Screening for anti-inflammatory components from Corydalis bungeana Turcz …
Effect of a alkaloid remove of CB on xylene-induced ear edema in mice
Utilizing a customary indication of inflammatory response (xylene-induced ear edema), a anti-inflammatory activity of a alkaloid remove of CB was investigated. The alkaloid remove of CB (40 and 80 mg/kg) showed poignant predicament of xylene-induced mice ear edema as compared with a control, with predicament of 32.1 and 45.1 % (P 0.01) respectively (Fig. 1).
Fig. 1. Anti-inflammatory outcome of a alkaloid remove of CB on ear flourishing (a) and ear weight (b) in xylene-induced mice. Values are voiced as mean ± SD (n = 10). *p 0.05 and **p 0.01 vs. indication group
Effect of a alkaloid remove of CB on carrageenan-induced hind-paw edema
Carrageenan-induced duke inflammation is an strident and rarely reproducible indication of strident inflammatory response [12]. Cardinal signs of inflammation rise immediately after subcutaneous injection and outcome from movement of pro-inflammatory agents. The inflammatory response is customarily quantified by boost in duke distance (edema) and is modulated by inhibitors within a inflammatory cascade [13]. A bar draft is given to illustrate a anti-inflammation outcome of a alkaloid remove of CB in Fig. 2. The formula prove that a flourishing volume increasing gradually and appearance during 3rd hour after carrageenin injection. However, a edema of rodent duke was significantly reduced by a diagnosis with a high-dose alkaloid remove of CB (60 mg/kg) during all phases of carrageenan-induced inflammation. And a predicament ratios of a CB (60 mg/kg) diagnosis with rats during 0.5, 1 and 3 h were 29.8 % (P 0.05), 28.3 % (P 0.05) and 28.6 % (P 0.01), respectively, compared to a indication control. Aspirin was used as a certain control and had matching effects (31.9, 32.5 and 30.0 % inhibition, respectively).
Fig. 2. Anti-inflammatory outcome of a alkaloid remove of CB on carrageenan-induced hind-paw edema in rats. Values are voiced as mean ± SD (n = 10). *p 0.05 and **p 0.01 vs. indication group
HPLC research for a alkaloid remove of CB
First, we dynamic a fingerprint of a alkaloid remove of CB as a credentials information of macrophage binding. There were 10 categorical peaks in a fingerprint of a alkaloid remove of CB during 289 nm (Fig. 3). Among these, 5 components were dynamic after comparison with standards (Table 1). All of a categorical peaks had good separation.
Fig. 3. HPLC fingerprint of a alkaloid remove of Corydalis bungeana Turcz
Table 1. RRT and a RPA of any rise in a fingerprint of a alkaloid remove of CB
Injection pointing was dynamic by 5 replicate injections of a same representation in one day. The relations customary deviations (RSDs) of relations influence time (RRT) and relations rise area (RPA) were 0.72 and 0.56 %, respectively, and a changes were not significant. Repeatability was assessed by examining 5 exclusively prepared samples of a alkaloid remove of CB. RSDs of RRT and RPA were 1.22, 1.57 %, respectively, and a changes were not significant. Sample fortitude was assessed by unbroken injections of a same representation during 0, 1, 2, 4, 8, 12 and 24 h. During this period, a resolution was stored during room temperature. RSDs of RRT and RPA were 1.72 and 2.05 %, respectively, and a changes were not significant. Results of injection precision, repeatability and a fortitude exam suggested that this process was adequate, current and applicable. RRT and RPA of any evil element was distributed regulating corynoline as a anxiety peak. The RRT and RPA of a 10 peaks are shown in Table 1.
Binding of a alkaloid remove of CB to RAW 264.7 cells
The alkaloid remove of CB exerted anti-inflammatory properties, so we used a contracting to RAW 264.7 cells to shade a active compounds within it. The alkaloid remove of CB was incubated with RAW 264.7 cells and firm components collected by centrifugation. After entirely soaking and eluting from dungeon pellets, defended components were injected into HPLC complement for analysis.
Two principal peaks (K-1 and K-2) were rescued in representation B (desorption eluate of a alkaloid remove of CB) (Fig. 4a). No allied peaks were rescued in a chromatograms of a dual control samples (sample A, final rinse eluate; representation C, car desorption eluate) (Fig. 4c and d). By comparison of a influence times and UV fullness profiles of K-1 and K-2 with a HPLC fingerprint of a alkaloid remove of CB, components K-1 and K-2 were identified as peaks 2 and 3 (Fig. 4b). These commentary identified a RAW 264.7 cell-binding molecules K-1 and K-2 as intensity anti-inflammatory components of CB.
Fig. 4. Detection of RAW 264.7 cells-binding molecules in a alkaloid remove of CB by HPLC analysis. a Comparative chromatograms of a desorption eluate of alkaloid remove of CB. b a fingerprint of a alkaloid remove of CB. c a final rinse eluate and d a car desorption eluate. Detection was during 289 nm
Identification of K-1 and K-2 by HPLC–MS
5 mg of K1 and K2 were respectively dissolved in d6-CDCl 3 as a samples, and rescued by Bruker AV-500. The MS spectrum of devalue K-2 is shown in Fig. 5. The mass spectrum of devalue K-2 was matching to a mass spectrum of 12-hydoxycorynoline. Identification was upheld serve by 1 H-NMR information and 13 C-NMR data: 1 HNMR (500 MHz, CDCl3): 6.661 (1H, s, 1-H), 7.023 (1H, s, 4-H), 2.177 (3H, s, 5-N-CH3), 3.437, 4.020 (2H, ABq, J = 15.5, 6-H), 6.808 (1H, d, J = 8.0, 9-H), 6.962 (1H, d, J = 8.0, 10-H), 3.878 (1H, s, 11-H), 4.902 (1H, s, 12-H), 1.242 (3H, s, 13-CH3), 3.310 (1H, s, 14-H), 5.995 ~ 5.942 (4H, m, 2-O-CH2-O-3, 7-O-CH2-O-8); 13 C-NMR (125 MHz, CDCl3): 107.8 (C-1), 125.9 (C-1a), 145.1 (C-2), 147.0 (C-3), 112.0 (C-4), 128.8 (C-4a), 43.1 (5-N-CH3), 54.0 (C-6), 116.3 (C-6a), 142.7 (C-7), 148.6 (C-8), 109.9 (C-9), 118.9 (C-10), 135.5 (C-10a), 74.0 (C-11), 81.4 (C-12), 39.7 (C-13), 23.7 (13-CH3), 70.2 (C-14), 101.4 (2-O-CH2-O-3), 101.4 (9-O-CH2-O-10).
Fig. 5. ESI-MS disastrous magnetism spectrum of K-2 performed from macrophage binding
Compound K-1 was presumed to be corynoline. Under matching chromatographic conditions, a teenager frigid compounds of K-1 and a influence time of a anxiety rise of corynoline were identical.
Anti-inflammatory activity of corynoline and 12-hydroxycorynoline in vitro
ELISA test was practical to inspect a outcome of corynoline and 12-hydroxycorynoline on IL-6, TNF-α, IL-10 and NO levels in LPS-treated cells. As showed in Figs. 6 and 7, LPS alone dramatically increasing a levels of IL-6, TNF-α, IL-10 and NO. However, corynoline or 12-hydroxycorynoline during a certain concentrations apparently indifferent LPS-induced IL-6 and TNF-α overproduction . (P 0.01 or P 0.05). Corynoline (100 μg/mL) significantly suppressed LPS-induced IL-10 overproduction (P 0.05) while 12-hydroxycorynoline had no apparent outcome on this index (Fig. 7a). In addtion, Corynoline (1, 100 μg/mL) and 12-hydroxycorynoline (100 μg/mL) significantly indifferent LPS-induced NO prolongation (Fig. 7b).
Fig. 6. Anti-inflammatory outcome of corynoline and 12-hydroxycorynoline on a turn of IL-6 (a) a nd TNF-α (b) prompted by LPS in RAW 264.7 cells. Data are voiced as mean ± SD (n = 10). *p 0.05 and **p 0.01 vs. indication group
Fig. 7. Anti-inflammatory outcome of corynoline and 12-hydroxycorynoline on a turn of IL-10 (a) and NO (b) prompted by LPS in RAW 264.7 cells. Data are voiced as mean ± SD (n = 10). *p 0.05 and **p 0.01 vs. indication group
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