Effects of Sanionia uncinata extracts in protecting against and inducing DNA cleavage by reactive oxygen species

Figures & data

Figure 1. Agarose gel electrophoresis of plasmid DNA (pUC18, 394 µg/ml) incubated for 1 hour at 37^oC with aqueous (A) or hydroalcoholic (B) extracts of S. uncinata (lanes 2–4). Lane 1 represents the negative control. Stannous chloride at 200 µg/ml was used as a positive control (lane 5). The direction of electrophoresis is top to bottom. Supercoiled (I) and relaxed circular (II) plasmid DNA forms are indicated. The gels are representative of experiments with similar results.

Figure 2. Agarose gel electrophoresis of plasmid DNA (pUC18, 394 μg/ml) incubated for 1 hour at 37^oC with aqueous and hydroalcoholic extracts of S. uncinata at 900 µg/ml in the presence and absence of ROS generation systems: (A) Pyrogalol 250 µM for O₂^•−, (B) Fe²⁺/H₂O₂ (200 µM, 50 mM) for ^•OH, via Fenton reaction, (C) Cu²⁺/H₂O₂ (200 µM, 50 mM) for ^•OH and O₂^−•, via Fenton-like reaction, (D) SnCl₂ (200 µg/ml) for ^•OH and O₂^−•, via in situ Fenton-like reaction. ROS inhibitors: DMSO 5%, except for SnCl₂ in which EDTA 25 mM was used. The direction of electrophoresis is top to bottom. Supercoiled (I), relaxed circular (II), and linear (III) forms are indicated. The gels are representative of experiments with similar results.

Table 1. Intensity (%, mean relative to total band density and standard deviation) of form II DNA (plasmid pUC18) treated with the aqueous (AE) and hydroalcoholic (HE) extracts of S. uncinata in the absence (–) or presence (+) of ROS generation systems and without (wo) or with (w) ROS inhibitors

Lane^a	Test		ROS generation systems (Figure)^b
			Pyrogallol	Fe^2+/H2O2	Cu^2+/H2O2	SnCl2
			(Fig. 2A)	(2B)	(2C)	(2D)
1	—	– (wo)	0 ± 0	0.6 ± 0.1	1.7 ± 0.5	5.0 ± 0.9
2	—	+ (wo)	44.8 ± 2.3^d*	86.1 ± 4.1^c,d	43.5 ± 5.7^d*	55.7 ± 2.0^d*
3	AE	+ (wo)	5.0 ± 2.9^e*	71.1 ± 4.8^d	84.6 ± 7.2^d,e*	90.2 ± 0.7^d
4	HE	+ (wo)	2.7 ± 1.2^e*	70.4 ± 4.1^d	97.8 ± 3.1^d,e*	87.1 ± 6.0^d
5	—	– (w)	2.0 ± 1.8^e*	1.3 ± 0.3^e	2.6 ± 3.7^e*	7.0 ± 3.1
6	—	+ (w)	2.3 ± 1.7^e*	4.1 ± 0.3^e	25.6 ± 4.5	22.7 ± 5.1
7	AE	+ (w)	1.8 ± 1.2^e*	0.6 ± 0.4^e,f	21.1 ± 14.3^f*	36.6 ± 16.2^f*
8	HE	+ (w)	0.6 ± 0.8^e*	0.3 ± 0.4^e,g	74.7 ± 9.8^d,g*	28.3 ± 3.0^g*

^aLane showed in the gels in Fig. 2.

^bDetails of ROS generation systems, incubation conditions, and ROS inhibitors are described in Fig. 2 and in the text.

^cForm III was observed only under these conditions and at a relative intensity of 13.9 ± 4.1. Statistically significant differences (z test for proportions, P < 0.01 or *P < 0.05) relative to

^dnegative control (lane 1),

^elane 2,

^flane 3,

^glane 4.

All experiments were performed in triplicate.

Figure 3. Agarose gel electrophoresis of plasmid DNA (pUC18, 394 µg/ml) incubated for 1 hour at 37^oC with aqueous (A) and hydroalcoholic (B) extracts of S. uncinata at 900 µg/ml in the presence of SnCl₂ (200 µg/ml) with ROS scavengers or antioxidants: l-histidine 50 mM (his), DMSO 5%, NaN₃ 100 mM, BHT 20 mM, and sodium benzoate 2 M (benz). The direction of electrophoresis is from top to bottom. Supercoiled (I) and relaxed circular (II) forms are indicated. The gels are representative of experiments with similar results.

Table 2. Intensity (%, mean relative to total band density and standard deviation) of form II DNA (plasmid pUC18) treated with the aqueous (AE) and hydroalcoholic (HE) extracts of S. uncinata in the absence (wo) or presence (w) of SnCl₂ and without (–) or with (+) ROS scavengers and antioxidants

Lane^a	SnCl2	ROS scavengers or antioxidants^b		Extract
				AE	HE
1	wo^c	—	wo	0.0 ± 0.0	0.0 ± 0.0
2	w	—	wo	54.1 ± 4.2^d*,f	76.7 ± 9.1^d
3	w	—	w	99.2 ± 1.1^d	99.2 ± 1.2^d,e
4	w	Histidine	w	11.7 ± 3.3^f	34.2 ± 0.5^f
5	w	DMSO	w	0.3 ± 0.0^f	0.2 ± 0.2^e,f
6	w	NaN3	w	8.0 ± 7.1^f	0.0 ± 0.0^e,f
7	w	BHT	w	1.2 ± 1.7^f	0.0 ± 0.0^e,f
8	w	Benz	w	15.0 ± 9.4^f	8.0 ± 3.3^e,f

^aLane showed in the gels in Fig. 3. No form III was observed.

^bDetails of ROS scavengers or antioxidants, incubation conditions, and ROS inductor are described in Fig. 3 and text.

^cAbsence (wo) or presence (w) of SnCl₂ or extracts. Statistically significant differences (z test for proportions, P < 0.01 or *P < 0.05) relative to

^dnegative control (lane 1),

^elane 2, and

^flane 3. All experiments were performed in triplicate.