8-shogaol derived from dietary ginger alleviated acute and inflammatory pain by targeting TRPV1.

Ginger, a well-known spice plant, has been used widely in medicinal preparations for pain relief. However, little is known about its analgesic components and the underlying mechanism. Here, we ascertained, the efficacy of ginger ingredient 8-Shogaol (8S), on inflammatory pain and tolerance induced by morphine, and probed the role of TRPV1 in its analgesic action using genetic and electrophysiology approaches. Results showed that 8S effectively reduced nociceptive behaviors of mice elicited by chemical stimuli, noxious heat as well as inflammation, and antagonized morphine analgesic tolerance independent on opioid receptor function. Genetic deletion of TRPV1 significantly abolished 8S' analgesia action. Further calcium imaging and patch-clamp recording showed that 8S could specifically activate TRPV1 in TRPV1-expressing HEK293T cells and dorsal root ganglion (DRG) neurons. The increase of [Ca2+]i in DRG was primarily mediated through TRPV1. Mutational and computation studies revealed the key binding sites for the interactions between 8S and TRPV1 included Leu515, Leu670, Ile573, Phe587, Tyr511, and Phe591. Further studies showed that TRPV1 activation evoked by 8S resulted in channel desensitization both in vitro and in vivo, as may be attributed to TRPV1 degradation or TRPV1 withdrawal from the cell surface. Collectively, this work provides the first evidence for the attractive analgesia of 8S in inflammatory pain and morphine analgesic tolerance mediated by targeting pain-sensing TRPV1 channel. 8S from dietary ginger has potential as a candidate drug for the treatment of inflammatory pain.

Epidemiological and Molecular Study on Tick-Borne Pathogens in Argun Port Area Near the Chinese-Russian Border.

Objective: We aim to investigate the species composition of ticks and the pathogen characteristics they carry in the Argun port area of the China-Russia border. Materials and Methods: Ticks were collected in surrounding grassland, mixed forest land, and other different habitats around the Argun port area at the Sino-Russian Border of Inner Mongolia in China in April 2019. The presence of 16 potential pathogens, including Yersinia Pestis, Francisella tularensis, Coxiella burnetii (Cb), Anaplasma sp. (Ap), spotted fever group rickettsiae (SFG Rk), Borrelia sp. (Bl), Leptospira, Bartonella spp., Babesia, Crimean-Congo hemorrhagic fever virus, tick-borne encephalitis virus, Bhanja virus, West Nile Virus, severe fever with thrombocytopenia syndrome bunyavirus, Hantaan virus, and bocavirus (boca) was analyzed by polymerase chain reaction. The DNA and amino acid sequences of tick-borne pathogens were compared for homology, and the phylogenetic trees were constructed by using Mega and Lasergene software. Results: A total of 210 ticks were collected and they belonged to three species: Dermacentor nuttalli, Ixodes persulcatus, and Haemaphysalis verticalis. Among them, 165 (78.57%) ticks tested positive for 5 pathogens, namely Ap, SFG Rk, Cb, Bl, and boca. Fifteen (7.14%) ticks were detected coinfection with two pathogens, and none were coinfected with three or more pathogens. Conclusion: This study shows the prevalence of at least five tick-borne pathogens in Argun, and there is a risk of coinfection by two pathogens in one tick. This study reveals the great importance of controlling tick-borne diseases in this region.

A red-light-activated sulfonamide porphycene for highly efficient photodynamic therapy against hypoxic tumor.

Photodynamic therapy (PDT) is an emerging alternative cancer treatment modality that utilizes photo-sensitivity to cause cell death upon photo-irradiation. However, PDT efficiency has been hampered by tumor hypoxia, blue-shifted excitation wavelengths, and the high dark toxicity of photo-sensitizers. We designed and synthesized two novel porphycene-based photosensitizers (TBPoS-OH and TBPoS-2OH) with potent photo-cytotoxicity and a LD50 in the nM range under both normoxic and hypoxic conditions in a variety of cell types after photo-irradiation (λ = 640 ± 15 nm). Further studies showed fast-cellular uptake for TBPoS-OH that localized lysosomes and subsequently induced cell apoptosis via the lysosomal-mitochondrial pathway. Moreover, TBPoS-OH significantly reduced tumor growth in two xenografted mouse models bearing melanoma A375 and B16 cells. Finally, TBPoS-OH exhibited no obvious immunogenicity and toxicity to blood cells and major organs in mice. These data demonstrated that these two porphycene-based photosensitizers, especially TBPoS-OH, could be developed as a potential PDT modality.

Umbelliferone ameliorates renal function in diabetic nephropathy rats through regulating inflammation and TLR/NF-κB pathway.

Diabetic nephropathy (DN) is a leading cause of renal failure, contributing to severe morbidity and mortality in diabetic patients. Umbelliferae (Umb) has been well characterized to exert protective effects in diabetes. However, the action and mechanism of Umb in DN remains unclear. In this work, we studied the effect of Umb in a streptozotocin (STZ)-induced DN rat model and explore its underlying mechanism. DN rats were treated withUmb (20, 40 mg·kg-1) orirbesartan (15 mg·kg-1) for 4 weeks. Levels of serum glucose, insulin, blood uric acid, creatinine, triglycerides (TG) and total cholesterol (TC) were measured bycommercial assay kits, respectively. Histopathological changes andinflammatory cytokine levels including IL-6, IL-1ß and TNF-α in the kidney were also evaluated. Alterations in the expression of podocin, CD2AP and TLR/NF-κB were assessed by western blotting. Our results showed that Umb reduced renal injury in DN rat model, as evidenced by the decrease in blood glucose, 24 h urinary protein, serum creatinine, and blood uric acid. Umb also significantly ameliorated the renal histopathological alteration, and down-regulated the expression of epithelial-to-mesenchymal transition-related molecular markers podocin and CD2AP. Moreover, Umb inhibited TLR2, TLR4, MyD88 expressions, NF-κB activation and considerably reduced levels of other downstream inflammatory molecules (TNF-α, IL-6, IL-1ß). These findings indicated that Umb improved renal function through regulating inflammation and TLR/NF-κB pathway, suggesting the potential efficacy of Umb in DN treatment.

Ultra-high performance liquid chromatography coupled with quadrupole/time of flight mass spectrometry based chemical profiling approach for the holistic quality control of complex Kang-Jing formula preparations.

The Kang-Jing (KJ) formula is a compound preparation made from 12 kinds of herbs. So far, four different methods (M1-M4) have been documented for KJ preparation, but the influence of preparation methods on the holistic quality of KJ have remained unknown. In this study, a strategy was proposed to investigate the influence of different preparation methods on the holistic quality of KJ using ultra-high performance liquid chromatography coupled with quadrupole/time of flight mass spectrometry (UHPLC-QTOF-MS/MS) based chemical profiling. A total of 101 compounds mainly belonging to flavonoids, tanshinones, monoterpene glycosides, triterpenoid saponins, alkaloids, phenolic acids and volatile oils, were identified. Among these compounds, glaucine was detected only in M3/M4 samples, while two dehydrocorydaline isomers merely detected in M2/M3/M4 samples. Tetrahydrocolumbamine, ethylic lithospermic acid, salvianolic acid E and rosmarimic acid were only detected in M1/M3/M4 samples. In the subsequent quantitative analysis, 12 major compounds were determined by UHPLC-MS/MS. The proposed method was validated with respect to linearity, accuracy, precision and recovery. It was found that the contents of marker compounds varied significantly in samples prepared by different methods. These results demonstrated that preparation method does significantly affect the holistic quality of KJ. UHPLC-QTOF-MS/MS based chemical profiling approach is efficient and reliable for comprehensive quality evaluation of KJ. Collectively, this study provide the chemical evidence for revealing the material basis of KJ, and establish a simple and accurate chemical profiling method for its quality control.

Anti-inflammatory and anti-arthritic effects of Guge Fengtong Formula: in vitro and in vivo studies.

Rheumatoid arthritis (RA) is the most common inflammatory arthritis and a major cause of disability. Presently, the clinical therapeutic medicines for inflammatory and arthritic diseases are unsatisfactory due to severe adverse effects or ineffectiveness. The Guge Fengtong formula (GGFT), containing the standardized extracts of Dioscoreae Nipponicae Rhizoma, Spatholobi Caulis, and Zingiberis Rhizoma, has long been used for RA treatment by Chinese doctorsin China. However, the detailed anti-inflammatory and anti-arthritic activity of GGFT has not been reported so far. In the present work, we aimed to evaluate the anti-inflammatory and anti-arthritic effects of GGFT using three in vivo animal models, and tried to uncover its preliminarythe underlying mechanism of action mechanism in RAW 264.7 macrophages. The obtained results indicated that GGFT significantly attenuated ear edema, decreased carrageenan-induced paw edema, reduced the arthritis score, and reversed the weight loss of the complete Freund's adjuvant (CFA)CFA-injected rats. Additionally, marked decrease of in synovial inflammatory infiltration and synovial lining hyperplasia in the joints and decline of inflammatory factors (TNF-α and IL-1ß) in the serum were observed in the GGFT-treated rats. In lipopolysaccharide-activated RAW264.7 macrophages, GGFT reduced the production of NO, PGE2, and IL-6, and inhibited the expression of iNOS, COX-2, and NF-κB expression. Our results demonstrated that GGFT possessed considerable anti-inflammatory activity and have had potential therapeutic effects on adjuvant induced arthritis in rats, which provided providing experimental evidences for its traditional application in the treatment of RA and other inflammatory diseases.

Ancient Chinese Formula Qiong-Yu-Gao Protects Against Cisplatin-Induced Nephrotoxicity Without Reducing Anti-tumor Activity.

Cisplatin is a highly effective anti-cancer chemotherapeutic agent; however, its clinical use is severely limited by serious side effects, of which nephrotoxicity is the most important. In this study, we investigated whether Qiong-Yu-Gao (QYG), a popular traditional Chinese medicinal formula described 840 years ago, exhibits protective effects against cisplatin-induced renal toxicity. Using a mouse model of cisplatin-induced renal dysfunction, we observed that pretreatment with QYG attenuated cisplatin-induced elevations in blood urea nitrogen and creatinine levels, ameliorated renal tubular lesions, reduced apoptosis, and accelerated tubular cell regeneration. Cisplatin-mediated elevations in tumor necrosis factor alpha (TNF-α) mRNA, interleukin-1 beta (IL-1ß) mRNA, and cyclooxygenase-2 (COX-2) protein in the kidney were also significantly suppressed by QYG treatment. Furthermore, QYG reduced platinum accumulation in the kidney by decreasing the expression of copper transporter 1 and organic cation transporter 2. An in vivo study using implanted Lewis lung cancer cells revealed that concurrent administration of QYG and cisplatin did not alter the anti-tumor activity of cisplatin. Our findings suggest that the traditional Chinese medicinal formula QYG inhibits cisplatin toxicity by several mechanisms that act simultaneously, without compromising its therapeutic efficacy. Therefore, QYG may be useful in the clinic as a protective agent to prevent cisplatin-induced nephrotoxicity.

Direct analysis of 18 flavonol glycosides, aglycones and terpene trilactones in Ginkgo biloba tablets by matrix solid phase dispersion coupled with ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry.

Analysis and quality control of Ginkgo biloba have been comprehensively studied. However, little attention has been devoted to the simultaneous extraction and analysis of flavonols and terpene trilactones, especially for direct quantification of flavonol glycosides. This work described a rapid strategy for one-step extraction and quantification of the components. A matrix solid phase dispersion (MSPD) method was designed for the extraction of ginkgo ingredients and compared with the heat-reflux and ultrasonic extraction methods. An ultra-high performance liquid chromatography (UHPLC)-tandem-triple-quadrupole-mass spectrometry (QQQ-MS) method was developed for detection of the 18 components, including 10 original flavonol glycosides, 3 aglycones, and 5 lactones. Subsequently, the proposed strategy was used for the analysis of 12 G. biloba tablets. Results showed that MSPD produced comparable extraction efficiency but consumed less time and required lower solvent volumes compared with conventional methods. Without hydrolysis, the concentration detected was much closer to the original in the sample. The total flavonol glycoside contents in ginkgo tablets ranged from 3.59 to 125.21µgmg(-1), and the terpene trilactone varied from 3.45 to 57.8µgmg(-1) among different manufacturers. In conclusion, the proposed MSPD and UHPLC-QQQ-MS is rapid and sensitive in providing comprehensive profile of chemical constituents especially the genuine flavonol glycosides for improved quality control of ginkgo products.

Tectorigenin Attenuates Palmitate-Induced Endothelial Insulin Resistance via Targeting ROS-Associated Inflammation and IRS-1 Pathway.

Tectorigenin is a plant isoflavonoid originally isolated from the dried flower of Pueraria thomsonii Benth. Although its anti-inflammatory and anti-hyperglycosemia effects have been well documented, the effect of tectorigenin on endothelial dysfunction insulin resistance involved has not yet been reported. Herein, this study aims to investigate the action of tectorigenin on amelioration of insulin resistance in the endothelium. Palmitic acid (PA) was chosen as a stimulant to induce ROS production in endothelial cells and successfully established insulin resistance evidenced by the specific impairment of insulin PI3K signaling. Tectorigenin effectively inhibited the ability of PA to induce the production of reactive oxygen species and collapse of mitochondrial membrane potential. Moreover, tectorigenin presented strong inhibition effect on ROS-associated inflammation, as TNF-α and IL-6 production in endothelial cells was greatly reduced with suppression of IKKß/NF-κB phosphorylation and JNK activation. Tectorigenin also can inhibit inflammation-stimulated IRS-1 serine phosphorylation and restore the impaired insulin PI3K signaling, leading to a decreased NO production. These results demonstrated its positive regulation of insulin action in the endothelium. Meanwhile, tectorigenin down-regulated endothelin-1 and vascular cell adhesion molecule-1 overexpression, and restored the loss of insulin-mediated vasodilation in rat aorta. These findings suggested that tectorigenin could inhibit ROS-associated inflammation and ameliorated endothelial dysfunction implicated in insulin resistance through regulating IRS-1 function. Tectorigenin might have potential to be applied for the management of cardiovascular diseases involved in diabetes and insulin resistance.

Highly efficient sample preparation and quantification of constituents from traditional Chinese herbal medicines using matrix solid-phase dispersion extraction and UPLC-MS/MS.

In this work, a rapid and simple method based on matrix solid-phase dispersion (MSPD) and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed. Guge Fengtong preparation (GGFT), a traditional Chinese herbal medicine, was investigated for validation, and eight major constituents were determined including four saponins (protodioscin, protogracillin, pseudoprotodioscin and dioscin) and four gingerols (6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol). Response surface methodology and desirability function were employed to optimize the extraction conditions, such as dispersant, dispersant/sample ratio, solvent concentration, and elution volume, of MSPD. Results showed that MSPD using C18 (1.75 g) as the dispersant material and methanol (89%, v/v) as the eluting solvent (12.00 mL) resulted in a high extraction efficiency. MSPD extraction had the advantages of combining extraction and clean-up in a single step, was less time consuming and required lower solvent volumes compared with conventional methods. Quantification of chemical compounds from GGFT preparations were performed using UPLC-MS/MS in multiple-reaction monitoring mode. The proposed method afforded a low limit of detection ranging from 0.02 to 0.40 ng for saponins and gingerols. For all the analytes, recoveries ranged from 80.9% to 103% and repeatabilities were acceptable with relative standard deviations of less than 6.81%. The proposed MSPD-UPLC-MS/MS method was successfully utilized to analyze five batches of GGFTs, and the results demonstrated that this method is simple, efficient and has potential to be applied for the quality control of herbal preparations.

Application of an efficient strategy for discovery and purification of bioactive compounds from Chinese herbal medicines, a case study on the Puerariae thomsonii Flos.

In this study, an efficient strategy based on bioassay-guided fractionation, high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q/TOF-MS) and high-speed counter-current chromatography (HSCCC) was established to screen and purify bioactive compounds from Chinese herbal medicines (CHMs). This screening system was efficient and successfully applied to reveal anti-prostate cancer candidates from Puerariae thomsonii Flos. As a result, an active fraction with strong in vitro anti-prostate cancer activity was obtained, and the main compounds in the fraction were purified by HSCCC, giving 82 mg of tectoridin, 36 mg of tectorigenin-7-O-[ß-D-xylopyranosyl-(1→6)-ß-D-glucopyranoside and 64 mg of tectorigenin. Among them, tectorigenin, possessing the highest anti-prostate cancer activity with IC50 value of 0.08 µM, has priority to be lead compound. The results of this work demonstrated that the developed method was efficient and could be employed for the rapid screening, identification and purification of active components from CHMs.

The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis.

Cervical cancer is one of the most common cancers among women in the world. 6-Shogaol is a natural compound isolated from the rhizome of ginger (Zingiber officinale). In this paper, we demonstrated that 6-shogaol induced apoptosis and G2/M phase arrest in human cervical cancer HeLa cells. Endoplasmic reticulum stress and mitochondrial pathway were involved in 6-shogaol-mediated apoptosis. Proteomic analysis based on label-free strategy by liquid chromatography chip quadrupole time-of-flight mass spectrometry was subsequently proposed to identify, in a non-target-biased manner, the molecular changes in cellular proteins in response to 6-shogaol treatment. A total of 287 proteins were differentially expressed in response to 24 h treatment with 15 µM 6-shogaol in HeLa cells. Significantly changed proteins were subjected to functional pathway analysis by multiple analyzing software. Ingenuity pathway analysis (IPA) suggested that 14-3-3 signaling is a predominant canonical pathway involved in networks which may be significantly associated with the process of apoptosis and G2/M cell cycle arrest induced by 6-shogaol. In conclusion, this work developed an unbiased protein analysis strategy by shotgun proteomics and bioinformatics analysis. Data observed provide a comprehensive analysis of the 6-shogaol-treated HeLa cell proteome and reveal protein alterations that are associated with its anticancer mechanism.

Platelet glycoprotein IIb HPA-3 a/b polymorphism is associated with native arteriovenous fistula thrombosis in chronic hemodialysis patients.

OBJECTIVE: The aim of this study was to investigate the association of Glycoprotein IIb (GPIIb) human platelet antigen-3 (HPA-3) a/b polymorphism with end-stage renal disease (ESRD) on hemodialysis (HD) and native Arteriovenous fistula (AVF) thrombosis. METHODS: The polymorphism in the GPIIb subunit of the receptor HPA-3 (a and b alleles) was identified by polymerase chain reaction with sequence-specific primers (PCR-SSP) in 145 HD patients and 120 healthy controls from a Chinese Han population. The HD patients were classified into two groups: G1 and G2. G1 included 65 HD patients presented at least one AVF thrombosis episode and G2 included 80 HD patients without any episode of AVF thrombosis. RESULTS: There were no significant differences in either HPA-3 a/b genotypes (aa, ab, and bb) frequency distribution (p = 0.396) or allele (a and b) frequency distribution (p = 0.146) between HD patients and control groups. However, there were significant differences in both HPA-3 a/b genotypes (aa, ab, and bb) distribution (χ(2) = 6.127, p = 0.047) and allele (a and b) frequency distribution (χ(2) = 5.954, p = 0.015) between G1 and G2. The relative risk of native AVF dysfunction in ab + bb patients compared with that of aa patients was 2.31 (95% confidence interval: 1.18-4.52). CONCLUSIONS: These findings suggest an association between AVF thrombosis and the HPA-3b allele, and it is likely that HPA-3 a/b polymorphisms could be useful markers for potential risk of native AVF thrombosis in HD patients.

Ultrasonic/microwave assisted extraction and diagnostic ion filtering strategy by liquid chromatography-quadrupole time-of-flight mass spectrometry for rapid characterization of flavonoids in Spatholobus suberectus.

Spatholobus suberectus is a widely used herb in traditional medicine for the treatment of blood stasis syndrome and related diseases. In this work, a potential ultrasonic/microwave assisted extraction (UMAE) method was developed for efficient sample pretreatment, and a diagnostic ion filtering strategy with liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) was established for rapid characterization of flavonoids in S. suberectus. The factors of UMAE influencing the extraction yield of flavonoids of S. suberectus were evaluated. The optimal conditions were determined as: microwave power of 300 W, extraction time of 450 s, 70% methanol as extraction solvent, solvent to solid ratio of 20 mL/g, ultrasound power of 50 W, extraction temperature of 80 °C, and one extraction cycle. Compared with commonly used extraction methods, UMAE showed higher efficiency and shorter extraction time for sample preparation. Subsequently, the major diagnostic ions and fragmentation pathways of flavonoids in Q-TOF-MS were summarized with available reference compounds. Using a new diagnostic ion filtering strategy, a rapid screening and identification of thirty-eight compounds was achieved in real S. suberectus samples. The results of this study clearly demonstrate the potential of UMAE for efficient extraction and LC-Q-TOF-MS for rapid and sensitive structural elucidation of flavonoids in S. suberectus, and open perspectives for similar studies on other medicinal herbs.

Oleanane-type triterpene glucuronides from the roots of Glycyrrhiza uralensis Fischer.

Investigation of characteristic constituents of the roots of Glycyrrhiza uralensis Fischer led to isolation of four new triterpene glucuronides, namely uralsaponins C-F (1-4), an artificial product, namely the methyl ester of glycyrrhizin (5), as well as six known triterpene glucuronides (6-11). These new compounds were identified by 1D and 2D NMR spectroscopic analysis. The cytotoxicity of the selected compounds and their aglycones were evaluated against HeLa and MCF-7 cancer cell lines, and the preliminary structure-activity relationship was also elucidated.

Simultaneous determination of twelve bioactive constituents in Buyang Huanwu decoction by HPLC-DAD-ELSD and HPLC-TOF/MS.

Buyang Huanwu Decoction (BYHYD) is a classical traditional Chinese medicinal prescription that has been widely used for treating cerebrovascular illnesses for hundreds of years. In this study, a comprehensive analytical method has been developed for quantitative analysis of the major constituents in BYHWD. This method was based on high-performance liquid chromatography coupled to a diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) on a common reverse-phase C(18) column. HPLC coupled with on-line time-of-flight mass spectrometry (HPLC-TOF/MS) was additionally adopted to provide further validation for the constituents. It was found that 0.3% aqueous formic acid and acetonitrile was the optimum mobile phase for gradient elution. This method, which showed excellent precision and accuracy, was successfully applied to quantify the bioactive constituents in six BYHWD products. The validated HPLC-DAD-ELSD method, together with the HPLC-TOF/MS analysis, provided a new basis for assessing the quality of traditional Chinese medicinal prescription consisting of many bioactive components.

Rapid separation and identification of 54 major constituents in Buyang Huanwu decoction by ultra-fast HPLC system coupled with DAD-TOF/MS.

Buyang Huanwu Decoction (BYHWD), is a well-known traditional Chinese preparation consisting of Radix Astragali, Radix Angelicae Sinensis, Rhizoma Ligustici Chuanxiong, Radix paeoniae Rubra, Flos Carthami, Semen Persicae and Lumbricus. An ultra-fast high-performance liquid chromatography (HPLC) method coupled with diode array detection (DAD) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF/MS) has been developed for rapid separation and structural identification of constituents in BYHWD. Using an ultra-fast HPLC system with short columns (4.6 x 50 mm, 1.8 microm), the total analysis time for this complex prescription is less than 30 min. With various fragmentor voltages in TOF/MS, accurate mass measurements (less than 5 ppm error) for molecular ions and characteristic fragment ions could represent reliable identification criteria for these compounds. Fifty-four major constituents from BYHWD sample, including four C-glycosyl quinochalcones, four flavonoid O-glycosides, sixteen isoflavones, six monoterpene glycosides, eight saponins, four organic acids and five amino acids, were identified or tentatively characterized based on their retention times, DAD and TOF/MS data. All the compounds were further assigned in the seven individual crude drugs. In conclusion, the ultra-fast HPLC with DAD-TOF/MS is a highly useful and efficient technique to separate and identify constituents in complex matrices of herbal medicines or preparations.

Qualitative and quantitative analysis of Radix Astragali products by fast high-performance liquid chromatography-diode array detection coupled with time-of-flight mass spectrometry through dynamic adjustment of fragmentor voltage.

A novel fast high-performance liquid chromatography (HPLC) method coupled with diode array detection (DAD) and time-of-flight mass spectrometry (TOF/MS) was developed for qualitative and quantitative analysis of Radix Astragali products. The potential of fast HPLC on 1.8-microm particles was compared with the performance of HPLC on conventional 5-microm particles columns. Significant advantages of fast HPLC include high-speed chromatographic separation, four times faster than HPLC with conventional columns, and great enhancement in sensitivity with limits of detection low to 0.001 ng. With dynamic adjustment of fragmentor voltage in TOF/MS, an efficient transmission of the ions was achieved to obtain the best sensitivity and abundant fragmentation. By accurate mass measurements within 5 ppm error for each molecular ion and subsequent fragment ions, a reliable identification and differentiation of six major saponins including two groups of isomers and twelve main isoflavonoids was described here for the first time. For quantitative analysis by fast HPLC-TOF/MS, linearity of response over two orders of magnitude was demonstrated (r(2)>0.99) for all analytes. Intra-day reproducibility was below 3% RSD and inter-day values were below 5% RSD. A good correlation (slope=1.1108, r(2)=0.9853) was observed for accuracy test. It is concluded that the fast and sensitive HPLC-DAD-TOF/MS is powerful in qualitative and quantitative analysis of complex herbal medicines in terms of time savings, sensitivity, selectivity, precision, accuracy as well as increasing sample throughout and lowering solvent consumption.

Rapid and sensitive screening and characterization of phenolic acids, phthalides, saponins and isoflavonoids in Danggui Buxue Tang by rapid resolution liquid chromatography/diode-array detection coupled with time-of-flight mass spectrometry.

A novel rapid resolution liquid chromatography (RRLC) method coupled with diode-array detection (DAD) and time-of-flight mass spectrometry (TOFMS) in both positive and negative modes has been developed for quick and sensitive identification of the major compounds in Danggui Buxue Tang (DBT) preparation. Significant advantages of the use of RRLC with 1.8-microm porous particles include the much higher speed of chromatographic separation and great enhancement in sensitivity, compared with the conventional high-performance liquid chromatography (HPLC). With dynamic adjustment of the key role as fragmentor voltage in TOFMS, an efficient transmission of the ions was achieved to obtain the best sensitivity for providing the molecular formula for each analyte, and abundant fragment ions for structural information. The structural characterization of the major compounds in DBT was elucidated with authentic standards by DAD-TOF/MS, including phenolic acids, phthalides, saponins and isoflavonoids. The targets were rapidly screened from the complicated DBT matrix using a narrow mass window of 0.01 Da to restructure extracted ion chromatograms. By accurate mass measurements within 3 ppm error for each molecular ion and subsequent fragment ions, ten phenolic acids and phthalides including three groups of isomers, thirteen major saponins with a 20,24-epoxy-9,19-cyclolanostane-3,6,16,25-tetrol skeleton, sixteen isoflavonoids, corresponding glycosides, malonylglycosides, and acetylglycosides were identified in DBT preparation. The appropriate fragmentation pathways for them were also proposed based on definite elemental composition of the fragment ions.