Cheng-Bo Gu

Antioxidant Activities of Extracts and Main Components of Pigeonpea [Cajanus cajan (L.) Millsp.] Leaves

Antioxidant activities of the aqueous and ethanol extracts of pigeonpea [Cajanus cajan (L.) Millsp.] leaves, as well as petroleum ether, ethyl acetate, n-butanol and water fractions and the four main compounds separated from the ethanol extract, i.e. cajaninstilbene acid (3-hydroxy-4-prenylmethoxystilbene-2-carboxylic acid), pinostrobin, vitexin and orientin, were examined by a DPPH radical-scavenging assay and a β-carotene-linoleic acid test. In the DPPH system, the antioxidant activity of the ethanol extracts was superior to that of the aqueous extracts, with IC50 values were 242.01 and 404.91 µg/mL, respectively. Among the four fractions, the ethyl acetate one showed the highest scavenging activity, with an IC50 value of 194.98 µg/mL. Cajaninstilbene acid (302.12 µg/mL) and orientin (316.21 µg/mL) showed more efficient radical-scavenging abilities than pinostrobin and vitexin. In the β-carotene-linoleic acid test, the inhibition ratio (%) of the ethyl acetate fraction (94.13%±3.41%) was found to be the highest, being almost equal to the inhibition capacity of the positive control BHT (93.89%±1.45%) at 4 mg/mL. Pinostrobin (>500 µg/mL) and vitexin (>500 µg/mL) showed insignificant antioxidant activities compared with cajaninstilbene (321.53 µg/mL) and orientin (444.61 µg/mL). In general, the ethyl acetate fraction of the ethanol extract showed greater activity than the main compounds in both systems, such results might be attributed to the synergistic effects of the components. The antioxidant activities of all the tested samples were concentration-dependent. Based on the results obtained, we can conclude that the pigeonpea leaf extracts may be valuable natural antioxidant sources and are potentially applicable in both medicine and the healthy food industry.

Ultrasound-assisted extraction of flaxseed oil using immobilized enzymes

An aqueous enzymatic process assisted by ultrasound extraction (AEP-UE) was applied to the extraction of oil from flaxseed (Linum usitatissimum L.). The highest oil recovery of 68.1% was obtained when ground flaxseed was incubated with 130 U/g of cellulase, pectinase, and hemicellulase for 12h, at 45°C and pH 5.0. The IC(50) values of oil obtained by AEP-UE and organic solvent extraction (OSE), as measured by DPPH scavenging activity essay, were 2.27 mg/mL and 3.31 mg/mL. The AEP-UE-derived oil had a 1.5% higher content of unsaturated fatty acids than the OSE-derived oil. AEP-UE is therefore a promising environmentally friendly method for large-scale preparation of flaxseed oil.

Dryofragin, a phloroglucinol derivative, induces apoptosis in human breast cancer MCF-7 cells through ROS-mediated mitochondrial pathway.

Dryofragin is a phloroglucinol derivative extracted from Dryopteris fragrans (L.) Schott. In this study, the anticancer activity of dryofragin on human breast cancer MCF-7 cells was investigated. Dryofragin inhibited the growth of MCF-7 cells in a time and concentration-dependent manner. The cell viability was measured using MTT assay. After treatment with dryofragin for 72, 48 and 24 h, the IC₅₀ values were 27.26, 37.51 and 76.10 μM, respectively. Further analyses of DNA fragmentation and Annexin V-PI double-labeling indicated an induction of apoptosis. Dryofragin-treatment MCF-7 cells had a significantly accumulation of reactive oxygen species (ROS), as well as an increased percentage of cells with mitochondrial membrane potential (MMP) disruption. These phenomena were blocked by pretreatment for 2 h of MCF-7 cells with the antioxidant compound N-acetyl-L-cysteine (NAC, 5 mM). These results speak for the involvement of a ROS-mediated mitochondria-dependent pathway in dryofragin-induced apoptosis. Western blot results showed that dryofragin inhibited Bcl-2 and induced Bax expression which led to an activation of caspases-9 and -3 in the cytosol, and further cleavage of poly ADP-ribose polymerase (PARP) in the nucleus, then induced cell apoptosis. In conclusion, the present study provides evidence that dryofragin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway.

Determination and quantification of astragalosides in Radix Astragali and its medicinal products using LC–MS

In the present study, a liquid chromatography-tandem mass spectrometry method was developed for the separation and simultaneous quantification of astragalosides I-IV in samples of Radix Astragali and a medicinal product thereof (Jinqi Jiangtang tablets). Chromatographic separation was achieved on an Agilent Eclipse XDB (ODS)-C18 column with a mobile phase consisting of acetonitrile and 0.05% formic acid aqueous solution by use of an efficient 17-min program. A triple quadrupole mass spectrometer was operated in positive ionization mode with multiple reaction monitoring for the detection of four astragalosides. The saponin ginsenoside Rg1 (similar structure to astralagosides) was used as an internal standard. All calibration curves showed excellent linear regressions (r(2) greater, not dbl equals 0.9912) within the range of tested concentrations. The intra- and inter-day variations were below 4.57% in terms of RSD. The recoveries were 94.38-103.53% with RSD of 1.39-3.58% for spiked Radix Astragali samples. The method was successfully used for the analysis of samples of Radix Astragali and Jinqi Jiangtang tablets. In conclusion, we have developed a rapid, efficient, and accurate LC-MS/MS method for the detection of astragalosides, which can be applied for quality control of Radix Astragali and related medicinal products.

Aqueous enzymatic process assisted by microwave extraction of oil from yellow horn (Xanthoceras sorbifolia Bunge.) seed kernels and its quality evaluation.

In this study, aqueous enzymatic process (AEP) assisted by microwave extraction (ME) of oil from yellow horn (Xanthoceras sorbifolia Bunge.) seed kernel was investigated. Central composite design (CCD) and response surface methodology (RSM) were used to optimise an enzyme cocktail (cellulase, hemicellulase, pectinase) for AEP. The main factors of ME were also studied. A maximal oil extraction yield of 55.8% was achieved under optimal conditions. Moreover, scanning electron microscope (SEM) was applied to characterise the extraction process. Analysing chemical composition of the extracted oil by GC-MS showed that the content of unsaturated fatty acids by this emerging method (91.18%) was similar to that by conventional organic solvent extraction (88.76%). In addition, the main physicochemical properties and antioxidant activities of yellow horn oil were measured to evaluate its quality. The present research supported necessary data for the green extraction method of edible oil in food industry.

Determination of paclitaxel and other six taxoids in Taxus species by high-performance liquid chromatography-tandem mass spectrometry.

A method of high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) has been developed for the trace analysis of paclitaxel and other six taxoids in three Taxus species including Taxus cuspidata, Taxus media and Taxus chinensis var. mairiei. Seven taxoids were separated using a gradient mode on an Eclipse XDB-C18 column (4.6x150 mm; i.d., 5 microm) at 20 degrees C. The compound separations were detected by an API 3000 mass spectrometer equipped with a TurboIonSpray interface. The compounds were detected using electrospray ionization (ESI) in positive-ion mode and quantified by multiple-reaction monitoring (MRM) mode using the transition mass of m/z 567.4-->445.4, 609.3-->549.5, 944.9-->286.4, 812.6-->286.1, 832.8-->264.1, 854.4-->286.1, and 812.6-->286.1 for 10-DAB III, baccatin III, 7-xyl-10-DAT, 10-DAT, cephalomannine, paclitaxel, and 7-epi-10-DAT, respectively. The ranges of limit of quantitation (LOQ) for taxoids were 32, 14, 26, 14, 20, 32, and 26 ng/mL for 10-DAB III, baccatin III, 7-xyl-10-DAT, 10-DAT, cephalomannine, paclitaxel, and 7-epi-10-DAT, respectively. Linearity was confirmed over the whole calibration range (0.07-45, 0.058-37.5, 0.058-37.5, 0.056-36.3, 0.053-33.8, 0.057-37.5, and 0.06-38.8 microg/mL for 10-DAB III, baccatin III, 7-xyl-10-DAT, 10-DAT, cephalomannine, paclitaxel, and 7-epi-10-DAT, respectively) with coefficients higher than 0.9903. The inter- and intra-day precision of taxoids ranged from 2.86% to 6.31% for retention time and ranged from 3.91% to 7.33% for peak area. The recovery rates of this method were higher than 94.32% for 10-DAB III, 94.68% for baccatin III, 93.65% for 7-xyl-10-DAT, 93.29% for 10-DAT, 92.91% for cephalomannine, 93.41% for paclitaxel, and 93.06% for 7-epi-10-DAT, respectively.

Biotransformation of polydatin to resveratrol in Polygonum cuspidatum roots by highly immobilized edible Aspergillus niger and Yeast.

A new biotransformation method of producing resveratrol with co-immobilized edible Aspergillus niger and Yeast (AY) was investigated. The biotransformation conditions were optimized for the resveratrol production under 30 °C, pH 6.5, 2 days, liquid-solid ratio 12:1 (mL/g), the yield of resveratrol reached 33.45 mg/g, which increased 11-fold to that of untreated one. The conversion rate of polydatin reached 96.7%. The residual activity of immobilized microorganism was 83.2% after used for 15 runs. The developed method could be an effectively alternative biotransformation method for producing resveratrol from the plants.

Endophytic fungi from pigeon pea [Cajanus cajan (L.) Millsp.] produce antioxidant Cajaninstilbene acid.

In this study, novel endophytic fungi producing cajaninstilbene acid (CSA) from pigeon pea [ Cajanus cajan (L.) Millsp.] were investigated and screened. CSA has prominent pharmacological activities. A total of 110 endophytic fungi isolates were grouped into 8 genera on the basis of morphological characteristics, and CSA-producing fungi were screened by liquid chromatography-tandem mass spectrometry (LC-MS/MS). According to ITS-rDNA sequences analysis, the CSA-producing fungi were identified as Fusarium solani (ERP-07), Fusarium oxysporum (ERP-10), and Fusarium proliferatum (ERP-13), respectively. The amount of CSA produced by the ERP-13 reached 504.8 ± 20.1 μg/L or 100.5 ± 9.4 μg/g dry weight of mycelium. In a DPPH radical-scavenging assay, when the concentration of fungal CSA was 500 μg/mL, inhibition percentage could reach 80%, which was almost the same as that of standard CSA. This study first reported the natural antioxidant CSA from endophytic fungi F. solani and F. proliferatum isolated from pigeon pea.

Cajaninstilbene acid (CSA) exerts cytoprotective effects against oxidative stress through the Nrf2-dependent antioxidant pathway.

Cajaninstilbene acid (CSA), an active compound separated from pigeon pea leaves, possesses the highly efficient antioxidant activities. Transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) is an important regulator of cellular oxidative stress. This study examined the role of Nrf2 in CSA-mediated antioxidant effects on human hepatocarcinoma (HepG2) cell line. The generation of reactive oxygen species (ROS) upon H2O2 and CSA treatment was lower than that of H2O2 alone. CSA activated Nrf2 as evaluated by Western blotting. A luciferase reporter assay also demonstrated that CSA-activated signaling resulted in the increased transcriptional activity of Nrf2 through binding to the antioxidant response element (ARE) enhancer sequence. Our study indicated that treatment of HepG2 cells with CSA induces Nrf2-dependent ARE activity and gene expression of heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase modifier subunits by activation of PI3K/AKT, ERK and JNK signaling pathways. Inhibition of Nrf2 by siRNA reduced CSA-induced upregulation of these Nrf2-related enzymes. These results suggest that the Nrf2/ARE pathway plays an important role in the regulation of CSA-mediated antioxidant effects in HepG2 cells.

Variation of active constituents and antioxidant activity in pyrola [P. incarnata Fisch.] from different sites in Northeast China

The variation of antioxidant activity and active components in pyrola [Passiflora incarnata Fisch.] from eight sites in Northeast China were investigated. Total phenolic and flavonoid contents were determined and varied within the range of 39.66-181.48 mg/g and 2.47-22.11 mg/g, respectively. Antioxidant activities were determined by scavenging activity against DPPH and ABTS, by a reducing power test and by a β-carotene-linoleic acid bleaching test. The IC50 of Tahe samples determined by the DPPH test was 0.106±0.006 mg/mL which was very close to that of Vc (0.076±0.004 mg/mL). The Tahe samples had good antioxidant activity. Principal component activity analysis indicated that the Tahe samples of P. incarnata had the highest potential antioxidant properties, and may be a valuable antioxidant natural resource in the northeast of China.