Ined by interpolation form the non-linear regression of plot of percentage of inhibition against the concentration of extracts, which is defined as the amount of extract needed to Acadesine site scavenge 50 of DPPH radicals.Hydrogen peroxide scavenging assayOil red O staining was used to monitor lipid accumulation in differentiated adipocytes. On day 8, cells were stained with Oil red O. The cells were fixed with 10 formalin for 30 min. After that the formalin was removed and washed with 60 isopropanol. Then the lipid droplets were stained for at least 30 min at room temperature in a PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28128382 freshly diluted Oil Red O solution [0.5 Oil Red O solution in 60:40 (v/v) isopropanol:water]. After Oil Red O stain, cells were photographed using a phase-contrast microscope (Olympus CK, Tokyo, Japan) in combination of digital camera at 100 ?magnifications. Finally, the dye retained in the 3 T3-L1 cells was eluted with isopropanol and quantified by measuring the absorbance at 510 nm.In vitro anti-inflammatory activityThe ability of different extracts to scavenge the hydroxyl SKF-96365 (hydrochloride) web radicals (OH.) was measured according to the method of Muller [21].RAW 264.7 cells were plated in a 24 well plate at a density of 106 cells/mL, 500 L in each well. Then after 24 hr incubation, the medium was changed and the samples (extracts of CA) were added. After 1 hr. of sample treatment LPS (final concentration: 1 g/ml) was added to both extracts treated as well as untreated wells. Amount of nitrite produced were measured using Griess reagent (1 sulfanilamide and 0.1 napthylethylenediamine dihhydorchloride in 2.5 phosphoric acid). 100 L of cell culture medium was mixed with 100 L of Griess reagent.Lamichhane et al. BMC Complementary and Alternative Medicine 2014, 14:342 http://www.biomedcentral.com/1472-6882/14/Page 4 ofSubsequently, the mixture was incubated at room temperature for 10 min and the absorbance at 540 nm was measured in a microplate reader. Fresh culture media was used as a blank.In vivo assay Preparation of samplesTable 2 DPPH radical assay of crude methanol extract and other fractions of CASample MeOH CHCl3 EtOAc BuOH Water Ascorbic acid IC50(g/ml) 19.55 ?2.83bc 38.0 ?6.03d 16.33 ?0.48b 24.66 ?1.35bc 27.80 ?3.52c 5.36 ?0.27aCrude methanol extract and phenolic fraction (mixture of butanol and ethyl acetate fraction in equal ratio of weight) were taken to prepare samples for the in vivo assay. The in vitro study evidenced the comparable activity for the butanol and ethyl acetate fraction. In addition, the TLC patterns for both fractions were similar. So we tried to see the in vivo effect of the combination of the two higher phenolic compound containing extracts. Required amount of extracts were weighed and suspended in PBS and homogenized using a homogenizer. The dose fed orally to each rat was 200 mg/kg per day.Animals and experiment designEach value is average of three analysis ?standard deviation. Values with different letters are significantly different (p < 0.05) based on one-way ANOVA post-hoc Ducan Multiple Range tests.Biochemical analysis on blood and evaluation of organ weightFour-week-old, male SD (Sprague Dawley) rats were purchased from Damool Science (Daejeon, Korea). The rats were maintained in accordance with the guidelines for the care and use of laboratory animals of Wonkwang University. All experiments complied with ethical standards and were approved by the Animal Ethics Committee at Wonkwang University (Iksan, Korea). They were housed under a 12-h light/1.Ined by interpolation form the non-linear regression of plot of percentage of inhibition against the concentration of extracts, which is defined as the amount of extract needed to scavenge 50 of DPPH radicals.Hydrogen peroxide scavenging assayOil red O staining was used to monitor lipid accumulation in differentiated adipocytes. On day 8, cells were stained with Oil red O. The cells were fixed with 10 formalin for 30 min. After that the formalin was removed and washed with 60 isopropanol. Then the lipid droplets were stained for at least 30 min at room temperature in a PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28128382 freshly diluted Oil Red O solution [0.5 Oil Red O solution in 60:40 (v/v) isopropanol:water]. After Oil Red O stain, cells were photographed using a phase-contrast microscope (Olympus CK, Tokyo, Japan) in combination of digital camera at 100 ?magnifications. Finally, the dye retained in the 3 T3-L1 cells was eluted with isopropanol and quantified by measuring the absorbance at 510 nm.In vitro anti-inflammatory activityThe ability of different extracts to scavenge the hydroxyl radicals (OH.) was measured according to the method of Muller [21].RAW 264.7 cells were plated in a 24 well plate at a density of 106 cells/mL, 500 L in each well. Then after 24 hr incubation, the medium was changed and the samples (extracts of CA) were added. After 1 hr. of sample treatment LPS (final concentration: 1 g/ml) was added to both extracts treated as well as untreated wells. Amount of nitrite produced were measured using Griess reagent (1 sulfanilamide and 0.1 napthylethylenediamine dihhydorchloride in 2.5 phosphoric acid). 100 L of cell culture medium was mixed with 100 L of Griess reagent.Lamichhane et al. BMC Complementary and Alternative Medicine 2014, 14:342 http://www.biomedcentral.com/1472-6882/14/Page 4 ofSubsequently, the mixture was incubated at room temperature for 10 min and the absorbance at 540 nm was measured in a microplate reader. Fresh culture media was used as a blank.In vivo assay Preparation of samplesTable 2 DPPH radical assay of crude methanol extract and other fractions of CASample MeOH CHCl3 EtOAc BuOH Water Ascorbic acid IC50(g/ml) 19.55 ?2.83bc 38.0 ?6.03d 16.33 ?0.48b 24.66 ?1.35bc 27.80 ?3.52c 5.36 ?0.27aCrude methanol extract and phenolic fraction (mixture of butanol and ethyl acetate fraction in equal ratio of weight) were taken to prepare samples for the in vivo assay. The in vitro study evidenced the comparable activity for the butanol and ethyl acetate fraction. In addition, the TLC patterns for both fractions were similar. So we tried to see the in vivo effect of the combination of the two higher phenolic compound containing extracts. Required amount of extracts were weighed and suspended in PBS and homogenized using a homogenizer. The dose fed orally to each rat was 200 mg/kg per day.Animals and experiment designEach value is average of three analysis ?standard deviation. Values with different letters are significantly different (p < 0.05) based on one-way ANOVA post-hoc Ducan Multiple Range tests.Biochemical analysis on blood and evaluation of organ weightFour-week-old, male SD (Sprague Dawley) rats were purchased from Damool Science (Daejeon, Korea). The rats were maintained in accordance with the guidelines for the care and use of laboratory animals of Wonkwang University. All experiments complied with ethical standards and were approved by the Animal Ethics Committee at Wonkwang University (Iksan, Korea). They were housed under a 12-h light/1.