[Research strategies and methods for precious animal medicine substitutes].

Animal medicine is a large category of Chinese medicinecommonly used in clinical practice and has important scientific and therapeutic value. Animal medicine isscarcer than herbal medicine. In recent years, with the vigorous development of traditional Chinese medicine(TCM),the contradiction between the increasing industrial demand andsupply of scarce and even endangered medicinal animals has become increasingly prominent. The continuous lack of medicinal animal resources affects the clinical demandandalso causes serious damage to the ecological environment. Only relying on artificial breeding is not enough to alleviate the current condition of depletion. In the face of this dilemma, it is a major challenge for the current industrial development to protect animal resources and meet clinical and industrial needs with "available medicines". The application of substitutes for animal medicines isthe key focus to alleviate this problem, and it is also the key scientific issue to be solved urgently in the modernization of TCM. This paper summarizedand reviewedthe history, current situation, strategies, and methods of animal medicinesubstitution and put forward the point of view of "similar chemical characteristics, similar efficacy, and higher safety" to provide references for scientific substitution and resource protection of rare animals.

A target lipidomics approach to investigate the acute inflammatory irritation induced by indolealkylamines from Chansu water fraction in rats.

Chansu has demonstrated adverse reactions in clinical settings, which is associated with its toxicity and limits its clinical applications. But there are methodological limitations for drug safety evaluation. In the current study, ultra-high performance liquid chromatography, lipidomic profiling, and molecular docking were used to systemically assess Chansu-induced acute inflammatory irritation and further identify the underlying drug targets. Compared with the EtOAc extract, Chansu water fraction containing indolealkylamines caused acute inflammatory irritation in rats, including acute pain (spontaneous raising foot reaction), and inflammation (paw edema). At the molecular level, lipids analysis revealed significantly higher levels of pro-inflammatory mediators of the COX and LOX pathways. However, anti-inflammatory mediators from the CYP 450, ALA, and DHA pathways markedly decreased after exposure to Chansu water fraction. Moreover, four indolealkylamines from Chansu showed a high theoretical affinity to a known irritation target, 5-HT2AR. These results suggest that Chansu-induced inflammatory irritation is related to the distinct dysregulation of inflammatory lipids, and peripheral 5-HT2AR is a potential target for irritation therapy. The strategy used in this study can be a crucial approach in the safety evaluation of natural medicinal substances.

Bufotenine and its derivatives: synthesis, analgesic effects identification and computational target prediction.

Natural product bufotenine (5) which could be isolated from Venenum Bufonis, has been widely used as a tool in central nervous system (CNS) studies. We present here its quaternary ammonium salt (6) which was synthesized with high yields using 5-benzyloxyindole as raw materials, and we firstly discover its analgesic effects in vivo. The analgesic evaluation showed that compounds 5 and 6 had stronger effects on the behavior of formalin induced pain in mice. Moreover, the combination of compound 6 and morphine has a synergistic effect. We intended to explain the molecular mechanism of this effect. Therefore, 36 analgesic-related targets (including 15 G protein-coupled receptors, 6 enzymes, 13 ion channels, and 2 others) were systemically evaluated using reverse docking. The results indicate that bufotenine and its derivatives are closely related to acetyl cholinesterase (AChE) or α4ß2 nicotinic acetylcholine receptor (nAChR). This study provides practitioners a new insight of analgesic effects.

[Comparison of chemical composition between fresh and processed Bufonis Venenum by UPLC-TQ-MS].

Toad venom is the Bufo bufo gargarizans or B. melanostictus after the ears of the gland secretion, used in the treatment of various cancers in recent years. Research shows that the main anti-tumor components in bufadienolide. Bufadienolide have free type structure and conjunct type structure. To identify and clarify the difference between bufogenin and bufotoxin contained in Bufonis Venenum, which was from B. bufo gargarizans, an UPLC-TQ-MS method has been established. UPLC-TQ-MS method was used to identify and quantify the major bufadienolides in Bufonis Venenum. UPLC-TQ-MS assay with positive ion mode was performed on a Waters ACQUITY UPLC BEH C, (2.1 mm x 100 mm, 1.7 µm) with the mobile phase consisting of 0. 1% aqueous formic and acidacetonitrile in gradient elution at a flow rate of 0.4 mL · min⁻¹ and the column temperature was set at 35 °C. By comparing their retention time and high resolution mass data of Bufonis Venenum extracts, 37 effective components were primarily identified by MS/MS analysis in positive ion mode. Twenty-six of them were free-type bufadienolides (bufogenin), 11 of them were conjugated bufadienolides. There were significant differences in the main composition between fresh and processed Bufonis Venenum. The study found that the chemical composition of toad venom through great changes after processing, conjunct type content is much less, free type content as well change.

[Molecular docking of chlorogenic acid, 3,4-di-O-caffeoylquinic acid and 3,5-di-O-caffeoylquinic acid with human serum albumin].

OBJECTIVE: To investigate the mechanism of binding of human serum albumin (HSA) with potential sensitinogen, including chlorogenic acid and two isochlorogenic acids (3,4-di-O-caffeoylquinic acid and 3,5-di-O-caffeoylquinic acid). METHODS: By using the docking algorithm of computer-aided molecular design and the Molegro Virtual Docker, the crystal structures of HSA with warfarin and diazepam (Protein Data Bank ID: 2BXD and 2BXF) were selected as molecular docking receptors of HSA sites I and II. According to docking scores, key residues and H-bond, the molecular docking mode was selected and confirmed. The molecular docking of chlorogenic acid and two isochlorogenic acids on sites I and II was compared based on the above design. RESULTS: The results from molecular docking indicated that chlorogenic acid, 3,4-di-O-caffeoylquinic acid and 3,5-di-O-caffeoylquinic acid could bind to HSA site I by high affinity scores of -112.3, -155.3 and -153.1, respectively. They could bind to site II on HSA by high affinity scores of -101.7, -138.5 and -133.4, respectively. In site I, two isochlorogenic acids interacted with the key apolar side-chains of Leu238 and Ala291 by higher affinity scores than chlorogenic acid. Furthermore, the H-bonds of isochlorogenic acids with polar residues inside the pocket and at the entrance of the pocket were different from chlorogenic acid. Moreover, the second coffee acyl of isochlorogenic acid occupied the right-hand apolar compartment in the pocket of HSA site I. In site I, the second coffee acyl of isochlorogenic acid formed the H-bonds with polar side-chains, which contributed isochlorogenic acid to binding with site II of HSA. CONCLUSION: The isochlorogenic acids with two coffee acyls have higher binding abilities with HSA than chlorogenic acid with one coffee acyl, suggesting that isochlorogenic acids binding with HSA may be sensitinogen.

[Analysis on competitive interaction between arenobufagin and verapamil hydrochloride with serum albumin].

OBJECTIVE: The present study was performed to investigate competitive interaction between arenobufagin and verapamil hydrochloride with serum albumin. METHODS: Equilibrium dialysis and high-performance liquid chromatography were used to analyze the binding rates of the two medicines to serum protein. The interactions based on bovine serum albumin (BSA) and human serum albumin (HSA) were investigated by using spectrofluorimetry. The interaction mode of arenobufagin and verapamil hydrochloride binding to serum proteins was simulated by molecular docking. RESULTS: The rate of arenobufagin (0.1µg/mL) binding to bovine serum was (61.1±0.2)%. Verapamil hydrochloride (0.025 to 0.1µg/mL) significantly reduced the bovine serum binding rate of arenobufagin, from (60.2±3.7)% to (36.9±3.4)%. However, arenobufagin at the tested doses had no marked effects on the binding rate of verapamil hydrochloride. Furthermore, the verapamil hydrochloride had an active effect on the arenobufagin-induced fluorescence quenching of BSA and HSA. The molecular docking results showed that verapamil hydrochloride and arenobufagin binded to HSA at site I. Molecular interaction energy of verapamil hydrochloride binding to site I was stronger than that of arenobufagin. CONCLUSION: Verapamil hydrochloride reduces the binding of arenobufagin to bovine serum. The mechanism may be a competitive interaction of arenobufagin and verapamil hydrochloride at site I on HSA.

The protective effects of the active fraction of Shaofu Zhuyu decoction on hydrogen peroxide-induced oxidative injury in vascular smooth muscle cells.

In this paper, the protective effects of the active fraction (SF-7) from Shaofu Zhuyu decoction (SFZYD) were tested on a hydrogen peroxide (H(2)O(2))-induced rat vascular smooth muscle cells (VSMCs) oxidative injury model. This active fraction (SF-7) shows potent antioxidant properties. The cell viability and oxidative damage markers of VSMCs were determined after exposure to H(2)O(2) for 16 hours. It was observed that SF-7 significantly increased cell survival and reduced apoptosis of H(2)O(2)-injured VSMCs. Moreover, SF-7 could markedly increase intracellular superoxide dismutase (SOD) activity and decrease the malondialdehyde (MDA) level in H(2)O(2)-injured VSMCs, and suppress the generation of intracellular reactive oxygen species (ROS), as well as intracellular Ca2+ concentration. Thus, SF-7 exhibits protective effects against H(2)O(2)-injury on VSMCs, which may be associated with its antioxidant properties. It is suggested that SF may be useful in the treatment of blood stasis syndrome in which oxidative injury is mainly implicated.

Evaluation of the anti-inflammatory and analgesic activities of Liu-Shen-Wan and its individual fractions.

Liu-Shen-Wan (LSW), a famous traditional Chinese medicine for treatment of upper respiratory tract inflammation, was evaluated for its anti-inflammatory and analgesic activities. Acetic acid-elevated vascular permeability, carboxymethylcellulose sodium (CMC-Na)-induced leukocyte migration and ear edema induced by picryl chloride were used to test anti-inflammatory activity. Moreover, acetic acid-induced writhing and hot-plate tests were used to determine analgesic effect. It was observed that LSW exerted significant anti-inflammatory and analgesic activities in these models at doses of 30 and 90mg/kg crude drug in vivo. In addition, LSW potently inhibited proliferation of human peripheral blood mononuclear cell (PBMC) stimulated by streptococcal pyrogenic exotoxin at doses of 0.5-5microg/ml in vitro. LSW was then partitioned with chloroform, methanol, water and mineral fraction. Several fractions inhibited inflammation and pain in varying degrees. Among them, chloroform fraction was the most active in hot-plate and writhing tests, and exerted the remarkable inhibitory effect on human PBMC proliferation. Methanol and water fractions had more suppressive activities in vascular permeability, leukocyte migration and PC-DTH tests. These results suggest that LSW has significantly anti-inflammatory and analgesic activities. The chloroform fraction is a key fraction of LSW to the overall anti-inflammatory and analgesic effects, while methanol and water fractions also partly contribute to anti-inflammatory activities of LSW.