Preliminary of Pharmacokinetics Study of Brown Seaweed ( Turbinaria decurrens Bory ) Extract in Colon Cancer Model Mice Induced by AOM ( Azoxymethane ) and DSS ( Dextran Sodium Sulphate )

Introduction: Colon Cancer is one of leading cancer that causes morbidity and mortality worldwide, causes 774,000 deaths. Fucoxanthin contained brown seaweed Bory) extracts showed cytotoxic activity by and study, including colon cancer. This study aims to examine the absorption profile of fucoxanthin in blood plasma on colon cancer model mice. Methods: Mice were induced by azoxymethane (AOM) and two cycle dextran sulfate sodium (DSS). Mice were given an extract of brown seaweed Bory that contain fucoxanthin. Fucoxanthin content in blood plasma analyzed using mobile phase methanol: acetonitrile by ReversedPhase High-Performance Liquid Chromatography (RP-HPLC) analysis. Results: The maximum concentration of fucoxanthin in blood was at 4 hours after administration, and the plasma concentration is 2.15 nmol/L, T1/2 6.25 h, and AUC 10.75 nmol/L/h Conclusions: The method was applied successfully in a pharmacokinetic study and the resulting oral brown seaweed bioavailability calculated.


Brown seaweed
Brown seaweed Turbinaria decurrens was collected from Binuangen Beach, Banten, Indonesia.The collected seaweeds were washed with tap water and distilled water to remove salt, and other debris then packed with ice to keep fresh during the transportation to the laboratory.Fresh seaweed Turbinaria decurrens Bory was extracted with ethanol (70%) and macerate for 3x24 hours.The filtrate was evaporated using vacuum evaporator.

AOM-DSS mouse model
The AOM-DSS mouse model was adopted based on the methods by Wang et al. (2016), with some modifications.Swiss Webster male mice (seven weeks old) were intraperitoneally injected with AOM at 12.5 mg/kg body weight.One week after AOM injection, the mice were treated with two DSS cycle, which comprised two steps in which mice were oral with 2.5% (w.v) DSS for five days followed by 16 days of regular water feeding, and then treated with DSS in one more cycle.All mouse model was treated by brown seaweed Turbinaria decurrens Bory extract (3.612, 7.224, 14.448 mg/mL 20 g body weight) from the first induced by AOM, except the normal group.In the last day of treatment, blood were collected into heparin tube, centrifuge at 1000 x g for 15 min to get the plasma.

Plasma sample preparation
Preparation procedure was adopted from Zhang et al. (2015) to 100 µL of plasma sample in a 1.5 mL tube, 20 µL of fucoxanthin standard solution and 680 µL methanol were added and mixed by vortexing for 1 min.After centrifugation at 25,000 x g for 10 min, the clear supernatant was filtered through a membrane (0.22-µm pore size) and injected into the HPLC system.

HPLC Condition
HPLC condition used the method as described by Noviendri et al. (2011).HPLC analysis carried out using a Shimadzu system equipped with a pump (LC-20AT), UV/Vis detector (SPD-20A).Reversed-phase HPLC (RP-HPLC) analysis was carried out using a C18 RP column, 5 µm particle size, 250 mm x 4,6 mm (YMC Co., LTD).Fucoxanthin content was determined with methanol-acetonitrile (7:3, v/v) as the mobile phase at a flow rate 0.5 ml/min.

Identification of Fucoxanthin
Figure 1A showed that Retention time of single compound of Fucoxanthin Standard at 7.905 min.Figure 1B showed that the peak with Rt 7.98 min almost similar to a peak on Fucoxanthin standard came from the ethanolic extract of T. decurrens.Figure 1C showed the chromatogram from normal mouse plasma after fed by ethanolic extracts of T. decurrens, the plasma showed fucoxanthin at the similar Rt of Fucoxanthin standard.Figure 1D showed the chromatogram of AOM-DSS mice after fed by ethanolic extract of T. decurrens, but the fucoxanthin peak cannot be found on mice model, but another major peak was observed at the Rt 2.5 min.We thought that peak was Fucoxanthinol, the major metabolite of Fucoxanthin.We then evaluate the plasma by LC-MS/MS and the result as shown in Figure 2.

Specificity and Selectivity of method
Specificity and selectivity were assessed by comparing chromatograms of six different batches of blank mouse plasma samples to corresponding spiked plasma samples.

Calibration Curve Linearity
Standard curves were established by plotting the value of chromatogram peak areas of fucoxanthin as shown in Figure 3 and Figure 4.

Studies of oral Fucoxanthin Administration on AOM-DSS model mouse.
The profiles of fucoxanthinol after a single dose of fucoxanthin in AOM-DSS mouse was shown in Figure 5.After calculation from Figure 5, we have pharmacokinetics data as shown in Table 1.

DISCUSSION
In the previous study, we evaluated the effect of fucoxanthin content brown seaweed on AOM-DSS mice.Fucoxanthin content brown seaweed could prevent the development of tumor in that model.Therefore, in the present, we studied the pharmacokinetics of fucoxanthin in the AOM-DSS mice.To this end, we evaluated the Fucoxanthin in the standard solution compare with Fucoxanthin in the ethanolic extract of T. decurrens and on the plasma of normal and AOM-DSS mice that fed by ethanolic extract of T. decurrens.We found as shown in Figure 1A-1C, the peak of Fucoxanthin that has Rt 7,903 as shown in standard, was found in those three samples.But the fucoxanthin in AOM-DSS mice was not found, probably in that model Fucoxanthin was metabolized faster to Fucoxanthinol.Fucoxanthinol has Rt faster than Fucoxanthin.Dietary fucoxanthin was mainly converted to fucoxanthinol.Fucoxanthin metabolites were accumulated in the body at a higher ratio than astaxanthin and were preferentially accumulated in the liver, heart and adipose tissues, suggesting that these tissues are targets of fucoxanthin metabolites.We then confirmed by LC-MS/MS and found that Fucoxanthin has MS 681.42, whereas Fucoxanthinol has 611,29.This result similar to Hashimoto et al. 2009.Then we evaluated Pharmacokinetics parameters of Fucoxanthinol in AOM-DSS mice and found that Tmax 4 hours, Cmax 2.15 nmol/L, T1/2 6.25 h, and AUC 10.75 nmol/L/h.

Figure 1 :
Figure 1: Chromatogram of Fucoxanthin in various conditions.Note: A. Chromatogram of Standard Fucoxanthin; B. Chromatogram of ethanolic extracts of T. Decurrens; C. Chromatogram of Normal Mouse Plasma that feed by ethanolic extracts T. Decurrens; D. Chromatogram of AOM-DSS mice plasma that feeds by ethanolic extracts T. Decurrens.

Figure 4 :
Figure 4: The linearity of Concentration of Fucoxanthin and its area.

Table 1 : Single dose oral Fucoxanthin in AOM-DSS mouse.
The profiles of fucoxanthinol after a single dose of fucoxanthin in AOM-DSS mouse.