Potent anti-glioblastoma effect of 4-methylthio-3-butenyl isothiocyanate from Raphanus sativus and antioxidant activity

Figures & data

Figure 1. Total phenols and flavonoid content of R. sativus aqueous and methanol extracts. Values are expressed as means ± SD. Radish aqueous extract is richer on phenols and flavonoids than methanol extract. All experiments were made in triplicates.

Figure 2. HPLC profiles of the obtained compounds. Five phenolic compounds were identified in radish aqueous extract.

Table 1. Retention time (min) and concentration of phenolics identified in R. sativus bulb aqueous extract bu HPLC DAD.

ID (mg/g)	RT	m/z	compound	concentration ± SD
1	24.63	197	syringic acid	9.15 ± 2.03
2	19.34	163	protocatchuic acid	6.23 ± 1.64
3	08.02	169	gallic acid	4.62 ± 0.98
4	27.42	163	p-coumaric acid	1.24 ± 0.54
5	99.87	343	cirsilineol	0.51 ± 0.06

All experiments were made in triplicates.

Values were expressed as means ± SD

Figure 3. GC-MS chromatogram of R. sativus methanol extract.

Figure 4. Chemical structure of 4-methylthio-3-butenyl isothiocyanate (raphasatin).

Table 2. IC₅₀ of DPPH, ABTS, hydroxyl radical scavenging activities, ferric-reducing power and iron chelation of R. sativus bulb extracts.

	DPPH (µg/mL)	ABTS (µg/mL)	OH radical (µg/mL)	FRAP (mM FeSO4/ g dw)	Iron (II) (mM/g dw)
aqueous extract	121.03 ± 5.71	31.46 ± 4.72	10.65 ± 1.88	91.27 ± 5.21	382.74 ± 10.72
methanol extract	34.87 ± 2.89	06.28 ± 1.04	1.84 ± 0.19	35.29 ± 3.93	56.16 ± 8.55
BHT	21.05 ± 0.40
TROLOX	15.52 ± 0.23	68.12 ± 0.15

All experiments were made in triplicates.

Values were expressed as means ± SD

Figure 5. Effect of R. sativus extracts on U-87 MG cells viability.. U-87 MG cells were incubated for 24, 48 and 72 h with different concentrations of R. sativus extracts (A) Methanol extract; (B) Aqueous extract. Doxorubicin (Dox) was used as positive control and water as negative one. All experiments were made in triplicates. Values were expressed as means ± SD, *Indicates significant differences (p < .05).

Figure 6. Radish methanol extract effect on various ECM in cell adhesion assays. Glioblastoma cells were preincubated with 10, 20, 50 and 100 µg/mL in radish methanol extract for 30 min at room temperature and then added to wells coated with 10 µg/mL fibronectin (Fn), 10 µg/mL fibrinogen (Fg) or 50 µg/mL (PLL) and allowed to attach for 1 h at 37°C. Values were expressed as means ± SD, *Indicates significant differences (p < .05).

Figure 7. Cell adhesion assay of (A) 4-methylthio-3-butenyl isothiocyanate and (B) positive control doxorubicin (Dox) on different ECM proteins. 4-methylthio-3-butenyl isothiocyanate and posititive control doxorubicin inhibited with the same manner the adhesion of U-87 MG to fibronectin (Fn), fibrinogen (Fg) and poly-L-Lysine (PLL).

Table 3. IC₅₀ (µg/mL) of radish extracts, raphasatin, and positive control doxorubicin against the tested ECM in dis-adhesion test.

	Fn	Fg	PLL
methanol extract	20.46 ± 2.52	12.23 ± 1.41	16.21 ± 1.92
aqueous extract	NA	NA	NA
raphasatin	0.63 ± 0.09	1.27 ± 0.18	0.95 ± 0.12
doxorubicin	0.42 ± 0.06	0.98 ± 0.015	0.69 ± 0.11

Fn (fibronectin), Fg (fibrinogene), PLL (poly-L-lysine), NA (not active)

All experiments were made in triplicates.

Values were expressed as means ± SD

Figure 8. 4-methylthio-3-butenyl isothiocyanate and doxorubicin (Dox) effects on U-87 MG cells proliferation. After 4 days of incubation with 0.5 µg/mL of raphasatin or doxorubicin, wells were washed with PBS and the cells were fixed with 1% glutaraldehyde, stained with 0.1% crystal violet and quantified by absorbance at 560 nm.