Bioassay guided fractionation and identification of active anti-inflammatory constituent from Delonix elata flowers using RAW 264.7 cells

Figures & data

Figure 1. Scheme for the fractionation of Delonix elata methanol extract.

Figure 2. Effect of Delonix elata flower methanol extract on LPS-induced RAW 264.7 cell line. (a) NO; (b) TNF-α and IL-1β; levels of LPS-induced RAW 264.7 cell lines. Values are mean ± SEM for triplicate independent experiments; values carrying the same alphabet did not vary significantly from each other (Tukey’s HSD; p ≤ 0.05).

Table 1. Effect of toluene (TO), diethylether (ET), ethyl acetate (EA), and methanol (MeOH) soluble fractions of Delonix elata flower methanol extract on LPS-induced RAW 264.7 cell line.

Groups	NO (µM)	TNF-α (pg/ml)	IL-1 β (pg/ml)
Normal cells	0.219 ± 0.09^a	89.27 ± 5.67^a	82.68 ± 4.36^a
LPS induced	0.774 ± 0.03^b	430.84 ± 20.21^b	756.68 ± 38.75^b
LPS + TO (10 µg/ml)	0.452 ± 0.01^d	692.71 ± 38.33^b	680.52 ± 64.23^b
LPS + TO (25 µg/ml)	0.388 ± 0.02^d,e	735.00 ± 13.48^b	692.72 ± 31.52^b
LPS + TO (50 µg/ml)	0.311 ± 0.02^a,e	681.24 ± 27.53^b	681.21 ± 27.52^b
LPS + ET (10 µg/ml)	0.450 ± 0.04^d	290.51 ± 22.69^c,d	466.36 ± 30.97^d,e
LPS + ET (25 µg/ml)	0.367 ± 0.08 ^d,e	227.64 ± 16.85^d.e	346.14 ± 14.45^e,f
LPS + ET (50 µg/ml)	0.303 ± 0.01^a,e	144.31 ± 10.45^a,d	292.96 ± 17.57^f
LPS + EA (10 µg/ml)	0.648 ± 0.03^b,c	648.65 ± 35.43^e,f	664.36 ± 56.45^b,c
LPS + EA (25 µg/ml)	0.688 ± 0.09^b,c	688.87 ± 8.92^e,f	692.83 ± 38.25^b,c
LPS + EA (50 µg/ml)	0.638 ± 0.03^c	639.04 ± 33.12^e,f	687.41 ± 40.59^b,c
LPS + MeOH (10 µg/ml)	0.653 ± 0.03^b,c	654.21 ± 33.45^e,f	649.89 ± 54.00^b,c
LPS + MeOH (25 µg/ml)	0.736 ± 0.03^b,c	737.18 ± 36.84^f	687.15 ± 40.65^b,c
LPS + MeOH (50 µg/ml)	0.609 ± 0.03^c	609.58 ± 37.75^e	679.05 ± 27.26^b,c

Values are mean ± SEM for triplicate individual experiments; values carrying the same letter did not vary significantly from each other (Tukey’s HSD; p ≤ 0.05).

Table 2. Effect of sub-fractions from the diethylether soluble fractions of Delonix elata flower methanol extract on LPS-induced RAW 264.7 cell line.

Groups	NO (µM)	TNF-α (pg/ml)	IL-1β (pg/ml)
Normal cells	0.227 ± 0.01^a	97.35 ± 6.35^a	85.77 ± 2.41^a
LPS induced	0.776 ± 0.03^b	464.45 ± 23.90^b	752.30 ± 39.15^b
LPS + SF-1 (10 µg/ml)	0.672 ± 0.05^b	468.45 ± 26.43^b	703.24 ± 40.23^b
LPS + SF-1 (25 µg/ml)	0.653 ± 0.03^b,c	492.55 ± 10.92^b	824.14 ± 27.94^b
LPS + SF-1 (50 µg/ml)	0.670 ± 0.03^b	493.45 ± 43.47^b	741.16 ± 34.20^b
LPS + SF-2 (10 µg/ml)	0.644 ± 0.03^b,c	472.95 ± 43.78^b	724.43 ± 57.86^b
LPS + SF-2 (25 µg/ml)	0.634 ± 0.05^b,c	454.76 ± 38.71^b	762.26 ± 48.12^b
LPS + SF-2 (50 µg/ml)	0.636 ± 0.04^b	499.23 ± 20.32^b	802.08 ± 40.42^b
LPS + SF-3 (10 µg/ml)	0.683± 0.01^b	474.54 ± 44.56^b	666.49 ± 30.41^b
LPS + SF-3 (25 µg/ml)	0.689 ± 0.09^b	473.64 ± 38.44^b	691.44 ± 28.21^b
LPS + SF-3 (50 µg/ml)	0.644 ± 0.03^b,c	484.29 ± 27.97^b	762.51 ± 47.26^b
LPS + SF-4 (10 µg/ml)	0.488 ± 0.03^c,d	286.29 ± 24.31^c	495.12 ± 26.89^c
LPS + SF-4 (25 µg/ml)	0.387 ± 0.02^a,d	210.39 ± 32.97^c,a	366.08 ± 24.84^c,d
LPS + SF-4 (50 µg/ml)	0.323 ± 0.01^a,d	126.42 ± 11.43^a	318.08 ± 18.56^d
LPS + SF-5 (10 µg/ml)	0.679 ± 0.01^b,c	470.50 ± 33.05^b	779.99 ± 35.80^b
LPS + SF-5 (25 µg/ml)	0.637 ± 0.05^b	474.17 ± 47.42^b	733.57 ± 35.81^b
LPS + SF-5 (50 µg/ml)	0.674 ± 0.06^b,c	488.83 ± 18.08^b	652.17 ± 30.05^b
LPS + SF-6 (10 µg/ml)	0.664 ± 0.03^b	531.88 ± 42.21^b	662.65 ± 24.22^b
LPS + SF-6 (25 µg/ml)	0.723 ± 0.01^b,c	543.05 ± 41.65^b	749.26 ± 39.88^b
LPS + SF-6 (50 µg/ml)	0.733 ± 0.01^b,c	431.61 ± 18.78^b	781.78 ± 49.21^b

Values are mean ± SEM for triplicate individual experiments; values carrying the same alphabet did not vary significantly from each other (Tukey’s HSD; p ≤ 0.05).

Table 3. Effect of sub-fractions from SF-4 of the diethylether soluble fractions of Delonix elata flower methanol extract on LPS-induced RAW 264.7 cell line.

Groups	NO (µM)	TNF-α (pg/ml)	IL-1β (pg/ml)
Normal cells	0.265 ± 0.09^a	97.80 ± 4.73^a	83.45 ± 4.09^a
LPS induced	0.766 ± 0.03^b	474.50 ± 20.11^b	715.49 ± 51.98^b
LPS + SF-1a (10 µg/ml)	0.688 ± 0.03^b	470.12 ± 27.17^b	663.28 ± 38.77^b
LPS + SF-1a (25 µg/ml)	0.597 ± 0.03^b	448.04 ± 35.29^b,c	736.01 ± 54.46^b
LPS + SF-1a (50 µg/ml)	0.709 ± 0.04^b	493.59 ± 14.05^b	712.61 ± 52.21^b
LPS + SF-2a (10 µg/ml)	0.635 ± 0.03^b	361.77 ± 25.16^c	389.57 ± 18.26^c
LPS + SF-2a (25 µg/ml)	0.412 ± 0.03^a	233.49 ± 19.31^d	342.99 ± 21.22^c
LPS + SF-2a (50 µg/ml)	0.313 ± 0.02^a	120.15 ± 14.88^a	334.00 ± 24.99^c
LPS + SF-3a (10 µg/ml)	0.672 ± 0.05^b	709.18 ± 30.82^e	769.18 ± 27.44^b
LPS + SF-3a (25 µg/ml)	0.692 ± 0.03^b	772.53 ± 35.46^e	722.07 ± 14.49^b
LPS + SF-3a (50 µg/ml)	0.646 ± 0.03^b	780.93 ± 20.38^e	681.65 ± 41.51^b

Values are mean ± SEM for triplicate individual experiments; values carrying the same alphabet did not vary significantly from each other (Tukey’s HSD; p ≤ 0.05).

Figure 3. Effect of active compound (hesperidin) isolated from Delonix elata flowers on LPS-induced RAW 264.7 cell line. (a) NO; (b) TNF-α and IL-1β; levels of LPS-induced RAW 264.7 cell lines. Values are mean ± SEM for triplicate individual experiments; values carrying the same alphabet did not vary significantly from each other (Tukey’s HSD; p ≤ 0.05).

Figure 5. The structure of hesperidin.