Detection of Astaxanthin at Different Regions of the Brain in Rats Treated with Astaxanthin Nanoemulsion.

Context: Astaxanthin (Ast), a compound used widely as a dietary supplement, has high antioxidant properties but poor oral bioavailability. To benefit from its nutritional values in cognitive function, Ast was formulated into a nanoemulsion which may improve its penetration through the blood-brain barrier (BBB). Aim: The present study aims to quantitate the Ast nanoemulsion in different regions of the brain tissue using the high-performance liquid chromatography method. Materials and
Methods: Sprague-Dawley rats were fed with Ast nanoemulsion formulation daily (40, 80, and 160 mg/kg body weight, bw) for 28 days before brain tissues were harvested, extracted, and quantified. A simple, sensitive, and reliable method using high-performance liquid chromatography with an ultraviolent detector was developed and validated to quantify Ast in the brain. Statistical Analysis: Data were analyzed using the ToolPak Data Analysis in Excel for t-test and analysis of variance single factor with Tukey post hoc analysis.
Results: The calibration curve demonstrated a linear regression with an r 2 of 0.9998 and absolute recovery ranging from 97.8% to 109.6%. The hippocampus of the 160 mg/kg bw treatment group showed a significantly higher concentration of Ast (77.9 ± 17.3 μg/g) compared to the cortex (22.3 ± 4.2 μg/g) and cerebellum (33.1 ± 5.4 μg/g). Ast was detected in the cerebellum of the 80 mg/kg bw (29.4 ± 7.8 μg/g) treatment group with the amount not being significantly different to the 160 mg/kg bw (33.1 ± 5.4 μg/g) treatment group. Conclusions: It was evident that the Ast nanoemulsion formulated was able to cross the BBB and may provide protective benefits. Copyright:

INTRODUCTION

Astaxanthin (Ast) is a natural red-orange carotenoid that belongs to the xanthophyll group. It possesses exceptional antioxidant properties compared to other carotenoids.[1] Ast can be classified into two different forms: The fatty acid ester that is extracted from Haematococcus pluvialis or krill and the non-fatty acid ester that is derived from the red yeast Phaffia rhodozyma or is chemically synthesized.[23] The conjugated double bonds that exist in the esterified Ast contribute to its strong antioxidant properties. It terminates free radical chain reactions within cells generated by cellular processes.[4] Ast has been widely used in the pharmaceutical, cosmetics, and food industries due to its anti-aging and antioxidant properties.[567] Clinical studies indicated that Ast showed promising effects in targeting cardiovascular disease, immune response, and neurological diseases.[8]

Ast possesses low oral bioavailability due to its lipophilic nature that resulted in its poor absorption by the intestine. Formulating Ast into a nanoemulsion has increased its solubility in water.[9] Previously, the Ast nanoemulsion showed better distribution in tissues such as the lung, kidney, spleen, heart, and liver when compared to oil and macro formulations.[10] However, its distribution in the brain has not been explored. According to Grimmig et al., Ast can pass through the blood– brain barrier (BBB), thus promoting its potential function in cognitive improvement and neurodegenerative diseases.[11] The potential of Ast as a treatment of neurological diseases is yet to be explored. Despite all of the findings, studies on the quantification of Ast from brain tissue are rare.[1213]

The quantitation of Ast from brain tissue can be taxing, with interferences in the chromatogram, probably due to the high-fat content (60%). Methods using high-performance liquid chromatography (HPLC) have been widely used to quantify β-carotenoids and xanthophylls in plasma.[1415] However, the information on Ast quantification in brain tissue is limited,[1213] perhaps due to the matrix effects exerted by brain tissue that alternatively opt for the quantification of Ast in plasma. This limits the knowledge on the ability of Ast as a potent antioxidant, and whether the compound can cross the BBB has not been well documented. To study a specific region of the brain where the protective effect of Ast takes place can be also challenging, with only small sample sizes being available, for example, the hippocampus. Previous methods utilized large amounts of brain samples but were unable to identify the specific regions in which Ast was detected.[12]

The present study aimed to prove the Ast nanoemulsion formulation distribution in different regions of the brain and to develop and validate a method that is sensitive to quantify Ast using a small size brain samples. A normal-phase HPLC with an ultraviolent (UV) detector method was developed and validated to quantify Ast nanoemulsion in the brain tissues, namely the cortex, hippocampus, and cerebellum. The samples were taken from Sprague–Dawley (SD) rats fed daily with Ast nanoemulsion for 28 days at concentrations ranging from 40 to 160 mg/kg body weight (bw). We hypothesized that the method developed is sensitive to detect Ast using small sample size and the Ast nanoemulsion formulated will to penetrate the BBB and be detected in different regions of the brain. This opens up the possibility of future studies to explore the potential of Ast in the management or treatment of neurological diseases.

MATERIALS AND METHODS

Materials

Ast used in this study was extracted from H. pluvialis algae containing 10% w/w of Ast standard (Astareal 10FC grade) and was purchased from Fuji Chemical Industries (Nakaniikawa, Toyama, Japan). All-trans-Ast analytical standard (purity ≥ 97%) and apocarotenal (β-Apo-8’-Carotenal) United States Pharmacopeia Reference Standard were purchased from Sigma-Aldrich (Sigma-Aldrich, Germany) and used as internal standards (IS), and hexane and acetone of HPLC grade were purchased from Merck (Merck, Germany). Other chemicals used were of analytical grade unless otherwise stated.

Astaxanthin nanoemulsion formulation

The formulation was developed as described by Affandi et al.[16] Ast extract (2% w/w) was mixed with lecithin (1.6% w/w), tween 80 (2.4% w/w), pure palm olein (20% w/w), and purified water (80% w/w). Samples were mixed using the high-speed homogenizer (Silverson, England) at 9000 rpm for 5 min with the start and end time recorded to obtain a macro-sized emulsion. Then, samples were further mixed using a high-pressure homogenizer (Nano DEBEE, USA) at 20,000 psi with six cycles of mixing to obtain nano-sized Ast emulsion. Next, the sample size was determined using a zetasizer (Malvern Instruments, Worcestershire, UK) and the size range <200 nm was accepted as nano.

Characterisation of astaxanthin nanoemulsion

Zetasizer

Dynamic light scattering by a zetasizer (Malvern Instruments, Worcestershire, UK) was used to determine the size and distribution of the Ast nanoemulsion by measuring the Brownian motion of the particles. The zeta potential (droplet surface charge) and polydispersity index (PDI) were also measured using the zetasizer. The zeta potential is often used for the characterization of double-layer particles such as nanoemulsions and indicates the stability of the colloidal dispersions system, whereby higher zeta potentials exude repulsion that resists aggregation and confers stability. Meanwhile, PDI measures the variation in particle size, showing either a monodispersed or polydispersed nanoemulsion system. In the study, the Ast nanoemulsion was diluted ×1000 (×10 dilution times 3) in distilled water before zetasizer measurement.

Field emission scanning electron microscopy

The Ast nanoemulsion physical morphology was analyzed using the field emission scanning electron microscope (FESEM) (JSM-6701F, JEOL, Netherlands). Here, 0.2 mL of Ast nanoemulsion was first mixed with 1% osmium tetraoxide in water. After 2 h, the sample was centrifuged at 10,000 rpm for 5 min followed by distilled water rinsing and vortexing. The rinsing process was repeated four times. Then, a drop of the sample was placed onto the aluminum slab and allowed to dry at room temperature. Dried samples were coated with platinum by an Emitech Magnetron Sputter Coater before imaging to avoid electric charge build-up. Samples were observed by FESEM at different magnifications.

Animal treatment and tissue extraction

The preparations for tissue extraction were based on a previous study by Choi et al., with some modifications.[12] Clearance for the use of animals was obtained from Universiti Teknologi MARA Committee on Animal Research and Ethics UiTM (UiTM CARE: 288/2019). Twenty male SD rats (300–320 g) were acclimatized (21°C ± 2°C, 12 h light-dark cycle) for 1 week and divided into four groups: Control (distilled water) and Ast treatment (40 mg/kg, 80 mg/kg and 160 mg/kg bw) (n = 5 in each group). Each group was orally fed with the designated treatment for 28 days. Rats were anesthetized with pentobarbital sodium (Dolethal, Vetoquinol S. A, France) 200 mg/mL (0.1 mL/100 mg body weight) prior to perfusion with cold 0.9% sodium chloride solution to remove blood in the brain. Immediately after perfusion, the brain was harvested and separated into the hippocampus, cerebellum, and cortex. Each individual tissue (approximately 0.08 g) was homogenized with acetone followed by hexane for 30 secs each in the ratio of 1:3 up to 1500 μl of solution. The sample was then centrifuged (10,000 rpm, 5 min, 4°C) and the pellet was discarded. The collected supernatant was dried under a nitrogen stream and reconstituted in 90 μl of mobile phase and 10 μl of IS.

Instrumentation

The HPLC (Agilent 1200, Germany) was operated at a wavelength of 470 nm with a silica column (Phenomenex, USA) for chromatographic separation. The mobile phase was comprised hexane: Acetone (75:25% (v/v). The flow rate was set at 1.0 mL/min, with an injection volume of 5 μl; the chromatogram was detected using a DAD-UV detector. The sample was quantified using the area under the curve ratio over IS.

Linearity

The ratio of Ast standard over IS (β-apo-8-carotenal; 1 μg/mL) was plotted to construct a calibration curve in the range from 19 to 2500 ng/mL.

Precision and accuracy

The precision and accuracy of the method (n = 6) were assessed within-day and between-day. Six sets of each concentration of standards were prepared and determined in a day. Meanwhile, another six sets of the same concentration were prepared and run daily for 6 days.

Limit of detection and limit of quantification

The limit of quantification (LOQ) was evaluated by five replicates of the lower concentrations of the calibration curve with the accepted deviation values of 20% using 200 μL of brain homogenate sample. Meanwhile, the limit of detection (LOD) was determined by the signal-to-noise ratio of 3:1.

Recovery

To assess the matrix effects and recovery, known concentrations of Ast standards (low, medium, and high) and IS were spiked in the nontreated brain tissue, homogenized, and centrifuged. Supernatant collected was run in the HPLC system in triplicates.

Statistical analysis

All data were analyzed using the ToolPak Data Analysis in Excel. Significant differences between different regions of the brain were analyzed using the single factor analysis of variance (ANOVA) and Tukey HSD post hoc test (https://astatsa.com/OneWay_ANOVA_with_TukeyHSD/). The significance between groups was analyzed using the two-sample t-Test assuming equal variances. All data were expressed as the mean ± standard error. Value of P < 0.05 was accepted as statistically significant.

RESULTS

Zetasizer and FESEM results of the freshly prepared Ast nanoemulsion were presented in Figure 1. The average droplet size measured by zetasizer showed that the nanoemulsion produced are nano-sized (112.65 ± 0.07 nm) with a good zeta potential (−30.55 ± 3.18 mV) and low PDI (0.1015 ± 0.0007) [Figure 1].

Figure 1

A typical profile of droplet size distribution (a) and zeta potential (b) in the astaxanthin nanoemulsion

The Ast nanoemulsion observed under the FESEM showed droplets which were spherical with an average size of 105 nm [Figure 2]. In general, the nanoemulsion produced is in nano sized with good zeta potential and PDI.

Figure 2

Field emission scanning electron microscope of astaxanthin nanoemulsion (a) at 20,000 magnification and (b) at 80,000 magnification showed a spherical shape with a mean size of 105 nm

Astaxanthin and internal standards chromatogram

Using the normal phase HPLC UV-DAD detection system, Ast and IS were well separated with no interference. Ast standard and IS injected in the HPLC system were eluted at approximately 6.2 and 3.2 min, respectively, as shown in Figure 3. Chromatograms obtained for a blank brain sample and a brain sample spiked with Ast and an internal standard showed no interference in the chromatogram [Figure 3].

Figure 3

Chromatogram of blank brain samples (a) and the chromatograms of brain samples spiked with Astaxanthin standard and internal standards (b) peaks in the hexane mobile phase: acetone (75:25%), wavelength of 470 nm, with an elution time of approximately 6.2 and 3.2 min, respectively

Validation of the method

The assessment of the validation method was based on the typical characteristics of analytical methods such as linearity, precision/repeatability, and accuracy/recovery.[17] In the present study, the linear regression of Ast was r = 0.9998, with an intercept of 0.0218 and a slope of 0.001, as shown in Figure 4; this showed a good linear relationship between the peak area ratio and concentration. The precision and repeatability of the method were assessed based on the range standard deviation (RSD) % results [Table 1]. Samples with a lower RSD (<10%) indicate that the method is accurate and reproducible. Ast LOD and LOQ were found to be 9.8 ng/mL and 19.5 ng/mL, respectively. The recovery of Ast was more than 95% for all concentrations, as shown in Table 2, thereby indicating that Ast was not lost during the extraction and the matrix effects are negligible. Modification of the sample extraction method using acetone and hexane had improved the recovery of Ast.

Figure 4

Linearity assessment of the high performance liquid chromatography method for astaxanthin quantification

Table 1

Within day and between day accuracy and precision range standard deviation (percentage) of astaxanthin (n=6)

Concentration (ng/mL)	Within day		Between day
	Accuracy (%)	RSD (%)	Accuracy (%)	RSD (%)
19	96.5	8.7	117.3	2.2
39	97.6	4.0	112.0	2.0
78	101.8	3.5	109.2	2.2
156	96.1	2.3	102.7	3.2
312	104.8	0.7	100.6	3.7
625	103.5	1.3	108.8	3.0
1250	101.6	1.0	99.3	2.3
2500	100.3	0.6	102.0	2.4

RSD: Range standard deviation

Table 2

Recovery of astaxanthin by spiking the analyte in blank brain matrices

Representative theoretical concentration (µg/mL)	Recovery
	Concentration (µg)	Mean (%)	RSD (%)
78	79.6	102.1	1.4
312	305.2	97.8	2.9
2500	2738.9	109.6	2.4

RSD: Range standard deviation

Ast quantitation in brain tissue

Table 3 shows the average content of Ast in the cortex, hippocampus, and cerebellum. The 40 mg/kg Ast treatment group was not able to detect Ast within the standard curve, probably due to the lower detection limit. There is a possibility that the lower concentration treatment group (40 mg/kg) would need larger brain samples (more than 0.08 g) for the chromatogram to be able to detect Ast. There was a significant difference in the average concentration of Ast between the three regions (F (2,12) = 7.5619, P < 0.01) of rats treated with 160 mg/kg bw of Ast nanoemulsion. The post hoc analysis revealed that the concentration of Ast in the hippocampus (77.9 ± 17.3 μg/g) was significantly higher compared to the cortex (22.3 ± 4.2 μg/g, P < 0.01) and cerebellum (33.1 ± 5.4 μg/g, P < 0.05). Meanwhile, Ast concentration in the cerebellum was not statistically significant when compared to the cortex. Treatment group 80 mg/kg bw showed no detection of Ast in cortex and hippocampus except for cerebellum. There was no significance difference of Ast concentration procured in cerebellum of group 80 mg/kg bw (29.4 ± 7.8 μg/g) and 160 mg/kg bw (33.1 ± 5.4 μg/g), t (8) = 0.3980, P = 0.3505 as shown in the t-test analysis.

Table 3

Amount of astaxanthin recovered (μg/g) in different regions of 0.08 g brain tissue after 28 days of oral feeding at 40, 80 and 160 mg/kg bw (n=5)

Tissues	Mean concentration (µg/g)
	40 mg/kg bw±SEM	80 mg/kg bw±SEM	160 mg/kg bw±SEM
Cortex	ND	ND	22.3±4.2a
Hippocampus	ND	ND	77.9±17.3b
Cerebellum	ND	29.4±7.8a	33.1±5.4a

Subscript with different letters indicates a significant difference of P<0.05. ND: Not detected, bw: Body weight, SEM: Standard error of mean

DISCUSSION

Ast nanoemulsion was successfully produced using the application of high energy. The HPH technique is very important for formation of a uniform and stable nanoemulsion due to its high energy applied in the homogenization process where, a very fine nanoemulsion with a low PDI value (monodispersed droplet) was produced.[1618] The physicochemical stability of the nanoemulsion droplet was analyzed by droplet size, zeta potential, PDI, and FESEM. As shown in [Figure 1], the droplet size distribution was unimodal, with an average size <200 nm, low PDI, and unimodal zeta potential results, which showed that the Ast nanoemulsion produced was stable, with dispersed droplets in the nanometer range.

The present study was able to quantify Ast in the cortex, hippocampus, and cerebellum of SD rats fed daily with the Ast nanoemulsion for 28 days using a small sample size (0.08 g). Ast was the highest when obtained from the hippocampus, followed by the cerebellum and cortex of rats administered 160 mg/kg bw of Ast nanoemulsion. The hippocampus and cerebellum are associated with memory, although they hold different types of memory, such as recognition memory and procedural memory.[1920] The identification of the accumulation of Ast nanoemulsion in these two regions of the brain is crucial since it will benefit patient undergoing treatment for neurological diseases that demonstrate memory impairment. Although the main function of the cortex is not memory (it still has a proportion of memory function), it contains layers of neurons that have neurotransmitters and glial cells which are also very important in information transmission.[2122] The accumulation of Ast in the cortex, although not the highest, will give possible protection to neurons from the cellular free radicals released by cells.

The quantification of Ast from brain tissue using HPLC has been reported earlier, although studies in the literature are scarce.[1213] Choi et al. showed that the distribution of Ast in rat organs was time-dependent, whereby the longer postoral administration of Ast (24 h) showed lower concentrations of Ast compared to the 8 h treatment in various organs, including the brain. The literature measured Ast concentrations in whole-brain recovered to be 0.260 and 0.123 μg/g after 8 h and 24 h of oral administration with tissue to plasma ratio of 1.96 and 2.14, respectively. The literature mainly discussed the pharmacokinetics and first-pass metabolism of Ast in rats being fed with Ast (100 and 200 mg/kg) dissolved in polyethylene glycol 400–N, and N-dimethylacetamide. Meanwhile, Manabe et al. showed that the Ast concentration was higher in the brain of rats fed for 5 days compared to a single dose which proved that Ast can accumulate in the brain. The author measured Ast in different regions of the brain, namely the hippocampus (10.5 ± 1.1 pmol/g), cerebral cortex (19.6 ± 1.8 pmol/g), and other regions (12.8 ± 1.6 pmol/g). However, these studies did not describe validation details, such as linearity, detection limit, mobile phase, recovery, and sensitivity of the instrument.

Although the methods used by Choi et al. and Manabe et al. successfully quantified Ast from brain tissue, they required 1 g of brain samples, which is approximately half of the brain sample compared to the present method (0.08 g brain tissue). The small sample size enabled us to quantify Ast in different regions of the brain and the smallest part of the brain is the hippocampus. This allowed the precise quantification of Ast in different brain regions, which would benefit researchers when designing their study using Ast in a possibly protective manner and as an option for treating neurological diseases.

Financial support and sponsorship

This study was supported by the Ministry of Higher Education (MoHE), Malaysia, through the Fundamental Research Grant Scheme (FRGS) (FRGS/1/2019/SKK08/UITM/02/5).

Conflicts of interest

There are no conflicts of interest.