Yichen Hu

GC–MS combined with chemometric techniques for the quality control and original discrimination of Curcumae longae rhizome: Analysis of essential oils

Curcumae longae rhizome is a widely used traditional herb in many countries. Various geographical origins of this herb might lead to diversity or instability of the herbal quality. The objective of this work was to establish the chemical fingerprints for quality control and find the chemical markers for discriminating these herbs from different origins. First, chemical fingerprints of essential oil of 24 C. longae rhizome from four different geographical origins in China were determined by GC-MS. Then, pattern recognition techniques were introduced to analyze these abundant chemical data in depth; hierarchical cluster analysis was used to sort samples into groups by measuring their similarities, and principal component analysis and partial least-squares discriminate analysis were applied to find the main chemical markers for discriminating these samples. Curcumae longae rhizome from Guangxi province had the highest essential oil yield (4.32 ± 1.45%). A total of 46 volatile compounds were identified in total. Consistent results were obtained to show that C. longae rhizome samples could be successfully grouped according to their origins, and turmerone, ar-turmerone, and zingiberene were the characteristic components for discriminating these samples of various geographical origins and for quality control. This finding revealed that fingerprinting analysis based on GC-MS coupled with chemometric techniques could provide a reliable platform to discriminate herbs from different origins, which is a benefit for quality control.

Quality evaluation of Salvia miltiorrhiza Bge. by ultra high performance liquid chromatography with photodiode array detection and chemical fingerprinting coupled with chemometric analysis

UNLABELLED An ultra high performance liquid chromatography with photodiode array detection method is developed for the simultaneous quantitative determination of five water-soluble compounds including danshensu, protocatechualdehyde, rosmarinic acid, salvianolic acid B, and salvianolic acid A in Salvia miltiorrhiza Bge. SAMPLES Through method optimization, the five compounds all expressed good linearity (R(2) > 0.9990) in a wide concentration range together with satisfactory accuracy, precision, and stability. Moreover, through qualitative analysis of the chemical fingerprint combined with similarity analysis, hierarchical cluster analysis, principle component analysis, and partial least-squares discriminate analysis, we determined that the 13 batches of Salvia miltiorrhiza Bge. were similar in internal quality and the differences resulted from various cultivation environments, recovery elements, and others. Seen from the results of hierarchical cluster analysis and principle component analysis, the classification of 13 batches was in accordance, and partial least-squares discriminate analysis technique was more suitable than the principle component analysis model to provide a distinct classification of test samples on the basis of their different components. Moreover, a permutation test verified the rationality of partial least-squares discriminate analysis and variable importance plot showed that peaks 37 and 38 were the most significant variables in distinguishing the Salvia miltiorrhiza Bge. SAMPLES The idea of the quantitative and qualitative analysis of Salvia miltiorrhiza Bge. was convenient, sensitive, and comprehensive, which could be applied to evaluate the quality of more traditional Chinese medicines.

Aptamer-affinity column clean-up coupled with ultra high performance liquid chromatography and fluorescence detection for the rapid determination of ochratoxin A in ginger powder

Aptamers are single-stranded oligonucleotides with high affinity and specificity and are widely used in targets separation and enrichment. Here, an aptamer-affinity column (AAC) was firstly prepared in-house through a covalent immobilization strategy. Then, ochratoxin A (OTA) in ginger powder was absorbed and enriched using the new aptamer-based clean-up technology for the first time, and was further analyzed by ultra high performance liquid chromatography with fluorescence detection. After optimization, the average recoveries for blank samples spiked with OTA at 5, 15, and 45 μg/kg ranged from 85.36 to 96.83%. Furthermore, the AAC exhibited a similar accuracy as an immunoaffinity column to clean up OTA in ginger powder. Above all, it exhibited better reusability, twice that of the immunoaffinity column, had lower toxicity and cost, and took less time. Of 25 contaminated ginger powder samples, OTA contamination levels ranged from 1.51 to 4.31 μg/kg, which were lower than the European Union (EU) regulatory limits. All the positive samples were further confirmed by ultra-fast LC with MS/MS. In conclusion, the method of clean-up based on the AAC coupled to ultra-HPLC with fluorescence detection was rapid, specific, and sensitive for the quantitative analysis of OTA in a complex matrix.

Mechanisms of antifungal and anti-aflatoxigenic properties of essential oil derived from turmeric (Curcuma longa L.) on Aspergillus flavus.

The antifungal activity and potential mechanisms in vitro as well as anti-aflatoxigenic efficiency in vivo of natural essential oil (EO) derived from turmeric (Curcuma longa L.) against Aspergillus flavus was intensively investigated. Based on the previous chemical characterization of turmeric EO by gas chromatography-mass spectrometry, the substantially antifungal activities of turmeric EO on the mycelial growth, spore germination and aflatoxin production were observed in a dose-dependent manner. Furthermore, these antifungal effects were related to the disruption of fungal cell endomembrane system including the plasma membrane and mitochondria, specifically i.e. the inhibition of ergosterol synthesis, mitochondrial ATPase, malate dehydrogenase, and succinate dehydrogenase activities. Moreover, the down-regulation profiles of turmeric EO on the relative expression of mycotoxin genes in aflatoxin biosynthetic pathway revealed its anti-aflatoxigenic mechanism. Finally, the suppression effect of fungal contamination in maize indicated that turmeric EO has potential as an eco-friendly antifungal agent.

UFLC-ESI-MS/MS analysis of multiple mycotoxins in medicinal and edible Areca catechu

A robust, sensitive and reliable ultra fast liquid chromatography combined with electrospray ionization tandem mass spectrometry (UFLC-ESI-MS/MS) was optimized and validated for simultaneous identification and quantification of eleven mycotoxins in medicinal and edible Areca catechu, based on one-step extraction without any further clean-up. Separation and quantification were performed in both positive and negative modes under multiple reaction monitoring (MRM) in a single run with zearalanone (ZAN) as internal standard. The chromatographic conditions and MS/MS parameters were carefully optimized. Matrix-matched calibration was recommended to reduce matrix effects and improve accuracy, showing good linearity within wide concentration ranges. Limits of quantification (LOQ) were lower than 50 μg kg(-1), while limits of detection (LOD) were in the range of 0.1-20 μg kg(-1). The accuracy of the developed method was validated for recoveries, ranging from 85% to 115% with relative standard deviation (RSD) ≤14.87% at low level, from 75% to 119% with RSD ≤ 14.43% at medium level and from 61% to 120% with RSD ≤ 13.18% at high level, respectively. Finally, the developed multi-mycotoxin method was applied for screening of these mycotoxins in 24 commercial samples. Only aflatoxin B2 and zearalenone were found in 2 samples. This is the first report on the application of UFLC-ESI(+/-)-MS/MS for multi-class mycotoxins in A. catechu. The developed method with many advantages of simple pretreatment, rapid determination and high sensitivity is a proposed candidate for large-scale detection and quantification of multiple mycotoxins in other complex matrixes.

Evaluation and structure-activity relationship analysis of a new series of arylnaphthalene lignans as potential anti-tumor agents.

Arylnaphthalene lignan lactones have attracted considerable interest because of their anti-tumor and anti-hyperlipidimic activities. However, to our knowledge, few studies have explored the effects of these compounds on human leukemia cell lines. In this study, five arylnaphthalene lignans including 6′-hydroxy justicidin A (HJA), 6′-hydroxy justicidin B (HJB), justicidin B (JB), chinensinaphthol methyl ether (CME) and Taiwanin E methyl ether (TEME) were isolated from Justicia procumbens and their effects on the proliferation and apoptosis of the human leukemia K562 cell line were investigated then used to assess structure-activity relationships. To achieve these aims, cytotoxicity was assayed using the MTT assay, while intracellular SOD activity was detected using the SOD Activity Assay kit. Apoptosis was measured by both the using a cycle TEST PLUS DNA reagent kit as well as the FITC Annexin V apoptosis detection kit in combination with flow cytometry. Activation of caspase-mediated apoptosis was evaluated using a FITC active Caspase-3 apoptosis kit and flow cytometry. The results indicated that HJB, HJA and JB significantly inhibited the growth of K562 cells by decreasing both proliferation and SOD activity and inducing apoptosis. The sequence of anti-proliferative activity induced by the five tested arylnaphthalenes by decreasing strength was HJB > HJA > JB > CME > TEME. HJB, HJA and JB also decreased SOD activity and induced apoptosis in a dose-dependent manner. Activation of caspase-3 further indicated that HJB, HJA and JB induced caspase-dependent intrinsic and/or extrinsic apoptosis pathways. Together, these assays suggest that arylnaphthalene lignans derived from Justicia procumbens induce apoptosis to varying degrees, through a caspase-dependent pathway in human leukemia K562 cells. Furthermore, analysis of structure-activity relationships suggest that hydroxyl substitution at C-1 and C-6′ significantly increased the antiproliferative activity of arylnaphthalene lignans while a methoxyl at C-1 significantly decreased the effect.

Rapid analysis and identification of multi-class mycotoxins in Morinda officinalis by UFLC-ESI-MS/MS

A simple, rapid and cost-effective analytical method based on dilute-and-shoot pretreatment coupled with ultra fast liquid chromatography-tandem mass spectrometry (DAS-UFLC-MS/MS) has been developed for simultaneous quantification and identification of multi-class mycotoxins in Morinda officinalis, using a hybrid triple quadrupole linear ion trap mass spectrometer (QTRAP®). Mycotoxins were extracted with a methanol/water/formic acid mixture and directly injected into the chromatographic system after a one-fold dilution with the initial mobile phase. One precursor ion and two product ions of all analytes were simultaneously characterized and quantified based on the scheduled multiple reaction monitoring-information-dependent acquisition-enhanced product ion (sMRM-IDA-EPI) mode with a turbo ion spray interface simultaneously operated in both positive and negative modes in one chromatographic run within 10 min. Matrix-matched calibration is recommended for reliable quantitation, with zearalanone as the internal standard. After careful optimization of the corresponding parameters, the DAS-UFLC-ESI-MS/MS method was validated to express satisfactory linearity (r > 0.9900), sensitivity (limits of detection, 0.02 and 4.00 ng mL−1 and limits of quantification, 0.06–10 ng mL−1), precision (intra-day and inter-day precision, <15%), stability (4.12–14.10%), repeatability (5.45–15.56%) and spiked recoveries (63.63–119.44%). The proposed method was applied for 40 M. officinalis samples, and two samples were detected with five classes of mycotoxins with concentrations below the regulatory maximum residue limits. This study highlighted the occurrence of multi-class mycotoxins in M. officinalis, which should be under safety control.

Dynamic variation of bioactive compounds and aflatoxins in contaminated Radix Astragali during extraction process

BACKGROUND Although increasing attention has been paid to the health threat caused by mycotoxins in commodities such as food or medicines, mycotoxin transfer processes from crude material to products have raised little concern so far. Radix Astragali is a commonly used edible and medicinal herbal plant that is susceptible to contamination with aflatoxins from Aspergillus flavus. There have been no studies on mycotoxin transfer into pharmaceutical preparations or derivative products. RESULTS To facilitate the aflatoxin reduction and bioactivity retention, the dynamic variations of aflatoxins as well as herbal compounds, namely calycosin-7-glucoside, astragaloside and formononetin, in Radix Astragali contaminated by A. flavus during water decoction and ethanol refluxing treatments were evaluated simultaneously by an ultra-fast liquid chromatography-triple quadrupole linear ion trap mass spectrometry method. After the extraction processes, although the amount of alfatoxins was reduced remarkably, aflatoxin residuals in preparation still exceed recommended limits, manifesting the great need to establish a limit for aflatoxins in herbal extractions or derivative products. Meanwhile, due to the hydrolysis of glucoside, water decoction period should be no longer than 4 h. CONCLUSIONS This investigation would benefit from the determination of the dynamic variation of aflatoxins in infected herbs in preparation treatments, in order to further develop aflatoxin limits in herbal preparations.

Uncovering the antifungal components from turmeric (Curcuma longa L.) essential oil as Aspergillus flavus fumigants by partial least squares

Highly efficient and eco-friendly antifungal fumigants are desirable in food and crop production. Although turmeric (Curcuma longa L.) essential oil is known to have a potent antifungal effect, the quality of whole essential oil can be unstable, leading to unreliable antifungal activities. The aim of the present study is to uncover the active individual compounds in turmeric essential oil that provide the antifungal properties using a convenient chemometric model. The Aspergillus flavus inhibition activities of essential oils derived from 24 batches of Curcuma longa L. were evaluated, with various fumigation concentrations from 50 μL to 500 μL essential oils per plate. Meanwhile, eighteen volatile compounds were identified by static headspace gas chromatography-mass spectrometry. To combine the antifungal activities and chemical profiles with the spectrum-effect relationship based on the partial least squares model, three volatile compounds (i.e., eucalyptol, beta-pinene and camphor) were identified and verified as the most potent antifungal compounds by their higher contribution factor to antifungal indexes. Thus, this research will provide a useful approach to screen bioactive compounds, and these three compounds are promising antifungal alternatives to conventional treatments for the control of A. flavus contamination.