Effect Of Procyanidin B₂ On Anti-aging And Identification Of Its Metabolites

Proanthocyanidins extraction possesses a strong antioxidant capacity, which has attracted scholars' attention in recent years. Procyanidin B2 is the main active ingredient of proanthocyanidins in food resources such as dragon fruit, grape seed,seabuckthorn fruit,and blueberry. But little reports were reported about the pure procyanidin B2 for identification of metabolites and its distribution. This paper studies the effect of procyanidin B2 on the anti-aging effect of mice by the way of biochemical indexes and spatial learning parameters by the establishment of senescence model using D-galactose, and describes the possible metabolites forms and distribution of procyanidin B2 in mice by ultra performance liquid chromatography with diode array detector and combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry method (UPLC-DAD-ESI-IT-TOF-MSn) technology. The results were as follows:(1) Mice were injected subcutaneously with D-galactose to establish the subacute aging model, and the anti-aging effect of procyanidin B2 on aging mice was studied by measuring the anti-oxidative indexes and learning abilities of mice. The results showed that. The rate of increase of body weight of mice in procyanidin B2 group was higher than that in senescence group (P<0.05). The index of Liver, brain and heart were significantly higher than those in the aged group (P <0.05). The activities of SOD, CAT, T-AOC, and GSH-PX in liver, kidney, and brain of mice were significantly increased (P <0.05), and the content of MDA was significantly decreased (P< 0.05). In the open field experiment, comparing with the control group,the number of access to the central area, and the total time, distance in the central area of the aging group were significantly lower (P<0.05). Comparing with the aging group, the number of access to the central area, and the total time, distance in the central area of the procyanidin B2 were significantly improved (P <0.05) . In the Y maze test, the total time in the aging group was significantly lower than those in the control group (P <0.05), and latency of the maze was significantly larger than those in control group (P<0.05). Comparing with the aged mice, the mice of procyanidin B2 group increased the retention time in the food arm, but there was no significant difference (P <0.05), and the latency was significantly decreased (P <0.05). In the Morries maze experiment, the incubation period of the aged group was significantly higher than that of the control group (P <0.05). The number of boarding the platforms and the total time and distance of the platform quadrant were significantly lower than those of the control group (P<0.05). Comparing with the aging group, the incubation period of the aged group was significantly lower in procyanidin B2 group(P<0.05). The number of boarding the platforms and the total time and distance of the platform quadrant were significantly higher than those of the in procyanidin B2 group (P<0.05). The results showed that procyanidin B2 could improve the oxidative stress in aging mice.(2) Mice were administered by procyanidin B2 (800 mg/kg body) to analyze the metabolites of plasma and urine samples. The 45 metabolites (16 new metabolites)of procyanidin B2 were found, including 32 sulfated metabolites, 14 methylated metabolites, 7 hydration metabolites, 2 hydroxylation metabolites, and 1 hydrogenation metabolites. Moreover 27 metabolites were formed by scission of the flavanol interflavanic bond C4-C8, including 16 aromatic acid products, 25 conjugated metabolites, 3 C-ring cleavage products,and 2 phenylvalerolactone metabolites. In addition,procyanidin B2 binding metabolites were 18 species,including 9 sulfonated products, 11 methylation products, 1 hydrogenation metabolites, 1 glucuronidation metabolites, 6 hydrated products, and 2 hydroxylation metabolites.(3) The metabolites profiles of procyanidin B2 in plasma, liver, heart, spleen,lung, kidney, brain, and small intestine were qualitatively described UPLC-DAD-ESI-IT-TOF-MSn technology. The results showed that the metabolites of procyanidin B2 in the lung, heart, brain,liver,small intestine, kidney,and spleen were 9?18?5?3?11?1?3, respectively,indicating that procyanidin B2 metabolites were widely distributed in mice. Futhermore, all the metabolites found in liver were conjugated metabolites of procyanidin B2 (Mr> 577.13). Epicatechin monomer metabolite only was detected in the small intestine. We found 36 metabolites in the urine of mice, and 25 kinds of metabolites were formed by scission of the flavanol interflavanic bond C4-C8,11 metabolites were conjugates of procyanidin B2,suggesting that the procyanidin B2 was directly absorbed and metabolized by tissues while procyanidin B2 could reach the small intestine where it was transformed by the intestinal microbiota before absorption, and was mainly eliminated by urine.In summary, the metabolites of procyanidin B2 in mice were varieties, and widely distributed in mice organ tissues. Procyanidin B2 possesses a strong antioxidant capacity, and significantly prevents the oxidative stress, which may be attributed to metabolites of procyanidin B2 and its widely distribution. Therefore,procyanidin B2 could improve the learning and memory abilities of aging mice.