A New Dimeric Protoberberine Alkaloid and Other Compounds from the Tubers of

: A new dimeric quaternary protoberberine alkaloid, bispalmatrubine ( 1 ), and thirteen known compounds ( 2 - 14 ) were purified from the tubers of Tinospora dentata . Their structures were determined by spectroscopic and spectrometric analytical methods. Among the isolates, eight compounds were examined for their in vitro anti-inflammatory potential and several tested alkaloids displayed moderate inhibitory effects of N -formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation and elastase release.

structures were determined by spectroscopic and spectrometric analytical methods. Among the isolates, eight compounds were examined for their in vitro anti-inflammatory potential and several tested alkaloids displayed moderate inhibitory effects of N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation and elastase release.

Preparation of human neutrophils
Human neutrophils study conducted according to the Declaration of Helsinki (2013)  and spun down at 400 g for 40 min at 20 °C. The granulocyte/erythrocyte pellets were resuspended in ice-cold 0.2 % sodium chloride to lyse erythrocytes. After 30 sec, the same volume of 1.6 % sodium chloride solution was added to reconstitute the isotonic condition.
Purified neutrophils were pelleted and then resuspended in a calcium-free Hank's balanced salt solution (HBSS) buffer at pH 7.4, and were maintained at 4 °C before use.

Measurement of superoxide anion generation and elastase release
The generation of superoxide anion assay was based on the superoxide dismutase Changes in the absorbance with a reduction in ferricytochrome c at 550 nm were continuously monitored in a double-beam spectrophotometer with constant stirring.
Calculations were based on differences in the reactions with and without SOD (100 U/mL) divided by the extinction coefficient for the reduction of ferricytochrome c (ε = 21.1/mM/10 mm). Degranulation of azurophilic granules was determined by elastase release as described previously (Yu et al., 2011;Yang et al., 2013). In this experiments, MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide was using as the elastase substrate. After supplementation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 µM), neutrophils (6 × 10 5 /mL) were equilibrated at 37 °C for 2 min and incubated with test compounds (10 μM) or a vehicle (0.1 % Dimethyl sulfoxide, negative control) for 5 min. Cells were activated by 100 nM fMLP and 0.5 µg/mL cytochalasin B. The changes in absorbance at 405 nm were continuously monitored to assay elastase release. The results were expressed as the percent of elastase release in the fMLP/CB-activated, drug-free control system. For compounds 5, 7, and genistein, IC 50 values were calculated from a series of different examined concentrations of 1-10 μM (5 and 7) and 1-50 μM (genistein), respectively.