LC-ESI-MS/MS Simultaneous Analysis Method Coupled with Cation-Exchange Solid-Phase Extraction for Determination of Pyrrolizidine Alkaloids on Five Kinds of Herbal Medicines.

BACKGROUND: Pyrrolizidine alkaloids (PAs) are naturally occurring plant toxins associated with potential hepatic and carcinogenic diseases in humans and animals. The concern over PAs has increased as the consumption of herbal medicines has increased. OBJECTIVE: This study aimed to develop and validate a sensitive analytical method to determine 28 PAs in five herbal medicines using liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). Additionally, this study identified and quantified the amount of PAs in 10 samples of each herbal medicine. METHODS: The pretreatment in the proposed LC-MS/MS analysis comprised solvent extraction using 0.05M H2SO4 in 50% methanol and clean-up step using an mixed-mode cationic exchange (MCX)-solid-phase extraction (SPE) cartridge. The PA contents in herbal medicines were measured by using the developed method. RESULTS: The proposed method had recoveries ranging from 72.5-123.7% for the Atractylodis Rhizoma Alba, 70.6-151.7% for Alba Chrysanthmi Flos, 80.6-130.9% for Leonuri Herba, 70.3-122.9% for Gastrodiae Rhizoma, and 67.1-106.9% for Glycyrrhizae Radix. Even though a few samples showed recoveries in unsatisfactory values, the proposed method indicated entirely sufficient recoveries and precision in most samples. In monitoring results, only Leonuri Herba contained two PAs, which indicated Retrorsine (4/10) of 84.7-120.9 µg/kg and Senkirkine (10/10) of 60.9-170.7 µg/kg. CONCLUSION: The results obtained from this study demonstrate that the proposed method is fit for purpose to determine 28 PAs in herbal medicines. Therefore it could serve as a regulatory method capable of being used for controlling the risks of PAs in certain medicinal plants and dietary supplements. HIGHLIGHTS: An LC-MS/MS method for the determination of 28 pyrrolizidine alkaloids in herbal medicines was developed and validated through this study. The proposed method is considered as an useful method for monitoring pyroolizidine alkaloids in herbal medicines.

Peripheral nerve injury and sensitization underlie pain associated with oral cancer perineural invasion.

Cancer invading into nerves, termed perineural invasion (PNI), is associated with pain. Here, we show that oral cancer patients with PNI report greater spontaneous pain and mechanical allodynia compared with patients without PNI, suggesting that unique mechanisms drive PNI-induced pain. We studied the impact of PNI on peripheral nerve physiology and anatomy using a murine sciatic nerve PNI model. Mice with PNI exhibited spontaneous nociception and mechanical allodynia. Perineural invasion induced afterdischarge in A high-threshold mechanoreceptors (HTMRs), mechanical sensitization (ie, decreased mechanical thresholds) in both A and C HTMRs, and mechanical desensitization in low-threshold mechanoreceptors. Perineural invasion resulted in nerve damage, including axon loss, myelin damage, and axon degeneration. Electrophysiological evidence of nerve injury included decreased conduction velocity, and increased percentage of both mechanically insensitive and electrically unexcitable neurons. We conclude that PNI-induced pain is driven by nerve injury and peripheral sensitization in HTMRs.

Polyphenol-enriched Vaccinium uliginosum L. fractions reduce retinal damage induced by blue light in A2E-laden ARPE19 cell cultures and mice.

Polyphenols exert beneficial effects on vision. We hypothesized that polyphenol components of Vaccinium uliginosum L. (V.U.) extract protect retinal pigment epithelial (RPE) cells against blue light-induced damage. Our aim was to test extracts containing polyphenol components to ascertain effects to reduce damage against blue light in RPEs. We measured the activity in fractions eluted from water, ethanol, and HP20 resin (FH), and found that the FH fraction had the highest beneficial activity. We isolated the individual active compounds from the FH fraction using chromatographic techniques, and found that FH contained flavonoids, anthocyanins, phenyl propanoids, and iridoids. Cell cultures of A2E-laden ARPE-19 exposed to blue light after treatment with V.U. extract fractions and their individual constituents indicated improvement. V uliginosum L extract fractions and constituent compounds significantly reduced A2E photo-oxidation-induced RPE cell death and inhibited intracellular A2E accumulation. Furthermore, Balb/c male mice were exposed to blue light at 10000 lux for 1 h/d for 2 weeks to induce retinal damage. One week after the final blue light exposure, retinal damage evaluated revealed that the outer nuclear layer thickness and nuclei count were improved. Histologic examination of murine photoreceptor cells demonstrated that FH, rich in polyphenols, inhibited the loss of outer nuclear layer thickness and nuclei. Our findings suggest that V.U. extract and eluted fractions are a potential source of bioactive compounds that potentially serve a therapeutic approach for age-related macular degeneration.

Extract of Salvia miltiorrhiza (Danshen) induces Nrf2-mediated heme oxygenase-1 expression as a cytoprotective action in RAW 264.7 macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Danshen (Salvia miltiorrhiza) is widely used in traditional herbal medicines for relief of a variety of symptoms related to complications arising from vascular diseases such as hypertension, diabetes, and atherosclerosis. Induction of heme oxygenase-1 (HO-1) expression protects against oxidative stress-induced cell damage, which plays an important role in cytoprotection in a variety of pathological models. MATERIALS AND METHODS: In the present study, we investigated the effect of Danshen on the up-regulation of HO-1, an inducible and cytoprotective enzyme in RAW 264.7 macrophages. Molecular mechanisms underlying the effects, especially protective effects, was elucidated by analyzing the activation of transcription factors and their upstream signalling, and by evaluating the inhibitory effect of HO-1 on ROS production. RESULTS: Danshen induced HO-1 mRNA expression and protein production, and nuclear translocation of NF-E2-related factor 2 in RAW 264.7 macrophages. Pharmacological inhibitors of PI3K/Akt and MEK1 attenuated HO-1 induction in Danshen-stimulated RAW 264.7 macrophages. Furthermore, Danshen pretreatment reduced intracellular production of reactive oxygen species after stimulation with hydrogen peroxide; this effect was reversed by the HO-1 inhibitor ZnPP. CONCLUSION: Danshen induced HO-1 expression through PI3K/Akt-MEK1-Nrf2 pathway and reduced intracellular production of reactive oxygen species via induction of HO-1 expression. The results support a role of HO-1 in the cytoprotective effect of Danshen.