Bioavailability ——- microlitres samples were introduced through

Bioavailability study

Instrumentation and HPLC conditions

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Chromatographic
analyses were performed on a —————HPLC system connected to a UV
detector. ——- microlitres samples were introduced through an injection
valve fitted with a 100 µl sample loop. Chromatographic separations were
achieved on a Phenomenex Luna™ C18 column (250 mm
length _ 4.6 mmi.d., 5 lmparticle size). The mobile phase was a mixture of 87.5%
v/v of a 1% v/v acetic acid solution and 12.5% v/v of acetonitrile, filtered
through a 0.22µ Millipore membrane filter. The mobile phase was pumped at a
flow rate of 1 ml/min under isocratic conditions at 25°C, with UV detection at
280 nm.

 

Dosing of rat and sample collection:
study design

A
single dose bioavailability study was designed in male Wistar rats under
fasting conditions. The oral bioavailability of the optimized SLN-EGCG
formulation and EGCG was estimated in

male
Wistar rats with an oral dose of 10 mg/kg
body weight. All experimental procedures were reviewed and approved by the institutional
animal ethical committee, VIT University  (Vellore, India). Male Wistar rats weighing
200–250 g were taken for the study (six animals per group). Blood samples were
withdrawn by retroorbital venous plexus puncture at 0, 0.5, 1, 2, 3, 4, 6, 8,
10, 12 and 24 h post-dose. About 1mL of blood samples was withdrawn in eppendorf tubes and centrifuged at 3000 rpm for
10 min.The resultant plasma solution was stored at -80°C until use. The tissues
were removed from animals on sacrifice, washed twice in ice-cold saline, and blot dried with tissue paper. For spleen,
kidney, liver and brain tissues were cut to open the cavity and washed
thoroughly. The tissues were weighed and immediately stored at ?80°C until use.
For the analysis of the samples, tissues were thawed and homogenized with
sodium buffer——-, the homogenates were centrifuged at 12,000rpm for 20
minutes and the supernatant was used for the analysis. The levels of EGCG in
the tissue were expressed as nanograms per gram of wet weight.

 

Quantification of Tea polyphenol

To
100 µl of blank plasma, 50 µl aliquots of the working standard solutions were
added to obtain EGCG plasma concentrations. 10 µl of 50 mM K2HPO4 buffer pH 6.8
was added and the samples incubated in a water bath set at 37°C for 45 min. The
addition of 10 µl of 50mM K2HPO4 was conducted in order
to mimic the addition of 5 µl of beta-glucuronidase and 5 µl of sulphatase
enzyme stock solutions, which are added to actual plasma samples (obtained from
dosed mice) to convert glucuronidated and sulphated
EGCG to parent EGCG. This conversion enables the determination of total EGCG in
plasma samples (Cai, Anavy, & Sherry Chow, 2002; Chen, Lee, Li, & Yang,
1997; Morand, Manach, Donovan, & Remesy, 2001). After the 45 min
incubation, EGCG was extracted from the plasma
through two successive additions of 500 µl of ethyl acetate. The plasma and
ethyl acetate mixture were vortex-mixed for 2 min and centrifuged at 16,000g
for 1 min before the upper layer of ethyl acetate was removed. Removal of the
ethyl acetate layer was conducted in a quantitative manner with 450 µl removed
after the first extraction and 550 µl after the second extraction. The ethyl
acetate fractions were pooled into a polyethylene tube, before being evaporated
under a gentle stream of nitrogen at ambient temperature. The residue obtained
after evaporation was reconstituted in 100 µl of mobile phase solution, and 20
µl of this solution was injected onto the HPLC column.

For
the analysis of free catechins, the mixture of ?-glucuronidase and sulfatase
was omitted from the incubation.

 

Toxicokinetic study:

Healthy male Swiss Albino rats (approximately 150–180 g body weight)
were housed in polypropylene cages placed
in a ventilated, temperature-controlled room. The standard conditions were
supplied and maintained at 20°C±2°C, 60%±10% relative humidity, and a 12-hour
light/dark cycle.

Acute toxicity study:

Swiss
albino mice (2 rats) were assigned to the following three test groups: The animals were administrated with higher doses  (1000, 2000, 3000 mg/kg) of Pure drug and
prepared nanoparticles and the respective doses were suspended in
PBS and were administered orally. Thereafter, the animals were monitored for
one days for clinical signs of toxicity or mortality.

Subchronic toxicity (OECD guideline 407;
OECD, 1995)

The
animals (3 rats) were assigned to the following four test groups: group I
(vehicle control), group II (EGCG – 10mg/kg/wt), group III (SLN-EGCG –
10mg/kg/wt). The animals were gavaged with the respective doses of  EGCG suspended in 0.5 mL of the vehicle, once
daily, for a period of 28 days. Body weights were recorded on days 0, 7, 14,
21, 27, and 28. Throughout the dosing, the animals were examined for any
clinical signs of morbidity, mortality, changes in body weight, and changes in
food consumption. At the end of the treatment, blood was collected from the
animals for clinical pathology assessment, which included analysis of various biochemical
parameters. Consequently, the animals were sacrificed by cervical dislocation and
necropsied for the gross evaluation of the various organs.

Estimation
of Serum Biochemical Parameters

Serum was
separated from the blood samples of animals and estimated for serum biochemical
parameters like glucose, protein, urea, bilirubin, lipid profile (cholesterol, triglycerides), creatinine, AST (aspartate
aminotransferase), ALT (alanine aminotransferase), ALP (alkaline phosphate) using
appropriate estimation kits.

Histopathological
Studies

Animals
were sacrifice after 28days, followed by
removal of the liver, spleen, and kidney, and placing the organs in a
fixative, i.e., 10% formalin (10% formaldehyde in water) which
stabilizes the tissues to prevent decay until processing. The processing of
tissues samples involved dehydration through a graded series of alcohols (70%,
80%, 95%, and 100%), followed by xylene and then infiltration with paraffin.
For obtaining thin sections (3–5 ?m), tissues were embedded on the edge of
paraffin blocks and were cut on a rotary microtome. These sections were deparaffinized, rehydrated with graded alcohols
(100%, 95%, 80%, and 75%), and stained with hemotoxylin/eosin
for microscopic examination.