Chemical derivatization strategies for enhancing the HPLC analytical performance of natural active triterpenoids.

Triterpenoids widely exist in nature, displaying a variety of pharmacological activities. Determining triterpenoids in different matrices, especially in biological samples holds great significance. High-performance liquid chromatography (HPLC) has become the predominant method for triterpenoids analysis due to its exceptional analytical performance. However, due to the structural similarities among botanical samples, achieving effective separation of each triterpenoid proves challenging, necessitating significant improvements in analytical methods. Additionally, triterpenoids are characterized by a lack of ultraviolet (UV) absorption groups and chromophores, along with low ionization efficiency in mass spectrometry. Consequently, routine HPLC analysis suffers from poor sensitivity. Chemical derivatization emerges as an indispensable technique in HPLC analysis to enhance its performance. Considering the structural characteristics of triterpenoids, various derivatization reagents such as acid chlorides, rhodamines, isocyanates, sulfonic esters, and amines have been employed for the derivatization analysis of triterpenoids. This review comprehensively summarized the research progress made in derivatization strategies for HPLC detection of triterpenoids. Moreover, the limitations and challenges encountered in previous studies are discussed, and future research directions are proposed to develop more effective derivatization methods.

Preclinical Investigations on Anti-fibrotic Potential of Long-Term Oral Therapy of Sodium Astragalosidate in Animal Models of Cardiac and Renal Fibrosis.

In traditional Chinese medicine, Radix Astragali has played a vital role in treating progressive fibrotic diseases. One of its main active components, astragaloside IV, is a promising anti-fibrotic treatment despite its extremely low bioavailability. Our study aimed to optimize sodium astragalosidate (SA) by salt formation to improve solubility and oral absorption for anti-fibrotic therapy in vivo. Isoproterenol-induced myocardial fibrosis rat models and obese BKS-db mice presenting diabetic kidney fibrosis were used in this study. Daily oral administration of SA (20 mg/kg) for 14 days ameliorated cardiac fibrosis by reducing collagen accumulation and fibrosis-related inflammatory signals, including TNF-α, IL-1ß, and IL-6. In db/db mice, SA (5,10, and 20 mg/kg per day for 8 weeks) dose-dependently alleviated lipid metabolism impairment and renal dysfunction when administered orally. Furthermore, Western blot and immunohistochemistry analyses demonstrated that SA treatment inhibited renal fibrosis by suppressing TGF-ß1/Smads signaling. Taken together, our findings provide the oral-route medication availability of SA, which thus might offer a novel lead compound in preclinical trial-enabling studies for developing a long-term therapy to treat and prevent fibrosis.

Role of simulated in vitro gastrointestinal digestion on biotransformation and bioactivity of astragalosides from Radix Astragali.

Radix Astragali (RA) is commonly used in Asian herbal therapy or food supply, and astragalosides and flavonoids are its major components with diverse pharmaceutical effects. To provide new information on the potential cardiovascular benefits of RA administered orally, the bioaccessibility of these compounds with relevant in vitro digestion parameters was determined for four digestion phases (oral, gastric, small and large intestines) by ultrahigh-performance liquid chromatography quadrupole time-of-flight-mass spectrometry (UPLC-Q-TOF/MS). Meanwhile, we compared the effects of digestion products on advanced glycation end products (AGEs)-induced intracellular reactive oxygen species (ROS) levels in a human arterial endothelial cells (HAECs) model, and studied the potential of RA against oxidative stress-related cardiovascular disease. The changes of saponins and flavonoids composition and antioxidant activity after digestion in intestines were mainly due to the astragaloside IV (AS-IV) biosynthesis involving saponins acetyl isomerization and deacetylation, and the flavonoid glycosides converted to aglycone by deglycosylation processes. All these results suggest that acetyl biotransformation of RA in small intestine directly influenced the response to oxidative stress, and might provide a reference for elucidation of the multi-component action after oral RA in cardiovascular health care.

Elucidation of the binding mechanism of astragaloside IV derivative with human serum albumin and its cardiotoxicity in zebrafish embryos.

LS-102 is a new derivative of astragaloside IV (AGS IV) that has been shown to possess potentially significant cardioprotective effects. However, there are no reports concerning its interaction with human serum albumin (HSA) and toxicology in vertebrates. The present investigation was undertaken to characterize the interaction of AGS IV and LS-102 with HSA using equilibrium dialysis and UHPLC-MS/MS methods, along with computational methods. Notably, the effects of AGS IV and LS-102 were studied in vivo using the zebrafish embryo model. Markers related to embryonic cardiotoxicity and thrombosis were evaluated. We showed that the plasma protein binding rate of AGS IV (94.04%-97.42%) was significantly higher than that of LS-102 (66.90%-69.35%). Through site marker competitive experiments and molecular docking, we found that AGS IV and LS-102 were located at the interface of subdomains IIA and IIIA, but the site I might be the primary binding site. Molecular dynamics revealed that AGS IV showed a higher binding free energy mainly due to the stronger hydrophobic and hydrogen bonding interactions. Moreover, the secondary structure implied no obvious effect on the protein structure and conformation during the binding of LS-102. LS-102 significantly ameliorated the astramizole-induced heart rate slowing, increased SV-BA spacing, and prevented arachidonic acid-induced thrombosis in zebrafish. To our knowledge, we are the first to reveal that LS-102 binds to HSA with reversible and moderate affinity, indicating its easy diffusion from the circulatory system to the target tissue, thereby providing significant insights into its pharmacokinetic and pharmacodynamic properties when spread in the human body. Our results also provide a reference for the rational clinical application of LS-102 in the cardiovascular field.

Advances in aptamer-based sensing assays for C-reactive protein.

C-reactive protein (CRP), a non-specific acute-phase indicator of inflammation, has been widely recognized for its value in clinical diagnostic applications. With the advancement of testing technologies, there have been many reports on fast, simple, and reliable methods for CRP testing. Among these, the aptamer-based biosensors are the focus and hotspot of research for achieving high-sensitivity analysis of CRP. This review summarizes the progress of in vitro aptamer screening for CRP and the recent advances in aptamer-based CRP sensor applications, thus developing insight for the new CRP aptasensor design strategy.

Determination of nine nucleosides in Rhizoma Paridis by quantitative analysis of multi-components via a single marker method.

In this work, a new quantitative analysis method of multi-components analysis via a single marker strategy coupled with high-performance liquid chromatography (HPLC) analysis, was proposed to analyze nine nucleosides (cytidine, uridine, 2'-deoxyuridine, inosine, guanosine, 2'-deoxyguanosine, thymidine, adenosine, and 2'-deoxyadenosine) as quality control markers in Rhizoma Paridis. Guanosine was set as the internal reference substance, whose content in Rhizoma Paridis was determined using conventional external standard method. Then, relative correction factors between guanosine and the other eight nucleosides were measured respectively. The amounts of the other eight components were calculated according to the relative correction factors by the quantitative analysis of multi-components via a single marker method. Finally, the result of vector angle cosine analysis showed that there was no significant difference of the contents between the external standard method and the quantitative analysis of multi-components via a single marker method, indicating that the quantitative analysis of multi-components via a single marker method can be applied for the quality control of Rhizoma Paridis. As far as we know, this is also the first report to analyze nucleosides by the quantitative analysis of multi-components via a single marker method, providing an efficient and promising quality assessment method for other traditional Chinese medicine containing nucleosides.

Astragaloside IV Derivative (LS-102) Alleviated Myocardial Ischemia Reperfusion Injury by Inhibiting Drp1Ser616 Phosphorylation-Mediated Mitochondrial Fission.

Our previous studies showed that Astragaloside IV derivative (LS-102) exhibited potent protective function against ischemia reperfusion (I/R) injury, but little is known about the mechanisms. Mitochondrial fission regulated by dynamin-related protein1 (Drp1) is a newly recognized determinant of mitochondrial function. This study aimed to investigate the protection of LS-102 on mitochondrial structure and function by regulating the activity of Drp1 using models of H9c2 cardiomyocyte injury induced by hypoxia-reperfusion (H/R), and rat heart injury induced by I/R. The results showed that LS-102 significantly decreased apoptosis, levels of ROS, CK, LDH, and calcium, upregulating MMP, and the Bax/Bcl-2 ratio in cardiomyocytes during I/R injury. Furthermore, LS-102 prevented I/R-induced mitochondrial fission by decreasing Drp1's mitochondrial localization through decreasing the phosphorylation of Drp1 at Ser616 (Drp1Ser616) and increasing the phosphorylation of Drp1 at Ser637 (Drp1Ser637) in H9c2 cells. Importantly, we also robustly confirmed Drp1Ser616 as a novel GSK-3ß phosphorylation site. GSK-3ß-mediated phosphorylation at Drp1Ser616 may be associated with mitochondrial fission during I/R of cardiomyocytes. In conclusion, LS-102 exerts cardio protection against I/R-induced injury by inhibiting mitochondrial fission via blocking GSK-3ß-mediated phosphorylation at Ser616 of Drp1.

A new strategy for the preparation of antibody against natural glycoside: With astragaloside IV as an example.

A new strategy for the hapten design of natural glycoside and application for the preparation of antibody is reported in this work. With astragaloside IV (AGS-IV) as an example, C6"-CH2OH on a glucosyl group was selectively oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation to C6"-COOH, which was subsequently condensed with -NH2 on bovine serum albumin to get artificial antigen. Then, the successful preparation of artificial antigen was verified by TCL, SDS-PAGE, UV, and MALDI-TOF-MS. Finally, rabbits were immunized with artificial antigen to obtain an antibody against AGS-IV. After tests of the titer, IC50, and cross-reactivity, the results showed that the antibody prepared by TEMPO oxidation in this work had higher specificity than that the antibody prepared by conventional sodium periodate (NaIO4) oxidation. The hapten, as a carboxylic acid derivative of AGS-IV, has better water solubility than AGS IV, which is more suitable for the synthesis of the hapten-carrier protein conjugate in aqueous phase, achieving another virtue of TEMPO oxidation over NaIO4 oxidation. This new strategy provides new ideas for the design of haptens of other natural glycosides, as well as the preparation of their antibodies.

A feasible image-based colorimetric assay using a smartphone RGB camera for point-of-care monitoring of diabetes.

Urinary glucose determination using a glucose test strip is simple and convenient in daily self-monitoring of diabetes. However, diabetic patients exhibit acquired impaired color vision (ICV), which results in the inability to discriminate between hues. Even with the assistance of a color chart, it is still not easy for these patients to read the urinary glucose results with the naked eye. In this study, a smartphone camera using an image-based colorimetric detection method was successfully developed for quantitative analysis of urine glucose. A horseradish peroxidase-hydrogen peroxide-3,3'5,5'-tetramethylbenzidine (HRP-H2O2-TMB) system was optimized for a reliable and gradual color fading process via a glucose oxidase (GOD) catalyzed oxidation reaction. The color changes of the peroxidase-H2O2 enzymatic reactions in the 96-well microplate were captured by a smartphone RGB camera with subsequent detection of red, green, and blue (RGB) intensities decreasing at each image pixel. The highly quantitative relationships between the glucose concentrations and the color characteristic values of the blue channel of the captured images were successfully established. The high accuracy of this method was demonstrated in urine glucose measurements with a linear response over the 0.039 mg mL-1 to 10.000 mg mL-1 glucose concentration range and a 0.009 mg mL-1 detection limit. The method has great potential as a point-of-need platform for diabetic patients with defective color vision and features high accuracy and low cost.

Colorimetric determination of the early biomarker hypoxia-inducible factor-1 alpha (HIF-1α) in circulating exosomes by using a gold seed-coated with aptamer-functionalized Au@Au core-shell peroxidase mimic.

An ultra-sensitive method is described here for the determination of HIF-1α (an early biomarker for myocardial infarction) in circulating exosomes in serum. Gold nanospheres were functionalized with a HIF-1α-binding aptamer via sulfydryl chemistry. The apt-AuNP-coated gold seeds were grown by seed-mediated growth, and this significantly increased the peroxidase-mimicking property the nanoparticles. A chromogenic system composed of 3,3'5,5'-tetramethylbenzidine and hydrogen peroxide was used. Absorbance at 652 nm increases linearly in the 0.3 to 200 ng L-1 HIF-1α concentration range, and the limit of detection is 0.2 ng L-1. The method was tested by analyzing rat serum from isoproterenol (ISO)-induced myocardial infarction. It allows HIF-1α to be directly determined in a 25 µL sample without preconcentration. The assay is not interfered by the polydispersity of exosomes released under either health and disease conditions. Graphical abstractGold nanospheres were functionalized with a HIF-1α-binding aptamer via sulfydryl chemistry. Nanosized gold seed particles were then modified with the functionalized gold nanospheres, and this strongly increases the peroxidase-mimicking activity of the nanomaterial. By using the tetramethylbenzidine/H2O2 chromogenic system, the absorbance at 652 nm increases linearly in the 0.3 to 200 ng L-1 HIF-1α concentration range.

Determination of Flavonoid Glycosides by UPLC-MS to Authenticate Commercial Lemonade.

So far, there is no report on the quality evaluation of lemonade available in the market. In this study, a sample preparation method was developed for the determination of flavonoid glycosides by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) based on vortex-assisted dispersive liquid-liquid microextraction. First, potential flavonoids in lemonade were scanned and identified by ultra-performance liquid chromatography-time of flight mass spectrometry (UPLC-TOF/MS). Five flavonoid glycosides were identified as eriocitrin, narirutin, hesperidin, rutin, and diosmin according to the molecular formula provided by TOF/MS and subsequent confirmation of the authentic standard. Then, an ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QqQ/MS) method was developed to determine these five flavonoid glycosides in lemonade. The results showed that the content of rutin in some lemonade was unreasonably high. We suspected that many illegal manufacturers achieved the goal of low-cost counterfeiting lemonade by adding rutin. This suggested that it was necessary for relevant departments of the state to make stricter regulations on the quality standards of lemonade beverages.

Competitive Protein Binding Assay of Naproxen by Human Serum Albumin Functionalized Silicon Dioxide Nanoparticles.

We have developed a new competitive protein binding assay (CPBA) based on human serum albumin functionalized silicon dioxide nanoparticles (nano-SiO2-HSA) that can be used for naproxen determination in urine. Compared with a conventional multi-well reaction plate, nano-SiO2 with a high surface-area-to-volume ratio could be introduced as a stationary phase, markedly improving the analytical performance. Nano-SiO2-HSA and horseradish peroxidase-labeled-naproxen (HRP-naproxen) were prepared for the present CPBA method. In this study, a direct competitive binding to nano-SiO2-HSAwas performed between the free naproxen in the sample and HRP-naproxen. Thus, the catalytic color reactions were investigated on an HRP/3,3'5,5'-tetramethylbenzidine (TMB)/H2O2 system by the HRP-naproxen/nano-SiO2-HSA composite for quantitative measurement via an ultraviolet spectrophotometer. A series of validation experiments indicated that our proposed methods can be applied satisfactorily to the determination of naproxen in urine samples. As a proof of principle, the newly developed nano-CPBA method for the quantification of naproxen in urine can be expected to have the advantages of low costs, fast speed, high accuracy, and relatively simple instrument requirements. Our method could be capable of expanding the analytical applications of nanomaterials and of determining other small-molecule compounds from various biological samples.

UPLC-QqQ/MS combined with similarity assessment of 17 nucleic acid constituents in 147 edible fungi from Sichuan Basin, China.

Nucleic acid constituents are the main functional ingredients in edible fungi, therefore understanding the nucleic acid constituents of edible fungi often eaten on the table by the Chinese people is of significant value. In this study, Sichuan Provincial Center for Disease Control and Prevention collected 147 samples of edible fungi (including certain species that the Chinese often eat) from different parts of the Sichuan Basin. A new UPLC-QqQ/MS method has been developed to determine the 17 nucleic acid constituents in these 147 samples, including guanosine, adenosine, uridine, cytidine, inosine, thymidine, xanthosine dehydrate, 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxyuridine, 2'-deoxycytidine, 2'-deoxyinosine, guanosine 5'-monophosphate, adenosine 5'-monophosphate, uridine 5'-monophosphate, cytidine 5'-monophosphate, and inosine 5'-monophosphate. Finally, similarity assessment of the main edible fungus was performed using vectorial angle cosine method, and hierarchical cluster analysis was used to classify all the 147 samples. The results showed that some edible fungi have high similarities, especially in Lentinula edodes (Berk.) Pegler, the monogenic nucleotides content of which (55.84 ±â€¯8.4 mg/100 g) is far greater than any other edible fungus, which is directly related to its taste. For quality control, this paper proposed to use the reference values of total nucleic acid compounds in edible fungi computed by percentile threshold method. This is the first time a comprehensive evaluation of nucleic acid constituents of different edible fungi of daily consumption was conducted for a large region, and the results is conducive to the quality evaluation and quality control of edible fungus.

Determination of a astragaloside IV derivative LS-102 in plasma by ultra-performance liquid chromatography-tandem mass spectrometry in dog plasma and its application in a pharmacokinetic study.

BACKGROUND: Astragalosidic acid (LS-102) is a new water-soluble derivative of astragaloside IV - a major effective component isolated from the Chinese herb Astragali Radix. Our previous study showed that LS-102 exhibited potent cardiovascular activity. PURPOSE: The objective of this study was to investigate the pharmacokinetic properties of LS-102 after single-dose, oral administration in beagle dogs by developing and validating an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. METHOD AND RESULT: The chromatographic separation was performed on a Acquity HSS C18 column (100 mm × 2.1 mm, 1.8 µm) by a gradient elution using a mobile phase consisting of water and acetonitrile at a flow rate of 0.35 ml/min. The analytes were detected with a triple quadrupole tandem mass spectrometry in multiple reaction monitoring mode. Method validation revealed a wide linearity over the range of 2.0-10,000 ng/ml together with satisfactory intra- and inter-day precision, accuracy, and recovery. Stability testing showed that LS-102 spiked into dog plasma was stable for 4 h at room temperature, for up to 2 weeks at -80 °C, and during three freeze-thaw cycles. The method was effectively and successfully applied to the pharmacokinetics of LS-102 after oral administration (5, 10 and 20 mg/kg) to beagle dogs. Peak plasma concentrations are attained within approximately 2 h after oral administration with a half-life ranging from 1.55 h to 4.49 h. The plasma concentration-time curve of LS-102 after oral administration presents the phenomenon of a double-peak absorption phase. The peak concentration and area under the concentration-time curve of LS-102 seemed to increase with the increasing doses proportionally, that suggesting linear pharmacokinetics in dogs. Meanwhile, the doxorubicin (Dox)-injured H9c2 cell model was prepared by incubating the cells in 1 µM Dox for 24 h. MTT assay and LDH release measurement showed that LS-102 protected against Dox-induced cardiomyocyte death. CONCLUSION: The obtained results may help to guide the further pre-clinical research of LS-102 as a potentially novel cardioprotective agent.

Pharmacokinetics Comparison, Intestinal Absorption and Acute Toxicity Assessment of a Novel Water-Soluble Astragaloside IV Derivative (Astragalosidic Acid, LS-102).

BACKGROUND AND OBJECTIVES: Astragaloside IV (AGS IV) is the most important bioactive constituent of Radix Astragali. However, its disappointing clinical application is mainly caused by its very low solubility in biologic fluids, resulting in poor bioavailability after oral administration. We recently obtained a novel water-soluble derivative of AGS IV (astragalosidic acid, LS-102) that displayed significant cardioprotective potential against hypoxia-induced injury. The objective of this study was to investigate the intestinal absorption, main pharmacokinetic parameters and acute toxicity of LS-102 in rodents compared with AGS IV. METHODS: An oral dose of LS-102 and AGS IV (20 mg/kg) was administered to Sprague-Dawley (SD) rats, and blood samples were collected at predetermined time points. The plasma concentrations were detected by a validated UHPLC-MS/MS method, and pharmacokinetic parameters were calculated using a compartmental model. In the intestinal permeability study, the transport of LS-102 across Caco-2 cell monolayers was investigated at six concentrations from 6.25 to 250 µM. Moreover, the acute toxicity of LS-102 (40-5000 mg/kg) via a single oral administration was investigated in BALB/c mice. RESULTS: LS-102 was rapidly absorbed, attaining a maximum concentration of 248.7 ± 22.0 ng/ml at 1.0 ± 0.5 h after oral administration. The relative bioavailability of LS-102 was twice that of AGS IV. LS-102 had a Papp (mean) of 15.72-25.50 × 10-6 cm/s, which was almost 500-fold higher than that of AGS IV, showing that LS-102 had better transepithelial permeability and could be better absorbed in the intestinal tract. The acute toxicity study showed no abnormal changes or mortality in mice treated with LS-102 even at the single high dose of 5000 mg/kg body weight. CONCLUSIONS: Oral LS-102 produced a pharmacokinetic profile different from AGS IV with higher bioavailability, while the toxic tolerance was similar to previous estimates. Thus, we speculated that LS-102 might provide better clinical efficacy and be a potential candidate for the new drug development of Radix Astragali.

Quality Evaluation of Tricholoma matsutake Based on the Nucleic Acid Compounds by UPLC-TOF/MS and UPLC-QqQ/MS.

So far, there has been no quality evaluation of Tricholoma matsutake. Nucleic acid compounds are a kind of functional ingredient in T. matsutake that is beneficial to human health. In this study, a UPLC-TOF/MS method was first used to scan and identify the potential nucleic acid compounds in T. matsutake. Based on the calculation of the molecular formula and subsequent confirmation by authentic standards, 15 nucleic acid compounds were unambiguously identified: adenosine, cytidine, guanosine, inosine, thymidine, uridine, xanthosine dehydrate, 2'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, 2'-deoxyuridine, adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate, and uridine 5'-monophosphate. Then, a UPLC-QqQ/MS method was developed for the subsequent quantitative analysis. After validating the limits of quantification, detection, precision, repeatability, and recovery through a calibration curve, the content of 15 nucleic acid compounds was determined by the proposed UPLC-QqQ/MS method in 80 T. matsutake samples collected from different regions in Sichuan province, Southwest China. After the statistical analysis, we suggest that the total content of nucleic acid compounds in the qualified T. matsutake should be higher than 24.49 mg/100 g. The results indicated that the combined use of UPLC-TOF/MS and UPLC-QqQ/MS is efficient for fast identification and determination of nucleic acid compounds to comprehensively evaluate the quality of T. matsutake.

Optimization of Vortex-Assisted Dispersive Liquid-Liquid Microextraction for the Simultaneous Quantitation of Eleven Non-Anthocyanin Polyphenols in Commercial Blueberry Using the Multi-Objective Response Surface Methodology and Desirability Function Approach.

In the present study, 11 non-anthocyanin polyphenols, gallic acid, protocatechuate, vanillic acid, syringic acid, ferulic acid, quercetin, catechin, epicatechin, epigallocatechin gallate, gallocatechin gallate and epicatechin gallate-were firstly screened and identified from blueberries using an ultra performance liquid chromatography⁻time of flight mass spectrography (UPLC-TOF/MS) method. Then, a sample preparation method was developed based on vortex-assisted dispersive liquid-liquid microextraction. The microextraction conditions, including the amount of ethyl acetate, the amount of acetonitrile and the solution pH, were optimized through the multi-objective response surface methodology and desirability function approach. Finally, an ultra performance liquid chromatography⁻triple quadrupole mass spectrography (UPLC-QqQ/MS) method was developed to determine the 11 non-anthocyanin polyphenols in 25 commercial blueberry samples from Sichuan province and Chongqing city. The results show that this new method with high accuracy, good precision and simple operation characteristics, can be used to determine non-anthocyanin polyphenols in blueberries and is expected to be used in the analysis of other fruits and vegetables.

A efficient method to identify cardioprotective components of Astragali Radix using a combination of molecularly imprinted polymers-based knockout extract and activity evaluation.

Although herb medicines have become the major source for new drug discovery, many of them are largely under-explored due to the purity-activity relationship. Efficient identification of bioactive compounds in conventional stepwise separation and isolation has not yet been elucidated. Therefore, we proposed a new separation strategy for holism understanding of herb pharmacology using molecularly imprinted polymers (MIPs). Astragali Radix (AR), important in traditional Chinese medicine, was chosen in this study for multicomponent knockout followed by bioactivity evaluation. We prepared calycosin molecularly imprinted polymers (calycosin-MIPs) which could selectively recognize flavonoid aglycons in AR. The molecular selectivity of calycosin-MIPs as a critical parameter was evaluated using the template and other high content compounds in AR. Based on it, using the calycosin-MIPs material via solid-phase extraction procedures was applied for the knockout of flavonoid aglycons in AR. Finally, hypoxia/reoxygenation model in H9c2 cells was used to evaluate the activity of the AR extract before and after knockout. The results showed that calycosin-MIPs as recognition materials for flavonoid aglycons in AR are applied in one-step separation with high selectivity and tunability. The subextract in the absence of flavonoid aglycons has been demonstrated to clarify the cardio-protective components of AR. In conclusion, this proof-of-principle study is the first one showing that molecular imprinting technology coupled with a bioassay can be used to explore the bioactive variability from the perspective of multicomponent separation of herbal medicine.

Neutral Loss Scan - Based Strategy for Integrated Identification of Amorfrutin Derivatives, New Peroxisome Proliferator-Activated Receptor Gamma Agonists, from Amorpha Fruticosa by UPLC-QqQ-MS/MS and UPLC-Q-TOF-MS.

Amorfrutins with a 2-hydroxybenzoic acid core structure are promising natural PPARγ agonists with potent antidiabetic activity. Owing to the complex matrix and low concentration in botanical material, the identification of unknown amorfrutins remains a challenge. In the present study, a combined application of UPLC-Q-TOF-MS and UPLC-QqQ-MS was developed to discover unknown amorfrutins from fruits of Amorpha fruticosa. First, reference compounds of amorfrutin A (AA), amorfrutin B (AB), and 2-carboxy-3,5-dihydroxy-4-geranylbibenzyl (AC) were analyzed using UPLC-Q-TOF-MS to reveal the characteristic fragment ions and the possible neutral loss. Second, the extract of A. fruticosa was separated and screened by UPLC-QqQ-MS using neutral loss scan to find out suspect compounds associated with the specified neutral fragment Δm/z 44. Third, the extract was re-analyzed by UPLC-Q-TOF-MS to obtain the exact mass of quasi-molecular ion and fragment ions of each suspect compound, and to subsequently calculate their corresponding molecular formulas. Finally, according to the molecular formula of suspect compound and its fragment ions and comparing with literature data, structure elucidation of four unidentified amorfrutins was achieved. The results indicated that the combination of QqQ-MS neutral loss scan and Q-TOF-MS molecular formula calculation was proven to be a powerful tool for unknown natural product identification, and this strategy provides an effective solution to discover natural products or metabolites of trace content. Graphical Abstract ᅟ.

Astragalosidic Acid: A New Water-Soluble Derivative of Astragaloside IV Prepared Using Remarkably Simple TEMPO-Mediated Oxidation.

There is an urgent need for a water-soluble derivative of astragaloside IV for drug R&D. In the present study, a remarkably simple method for the preparation of such a water-soluble derivative of astragaloside IV has been developed. This protocol involves oxidative 2,2,6,6-tetramethylpiperidine-1-oxyl free radical (TEMPO)-mediated transformation of astragaloside IV to its carboxylic acid derivative, which is a new compound named astragalosidic acid. The structure of astragalosidic acid was elucidated by means of spectroscopic analysis. Its cardioprotective activity was investigated using an in vitro model of cardiomyocyte damage induced by hypoxia/reoxygenation in H9c2 cells. The oxidative TEMPO-mediated transformation proposed in the present study could be applied to other natural saponins, offering an effective and convenient way to develop a new compound with greatly improved structure-based druggability.