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.

Detection of Arsenic(V) by Fluorescence Sensing Based on Chlorin e6-Copper Ion.

The high toxicity of arsenic (As) can cause irreversible harm to the environment and human health. In this study, the chlorin e6 (Ce6), which emits fluorescence in the infrared region, was introduced as the luminescence center, and the addition of copper ion (Cu2+) and As(V) provoked a regular change in fluorescence at 652 nm, whereas that of As(III) was 665 nm, which was used to optionally detect Cu2+, arsenic (As(III), and As(V)). The limit of detection (LOD) values were 0.212 µM, 0.089 ppm, and 1.375 ppb for Cu2+, As(III), and As(V), respectively. The developed method can be used to determine Cu2+ and arsenic in water and soil with good sensitivity and selectivity. The 1:1 stoichiometry of Ce6 with Cu2+ was obtained from the Job plot that was developed from UV-visible spectra. The binding constants for Cu2+ and As(V) were established to be 1.248 × 105 M-1 and 2.35 × 1012 M-2, respectively, using B-H (Benesi-Hildebrand) plots. Fluorescence lifetimes, B-H plots, FT-IR, and 1H-NMR were used to postulate the mechanism of Cu2+ fluorescence quenching and As(V) fluorescence restoration and the interactions of the two ions with the Ce6 molecule.

A Sensitive and Selective Colorimetric Method Based on the Acetylcholinesterase-like Activity of Zeolitic Imidazolate Framework-8 and Its Applications.

In this study, a simple colorimetric method was established to detect copper ion (Cu2+), sulfathiazole (ST), and glucose based on the acetylcholinesterase (AChE)-like activity of zeolitic imidazolate framework-8 (ZIF-8). The AChE-like activity of ZIF-8 can hydrolyze acetylthiocholine chloride (ATCh) to thiocholine (TCh), which will further react with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) to generate 2-nitro-5-thiobenzoic acid (TNB) that has a maximum absorption peak at 405 nm. The effects of different reaction conditions (buffer pH, the volume of ZIF-8, reaction temperature and time, and ATCh concentration) were investigated. Under the optimized conditions, the value of the Michaelis-Menten constant (Km) is measured to be 0.83 mM, which shows a high affinity toward the substrate (ATCh). Meanwhile, the ZIF-8 has good storage stability, which can maintain more than 80.0% of its initial activity after 30 days of storage at room temperature, and the relative standard deviation (RSD) of batch-to-batch (n = 3) is 5.1%. The linear dependences are obtained based on the AChE-like activity of ZIF-8 for the detection of Cu2+, ST, and glucose in the ranges of 0.021-1.34 and 5.38-689.66 µM, 43.10-517.24 µM, and 0.0054-1.40 mM, respectively. The limit of detections (LODs) are calculated to be 20.00 nM, 9.25 µM, and 5.24 µM, respectively. Moreover, the sample spiked recoveries of Cu2+ in lake water, ST in milk, and glucose in strawberry samples were measured, and the results are in the range of 98.4-115.4% with the RSD (n = 3) lower than 3.3%. In addition, the method shows high selectivity in the real sample analysis.

[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.

A portable personal glucose meter method for enzyme activity detection and inhibitory activity evaluation based on alkaline phosphatase-mediated reaction.

In this study, an effective and portable method for enzyme activity detection and inhibitory activity evaluation was developed based on the alkaline phosphatase (ALP)-mediated reaction in a personal glucose meter (PGM). In this method, ALP catalyzes the hydrolysis of substrate amifostine (WR-2721) to produce ethanethiol (WR-1065), which can trigger the reduction of ferricyanide (K3[Fe(CN)6]), an electron transfer mediator in glucose test strips, to ferrocyanide ([K4Fe(CN)6]) and generate a PGM-detectable signal. Thus, WR-1065 can be directly quantified by a PGM as simply as detecting glucose in blood. After being systematically optimized, the method was applied to evaluate the inhibitory activity of ten small-molecule compounds and six Cordyceps sinensis (CS) extracts on ALP. The results showed that adenosine-5-monophosphate and theophylline had high inhibitory activity, but two CS extracts have promotion potency on ALP with the values of -20.7 ± 1.3% and -46.6 ± 2.1%, respectively. Moreover, the binding sites and modes of small-molecule compounds to ALP were investigated by molecular docking, while a new substrate competitor with theoretically good inhibitory activity against ALP was designed by scaffold hopping. Finally, the accuracy of the PGM method for enzyme activity detection was assessed by detecting ALP from milk samples, and the recovery ranged from 87.7% to 116.9%. These results indicate that it is feasible to evaluate enzyme activity and the inhibitory activity of small-molecule compounds and CS extracts on ALP using a PGM based on ALP-mediated reaction. Graphical abstract.

[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.

SDS-PAGE and 2-DE protein profiles of Ganoderma lucidum from different origins.

Ganoderma lucidum (Chizhi in Chinese) is one of the most valuable and widely used medicinal fungi in traditional Chinese medicines (TCMs). Most of previous studies were focused on the triterpenoids and polysaccharides of G. lucidum, whereas less attention had been paid on the protein, which is another bioactive compound in it. In the present study, protein maps of fourteen G. lucidum samples were comprehensively analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE). The results indicated that there were significant differences in protein profiles of G. lucidum samples from different origins. Furthermore, previous reported bioactive proteins from the fruiting bodies of G. lucidum, were mainly distributed in 4 taxa (A, B, C and D) based on their molecular weights on the 2-DE maps. The proteins should be considered as marker for the quality control of G. lucidum, because the proteomic variation may affect on their pharmacological activities.

[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.

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.

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.

On-line pre-column FRAP-based antioxidant reaction coupled with HPLC-DAD-TOF/MS for rapid screening of natural antioxidants from different parts of Polygonum viviparum.

Polygonum viviparum L. (PV) is a widely used resource plant with high medicinal, feeding and ecological values. Our studies show that PV has strong antioxidant activity. However, up to date, the antioxidant activity and components in other parts were not fully elucidated. In the present study, a new online pre-column ferric ion reducing antioxidant power (FRAP)-based antioxidant reaction coupled with high performance liquid chromatography-diode array detector-quadrupole-time-of-flight mass spectrometry (HPLC-DAD-TOF/MS) was developed for rapid and high-throughput screening of natural antioxidants from three different parts of PV including stems and leaves, fruits and rhizomes. In this procedure, it was assumed that the peak areas of compounds with potential antioxidant activity in HPLC chromatograms would be greatly diminished or vanish after incubating with the FRAP. The online incubation conditions including mixed ratios of sample and FRAP solution and reaction times were firstly optimized with six standards. Then, the repeatability of the screening system was evaluated by analysis of the samples of stems and leaves of PV. As a result, a total of 21 compounds mainly including flavonoids and phenolic acids were screened from the three parts of PV. In conclusion, the present study provided a simple and effective strategy to rapidly screen antioxidants in 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.

Quantitative 1H NMR with global spectral deconvolution approach for quality assessment of natural and cultured Cordyceps sinensis.

Cordyceps sinensis is a precious medicinal food which has been successfully cultivated indoors. It remains to be investigated for a simultaneous comparison on aqueous components of natural and cultivated samples. Herein, an approach of quantitative nuclear magnetic resonance (qNMR) analysis combined with global spectral deconvolution (GSD) was established for simultaneous quantification of 26 aqueous components in C. sinensis. Processed by GSD, the distorted baselines of 1H NMR spectra were greatly improved, and overlapped signals were also well separated so as to achieve accurate identification and quantitation of components in C. sinensis. Method validation by UHPLC-QTOF-MS and TOF-SIMS analysis revealed that qNMR combined with GSD is a reliable approach for simultaneous quantification of multiple components including characteristic markers of glutamine, GABA and trehalose in authentic and fake C. sinensis. The well-established qNMR approach can be used for quality assessment of natural and cultivated C. sinensis as well as differentiation from fake ones.

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.

In situ Chemical Profiling and Imaging of Cultured and Natural Cordyceps sinensis by TOF-SIMS.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a sensitive surface analytical technology, which can simultaneously acquire diverse chemical components and their precise locations on the surfaces of samples without any requirements for chemical damage pretreatments or additional matrices. Commonly, the quality control of TCMs (traditional Chinese medicines) is limited by the qualitative and quantitative evaluations of the specifically extractive constituents. In this study, a practical sample preparation strategy named two-layered media embedding sample preparation was developed to obtain ideal freezing sections of dried materials of Cordyceps sinensis. Meanwhile, the well-established sample preparation method was applied for in situ chemical profiling and imaging of natural (NCS) and cultured Cordyceps sinensis (CCS) by using TOF-SIMS. More than 200 components were tentatively identified and imaged in NCS and CCS at the same time. Mass spectrometry imaging revealed that most components have even distributions in caterpillars of Cordyceps sinensis, while TAGs, DAGs, MAGs, and FAs only have distributions outside caterpillars' digestive chambers. This is the first time that components were in situ imaged for Cordyceps sinensis to exhibit the chemical distributions which have never been achieved by other analytical techniques so far. In addition, chemometrics was used to simplify and explain the massive TOF-SIMS mass data sets, which revealed the high chemical similarity between CCS and NCS. Furthermore, the relative quantification of TOF-SIMS data showed that CCS has comparable proportions of amino acids, nucleosides, monosaccharides, sphingolipids, sterols and other principles to NCS except for fatty acids, glycerides and glycerophospholipids. The higher amounts of TAGs and DAGs in CCS were confirmed by quantitative 1H-NMR, indicating reliable relative quantification of TOF-SIMS. In general, our research developed a novel approach of TOF-SIMS for in situ chemical analysis of TCMs, and its successful application in comparative study of CCS and NCS suggested that TOF-SIMS is an advanced and promising analytical technology for the research of TCMs.

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.

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.