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Abstract
C60 is a potent antioxidant that has been reported to substantially extend the lifespan of rodents when formulated in olive oil (C60-OO) or extra virgin olive oil (C60-EVOO). Despite there being no regulated form of C60-OO, people have begun obtaining it from online sources and dosing it to themselves or their pets, presumably with the assumption of safety and efficacy. In this study, we obtain C60-OO from a sample of online vendors, and find marked discrepancies in appearance, impurity profile, concentration, and activity relative to pristine C60-OO formulated in-house. We additionally find that pristine C60-OO causes no acute toxicity in a rodent model but does form toxic species that can cause significant morbidity and mortality in mice in under 2 weeks when exposed to light levels consistent with ambient light. Intraperitoneal injections of C60-OO did not affect the lifespan of CB6F1 female mice. Finally, we conduct a lifespan and health span study in males and females C57BL/6 J mice comparing oral treatment with pristine C60-EVOO and EVOO alone versus untreated controls. We failed to observe significant lifespan and health span benefits of C60-EVOO or EVOO supplementation compared to untreated controls, both starting the treatment in adult or old age. Our results call into question the biological benefit of C60-OO in aging.
Keywords: Antioxidant;; C60-OO;; Fullerene.
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Figures
Fig. 1
Basic characterization of C 60…
Fig. 1
Basic characterization of C 60 -OO preparations. a Photographs of commercial C 60…
Fig. 1 Basic characterization of C60-OO preparations. a Photographs of commercial C60-OO preparations as received from the manufacturer in comparison to pristine C60-OO. b Representative HPLC chromatograms of extracted C60 from C60-OO preparations. Inset is zoomed to aid in visualization of impurity peaks. Note the decreased peak height of the main C60 peak and increased non-C60 impurity peaks seen in the commercially sourced C60-OO. c Measured C60 concentrations in C60-OO samples relative to their label claims. In-house C60-OO is shown without a labeled concentration. Data are mean ± SEM. d ROS protection activity of C60-OO preparations as measured by 4-NBP assay. Only in-house pristine C60-OO exhibits protection. Data are mean ± SEM. Data were analyzed with one-way ANOVA with Bonferroni post-hoc correction. ***p < 0.001, ****p < 0.0001, ns = not significant
Fig. 2
Light-induced toxic byproduct formation of…
Fig. 2
Light-induced toxic byproduct formation of C 60 -OO. a Degradation of pristine C…
Fig. 2 Light-induced toxic byproduct formation of C60-OO. a Degradation of pristine C60-OO by light under the indicated atmosphere calculated by AUC of the C60 peak on HPLC. Data were fit with single-exponential decays. t1/2 were as follows: b Mice remaining alive at the end of the 14-day observation period with samples taken from the open-container sample shown in a at the given time interval shown in days. T = 8 OO indicates OO only exposed to light for 8 days. n = 4 per group
Fig. 3
Lifespan study with IP injections…
Fig. 3
Lifespan study with IP injections of C 60 in aged mice CB6F1 mice.…
Fig. 3 Lifespan study with IP injections of C60 in aged mice CB6F1 mice. There was no significant difference in mice treated with C60-OO vs OO alone. Data show survival curves estimated by Kaplan-Meier of female CB6F1 mice randomly distributed into two groups of 11 mice and starting the treatment at 100–109 weeks of age
Fig. 4
Replication of lifespan study with…
Fig. 4
Replication of lifespan study with oral supplementation of C 60 in C57BL/6 J.…
Fig. 4 Replication of lifespan study with oral supplementation of C60 in C57BL/6 J. Left panel: survival curves estimated by Kaplan-Meier in all C57BL/6 J mice treated with C60-EVOO or EVOO versus untreated controls. Right panel: survival curves of the same mice subdivided in those starting the treatment with C60-EVOO or EVOO at the age of 9 months (C60 + EVOO_Y and EVOO_Y, respectively) and those starting the treatment at the age of 23 months (C60 + EVOO_O and EVOO_Y, respectively) versus untreated mice (Control). No significant differences in survival were detected among the experimental groups and between females and male mice. Analysis of survival curves starts at 21 months of age for all experimental groups. Respective numbers of mice at start are as follows: Controls n = 24 (12 F, 12 M); EVOO_Y n = 8 (4 F, 4 M); C60-EVOO_Y n = 8 (3 F, 5 M); EVOO_O n = 9 (5F, 4 M); C60-EVOO_O n = 10 (5F, 5 M)
Fig. 5
Health span assessment in C57BL/6…
Fig. 5
Health span assessment in C57BL/6 J. Graphical presentation of age-related changes of body…
Fig. 5 Health span assessment in C57BL/6 J. Graphical presentation of age-related changes of body weight (panel a), surface temperature (panel b), grip strength score (panel c) as well as of three parameters obtained from a 5-min locomotor activity test [track length (panel d), activity (panel e) and max speed kept for 3 s (Vmax3s, panel f)] in C57BL/6 J mice. C60 + EVOO_Y and EVOO_Y represent the groups of mice starting the treatment with C60-EVOO or EVOO, respectively, at the age of 9 months. C60 + EVOO_O and EVOO_O represent the groups of mice starting the treatment with C60-EVOO or EVOO, respectively, at the age of 23 months. The graphs are plotted using the mean estimates obtained by linear mixed models using age (months) as categorical (ordinal) variable, sex (categorical), experimental group (categorical) and the interaction between age and experimental groups. Transparent grey intervals overlaid with the means represents 95% confidence interval. Pairwise comparisons (sequential Bonferroni) are shown only for those experimental groups displaying significant changes from controls also in linear mixed model using age as continuous variables. # p < 005 for C60 + EVOO_Y compared to controls. * p < 0.05 for EVOO_O compared to controls. Comparison of health span data starts at 21 months of age. Respective numbers of mice at 21 months are as follows: Controls n = 24 (12 F, 12 M); EVOO_Y n = 8 (4 F, 4 M); C60-EVOO_Y n = 8 (3 F, 5 M); EVOO_O n = 9 (5F, 4 M); C60-EVOO_O n = 10 (5F, 5 M). Since the number of mice decreases with aging due to age-related mortality, a detailed description of the sample size is reported in Supplementary table 2 See this image and copyright information in PMC
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