Direct assessment by electron spin resonance spectroscopy of the antioxidant effects of French maritime pine bark extract in the maxillofacial region of hairless mice

Abstract

Flavangenol, one of extract of French maritime pine bark, is a complex mixture of bioflavonoids with oligometric proanthocyanidins as the major constituents. These constituents, catechin and procyanidin B(1), are water-soluble derivatives of flavangenol. In this study, we investigated the antioxidant effects of flavangenol on reactive oxygen species such as hydroxyl radical, superoxide anion and singlet oxygen using electron spin resonance and spin trapping. The effect of flavangenol on oxidative stress in the skin from the maxillofacial region of hairless mice was investigated using an in vivo L-band electron spin resonance imaging system. Flavangenol attenuated oxidative stress in the maxillofacial skin by acting as a reactive oxygen species scavenger, as demonstrated by in vitro and in vivo electron spin resonance imaging analysis. The absorption and metabolism of flavangenol were also examined. After oral administration of flavangenol in human and rat, most of the catechin in plasma was in the conjugated form, while 45% to 78% of procyanidin B(1) was unconjugated, indicating that non-conjugated procyanidin B(1) would be active in the circulation. The ability of flavangenol to reduce reactive oxygen species levels in the circulation of the maxillofacial region suggests that this extract may be beneficial for skin protection from exposure to ultraviolet irradiation.

Keywords: French maritime pine bark extract; antioxidants; electron spin resonance (ESR); oxidative stress; reactive oxygen species.

Fig. 1

Structure of oligometric proanthocyanidins, the major constituents of FVG.

Fig. 2

Experimental setup in the maxillofacial region of hairless mice exposed to UVB irradiation.

Fig. 3

Effects of FVG, PB₁ and catechin on HO^• generation via the Fenton reaction. A, ESR spin trapping measurement of HO^• generation by H₂O₂ (20 µM) and FeSO₄ (20 µM) in 0.1 M PBS (pH 7.2) with CYPMPO (5 mM) as the spin trap (a) in the absence of antioxidant; FVG (b; 100 µg/ml), catechin (c; 100 µg/ml) and PB₁ (d; 100 µg/ml), respectively. B, the effects of FVG, catechin and PB₁ on HO^• generation from the Fenton reaction. The signal intensity of the seventh peak of the spectrum was normalized as the relative height against the signal intensity of control. Results are expressed as percent of control and are represented as mean ± SEM. * indicates a significant difference (p<0.05) versus the corresponding control value.

Fig. 4

Effects of FVG, PB₁ and catechin on O₂^•− generated by xanthine-XO system. A, ESR spin trapping measurement of O₂^•− generation by xanthine (1 mM) and XO (0.5 U/ml) in distilled water with CYPMPO (10 mM) as the spin trap (a) in the absence of antioxidant; FVG (b; 100 µg/ml), catechin (c; 100 µg/ml), and PB₁ (d; 100 µg/ml), respectively. B, the effects of FVG, catechin, and PB₁ on O₂^•− generated by xanthine-XO. The signal intensity of the seventh peak of the spectrum was normalized as the relative height against the signal intensity of a control. Results are expressed as percent of control and are represented as mean ± SEM. * indicates a significant difference (p<0.05) versus the corresponding control value.

Fig. 5

Effects of FVG, PB₁ and catechin on ¹O₂ generated by illuminated rose bengal. A, ESR spin trapping measurement of ¹O₂ produced by the photochemical reaction of rose bengal illuminated for 2 min (18,000 lux) with 4-oxo-TEMP (40 mM) as the spin trap (a) in the absence of antioxidant; control with FVG (b; 100 µg/ml), catechin (c; 100 µg/ml), and PB₁ (d; 100 µg/ml) pre-treatment, respectively. B, the effects of FVG, catechin, and PB₁ on ¹O₂ generated by illuminated rose bengal. The signal intensity of the second peak of the spectrum was normalized as the relative height against the signal intensity of a control. Results are expressed as percent of control and are represented as mean ± SEM. * indicates a significant difference (p<0.05) versus the corresponding control value.

Fig. 6

2D ESR projection images (z-x plane) of C-PROXYL distributions in the maxillofacial region of hairless mice. The ESR was measured at 1, 3, 5 and 7 min after i.v. treatment with C-PROXYL. A; control (non-UVB irradiation); B; UVB irradiation; C; 200 mg/kg or D; 600 mg/kg FVG pretreatment for 4 days with UV irradiation, respectively. As indicated by the attached color scale (32 colors; white and 100 being the maximum ESR signal), ESR images were reproduced in 32 colors and signals lower than 10% of the maximal signal intensity detected in all slices were regarded as noise. Experimental conditions were as described under ”Methods”.

Fig. 7

Effects of FVG on UVB-induced oxidative stress in the maxillofacial region of hairless mice. Mice were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). L-band ESR was used to determine the signal decay of C-PROXYL in the maxillofacial skin of hairless mice. A, Decay rate constants of (K₁ and K₂) of C-PROXYL after UVB irradiation (open circle); control (non-UVB irradiation, closed circle). B, the columns represent: K₁ of C-PROXYL after UVB irradiation, control (non-UVB irradiation); UVB irradiation with FVG (200 mg/kg or 600 mg/kg) pretreatment for 4 days respectively. The results are expressed as mean ± SD of hairless mice (n = 7). * indicates a significant difference (p<0.05) versus the corresponding value in control group.

Fig. 8

Alteration in the concentration of PB₁ and catechin in rat serum and human plasma following oral administration of FVG. A, Changes in the plasma concentration of PB₁ and catechin in rats. FVG was given to rats (n = 5) by oral administration (600 mg/kg). Mean concentration of total (closed rhombus) and non-glucuronate/sulfate–conjugated (open rhombus) PB₁ (a) and catechin (b) in serum. B, Changes in the plasma concentration of PB₁ and catechin in human subjects. FVG (5 g) with 100 ml water was given to subjects (n = 6–7). Mean of total (closed rhombus) and non-glucuronate/sulfate–conjugated (open rhombus) PB₁ (a) and catechin (b) in plasma.