Rapid screening and evaluation of natural antioxidants from leaf, stem, and root of Artemisia argyi by online liquid microextraction combined with HPLC-based antioxidant assay system coupled with calibration quantitative analysis.

To reveal the utilization value of leaf, stem, and root of Artemisia argyi, a rapid online liquid microextraction combined with a high-performance liquid chromatography coupled with 2,2-nitrogen-di (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt antioxidant assay system was established for analysis of antioxidants in the leaf, stem, and root of A. argyi, and a calibration quantitative method of antioxidant activity with equivalent chlorogenic acid was proposed. Thirty-three positive peaks were identified; among them, 12 compounds were found that possess good antioxidant activity including eleven organic acids (components 2-4, 8, 11-14, 17, 19, and 21) and one flavonoids (component 22). The proposed calibration quantitative method avoided the influence of content of compound and compared the extent of radical scavenging capacity of five antioxidant compounds, which were ranked as follow: 3,5-dicaffeoylquinic acid > 3,4-dicaffeoylquinic acid ≈ 4,5-dicaffeoylquinic acid > 1,4-dicaffeoylquinic acid > chlorogenic acid. In conclusion, this study provided composition and biological potential for the future development of the leaf, stem, and root of A. argyi. It is believed that the online liquid microextraction combined with high-performance liquid chromatography based antioxidant assay system can be widely used for the rapid screening of natural antioxidant components in the different parts of natural products.

[Rapid determination of aesculin and aesculetin in Fraxini Cortex by high performance liquid chromatography-ultraviolet at equal absorption wavelength].

Fraxini Cortex is a traditional Chinese herbal medicine that has been used for thousands of years to treat dampness-heat diarrhea, dysentery, red or white vaginal discharge, painful swelling or redness of the eyes, and nebula. It contains various chemical components, including coumarins, iridoids, phenolic acids, and flavonoids. Coumarins are important active ingredients in Fraxini Cortex and have antibacterial, anti-inflammatory, antioxidant, antitumor, and antiviral activities. Aesculin and aesculetin are two major coumarin components of Fraxini Cortex that are widely used in its quality evaluation. Previous HPLC methods for determination of aesculin and aesculetin present several limitations, such as long analysis times and high solvent and reference compound consumption. In this study, a rapid, eco-friendly and cost saving HPLC method for the determination of aesculin and aesculetin in Fraxini Cortex was established by using the core-shell column and equal absorption wavelength (EAW). Different factors influencing the extraction process, such as the extraction solvent, temperature, and time, were assessed to obtain the optimal extraction conditions. The results showed that Fraxini Cortex samples could be well extracted by ultrasonic extraction for 5 min with a 25% ethanol aqueous solution. A core-shell column was used, and different mobile phases and flow rates were investigated to obtain the best rapid-HPLC separation conditions. The optimized HPLC conditions were as follows: a Poroshell 120 EC-C18 column (50 mm×4.6 mm, 2.7 µm), acetonitrile-0.1% formic acid aqueous solution (6∶94, v/v) as the eluent, a flow rate of 1.5 mL/min, and a column temperature of 25 ℃. The EAW of aesculin and aesculetin was a key factor in their determination using a single reference compound. EAW selection was performed in two steps. First, the UV spectra of two equimolar concentrations of the reference compounds (aesculin and aesculetin) were compared to determine the EAW of the two analytes. The EAW results were then verified by the HPLC analysis of the reference compound solutions. The final EAW of aesculin and aesculetin was 341 nm. The determination of aesculin and aesculetin using only one reference compound (i. e., aesculin) was achieved by HPLC-UV at this EAW. The newly developed HPLC method revealed a good linear relationship between the two target analytes (r=1.0000). The limits of detection (LODs) and limits of quantification (LOQs) were 1.5 µmol/L and 3.0 µmol/L, respectively, and the average recoveries of aesculin and aesculetin were 99.0% and 97.5%. The stabilities of the sample solutions were examined, and the two analytes demonstrated good stability for 24 h. The contents of the target analytes in 10 batches of Fraxini Cortex were determined using the proposed EAW method and the classic external standard method (ESM), and comparable concentrations were obtained. The contents of aesculin and aesculetin in the 10 batches of Fraxini Cortex were 0.26%-2.80% and 0.11%-1.47%, respectively. A t-test was conducted to compare the results of the proposed EAW technique with those obtained via the method reported in the Chinese Pharmacopoeia, and no significant difference between the two assay methods was noted (P>0.05). Comparison of the newly established EAW method with those reported in the literature revealed that our method required only 10 min to complete and used as little as 0.5 mL of the solvent and only one standard. Therefore, the developed EAW method is a rapid, simple, eco-friendly, and cost-effective analytical method that is suitable for the determination of aesculin and aesculetin in Fraxini Cortex and its related products. The proposed technique is an improved method for determining aesculin and aesculetin and contributes to the enhancement of the quality evaluation of Fraxini Cortex.

Cordythiazole A, the first member of thiazole alkaloids from Chinese cordyceps, with α-glucosidase inhibitory activity.

Chinese cordyceps, also known as Dong-Chong-Xia-Cao, is widely recognized as a famous precious tonic herb, and used as traditional Chinese medicine for centuries. It is mainly used for regulating the immune system and improving functions of the lung and kidney, with anti-tumor, anti-inflammatory, and anti-diabetic activities. Due to its rarity and preciousness, a few chemical components are isolated and identified. Moreover, most of them are common chemical components and widely distributed in other natural resources, such as nucleosides, sterols, fatty acids, sugar alcohols, and peptides. Therefore, a large number of active substances of Chinese cordyceps is still unclear. During our search for chemical constituents of Chinese cordyceps, a new thiazole alkaloid, cordythiazole A (1), was isolated and identified. Its structure was elucidated by comprehensive spectroscopic analysis and single-crystal X-ray diffraction analysis. This is the first report of the presence of thiazole alkaloid in Chinese cordyceps, which adds a new class of metabolite of Chinese cordyceps. Furthermore, a putative biosynthesis pathway of cordythiazole A was proposed based on possible biogenic precursor, genes, and literatures. In addition, it showed α-glucosidase inhibitory activity with potency close to that of acarbose. The discovery of cordythiazole A with α-glucosidase inhibitory activity adds a new class of potential anti-diabetes ingredient in Chinese cordyceps.

Online Microextraction Coupled with HPLC-ABTS for Rapid Analysis of Antioxidants from the Root of Polygonum bistorta.

The root of Polygonum bistorta (PB) is a traditional Chinese medicinal plant material widely used in China. It has been commonly used for the treatment of hemostasis, detumescence, diarrhea, snake bite, and acute gastroenteritis. However, the research on the antioxidant properties and bioactive compounds from PB is inadequate. In the current research, an online microextraction (OLME) coupled with a high-performance liquid chromatography coupled with the 2,2-nitrogen-di (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt antioxidant assay (HPLC-ABTS) system for rapid analysis of antioxidants from PB was proposed. The PB sample (0.17 mg) was online extracted by mobile phase (acetonitrile and 0.2% acetic acid); a Poroshell 120 SB-Aq column was used for separation; then, an online ABTS assay system was used for screening the antioxidants. Finally, ten components were found in PB, and among them, eight components possessed antioxidant activities. Furthermore, five components (gallic acid, neochlorogenic acid, caffeic acid, chlorogenic acid, and an unknown compound) were proved as major antioxidants when compared with rutin as an antioxidant marker. The results showed that the developed OLME-HPLC-ABTS system was a simple, rapid, green, and efficient instrument for the screening of antioxidants from PB, which provides a powerful tool for the discovery of natural antioxidants in Chinese medicines.

Fractional extraction and structural characterization of glycogen particles from the whole cultivated caterpillar fungus Ophiocordyceps sinensis.

Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a valuable medicinal fungus in traditional Chinese medicine, and one or more polysaccharides are the key constituents with important medical effects. Glycogen as a functional polysaccharide is widely identified in eukaryotes including fungi. However, there is no definitive report of glycogen presence in O. sinensis. In this study, we carefully fractionated polysaccharides from cultivated caterpillar fungus O. sinensis, which were then characterized via methods for glycogen analysis. According to the results, 1.03 ± 0.43 % of polysaccharides were quantified via amyloglucosidase digestion in the whole cultivated caterpillar fungus, which had a typical spherical shape under transmission electron microscope with an average peak radius of 37.63 ± 0.57 nm via size exclusion chromatography and an average chain length of 12.47 ± 0.94 degree of polymerization via fluorophore-assisted capillary electrophoresis. Taken together, this study confirmed that the polysaccharides extracted form O. sinensis were mostly glycogen.

Trilobatin alleviates non-alcoholic fatty liver disease in high-fat diet plus streptozotocin-induced diabetic mice by suppressing NLRP3 inflammasome activation.

Diabetes mellitus (DM) is a factor with great risk in the course of non-alcoholic fatty liver disease (NAFLD) due to its high glucotoxicity and lipotoxicity. Trilobatin, a glycosylated dihydrochalcone derived from the leaves of the Chinese sweet tea Lithocarpus polystachyus Rehd, is reported to possess various pharmacological activities. Nevertheless, it is still unclear regarding if trilobatin can alleviate liver injury in diabetic mice with NAFLD and its mechanism. Our aim was to investigative the protective effects of trilobatin against DM with NAFLD and its mechanism of action. A DM mice model was established by high-fat diet (HFD) feeding with streptozocin (STZ) injections, and treated with trilobatin for 10 weeks. The biochemical results showed that trilobatin restored glucose metabolic disorder and liver function in diabetic mice. The histopathological evaluation revealed that trilobatin improved liver injury by alleviating lipid accumulation and liver fibrosis. Mechanistically, trilobatin decreased expression of NLRP3, p65 NF-κB, cleaved-Caspase-1 and N-GSDMD, as well as the release of IL-18 and IL-1ß, leading to a alleviation of inflammation and pyroptosis. Taken together, we determined for the first time found that trilobatin could prevent liver injury in diabetic mice with NAFLD by suppressing NLRP3 inflammasome activation to reduce inflammation and pyroptosis.

Non-targeted metabolomic analysis of variation of volatile fractions of ginseng from different habitats by HS-SPME-GC-MS coupled with chemometrics.

Cultivated ginseng (CG), transplanted ginseng (TG) and mountain cultivated ginseng (MCG) classified by the habitat type all belong to Panax ginseng and were reported to have similar types of secondary metabolites. Nonetheless, owing to the distinctly diverse habitats in which these ginseng types grow, their pharmacological effects differ. In the present study, an emerging analytical approach involving headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) was established to effectively distinguish among CG, TG and MCG. First, the volatile components were analysed and identified by using the NIST library combined with measured retention indices (Kovats', RI), and a total of 78 volatile components were finally characterized, which included terpenes, alcohols, esters, aldehydes and alkynols. Furthermore, multivariate statistical approaches, principal component analysis (PCA) and orthogonal partial least-squares discrimination analysis (OPLS-DA) were subsequently utilized to screen for compounds of significance. Under optimized HS-SPME-GC-MS conditions, 12, 16, and 16 differential markers were screened in the CG-TG, CG-MCG and TG-MCG groups, respectively. Our study suggested that HS-SPME-GC-MS analysis combined with metabolomic analytical methods and chemometric techniques can be applied as potent tools to identify chemical marker candidates to distinguish CG, TG and MCG.

[Form and valence of arsenic in dry and fresh Cordyceps breeding products based on HPLC-ICP-MS and its risk assessment].

A comparative study was conducted for the first time on the form and valence of arsenic in the dry and fresh Cordyceps breeding products to clarify the specific morphology and valence of arsenic in Cordyceps breeding products and its safety. Arsenic betai-ne(AsB), arsenite(AsⅢ), dimethyl arsenic(DMA), arsenocholine(AsC), monomethyl arsenic(MMA), and arsenate(AsⅤ) in the dry and fresh samples were investigated using a bionic extraction method combined with HPLC-ICP-MS. The HPLC separation was performed on a DioncxIonPac~(TM) AS7 anion exchange column with a mobile phase of 100 mmol·L~(-1) ammonium carbonate-water for gradient elution at room temperature and the flow rate of 0.8 mL·min~(-1). HPLC was coupled with ICP-MS for the determination. The arsenic content was characterized in combination with chemometrics. The health safety risk of inorganic arsenic in the samples was assessed using the margin of exposure(MOE). The results of methodological validation showed that the six arsenic compounds showed good linearity(R~2>0.999) from 10 to 500 ng·mL~(-1), with precision RSDs of 1.8%-3.0%, recoveries(n=6) of 84.15%-98.28%, reproducibility RSDs of 6.4%-7.7%, and sample stability RSDs of 8.3%-14% within 10 h. Trace AsⅢ and AsⅤ were detected in 30 batches of dry and fresh Cordyceps breeding products, while arsenic compounds in other forms and valence were not detected. In the dry products, AsⅢ was 0.019-0.040 mg·kg~(-1) and AsV was 0.024-0.061 mg·kg~(-1), while in the fresh products, AsⅢ was 0.002 3-0.006 1 mg·kg~(-1) and AsⅤ was 0.008-0.016 mg·kg~(-1). The risk assessment results showed that the MOE of inorganic arsenic was much higher than 1 in both dry and fresh products, and the potential health safety risk of inorganic arsenic was low. The HPLC-ICP-MS method established in this study was efficient, rapid, accurate, and stable for the determination of six arsenic compounds in Cordyceps breeding products. The results of this study provide a basis for the safety and quality control of Cordyceps breeding products.

Effects of natural dihydrochalcones in sweet tea (Lithocarpus polystachyus) on diabetes: a systematical review and meta-analysis of animal studies.

Sweet tea (Lithocarpus polystachyus Rehd.), a natural functional food highly rich in dihydrochalcones including trilobatin, phlorizin and phloretin, is reported to possess numerous biological activities especially for treating diabetes. Here, the aim of this systematical review and meta-analysis is to assess the effect of dihydrochalcones in sweet tea (DST) on diabetes and summarize their possible mechanisms. We searched in eight databases including Embase, PubMed, Cochrane, Web of Science, WanFang database, VIP database, China National Knowledge Infrastructure and China Biology Medicine from Jan 2000 to Nov 2021 and ultimately included 21 animal studies in this review. A total of 10 outcome measurements including blood lipid indexes, blood glucose, insulin resistance indicators and oxidative stress biomarkers were extracted for meta-analysis using RevMan 5.4 software. DST significantly decreased the levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), blood glucose (BG), homeostasis model assessment of insulin resistance (HOMA-IR) and malondialdehyde (MDA), and increased high-density lipoprotein cholesterol (HDL-c), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity in diabetic animal models. In summary, DST could treat diabetes by regulation of blood glucose/lipid metabolism, oxidative/carbonyl stress, inflammatory response etc.

Development of an eco-friendly and fast HPLC method for quantitative analysis of four nucleosides in Cordyceps and related products.

An eco-friendly and fast HPLC method was developed for the determination of adenosine, inosine, guanosine and uridine in Cordyceps and related products (fermented mycelia of Hirsutella sinensis andPaecilomyces hepiali). The sample was ultrasonically extracted using 0.5% phosphoric acid solutions for 2.5 min. Sample separation was performed on a Poroshell SB-Aq column (50 mm × 4.6 mm, 2.7 µm) using eco-friendly mobile phase consisting of formic acid and ammonium formate aqueous solution at a flow rate of 1.0 mL·min-1. The detection wavelength was 260 nm. The developed HPLC method showed good linearity with correlation coefficients of 1.0000 in the test range. Good precision, repeatability and stability of this method were also observed (RSD ≤ 2.81%). The recovery ranged from 91.84%-105.19% (RSD ≤ 2.59%). Compared with reported methods, the current method did not use harmful organic solvent and took only 10.5 min. It obtained a high eco-score of 91 by the "Analytical Eco-Scale" tool. The developed method is eco-friendly and fast, which is suitable for the quality evaluation of Cordyceps and related products.

Online Liquid Microextraction Coupled with HPLC-ABTS for Rapid Screening of Natural Antioxidants: Case Study of Three Different Teas.

In the present study, an online liquid extraction coupled with high-performance liquid chromatography-2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (HPLC-ABTS) system for rapid screening of antioxidants in tea samples was proposed. As an example, the tea samples were firstly extracted by online HPLC extractor with mobile phase at 70°C, then the hyphenated HPLC-ABTS was used for the chromatographic separation on a Poroshell EC C18 column by 0.3% aqueous formic acid and acetonitrile with a gradient elution at 1.5 mL·min-1, and the UV and antioxidant chromatograms with detection wavelengths at 270 nm and 750 nm were recorded, respectively. The established system integrated the processes of online HPLC sample extraction, HPLC separation and online antioxidants detection, the total analysis time of which was <20 min. The developed method was successfully applied to samples of green tea, oolong tea and black tea. As a result, 11 antioxidants were found in tea samples, including gallocatechin, epigallocatechin, catechin, chlorogenic acid, epicatechin, epigallocatechingallate, epicatechingallate, rutin, 1,4,6-trigalloylglucose, quercetin-3-glycoside and kaempferol-3-glucoside. The combined online liquid microextraction and online HPLC-ABTS method is a rapid and green approach for the quality evaluation of tea.

Rapid screening and characterization of natural antioxidants in Polygonum viviparum by an on-line system integrating the pressurised liquid micro-extraction, HPLC-DAD-QTOF-MS/MS analysis and antioxidant assay.

Rapid discovery of active ingredients from complex matrices is one of great challenges for modern drug development. Traditional methods often require many sample treatment steps, including an extraction step with exclusively dedicated solvents followed by repeated separation and activities assessment. This present work described an integrated analytical setup for natural antioxidants discovery in which the online extraction (OLE) of a solid sample is directly coupled to its analysis by high performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry and 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) antioxidant assay (OLE-HPLC-DAD-QTOF-MS/MS-ABTS). This developed approach makes sample extraction, chromatographic separation and chemical detection, and antioxidant assay integrated into a single HPLC injection and was successfully applied for the rapid discovery of natural antioxidant bioactives from Polygonum viviparum. A total of 21 secondary metabolites were characterized according to their retention times, ultraviolet (UV) spectra, exact mass and fragmentation ions in MS/MS spectra, and 18 of them displayed antioxidant activity (response as negative peaks in antioxidant assay). This work describes a simple, green and efficient approach to minimize the sample consumption (only 0.4 mg was required) and eliminate complex sample treatment procedures. The developed OLE-HPLC-DAD-QTOF-MS/MS-ABTS system offers new perspectives for rapid chemical profiling of natural products and their antioxidants discovery.

Profiling and identification of aqueous extract of Cordyceps sinensis by ultra-high performance liquid chromatography tandem quadrupole-orbitrap mass spectrometry.

Characterization of aqueous extract in traditional Chinese medicine (TCM) is challenging due to the poor retention of the analytes on conventional C18 columns. This study presents a systematic characterization method based on a rapid chromatographic separation (8 min) on a polar-modified C18 (Waters Cortecs T3) column of aqueous extract of Cordyceps sinensis. UHPLC-HRMS method was used to profile components in both untargeted and targeted manners by full MS/PIL/dd-MS2 acquisition approach. The components were identified or tentatively identified by reference standards comparison, fragmentation rules elucidation and available databases search. A total of 91 components, including 10 nucleobases, 20 nucleosides, 39 dipeptides, 18 amino acids and derivatives and 4 other components, were characterized from the aqueous extract of C. sinensis. And this was the first time to systematically report the presence of nucleosides and dipeptides in C. sinensis, especially for modified nucleosides. The chemical basis inquiry of this work would be beneficial to mechanism exploration and quality control of C. sinensis and related products. Meanwhile, this work also provided an effective solution for characterization of aqueous extract in TCM.

[Analysis of chemical components in Cordyceps by online gradient extraction-HPLC].

Online gradient extraction-high performance liquid chromatography( HPLC) method was developed for simultaneous determination of high and low polar components in Cordyceps. The sample powder of Cordyceps was uniformly mixed with diatomaceous earth,packed into extraction tank,and installed into the HPLC system. Online gradient extraction was conducted with mobile phase at 70 ℃. The separation was performed on Zorbax SB-AQ( 4. 6 mm×150 mm,5 µm) column with 0. 1% formic acid solution-methanol as the mobile phase for gradient elution at 1. 0 mL·min~(-1). The column temperature was 30 ℃,and detection wavelength was set at 260 nm. The results showed that the high and low polar components in Cordyceps could be simultaneously extracted and separated by the developed method. Meanwhile,six high polar compounds( uracil,uridine,thymine,inosine,guanosine and adenosine) and one low polar compound( ergosterol) were identified by comparison with the reference peaks. The established method is rapid,stable and environment friendly,which is helpful to improve the quality evaluation level for Cordyceps.

[Effects of different drying conditions on protein in Cordyceps].

In this study,the protein in different Cordyceps samples,which include fresh sample( S1),22 ℃ drying sample( S2),37 ℃ drying sample( S3) and 60 ℃ drying sample( S4),were analyzed by sodium dodecylsupinate-polyacrylamide gel electrophoresis( SDS-PAGE) and two-dimensional electrophoresis( 2-DE). The total protein contents in Cordyceps samples were from 1. 655-4. 493 mg·g~(-1) and the protein contents in fresh sample was the highest. The results of SDS-PAGE showed that the mainly ranges of protein molecular weight of Cordyces samples were 10-100 kDa and the numbers of protein bands were 28 to 41,the fresh sample had the maximum number of protein bands. The 2-DE profiles were analyzed by PDQuest software. The resulted indicated that 488-876 protein spots were detected in different Cordyceps samples and the isoelectric point( pI) was distributed between 4. 5 and 6. 5,the protein molecular weight was distributed in 10-20 kDa and 25-100 kDa,the fresh sample had the maximum number of protein spots. Therefore,the drying process could decrease contents and species of protein in Cordyceps,and the different drying conditions had different effects on protein. These results provide a reference for improving the drying process of Cordyceps.

[Comparative study on specific chromatograms and main nucleosides of cultivated and wild Cordyceps sinensis].

This study is to establish the HPLC specific chromatogram and determine four main nucleosides of wild and cultivated Cordyceps sinensis. Uridine, inosine, guanosine and adenosine were selected as reference substance. HPLC analysis was performed on a Waters XSelect HSS T3 C18 (4.6 mm×250 mm, 5 µm), with a mobile phase consisting of water(A)-acetonitrile (B) at a flow rate of 0.6 mL•min⁻¹ (0-5 min,0% B;5-15 min,0%-10% B, 15-30 min,10%-20% B, 30-33 min, 20%-50% B, 33-35 min, 50%-0% B, 35-40 min, 0% B). The detection wavelength was 260 nm and the column temperature was controlled at 30 ℃, and the injection volume was 5 µL. HPLC specific chromatogram of wild and cultivated C. sinensis was established and four main nucleosides were simultaneously determined by the above method. Specific chromatograms and contents of four main nucleosides showed no significant differences between cultivated and wild C. sinensis. These results can provide scientific evidences for further development and utilization of cultivated C. sinensis.

[Simultaneous determination of cordycepin and 2'-deoxyadenosine in Cordyceps genus by online SPE-HPLC].

An online SPE-HPLC method for simultaneous determination of cordycepin (3'-deoxyadenosine) and 2'-deoxyadenosine in Cordyceps genus (C. sinensis,C. militaris,Hirsutella sinensis and C. sobolifera) was developed. The samples were enriched on a ZORBAX SB-AQ (4.6 mm×12.5 mm,5 µm) column with isocratic elution by 9% methanol solution. The separation of analytes was performed on a ZORBAX SB-AQ (4.6 mm×150 mm,5 µm) column with gradient elution by 0.1% formic acid solution and methanol (91∶9). The flow rate was 1.0 mL•min⁻¹. Column temperature was 40 ℃ and detection wavelength was 260 nm. This method has been applied for analysis of different Cordyceps genus. The 2'-deoxyadenosine was detected in C. sinensis,Hirsutella sinensis and C. sobolifera. The cordycepin was detected in C. militaris. In summary,the cordycepin chromatographic peak from C. sinensis in some past reports may be the 2'-deoxyadenosine chromatographic peak or the mixture peak of 2'-deoxyadenosine and cordycepin in which 2'-deoxyadenosine content was higher than cordycepin. The developed method is suitable for analysis of cordycepin and 2'-deoxyadenosine in Cordyceps genus.

[Quantitative analysis of nucleosides in four Cordyceps genus by HPLC].

To compare the main nucleosides in Cordyceps genus herbs (C. sinensis, C. millitaris, Hirsutella sinensis and C. sobolifera), an HPLC method for simultaneous determination of uridine, inosine, guanosine, adenosine and cordycepine in Cordyceps genus herbs was developed. The sample was extracted with 0.5% phosphoric acid solution to prepare test solution. The separation was performed on a Zorbax SB-Aq (4.6 mm×150 mm, 5 µm) column with gradient elution by 0.04 mol•L⁻¹ potassium dihydrogen phosphate solution and acetonitrile, column temperature 30 ℃,flow rate 0.8 mL•min⁻¹,and detection wavelength 260 nm. The content of nucleosides in four Cordyceps genus herbs was evaluated by fingerprint analysis and hierarchical cluster analysis (HCA). The calibration curves of five nucleosides showed good linear regression (r>0.99) and the average recoveries were between 95.0% and 105.0%. The contents of the five nucleosides in the four Cordyceps genus herbs were different and could be obviously distinguished by HCA. The fingerprint analysis result showed that the similarity between C. sinensis and the others was less than 0.9. The method was accurate and reliable, which can be used for quality control of Cordyceps genus herbs.

Inhibition of three selected beverage extracts on alpha-glucosidase and rapid identification of their active compounds using HPLC-DAD-MS/MS and biochemical detection.

Inhibition of alpha-glucosidase is a therapeutic approach for diabetes. In this study, a method based on online liquid chromatography-diode array detection-tandem mass spectrometry and biochemical detection (LC-DAD-MS/MS-BCD) was developed to screen and identify alpha-glucosidase inhibitors from selected beverage extracts, including pu-erh tea ( Camellia sinensis var. assamica), eagle tea ( Litsea coreana Levl.), and radix glycyrrhizae ( Glycyrrhiza uralensis Fisch.). As a result, two components, (-)-epigallocatechingallate (EGCG) and (-)-epicatechingallate (ECG), as potent alpha-glucosidase inhibitors, were found in pu-erh tea. The IC(50) values of EGCG and ECG on alpha-glucosidase (EC 3.2.1.20, from Saccharomyces cerevisiae ) were 175.1 and 246.9 microM, respectively, and both were lower than that of acarbose (IC(50) = 3553.0 microM), a commercial alpha-glucosidase inhibitor. Kinetic studies revealed that both EGCG and ECG inhibited alpha-glucosidase activity in a noncompetitive manner. The study suggests that the developed LC-DAD-MS/MS-BCD system is a powerful tool for rapid screening and identification of alpha-glucosidase inhibitors in complex samples and that EGCG and ECG may be good candidates as alpha-glucosidase inhibitors.

Fast determination of five components of coumarin, alkaloids and bibenzyls in Dendrobium spp. using pressurized liquid extraction and ultra-performance liquid chromatography.

A fast method for simultaneous determination of five compounds (one coumarin, two alkaloids and two bibenzyls) in Dendrobium spp. using pressurized liquid extraction and ultra-performance liquid chromatography coupled with photo diode array detection was developed. The separation was performed on an Acquity UPLC BEH C(18) column (50 mm x 2.1 mm id, 1.7 microm) with gradient elution of phosphate buffer (pH=7.1, 20 mM) and acetonitrile within 6 min. The method was validated for linearity, LOD and LOQ, precision and accuracy. All calibration curves showed good linearity (R(2) > or = 0.9999) within test ranges. The overall intra- and inter-day variations (RSDs) of five analytes were less than 2.5%, and the detection recoveries were between 95.6 and 102.3%. The developed method was successfully applied to quantitative analysis of five investigated compounds in different species of Dendrobium. The results showed that there were great variations of their contents, though all these materials were officially used as Chinese herb, Shihu. In addition, the chromatographic fingerprints of Dendrobium spp. were also investigated.