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Photo: SHVETS production
Quercetin suppresses the clinical symptoms of arthritis by inhibiting the release of inflammatory cytokines, decreasing lipopolysaccharide-induced cyclooxygenase (COX-2) levels, and antagonizing and bone resorption by suppressing of nuclear factor-kappa β (NF-kβ) and AP-1 activity (Kim et al. 2019).
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Learn More »Quercetin (QUE) inhibits the oxidation of biomolecules and activates or inhibits several proteins. For instance, QUE inhibits the non-specific protein kinase enzyme (Williams et al. 2004). QUE has also been shown to act as an agonist of the G protein-coupled receptor (Prossnitz and Barton 2014). Previous studies have demonstrated that QUE is a potential agent for the treatment of RA, however, the underlying antiarthritic mechanism of QUE has not been fully elucidated. Therefore, the present study addressed the molecular and biochemical mechanisms of QUE alone or in combination with MTX on the ADA enzyme in an arthritic rat model. According to the in-silico study, QUE showed favorable binding with the ADA enzyme. QUE was isolated from Fenugreek seeds extract using silica gel column chromatography and it was resolved into fractions containing hexane/ethyl acetate/ethanol. This was done in accordance with Sambandam et al. (2016) who isolated QUE from the leaves of Trigonella foenum-graecum (Sambandam et al. 2016). The structure of the isolated QUE was confirmed by several technical methods. TLC revealed the same brown spot for the isolated QUE and the standard with R f value of 0.52 after the plate was sprayed with iodine and 5% FeCl 3 . This result was similar to that of Singh et al. (2012) in which the R f value of QUE was confirmed. Moreover, the UV–vis spectroscopy initially confirmed the isolation of quercetin from Fenugreek extract with peaks at 386, 296 and 262 nm compared with standard quercetin. Also, the isolated quercetin was analyzed by FTIR and revealed the presence of a C–O–C bond at 1009 cm−1, C=C bond at 1610 cm−1, C=O bond at 1667 cm−1 and an O–H bond at 3406 cm−1 compared with the standard quercetin. The results of UV–visible and FTIR of isolated QUE confirmed that its structure was similar to that previously described (Deore et al. 2013; Tiwari et al. 2015). Therefore, the characterization measurements elucidated the effective isolation of quercetin from Fenugreek extract with a high degree of purity. To compare the in-silico theoretical study with practical data, the ADA enzyme was isolated from rat joints and assayed in the absence and presence of Fenugreek QUE. The ADA activity was inhibited in rat joints by Fenugreek QUE with an IC 50 value of 0.17 mM, whereas previous study reported that the IC 50 value of Reynoutria japonica which represented a natural inhibitor of ADA was 0.629 mM (Zhang et al. 2019). The fenugreek QUE acts as a non-competitive inhibitor with a K i value of 55.5 mM. Moreover, the K i result is novel because there is no evidence in the literatures to compare the results with however, some studies are not consistent with our results (Singh and Sharma 2000; Adamek et al. 2020; Kumar and Sharanya 2020; Kutryb-Zajac et al. 2020). Thus, our results indicated that Egyptian Fenugreek QUE may act as a natural inhibitor of ADA which is a key inflammatory enzyme. This was confirmed theoretically in-silico and in practical studies. The results showed that combining QUE and MTX therapy had a greater effect on reducing inflammatory symptoms compared with either MTX or QUE treatment alone. The progression of arthritis led to dramatic decrease in rat body weight and an increase in the PV, this was consistent with previous studies reported RA in experimental animals (Rasool et al. 2006; Egan et al. 2004; Granado et al. 2005; Roy et al. 2017). When arthritic rats were compared with their normal control counterparts, they showed signs of arthritis. In contrast to their arthritic control counterparts, QUE-treated arthritic rats reported a marked increase in body weight and decrease in PV. These results are consistent with that of other studies (Rasool et al. 2006; Roy et al. 2017; Kumar et al. 2009; Haleagrahara et al. 2017).
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Learn More »The anti-inflammatory efficacy of QUE alone or in combination with MTX was further confirmed by live imaging and X-ray examination of the animals. RA control rats showed mild to moderate cartilage damage and, bone erosion indicating bone destruction. This may result from the chronic exposure of ankle joints to proinflammatory mediators such as TNF-α and IL-1β, which stimulate the production of proteolytic enzymes which result in the degradation of cartilage (Roy et al. 2017). We observed that small joints such as the tarsal, metatarsal, and interphalangeal were more affected in RA rat control group. However, in case of QUE-treated and MTX alone or in the combinational groups, these abnormalities were ameliorated, and in the QUE/MTX combined group, it is bearing more resemblance to radiographic pattern of the joints of normal group. This indicates the enhanced anti-inflammatory efficacy of QUE and MTX. Local injection of CFA resulted in an increase of TNF-α, IL-1β, and IL-6 levels due to activation of autoimmunity cells which aggravated complete inflammation. In contrast, treatment with QUE and/or MTX, showed a reduction in serum TNF-, IL-1β, and IL-6 with a minimal reduction in the combined treated group compared with the RA untreated group. Thus, the combination treatment of QUE and MTX was more effective at limiting inflammatory cell infiltration and halting or delaying bone loss. This is largely consistent with the presence of the different pathological patterns observed previously (Haleagrahara et al. 2017). The immunological markers C-reactive protein (CRP), anti-cyclic citrullinated peptide (anti-CCP) or rheumatoid factor (RF) and ADA enzyme are released when cartilage and bone become inflamed. The presence of these immunological markers in the blood is associated with the onset and progression of arthritis. They are thought to play a part in the progression of RA (Nagatomo et al. 2010; Shadick et al. 2006). Contrary to what was mentioned previously (Shen et al. 2015), our results showed that after 28 days, QUE and MTX therapy significantly decreased CRP, anti-CCP, RF and ADA enzyme with the lowest value for the combined treatment. Recently, Sangeetha et al. reported a link between RA and oxidative stress. They demonstrated that elevated levels of ROS and oxidative stress caused liver, brain, and cartilage damage in CFA rats (Sangeetha et al. 2020). The data from the present study revealed that arthritic rats and MTX-administrated rats exhibited an impairment in oxidant/antioxidant homeostasis, resulting in a marked increase in MDA levels and degradation of the antioxidant enzyme GPx in the joint supernatant. This finding is consistent with that of a previously study (Elmansy et al. 2021). Furthermore, the combined therapy of QUE and MTX reduced MTX-induced oxidative damage and increased antioxidant enzyme protection in joint tissue as QUE could reduce the production of free radicals. This is in accordance with previous report by Eshwarappa et al. who found that QUE had a hepatoprotective activity against hexachlorocyclohexane and thioacetamide-induced oxidative injury (Eshwarappa et al. 2015).
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Learn More »Previous studies have suggested correlation between ADA activity and RA joint inflammation (Gao et al. 2020; Nair et al. 2020; Gangadharan et al. 2021). The results demonstrated that there was significant increase in ankle joint ADA activity and mRNA levels in RA rats compared with control rats. After 28 days, QUE and MTX significantly decreased the activity and expression levels of ADA enzyme compared with the untreated controls. This may be due to the anti-inflammatory, immunomodulatory, antioxidant, and the regulatory effects of QUE on cell signaling (Almatroodi et al. 2021; Zaragozá et al. 2020; Marefati et al. 2021). Thus, QUE represents a targeted treatment for RA due to its effect on inflammatory cells and molecules and this effect results from its role in inhibition of the ADA inflammatory enzyme. The histopathology of the arthritic rat ankle joint revealed marked inflammatory conditions. QUE and MTX alone treated, or the combination groups showed a decrease in the sub—periosteum region, destruction of cartilage, synovial membrane and vascular proliferation with reduced levels of chondrocytes and synovial joint space. These observations were seen to a greater extent in the combination drug-treated group compared with the alone treated group indicating a synergistic effect of the combination. Furthermore, X-ray analysis and histopathological assessment also provided evidence for the proper induction of RA in the rats following adjuvant injection and the role of QUE and/or MTX as effective therapy for RA inflammation. This effect resulted from their role in inhibition of ADA inflammation enzyme. These results was in accordance with that of previous studies (Roy et al. 2017; Ebrahimzadeh et al. 2008).
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