The chemometrics analysis and integrated pharmacology approach to decipher the effect and mechanism between raw and processed cistanche tubulosa.

ETHNOPHARMACOLOGICAL RELEVANCE: Cistanche tubulosa (CT) is the dried fleshy stem with scaly leaves of Cistanche tubiflora (Schenk) Wight, which has the effects of tonifying the kidney-yang, benefiting the vital essence and blood, and moisturizing the intestines and laxatives. There are differences in the activity of CT before and after processing, but the mechanism of processing is not clear. AIM OF THE STUDY: The study aimed to compare the strength of action of CT before and after yellow-wine processing in the treatment of constipation and kidney yang deficiency and to identify the active ingredients responsible for the differences in activity before and after yellow-wine processing. MATERIALS AND METHODS: This study established the fingerprints of CT and PCT using HPLC to identify their shared components. Then efficacy of KYDS and FC were carried out to compare the differences between CT and PCT in terms of efficacy. Next, this study established the spectrum-effect relationship between the shared chemical components and the medical effects of CT and PCT using the gray correlation analysis and entropy methods. Ultimately, the activity of the analyzed chemical components was verified using the zebrafish model. RESULTS: CT was more effective than PCT in promoting intestinal peristalsis, regulating gastrointestinal hormone levels, and thus treating FC. PCT was more effective than CT in improving the level of hormone indexes of the hypothalamus-pituitary-target gland axis, replenishing blood, and enhancing immunity. Through the analysis of the spectrum-effect relationship, it was finally found that 5, 6, 12 (tubuloside A), and 13 (isoacteoside) might be more closely related to the activity of tonifying kidney yang, and peaks 9, 10, and 11 (acteoside) are more closely associated with the treatment of constipation, and peaks 3 (salidroside), 4, 1, 2 (geniposidic acid), and 8 (echinacoside) were associated with both kidney yang tonic and treatment of constipation. At the same time, an activity verification experiment showed that echinacoside, geniposidic acid, and salidroside were effective in the treatment of FC and KYDS, while acteoside was very effective in the treatment of FC, and tubuloside A was significant in supplementing the blood, which validated the spectrum-effect relationship analysis. CONCLUSION: This study proved that the raw CT had a better laxative effect, while the yellow-wine processed CT had a better kidney-yang tonic effect; moreover, spectrum-effect relationships were established to analyze the chemical components leading to changes in the activity of CT before and after yellow-wine processing.

Lysimachiadanxiashanensis, a new species of Primulaceae from Guangdong, China.

Lysimachiadanxiashanensis, a new Primulaceae species, endemic to the Danxia landscape in Guangdong Province, China, is described and illustrated. This new species is morphologically similar to L.pseudohenryi, L.phyllocephala, L.congestiflora and L.kwangtungensis, but it differs from the similar species by its purplish-red plants, petiole without wings, calyx with orange glandular and the corolla margin serrated on upper half with orange-red glandular punctates. This new species belongs to Lysimachiasubgen.Lysimachiasect.Nummularia. Phylogenetic analysis confirmed that L.danxiashanensis is a distinct clade, based on the combined data of ITS and rbcL sequences. The conservation status of the new species was evaluated as Endangered (EN) according to IUCN Red List Categories and Criteria.

Crystal Violet-Sensitized Direct Z-Scheme Heterojunction Coupled with a G-Wire Superstructure for Photoelectrochemical Sensing of Uracil-DNA Glycosylase.

Dye sensitization achieving photoelectrochemical (PEC) signal amplification for ultrasensitive bioanalysis has undergone a major breakthrough. In this proposal, an innovative PEC sensing platform is developed by combining Z-scheme WO3@SnS2 photoactive materials and a G-wire superstructure as well as a dye sensitization enhancement strategy. The newly synthesized WO3@SnS2 heterojunction with outstanding PEC performance is employed as a photoelectrode matrix. Due to the formation of the Z-scheme heterojunction between WO3 and SnS2, the migration dynamics of the photogenerated carrier is evidently augmented. To improve sensitivity, the target excision-driven dual-cycle signal amplification strategy is introduced to output exponential c-myc fragments. Crystal violet is then conjugated into the G-quadruplex to amplify the PEC signal, where crystal violet generates excited electrons by capturing visible light and rapidly injects electrons into the conduction band of SnS2, suppressing the recombination of the photo-induced carrier. Moreover, the G-wire superstructure acts as a universal amplification pathway, ensuring adequate crystal violet loads. Specifically, the biosensor for uracil-DNA glycosylase quantification displays a wide detection range (0.0005-1.0 U/mL) and a lower detection limit (0.00025 U/mL). Furthermore, the Z-scheme electron migration mechanism and the crystal violet sensitization effect are discussed in detail. The construction of the PEC sensor provides a new consideration for signal amplification and material design.

Signal-off photoelectrochemical determination of miRNA-21 using aptamer-modified In2O3@Cu2MoS4 nanocomposite.

In2O3@Cu2MoS4 nanocomposite with superior photoelectrochemical (PEC) performance is used for the first time as a photoactivity material, and a signal-off PEC biosensing platform for miRNA detection has been successfully constructed. Firstly, the Cu2MoS4 nanosheets are synthesized by a hydrothermal method, and then, the homogeneous In2O3 nanoparticles (In2O3 NPs) are synthesized by calcination in the air. The In2O3@Cu2MoS4 nanocomposite is constructed with the Cu2MoS4 nanosheets as matrix and In2O3 NPs as sensitizer through a layer-by-layer assembly strategy. The nanocomposite with a tight interface and the matched band structure restrains the electron-hole pair recombination. Under visible light (400-700 nm), the nanocomposite exhibits a strong initial signal. With the catalyzed hairpin assembly, dozens of PbS quantum dots (QDs) are introduced on the surface of an electrode, significantly reducing the photocurrent of n-type In2O3@Cu2MoS4. Since PbS QDs can compete with the nanocomposite for light energy and electron donors, the signal decreased. Under optimal conditions, the biosensor manifests a broad linear range (1 fM-1 nM) and a low detection limit of about 0.57 fM, at a working potential of 0 V (vs. Ag/AgCl). The recovery of spiked human serum is between 94.0 and 102%, and the relative standard deviation (RSD) is between 1.3 and 2.7%. Therefore, the as-fabricated biosensor exhibits a potential for the determination of miRNA-21 in practical applications.Graphical abstract The In2O3@Cu2MoS4 nanocomposite owns a strong anode photocurrent signal, which can be used as a photoactive material to construct a "signal-off" biosensor for the detection of miRNA in non-enzymatically catalyzed hairpin assembly (CHA) reaction.

Ratiometric assay of mercury ion based on nitrogen-doped carbon dots with two different optical signals: second-order scattering and fluorescence.

Ratiometric assays, which can effectively surmount external interference, have attracted extensive research interests. Herein, a novel ratiometric sensing platform for Hg2+ is designed based on nitrogen-doped carbon dots (N-CDs) with two different optical signals. Under a single excitation, N-CDs have two emission peaks around 668 nm and 412 nm, which are second-order scattering and fluorescence, respectively. Upon the addition of Hg2+, the weak scattering emission at 668 nm can be increased apparently, while the strong fluorescence intensity at 412 nm is weakened. Moreover, the ratio of scattering intensity to fluorescence intensity is linearly dependent on Hg2+ concentration (0.1-10 µM and 10-30 µM, respectively), and the detection limit is 66 nM. In addition, the ratiometric sensing mechanism is investigated in detail, which is due to the combined effect of aggregation-induced fluorescence quenching and scattering enhancement. Furthermore, the developed sensing approach holds a promising application for Hg2+ detection in actual samples. Graphical abstract.

Protective effects of acacetin isolated from Ziziphora clinopodioides Lam. (Xintahua) on neonatal rat cardiomyocytes.

BACKGROUND: The total flavonoids from ethanol extract of the aerial part of Ziziphora clinopodioides Lam. (Lamlaceae) (Xintahua) showed protective activities against rat acute myocardial ischemia in rats. This study aims to isolate acacetin, a flavonoid, from the aerial part of Z. clinopodioides, to develop an HPLC method for its detection, and to evaluate its protective effects on neonatal rat cardiomyocytes. METHODS: Sephadex LH-20 silicagel and pillar layer chromatography silica gel were applied for the isolation and purification of acacetin and its structure was elucidated on the basis of (1)H and (13)C NMR spectroscopy. The content of acacetin in Z. clinopodioides collected from three different origins was determined by HPLC. The neonatal rat cardiomyocytes were isolated and cultured in vitro to establish a hypoxia/reoxygenation injury model. The viability of cardiomyocytes was measured by the MTT method. Changes of malondialdehyde (MDA) content in the medium were also determined. RESULTS: The acacetin content in various batches of Z. clinopodioides ranged from 45.50 to 47.41 µg/g. Acacetin of 25, 10, 5 µg/mL significantly decreased the MDA content in a model of hypoxia/reoxygenation injury (P < 0.001, P < 0.001 and P = 0.033, respectively). CONCLUSIONS: Acacetin protects neonatal cardiomyocytes from the damage induced by hypoxia/reoxygenation stress through reduction of lipid peroxidation and enhancement of the antioxidant activity.

Identification and functional characterization of hCLS1, a human cardiolipin synthase localized in mitochondria.

In eukaryotic cells, CLS (cardiolipin synthase) is involved in the final step of cardiolipin synthesis by catalysing the transfer of a phosphatidyl residue from CDP-DAG (diacylglycerol) to PG (phosphatidylglycerol). Despite an important role of cardiolipin in regulating mitochondrial function, a gene encoding the mammalian CLS has not been identified so far. We report in the present study the identification and characterization of a human cDNA encoding the first mammalian CLS [hCLS1 (human CLS1)]. The predicted hCLS1 peptide sequence shares significant homology with the yeast and plant CLS proteins. The recombinant hCLS1 enzyme expressed in COS-7 cells catalysed efficiently the synthesis of cardiolipin in vitro using CDP-DAG and PG as substrates. Furthermore, overexpression of hCLS1 cDNA in COS-7 cells resulted in a significant increase in cardiolipin synthesis in intact COS-7 cells without any significant effects on the activity of the endogenous phosphatidylglycerophosphate synthase of the transfected COS-7 cells. Immunohistochemical analysis demonstrated that the recombinant hCLS1 protein was localized to the mitochondria when transiently expressed in COS-7 cells, which was further corroborated by results from subcellular fractionation analyses of the recombinant hCLS1 protein. Northern-blot analysis showed that the hCLS1 gene was predominantly expressed in tissues that require high levels of mitochondrial activities for energy metabolism, with the highest expression in skeletal and cardiac muscles. High levels of hCLS1 expression were also detected in liver, pancreas, kidney and small intestine, implying a functional role of hCLS1 in these tissues.

A novel and selective beta-melanocyte-stimulating hormone-derived peptide agonist for melanocortin 4 receptor potently decreased food intake and body weight gain in diet-induced obese rats.

alphaMSH has generally been accepted as the endogenous ligand for melanocortin 4 receptor (MC4R), which plays a major role in energy homeostasis. Targeting MC4R to develop antiobesity agents, many investigators have performed a structure-activity relationship (SAR) studies based on alphaMSH structure. In this report, we performed a SAR study using human betaMSH (5 - 22) (DEGPYRMEHFRWGSPPKD, peptide 1) as a lead sequence to develop potent and selective agonists for MC4R and MC3R. The SAR study was begun with a truncation of N terminus of betaMSH (5 - 22) together with acetylation of the N terminus and amidation of the C terminus of the peptide. Introduction of a cyclic disulfide constrain and replacement of L-Phe with D-Phe afforded a super potent agonist (peptide 5). Furthermore truncation at the C terminus generated a small and potent MC4R and MC3R agonist (Ac-YRcyclo[CEHdFRWC]amide, peptide 6), which exhibited no MC5R and greatly reduced MC1R activity. Molecular modeling of Ac-YRcyclo[CEHdFRWC]amide (peptide 6) revealed that Arg2 in the peptide formed a salt bridge with Glu4. Subcutaneous or intracerebroventricular administration of peptide 6 in rats showed potent in vivo efficacy as evidenced by its effects in reducing energy balance, increasing fat use, and decreasing weight gain in both acute and chronic rat metabolic studies. Furthermore, the antiobesity effect by peptide 6 was manifested only in wild-type but not MC4R-deficient mice, indicating that antiobesity effects of the peptide were attributed largely through MC4R but not MC3R agonist activity of the peptide.

Potent peptide agonists for human melanocortin 3 and 4 receptors derived from enzymatic cleavages of human beta-MSH(5-22) by dipeptidyl peptidase I and dipeptidyl peptidase IV.

Human beta-MSH(1-22) was first isolated from human pituitary as a 22-amino acid (aa) peptide derived from a precursor protein, pro-opiomelanocortin (POMC). However, Bertagna et al. demonstrated that a shorter human beta-MSH(5-22), (DEGPYRMEHFRWGSPPKD), is a true endogenous peptide produced in human hypothalamus. In this report, we demonstrated that in vitro enzymatic cleavage of native human beta-MSH(5-22) with two ubiquitous dipeptidyl peptidases (DPP), DPP-I and DPP-IV, generated two potent MC3/4R peptide analogues, beta-MSH(7-22) (GPYRMEHFRWGSPPKD) and beta-MSH(9-22) (YRMEHFRWGSPPKD). In fact, the MC4R binding affinity and functional potency of beta-MSH(7-22) (Ki=4.6 nM, EC50=0.6 nM) and beta-MSH(9-22) (Ki=5.7 nM, EC50=0.6 nM) are almost an order of magnitude greater than those of their parent peptide, beta-MSH(5-22) (MC4R, Ki=23 nM, EC50= 3nM). Furthermore, the DPP-I/DPP-IV cleaved peptide, beta-MSH(9-22), when administered intracerebroventricularly (ICV) at a dose of 3 nmol/rat, potently induced an acute negative energy balance in a diet-induced obese rat model, while its parent molecule, beta-MSH(5-22), administered at the same dose did not have any effect. These data suggest that DPP-I and DPP-IV may play a role in converting the endogenous beta-MSH(5-22) to more potent peptides that regulate energy homeostasis in the hypothalamus.

Discovery of a beta-MSH-derived MC-4R selective agonist.

A series of novel, disulfide-constrained human beta-melanocyte stimulating hormone (beta-MSH)-derived peptides were optimized for in vitro melanocortin-4 receptor (MC-4R) binding affinity, agonist efficacy, and selectivity. The most promising of these, analogue 18, was further studied in vivo using chronic rat food intake and body weight models.