concentrations were measured in the brain using ELISA technique. Our result showed that brain TNF-α was significantly (P < 0.05) increased in aged water-treated mice (negative control) (21.6 ± 2.3 pg/μg protein) compared with young water-treated group (positive control) (16.4 ± 1.7 pg/μg protein) (Figure 9A). However, CA-treated group showed a significant decrease (17.2 ± 1.2 pg/μg protein) of TNF-α levels in brain compared with aged water-treated group (negative control) (P < 0.05, Figure 9A).

Figure 9. Effect of CA on levels of (A) tumor necrosis factor-α (TNF-α) and (B) interleukin 6 (IL-6) in C57BL/6J mice brain. C57BL/6J mice were administrated with CA for 30 days. The control group was administered with distilled water. Each bar represents the mean ± SD. * P < 0.05, ** P < 0.01 as compared to negative control group. NC: negative control (aged water-treated group), PC: positive control (young water-treated group), CA: Cuminaldehyde treated group.

Moreover, brain IL-6 level in aged water-treated group (negative control) was significantly (P < 0.01) increased compared with young water-treated group (positive control) (6.7 ± 0.8 pg/μg protein) (Figure 9B). Similarly, IL-6 level in the CA-administered group (7.6 ± 1.3 pg/μg protein) was significantly increased (P < 0.01) as compared to that of aged water-treated group (negative control) (13.1 ± 1.3 pg/μg protein).

Chemicals and reagents

CA (Figure 1), Dulbecco’s Modified Eagle Medium (DMEM), Ham and Fetal bovine serum (FBS) were purchased from Sigma Aldrich Co., Ltd. (Irvine, United Kingdom). Penicillin and Streptomycin were purchased from Lonza Inc. (Walkersville, MD), while Eagle’s minimum essential medium (OPTI-MEM) was purchased from Gibco (Japan), and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) from DOJINDO, (Japan). SDS was purchased from GE Healthcare (Sweden).

Cell culture

The human neuroblastoma SH-SY5Y cell line was obtained from American Type Culture Collection (ATCC). This cell line is widely used in the field of neuroscience for the research on Alzheimer's disease, neurotoxicity, ischemia, and amyotrophic lateral sclerosis [50]. According to our previous study [51], SH-SY5Y neuroblastoma cells were cultured in 100 mm petri dish or in 96-well plates with a 1:1 (v/v) mixture of Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F-12 nutrient mixture supplemented with 15% fetal bovine serum (FBS), and 1% penicillin (5000 μg/mL) streptomycin (5000 IU/mL) solution at 37° C in a 95% humidified air of 5% CO2 incubator. A serum-free Eagle’s minimum essential medium (Opti-MEM) was used to culture the cells for cell viability assay.

MTT assay

To assess the cell viability of SH-SY5Y cells, MTT assay was performed as previous our study [51]. SH-SY5Y cells were cultured at a density of 2 x 10⁵ cells/well in a fibronectin coated 96-well micro-plate (BD, BioCoat). After a period of 24 h incubation at 37° C (5% CO), cells were treated with CA, at concentration of 5, 50 or 100 μM, dissolved in Opti-MEM. After 72 h of incubation, 10 μL of MTT (5 mg/mL) dissolved in 100 μL of Opti-MEM was added to each well and the plate was incubated for further 6 h. The colored formazan was dissolved in 100 μL of 10% SDS. The cell viability was determined using a multi-detection micro plate reader (Powerscan_1-42, HT, Dainippon Pharmaceutical, Japan) using absorbance at 570 nm, and the results were represented as percentage compared to control.

Neuroprotective assay

To investigate the neuroprotective effect of CA, Dexa, a synthetic glucocorticoid used as an inducer of apoptosis, was used according to our previous study [51]. SH-SY5Y cells were cultured in 96-well plate (fibronectin-coated plate) and treated with CA and co-treated with Dexa (500 μM) for 24 h and 72 h. After the treatment, 100 μL of Opti-MEM and 10 μL of MTT (5 mg/mL) were added, and cell viability assay was performed as described above.

Animals

C57BL/6J male mice aged 10 weeks and 9 months, weighting between 25 g and 35 g were used in the experiment. The animals were obtained from Japan (Japan SLC Company, Japan). Mice were housed individually, one mouse per cage and had free access to food and water, under a 12 h light/12 h dark cycle in a controlled environment of 56% humidity and 23° C temperature.

After 7 days of acclimatization to the laboratory conditions, mice were divided into 3 groups: a positive control group composed of 10 weeks-old mice (young water-treated group) (n = 7), a negative control group composed of 9 months-old mice (aged water-treated group) (n = 5), and CA-treated group composed of 9 months-old mice (n = 8) which were treated by oral administration of CA (25 mg/kg) for 30 days. The concentration dose (25 mg/kg) of CA for in vivo study was determined based on the previous study using cuminum cyminum extract [52]. The respective control groups (positive and negative groups) were administered an equivalent volume of distilled water. This animal experiment was approved by the Animal Care and Use Committee of the University of Tsukuba.

Morris water maze (MWM) test

The MWM test was carried out using a circular pool of 120 cm in diameter and 45 cm in height as reported previously [53]. On the inner surface of the circular pool four symbols (a square, a circle, a cross mark, and a triangle) were attached. The pool was divided into four quadrants (north, east, west, and south) and filled with water (23 ± 2° C). A Plexiglas platform (10 cm of diameter) was placed exactly in the northeast quadrant, submerged 1 cm below the water surface. The platform was fixed in the same place in the pool throughout the experiment. The mice were given a trial session (60 s) 4 times each day for 7 days, then each mice were placed on the platform for 30 s. CA was administered to the treatment group 30 min before the experiment.

After 7 days, each mouse performed one probe trial, in which the platform is removed from MWM pool. Probe trials are performed to validate the strategy followed by the animals when they notice an absence of the platform. The animals were released into the pool from a single point and the number of times the animal crosses the quadrant of the platform in 30 s was recorded.

Tissue preparation

Mice were sacrificed by cervical dislocation 24 h after the last trial of the MWM test and 30 min after the last oral administration of the pure compound. The brain samples were dissected and fixed in liquid nitrogen, and stored in -80° C until use.

RNA extraction from mouse brain

A total of 100 mg of the cerebral tissue was removed and washed with ice-cold phosphate-buffered solution (PBS). The total RNA was extracted from it using the ISOGEN kit (Nippon Gene Co. Ltd., Toyama, Japan), following the manufacturer’s instruction. Total RNA was quantified and assessed for its purity with the NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

RT-PCR

The Superscript III reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA) allowed us to carry out reverse transcription reactions [54]. Briefly, the RNA was denatured at 65° C for 5 min, incubated with 1 ml of oligo primer (dT) and cooled to 4° C. After adding the Superscript III reverse transcriptase (200 units), the reaction mixture was incubated at 42° C for 60 minutes, then for 10 minutes at 70° C. The cDNA was used to evaluate the gene expression of Bdnf, ApoE, Icam, and IL-6. This experiment was conducted using TaqMan Universal PCR mix and TaqMan Probes, and the amplification reaction was performed in a 7500 Fast Real-time PCR (Applied Biosystems, USA) with the following conditions: 50° C for 2 min, followed by 95° C for 10 min, and 40 cycles of 95° C for 15 s followed by 60° C for 1 min.

Dopamine, noradrenaline and adrenaline levels in mice brain

Dopamine, noradrenaline, and adrenaline levels in the mice brains were measured by Dopamine, Adrenaline, and Noradrenaline Research ELISA kits (ImmuSmol, Pessac, France) as reported in the previous study [54]. Briefly, the cerebral cortex (100 mg) was isolated from brain tissue of mice and homogenized in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor (Santa Cruz Biotechnology, Inc., Tokyo, Japan). Further, 50 μL of protein samples or 10 μL standards were used for catecholamine extraction, acylation, and determination according to manufacturer’s instructions. After optimal color development, the reaction was stopped and the absorbance was measured at 450 nm. Protein concentration was determined using the 2-D Quant kit (GE Healthcare Inc., Tokyo, Japan) and the data were expressed as ng/μg protein.

Brain tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) quantification

Brain tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) levels in the brain were measured using ELISA kit (TNF-α: Proteintech, Japan; IL-6: Invitrogen, Japan) in accordance with the manufacturer’s instructions. Briefly, 100 mg of the limbic area was isolated from the brain tissue and homogenized in RIPA buffer with protease inhibitor (Santa Cruz Biotechnology, inc., Tokyo, Japan). Following centrifugation (1000 × g, 20 min), the supernatants or standards were used to determine the TNF-α and IL-6 levels in brain. After the addition of TNF-α or IL-6 antibody, a second incubation was performed with streptavidin-horseradish peroxidase conjugate solution. After addition of substrate and stop solution, TNF-α and IL-6 levels were determined by measuring absorbance at 450 nm.

Statistical analysis

All results are expressed as mean ± standard deviation (SD), and statistical analyses were performed using a Student’s t-test using SigmaPlot 12.0 software. Results were considered statistically significant when P-value was less than 0.05.