Enhanced Antioxidant Activity under Biomimetic Settings of Ascorbic Acid Included in Halloysite Nanotubes

Abstract

Antioxidant activity of native vitamin C (ascorbic acid, AH₂) is hampered by instability in solution. Selective loading of AH₂ into the inner lumen of natural halloysite nanotubes (HNT) yields a composite nanoantioxidant (HNT/AH₂), which was characterized and investigated for its reactivity with the persistent 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radical and with transient peroxyl radicals in the inhibited autoxidation of organic substrates, both in organic solution (acetonitrile) and in buffered (pH 7.4) water in comparison with native AH₂. HNT/AH₂ showed excellent antioxidant performance being more effective than native ascorbic acid by 131% in acetonitrile and 290% (three-fold) in aqueous solution, under identical settings. Reaction with peroxyl radicals has a rate constant of 1.4 × 10⁶ M^-1 s^-1 and 5.1 × 10⁴ M^-1 s^-1, respectively, in buffered water (pH 7.4) and acetonitrile, at 30 °C. Results offer physical understanding of the factors governing HNT/AH₂ reactivity. Improved performance of HNT/AH₂ is unprecedented among forms of stabilized ascorbic acid and its relevance is discussed on kinetic grounds.

Keywords: antioxidant; ascorbic acid; biomimetic; halloysite; nanoantioxidant; nanotube; peroxyl radicals; rate constant; stability; vitamin C.

Scheme 1

Preparation of ascorbic acid loaded halloysite nanotubes (HNT/AH₂).

Figure 1

(A,B) TGA (solid line) and derivative DTG (broken line) thermograms of: (A) ascorbic acid (AH₂) under air atmosphere at heating rate of 10 °C/min; and (B) HNT (▬▬) and HNT/AH₂ (▬▬). The temperature profile applied during the measurement is reported. (C,D) DLS intensity distribution ofHNT (solid line) and HNT/AH₂ (broken line) (C) in acetonitrile; and (D) in water solution (0.5 mg/mL).

Figure 2

Transmission electronic microscopy (TEM) images of (a,c) pristine HNT; (b,d) of HNT/AH₂ showing the nanotubular structure. Scale bars: (a,b) 500 nm; and (c,d) 100 nm.

Scheme 2

Aerobic degradation of ascorbic acid (AH₂) in aqueous solution.

Figure 3

Stability of 100 µM ascorbic acid (AH₂) in acetonitrile (red), water buffer pH = 7.4 (green) and methanol (black) at 298 K, in the presence of atmospheric oxygen. Insert shows the full time-course of AH₂ decay expressed as remaining % of the starting concentration, while main graph represents the scale expansion for the first 100 min (kinetic analysis is shown in Figures S4–S6).

Figure 4

Oxygen consumption during the autoxidation of: (black) cumene (1.8 M) initiated by AIBN (0.05 M) at 30 °C in dry acetonitrile, or (red) with addition of 1% wt water (A,C), and (blue) during the autoxidation of THF (3.1 M) initiated by AAPH (25 mM) in phosphate buffer (0.1 M pH = 7.4) at 30 °C (B,D) without inhibitors (dash line); or in the presence of AH₂ 2.5 × 10⁻⁵ M (solid line, A); or of AH₂ 2.2 × 10⁻⁵ M (solid line, B); or in the presence of HNT/AH₂ 0.18 mg/mL (corresponding to AH₂ 2.5 × 10⁻⁵ M, solid line, C); or in the presence of HNT/AH₂ 0.16 mg/mL (corresponding to AH₂ 2.2 × 10⁻⁵ M, solid line, D).

Figure 5

Variation of stoichiometric factor n: (A) during the autoxidation of Cumene (1.8 M) initiated by AIBN (0.05 M) in acetonitrile at 30° C (red), and upon addition of: 1% water (black), HNTs 0.25 mg/mL (green), 1% water and HNTs 0.25 mg/mL (pink) inhibited by variable amount of AH₂ (7.0 × 10⁻⁷ M to 6.4 × 10⁻⁵ M, red, black, green, and pink), or inhibited by HNT/AH₂ (blue); (B) during the autoxidation of THF (3.1 M) initiated by AAPH (25 mM) in phosphate buffer 0.1 M pH = 7.4 at 30 °C (black) and upon addition of HNTs 0.25 mg/mL (green), inhibited by variable amount of antioxidant AH₂ (1.4 × 10⁻⁶ M to 8.1 × 10⁻⁵ M, black and green) or inhibited by HNT/AH₂ (blue).