Quantification of flavonoids in buckwheat sprouts and their antioxidative, anti-inflammatory, and immunomodulatory effects
Buckwheat sprouts T lymphocyte Quercetin-3-O-robinobioside Rutin;
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일반메밀새싹의 대표적 플라보노이드는 orientin, isoorientin, vitexin, isovitexin, rutin로 알려져 있다. 본 연구에서는 Q-TOF MS/MS와 NMR을 이용하여 위 언급된 플라보노이드 이외에 quercetin-3-O-robinobioside를 추가적으로 분석하였다. Quercetin-3-O-robinobioside는 rutin의 이성질체이며 기존에 보고된 연구 결과는 충분히 두 물질을 분리하고 분석하지 못하였다. 본 연구는 최적화된 HPLC 분리 조건을 이용하여 메밀새싹내 플라보노이드 분석법을 확립하였으며, 메밀새싹에서 quercetin-3-O-robinobioside를 분리하고 정성, 정량을 한 최초의 실험이다. 메밀새싹 발아시 blue LED, red LED, 형광등과 같은 광원을 이용하였으며, 일반메밀새싹의 플라보노이드 함량은 광원과 밀접한 관련이 있었다. 광원을 이용하여 생장시킨 메밀새싹의 플라보노이드 함량은 암조건에 비하여 높게 나타났다. 클로로필 함량은 형광등과 red LED에서 생장시킨 새싹에서 높게 나타났으며 red LED는 메밀새싹의 상층부 생육에 밀접한 영향을 주는 것으로 나타났다. 다양한 광원에서 재배한 메밀새싹의 항산화능을 측정하였으며 blue LED에서 재배한 메밀새싹에서 가장 높은 항산화능을 보였다. Blue LED에서 재배한 메밀새싹은 가장 높은 플라보노이드 함량을 보였으며 그 중, orientin, isoorientin, rutin이 강한 항산화능을 보였다. 메밀새싹은 과산화수소로부터 세포를 보호하였으며 AAPH로 야기시킨 세포내 활성산소종을 효과적으로 제거하였다. 또한 세포내 항산화효소의 활성을 증가시켰다. 또한 일반메밀새싹의 항염증 효과에 대한 연구를 수행하였다. 쓴메밀새싹을 함께 사용하였으며, 쓴메밀새싹은 일반메밀새싹과 달리 rutin이 다량 함유되어 있으며, 위 언급한 플라보노이드는 검출되지 않았다. 항염증 연구모델에서는 RAW 264.7 세포와 쥐에서 적출한 대식세포를 lipopolysaccharide로 자극시켜 사이토카인을 유도시켰다. 일반메밀새싹과 쓴메밀새싹 추출물 모두 두종의 세포에서 효과적으로 사이토카인 양을 감소시키는 것을 알 수 있었다. 면역조절연구에서 일반메밀새싹과 쓴메밀새싹을 4주 동안 쥐에게 경구 투여하였다. 4주 후 쥐의 비장세포를 분리하여 T 세포 분포(%)를 분석하였으며 메밀새싹처리군에서 T 세포 분포를 증가시키는 것을 알 수 있었다. 또한 일반메밀새싹과 쓴메밀새싹 처리군 모두 혈액내 사이토카인이 효과적으로 감소하는 것을 알 수 있었다.
Previous phytochemical studies in buckwheat sprouts have presented that the sprouts contain several flavonoids, including orientin, isoorientin, vitexin, isovitexin, rutin, and quercetrin, whereas tartary buckwheat sprouts contain only rutin. In this study, we evaluated flavonoids present in buckwheat sprouts and identified a previously unreported flavonoid. Simultaneous detection by HPLC was used to separate rutin and a compound not separated in previous studies. We used a novel HPLC elution gradient method to successfully separate rutin and the previously unidentified compound, for which we performed structural analysis. The identification of six flavonoids by HPLC was confirmed using HPLC-ESI-MS/MS analysis. The newly identified compound, [M+H]+ = 611.1736, was identified by NMR as the rutin epimer quercetin-3-O-robinobioside. Unlike common buckwheat sprout, tartary buckwheat sprout contained rutin as a main flavonoid, whereas other flavonoids appeared only in trace amounts or were not detected. Quercetin-3-O-robinobioside was not detected in tartary buckwheat sprout. Effects of blue LED (BL), red LED (RL), and fluorescent light (FL) on sprout growth, chlorophyll contents, levels of flavonoids and antioxidant capacity in common buckwheat (F. esculentum Möench) sprouts were evaluated. Germinated seeds were planted in a light supplemented growth chamber from 1 to 9 days after germination (DAG). Length of shoots cultivated in RL and dark displayed increases compared to FL and BL. Light irradiation did not induced change the length of roots except for FL. The length of roots grown under FL was significantly increased during seedling period. Contents of chlorophyll in buckwheat sprouts were greatly enhanced by light irradiation. FL and RL induced large increment of chlorophyll production within 5 and 7 DAG, respectively. FL increased the accumulation of orientin and isoorientin than the other light sources at 5 DAG. RL and BL increased more the contents of vitexin and rutin than those of FL and dark conditions at 5 DAG. Leaves of buckwheat sprouts are relatively rich source of C-glycosyl flavones, quercetin-3-O-robinobioside and rutin compared with stems. The levels of antioxidant capacity of buckwheat sprouts cultivated by BL and FL were significantly increased until 5 DAG. There was a positive correlation between contents of flavonoids and antioxidant capacity. Results of this study indicate that accumulation of flavonoids and antioxidant capacity are influenced by light sources and seedling period. To investigate antioxidant capacity, common buckwheat sprouts grown under blue LED, red LED, fluorescent light, and dark were extracted. Through HPLC analysis, orientin, isoorientin, vitexin, isovitexin, quercetin-3-O-robinobioside, and rutin were quantified. Buckwheat sprouts grown under blue LED showed the highest antioxidant capacity among the other light sources using radical scavenging assays. Also, orientin, isoorientin, and rutin effectively scavenged artificial radicals. The protective effect of extracts of buckwheat sprouts on oxidative stress in vitro was investigated. Pre-treatment of extracts of common buckwheat sprouts effectively inhibited neuronal PC-12 cell death induced by H2O2 in MTT assays. Buckwheat sprouts and their flavonoids such as orientin, vitexin, and rutin effectively reduced intracellular oxidative stress level. Buckwheat sprouts and individual flavonoids significantly increased intracellular antioxidant capacity and bioactivity of superoxide dismutase (SOD) in PC-12 cells. These results suggest that common buckwheat sprouts and their bioactive compounds may enhance cytoprotective effect against oxidative insults-induced neuronal PC-12 cells death and upregulated antioxidant enzyme such as SOD. Anti-inflammatory effect of common buckwheat sprouts (CBS) and tartary buckwheat sprouts (TBS) grown under blue LED (BL) and dark were evaluated in LPS-stimulated peritoneal macrophages and RAW 264.7 cells. The extracts of CBS and TBS grown under dark and BL effectively attenuated interleukin (IL)-6 and IL-12 production in LPS-stimulated peritoneal macrophages and RAW 264.7 cell. Similar results were obtained for the suppression of TNF-α release. The extracts of TBS grown under BL effectively reduced TNF-α and IL-12 secretion in stimulated peritoneal macrophages. CBS contained C-glycosyl flavone (orientin, vitexin and their isomers) and rutin, whereas TBS had high amounts rutin mainly. It is considered that flavonoids in tartary buckwheat sprouts, at least partially, may contribute to suppression of IL-6, IL-12, and TNF-α production in LPS-stimulated peritoneal macrophages and RAW 264.7 cells. Overall, the results indicate that buckwheat sprouts serve as natural dietary source of bioactive phytochemicals for anti-inflammatory compounds. Effects of common buckwheat sprouts (CBS) and tartary buckwheat sprouts (TBS) extracts on lymphocytic population of splenocyte isolated from Balb/c mice were determined. Buckwheat sprouts were orally administered to mice for 28 days and then injected with anti-CD3 mAb intravenously. Oral administration of CBS and TBS extracts significantly increased the population of T lymphocytes (CD3+) and CD4+ T lymphocytes in spleen compared to normal group. Moreover, CBS and TBS extracts increased CD8+ T-lymphocyte. Increment of CD8+ T-lymphocyte was the highest treated with high concentration of TBS extract among treated samples. After stimulation with anti-CD3, buckwheat sprouts significantly reduced expression of CD69. CBS and TBS extracts significantly decreased IFN-γ level in serum. These data indicated that buckwheat sprouts increased population of CD3+, CD4+, and CD8+ T-lymphocyte and down-regulated expression of CD69 and IFN-γ level as an immunomodulatory agent.
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