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Journal of Chinese Integrative Medicine ›› 2013, Vol. 11 ›› Issue (4): 246-252.doi: 10.3736/jintegrmed2013036

• Research Article • Previous Articles     Next Articles

Extract of buckwheat sprouts scavenges oxidation and inhibits pro-inflammatory mediators in lipopolysaccharide-stimulated macrophages (RAW264.7)

Rajendra Karkia,b, Cheol-Ho Parkc, Dong-Wook Kima()   

  1. a. Department of Oriental Medicine Resources, Mokpo National University, Muan-gun, Jeollanam-do 534-729, South Korea
    b. Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, KansasCity, MO 64108, USA
    c. Department of Bio-Health Technology, Kangwon National University, Chuncheon, Gangwondo 200-701,South Korea
  • Received:2013-04-21 Accepted:2013-06-17 Online:2013-07-10 Published:2013-07-15

Objective

Buckwheat has been considered as a potential source of nutraceutical components on the world market of probiotic foodstuffs. The purpose of this study was to evaluate the effects of tartary buckwheat (Fagopyrum tataricum) sprouts on oxidation and pro-inflammatory mediators.


Methods

The anti-oxidant effects of buckwheat extract (BWE) and rutin were evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH)- and nitric oxide (NO)-scavenging activities, serum peroxidation and chelating assays. Lipopolysaccharide (LPS)-stimulated RAW264.7 cells were used to evaluate anti-inflammatory activities of buckwheat and rutin. NO production in LPS-stimulated RAW264.7 cells was determined by using Griess reagent. The expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB) p65 subunit in cytosolic and nuclear portions were determined by Western blot analysis. Also, the production of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was determined by enzyme-linked immunosorbent assay.


Results

Inhibitory concentration 50 values for DPPH- and NO-scavenging activities of BWE were 24.97 and 72.54 μg/mL respectively. BWE inhibited serum oxidation and possessed chelating activity. Furthermore, BWE inhibited IL-6 and TNF-α production in LPS-stimulated RAW264.7 cells. Also, BWE inhibited iNOS and COX-2 expression and NF-κB p65 translocation.


Conclusion

Buckwheat sprouts possessed strong antioxidant activity and inhibited production of pro-inflammatory mediators in the applied model systems. Thus, buckwheat can be suggested to be beneficial in inflammatory diseases by inhibiting the free radicals and inflammatory mediators.

Key words: Fagopyrum, Plant extracts, Oxidation, Nitric oxide synthase type II, Cyclooxygenase 2

Figure 1

DPPH-scavenging activity, NO-scavenging activity, metal chelation and serum oxidationA: DPPH-scavenging activity of BWE and rutin. Ascorbic acid was used as the positive control, and values are expressed as mean ± standard deviation, n = 5. B: Nitric oxide-scavenging activity of BWE and rutin. Ascorbic acid was used as the positive control, and values are expressed as mean ± standard deviation, n = 5. C: Chelating activity of BWE and rutin; values are expressed as mean ± standard deviation, n = 5. D: Effects of BWE and rutin on serum lipid oxidation. *P<0.05, **P<0.01, vs control (sample-untreated but CuSO4-treated). Butylated hydroxytoluene was used as positive control; values are expressed as mean ± standard deviation, n = 5. DPPH: 1,1-diphenyl-2-picryl hydrazyl; NO: nitric oxide; BWE: buckwheat extract; BHT: butylated hydroxytoluene; TBARS: thiobarbituric acid reactive substance."

Figure 2

Viability of RAW264.7 cells and NO production RAW264.7 cells were pre-treated with samples for 1 h followed by stimulation of the cells with LPS for additional 24 h. A: The cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. B: NO production was measured by using Griess reagent. Values are expressed as mean ± standard deviation, n = 3. *P<0.05, **P<0.01, vs control (LPS-treated, sample-untreated). NO: nitric oxide; BWE: buckwheat extract; LPS: lipopolysaccharide."

Figure 3

iNOS and COX-2 expressionRAW264.7 cells were pre-treated with samples for 1 h followed by stimulation of the cells with LPS for additional 24 h. Then, the expression of iNOS and COX-2 was determined by Western blot analysis. b-actin was used for normalization. Values are expressed as mean ± standard deviation, n = 3. *P<0.05 , **P<0.01, vs control (LPS-treated, sample-untreated). iNOS: inducible nitric oxide synthase; COX-2: cyclooxygenase-2; BWE: buckwheat extract; LPS: lipopolysaccharide."

Figure 4

TNF-α and IL-6 production RAW264.7 cells were pre-treated with samples for 1 h followed by stimulation of the cells with LPS for additional 24 h. The levels of TNF-α and IL-6 in supernatant were measured by using enzyme-linked immunosorbent assay. Values are expressed as mean ± standard deviation, n = 3. *P<0.05, **P<0.01, vs control (LPS-treated, sample-untreated). TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; BWE: buckwheat extract; LPS: lipopolysaccharide."

Figure 5

NF-κB translocationRAW264.7 cells were pre-treated with samples for 1 h followed by stimulation of the cells with LPS for additional 30 min. The expression of NF-κB p65 subunit in the cytosolic and nuclear fractions was determined by Western blot analysis. Values are expressed as mean ± standard deviation, n = 3. *P<0.05, **P<0.01, vs control (LPS-treated, sample-untreated). NF-κB: nuclear factor-kappa B; PCNA: proliferating cell nuclear antigen; BWE: buckwheat extract; LPS: lipopolysaccharide."

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