Fig 1.
Diagrammatic representation of various treatment groups made for in-vivo studies.
Fig 2.
Comparative effect of EGCG and inulin on the growth of L. plantarum.
Inset-Enhancing effect of various doses of EGCG on the growth of L. plantarum. Values are expressed as mean ± standard deviation of three individual values. $, 50mg dose displays most significant effect (P>0.05), *, P <0.05 versus log10 CFU of L. plantarum after 24 hour in the absence of EGCG and inulin (control); #, P <0.05 versus log10 CFU of L. plantarum after 24 hour in the presence of inulin.
Fig 3.
Characterization of formulated beads on the basis of entrapment efficiency, SEM, stability of EGCG in alkaline pH, shelf life of probiotic for 6 months.
A) Scanning electron micrographs (a) Probiotic-EGCG beads at 50X (b) Probiotic-EGCG beads at 1000X. B) Drug entrapment efficiency of probiotic and EGCG co-encapsulated in alginate beads (n = 6). C) In-vitro release of entrapped probiotic and EGCG from probiotic-EGCG beads upto 6 hours. D) Stability of EGCG in probiotic- EGCG alginate beads as compared to free EGCG in alkaline pH after 6 hours.(n = 6) * represents p<0.05 versus free EGCG. E) Shelf-life of probiotic for 6 months. Values are represented as ± SD. * represents p<0.05 versus probiotic at the start of 6 months.
Fig 4.
Representative photomicrographs of hematoxylin-eosin stained rat liver sections.
(A) Normal rat liver (100x); B,C) Liver section from rat administered 10-14g/kg body weight of 35% alcohol orally for 12 weeks showing vacuolar degeneration, microvesicular fatty change, focal collection of lymphocytes and vascular congestion (200X, 400X respectively); D) Photomicrograph of alcohol administered co-supplemented with free probiotic-EGCG group showing normal histology (100X); E) Photomicrograph of alcohol administered co-supplemented with encapsulated probiotic—EGCG group showing normal histology (100X); F) Photomicrograph of Free probiotic-EGCG per se group showing normal histology (100X); G) Encapsulated probiotic +EGCG per se group showing normal histology (100X).
Fig 5.
Effect of probiotic- EGCG combination (free and encapsulated) on PAO levels, *p<0.05 vs. control, #p<0.05 vs. alcohol group, $p<0.05 vs. alcohol + free probiotic-EGCG group.
Fig 6.
HPTLC Chromatogram (inset) and chromatograph showing the differential sugars i.e. lactulose and mannitol released as a marker of intestinal permeability.
Inset- A,G–lactulose standard; B,H—mannitol standard; C- Alcohol group showing the presence of both the sugars in the serum due to increased permeability; D- Alcohol + Encapsulated probiotic-EGCG group showing only mannitol, no disruption of gut permeability; E- Alcohol fed; F- Alcohol + Free probiotic-EGCG group showing only mannitol, confirming intact gut permeability
Fig 7.
Effect of probiotic- EGCG combination (free and encapsulated) on A) NF- B/p50, B) TNF-α levels, and C) IL-12/p40 levels in alcohol administered rats with and without treatment. Values are expressed as mean± S.D. of eight different observations. *p<0.05 vs. control, #p<0.05 vs. alcohol group, $p<0.05 vs. alc+ free pro-EGCG group.
Fig 8.
RT-PCR analysis of liver TLR4, CD14, MD2, MyD88 COX-2 mRNA expressions.
Alc- alcohol group; Alc+ FPE- Alcohol+ free probiotic- EGCG group, Alc + EPE- Alcohol+ co-encapsulated probiotic-EGCG group. *p<0.05 vs. control.
Fig 9.
Micronuclei analysis in the hepatocytes of alcohol-fed rats.
Effect of co-enapsulated Probiotic-EGCG on the extent of micronuclei formation in hepatocytes of alcohol administered rats. Values are expressed as percentage of micronucleated cells. *p<0.001 vs. control, #p<0.01 vs. control, #$p<0.05 vs. control; Inset- Dividing cells showing binuclei (BN) and micronuclei (MN) in hepatocytes of alcohol-fed rats.