HBB contributes to individualized aconitine-induced cardiotoxicity in mice via interfering with ABHD5/AMPK/HDAC4 axis.

The root of Aconitum carmichaelii Debx. (Fuzi) is an herbal medicine used in China that exerts significant efficacy in rescuing patients from severe diseases. A key toxic compound in Fuzi, aconitine (AC), could trigger unpredictable cardiotoxicities with high-individualization, thus hinders safe application of Fuzi. In this study we investigated the individual differences of AC-induced cardiotoxicities, the biomarkers and underlying mechanisms. Diversity Outbred (DO) mice were used as a genetically heterogeneous model for mimicking individualization clinically. The mice were orally administered AC (0.3, 0.6, 0.9 mg· kg-1 ·d-1) for 7 d. We found that AC-triggered cardiotoxicities in DO mice shared similar characteristics to those observed in clinic patients. Most importantly, significant individual differences were found in DO mice (variation coefficients: 34.08%-53.17%). RNA-sequencing in AC-tolerant and AC-sensitive mice revealed that hemoglobin subunit beta (HBB), a toxic-responsive protein in blood with 89% homology to human, was specifically enriched in AC-sensitive mice. Moreover, we found that HBB overexpression could significantly exacerbate AC-induced cardiotoxicity while HBB knockdown markedly attenuated cell death of cardiomyocytes. We revealed that AC could trigger hemolysis, and specifically bind to HBB in cell-free hemoglobin (cf-Hb), which could excessively promote NO scavenge and decrease cardioprotective S-nitrosylation. Meanwhile, AC bound to HBB enhanced the binding of HBB to ABHD5 and AMPK, which correspondingly decreased HDAC-NT generation and led to cardiomyocytes death. This study not only demonstrates HBB achievement a novel target of AC in blood, but provides the first clue for HBB as a novel biomarker in determining the individual differences of Fuzi-triggered cardiotoxicity.

Occurrence of Stem Rot on Acacia mangium Caused by Amauroderma subresinosum.

Acacia mangium has the characteristics of developed root system, nitrogen fixation and soil improvement, fast growth and high yield, and improvement of soil fertility. It is often used as a windbreak tree species in rubber plantations, a highway shade tree, for coastal and mountain restoration in Hainan . In October 2021, a stem rot disease with an incidence of 3% was found in Baisha city(19°22'18″N,109°16'58″E), Hainan Province, China. In the early stage of the disease, the crown showed chlorotic leaves, followed by defoliation. In later stages, whole tree dieback was observed. The basal tissue of the stem of the diseased tree had white rot, and black-brown basidiocarps were observed about 1 m away from the ground. The basidiocarps surface of fresh was disinfected with 75 % ethanol, the epidermal tissue was removed, and the inner tissue blocks were transferred to PDA medium. After culturing in dark at 28°C for 3 days, a colony with white aerial mycelium was isolated and designated: HNBSMZXS20211011001. The basidiocarp was dark brown, sessile, mostly one-year-old, and the cap is nearly semi-circular, wooden, slightly shiny, with a size of 14.7 to 18.1cm × 9.5 to 10.1 cm. The base is thick (5 to 6.5 cm) and the edge is thin (0.3 to 0.7 cm). The basidiospores are oval, 10.7 to 13.65µm × 6.7 to 9.06µm in size, with a double-layer wall. The outer wall is transparent and the inner wall is light yellow. The basidiospores contain 1~2 oil droplets. The morphological features are consistent with those of Amauroderma subresinosum (Murrill) Corner (Zhang et al., 2000). The basidiocarps and type strain cultures were stored as accessions in the Laboratory of Plant Pathogen Fungus Biology, Hainan University. For pathogenicity tests, sawdust culture medium was used (soft sawdust 82%, wheat bran 15%, glucose 2%, gypsum 1%, mixed with water in proportion, sterilized at 121°C for 40min). The mycelium plug from a fresh culture (d=5mm) was taken from the edge of the colony of type strain, and transferred to the sterilized sawdust medium. When mycelium has colonized the media, it was used to inoculate plants. Media without mycelium was used as a control. Naturally growing seedlings (three year old) of A.mangium were selected from the teaching nurseries of Hainan University (20°6'25''N,110°32'24''E). First, 75 % alcohol was sprayed on the stem of the base of A.mangium for surface disinfection. After the surface was dried, a slight wound (about 4×2cm) was made on the surface with a sterilized scalpel. A inoculated and control sawdust media rods were tightly attached to the wound, moistened with cotton balls soaked in sterile water, and then fixed with plastic wrap, and the outer layer was wrapped with newsprint. Inoculation and controls were replicated three times. Two months after inoculated, the stems of the plants inoculated with the isolated fungus grew white hyphae and showed white rot symptoms, and the leaves became chlorotic and defoliated with complete tree decline in six months, which was consistent with the original symptoms observed. By comparison, white callus had grown on the edge of the stem wounds of the control plants. The same fungus was re-isolated from the inoculated plants and confirmed as A.subresinosum based on the internal transcribed spacer (ITS), the ribosomal large subunit(LSU), and the translation elongation factor 1-α(EF1-α) gene sequence, the fungus was not isolated from control plants thus fulfilling Koch's postulates. The ITS region of r-DNA, the ribosomal large subunit(LSU), the translation elongation factor 1-α gene(EF1-α) were amplified using ITS1/ITS4(White et al. 1990), LR0R/LR5(Hu et al. 2021), EF1-983F/EF1-1567R(Buckley et al. 2005) primers, respectively. The sequences of ITS (OQ674500), LSU (OQ674502) and EF1-α gene (OQ883944) were submitted to GenBank. Through with BLAST, the identities of the ITS, LSU and EF1-α sequences to A.subresinosum (GenBank Accession no. ITS: LC176755; LSU: MK119903 and EF1-α: MK121572) was 99.82%; 99.15% and 99.82%, respectively, the identities were more than 99 %. It was reported that A.subresinosum could infect Casuarina equisetifolia and Areca catechu(Chen et al., 2016; Cheng. 2017; Wu et al., 2019). However, this is the first report of Amauroderma subresinosum causing stem rot of Acacia mangiumin Hainan, China. This report will facilitate field diagnosis and provide scientific reference for further research on the disease.

Two cardenolide glycosides from the seed fairs of Asclepias curassavica and their cytotoxic activities.

Two cardenolide glycosides, corotoxigenin 3-O-[ß-D-glucopyranosyl-(1→4)-6-deoxy-ß-D-glucopyranoside] (1) and coroglaucigenin 3-O-[ß-D-glucopyranosyl-(1→4)-6-deoxy-ß-D-glucopyranoside] (2), were isolated from the seed fairs of Asclepias curassavica. The structures of 1-2 were determined based on the combination of the analysis of their MS, NMR spectroscopic data and acid hydrolysis. The inhibitory effects of compounds 1 and 2 on human colorectal carcinoma cells (HCT116), non-small cell lung carcinoma cells (A549) and hepatic cancer cells (SMMC-7721) were evaluated. The results showed that both compounds 1 and 2 significantly inhibited the viability, proliferation, and migration of A549, HCT116 and SMMC-7721 cells, suggesting that compounds 1 and 2 can be applied in the treatment of lung, colon and liver cancers in clinical practice. This study may not only provide a scientific basis for clarifying the active ingredients in A. curassavica, but also help to understand its antitumor activity, which can promote the application of A. curassavica in clinical treatment of various cancers.

(+/-)-Dievodialetins A-G: Seven pairs of enantiomeric coumarin dimers with anti-acetylcholinesterase activity from the roots of Evodia lepta Merr.

Seven pairs of undescribed enantiomeric bis-coumarins, (±)-dievodialetins A-G, were separated from the roots of Evodia lepta Merr. Two coumarin nuclei were linked via a 1,4-dimethyl4-vinylcyclohexene moiety in (±)-dievodialetins C-G. The structures of the undescribed compounds, including their absolute configurations were elucidated by spectroscopic analyses, X-ray diffraction, and computational calculations. In the biosynthetic pathways, these bis-coumarins were presumably derived from the precursors demethylsuberosin and 3-(3-methylbut-2-enyl)umbelliferone via a [4 + 2] Diels-Alder reaction. Besides, all compounds exhibited neuroprotective effects by inhibiting acetylcholinesterase (AChE) activity with IC50 values ranging from 7.3 to 12.1 nM and they also suppressed oxidative stress (MDA and SOD) and neuroinflammation (IL-1ß and IL-6).

Chinese herbal medicines for prevention and treatment of colorectal cancer: From molecular mechanisms to potential clinical applications.

Worldwide, colorectal cancer (CRC) is one of the most common malignant tumors, leading to immense social and economic burdens. Currently, the main treatments for CRC include surgery, chemotherapy, radiotherapy and immunotherapy. Despite advances in the diagnosis and treatment of CRC, the prognosis for CRC patients remains poor. Furthermore, the occurrence of side effects and toxicities severely limits the clinical use of these therapies. Therefore, alternative medications with high efficacy but few side effects are needed. An increasing number of modern pharmacological studies and clinical trials have supported the effectiveness of Chinese herbal medicines (CHMs) for the prevention and treatment of CRC. CHMs may be able to effectively reduce the risk of CRC, alleviate the adverse reactions caused by chemotherapy, and prolong the survival time of patients with advanced CRC. Studies of molecular mechanisms have provided deeper insight into the roles of molecules from CHMs in treating CRC. This paper summarizes the current understanding of the use of CHMs for the prevention and treatment of CRC, the main molecular mechanisms involved in these processes, the role of CHMs in modulating chemotherapy-induced adverse reactions, and CHM's potential role in epigenetic regulation of CRC. The current study provides beneficial information on the use of CHMs for the prevention and treatment of CRC in the clinic, and suggests novel directions for new drug discovery against CRC.

Potential of herb-drug / herb interactions between substrates and inhibitors of UGTs derived from herbal medicines.

Herbal medicines are widely used as alternative or complementary therapies worldwide to treat and prevent chronic diseases. However, herbal medicines coadministration with therapeutic drugs may cause dramatic clinical herb-drug/herb interactions (HDIs/HHIs) that may result in low drug efficacy or serious toxic reactions. Phase II metabolism enzyme UDP-glucuronosyltransferases (UGTs) play a significant detoxification role in vivo. Most drugs and non-drug xenobiotics undergo phase II metabolic transformations to be more polar compounds that are more easily excreted. Herbal medicines are a mixed and chemically varied group that includes flavonoids, stilbenes, coumarins, quinones, and terpenes, which are potential substrates and inhibitors of UGTs. Although increasing studies about glucuronidation metabolism and the inhibition toward UGTs of many herbal medicines have been reported, it is still difficult to determine which compounds from herbal medicines are substrates or inhibitors of UGTs. This article gives an overview of UGTs studies, which mainly focuses on glucuronidation of herbal constituents as substrates catalyzed by UGTs, potential herbal inhibitors for UGTs. We summarize the negative effects of UGT1A polymorphism and single nucleotide polymorphisms (SNPs), relevant clinical situations of HDIs/HHIs induced by inhibition of UGTs, and propose establishing classification criteria for inhibitors. Finally, we also discuss future research and strategic directions to advance the understanding of the potential HDIs/HHIs and suggest some additional studies revealing more information on UGT-mediated HDIs/HHIs.

Synthesis and Biological Evaluation of Genistein-IR783 Conjugate: Cancer Cell Targeted Delivery in MCF-7 for Superior Anti-Cancer Therapy.

The flavonoid-based natural product genistein is a biologically active compound possessing promising anti-oxidant and anti-cancer properties. Poor pharmacokinetics along with low potency limit however the therapeutic application of genistein in cancer therapy. In order to overcome those limitations and to expand its therapeutic window of efficacy, we sought to covalently attach genistein with a heptamethine cyanine dye-IR 783-for cancer cell targeting and enhanced delivery to tumors. Herein we report the synthesis, a selective detailed characterization and preliminary in vitro/in vivo biological evaluation of genistein-IR 783 conjugate 4. The conjugate 4 displayed improved potency against human breast cancer MCF-7 cells (10.4 ± 1.0 µM) as compared with the parent genistein (24.8 ± 0.5 µM) or IR 783 (25.7 ± 0.7 µM) and exhibited selective high uptake in MCF-7 as against the normal mammary gland MCF-10A cells in various assays. In the cell viability assay, conjugate 4 exhibited over threefold lower potency against MCF-10A cells (32.1 ± 1.1 µM) suggesting that the anti-cancer profile of parent genistein is significantly improved upon conjugation with the dye IR783. Furthermore, the genistein-IR783 conjugate 4 was shown to be especially accumulated in MCF-7 cancer cells by fluorescent intensity measurements and inverted fluorescence microscopy in fixed cells as well as in live cells with time via live cell confocal fluorescence imaging. The mechanism-based uptake inhibition of conjugate 4 was observed with OATPs inhibitor BSP and in part with amiloride, as a macropinocytosis inhibitor. For the first time we have shown amiloride inhibited uptake of cyanine dye by about ~40%. Finally, genistein-IR 783 conjugate 4 was shown to be localized in MCF-7 tumor xenografts of mice breast cancer model via in vivo near infrared fluorescence (NIRF) imaging. In conclusion, conjugation of genistein with cyanine dye IR783 indeed improved its pharmacological profile by cancer cell selective uptake and targeting and therefore warrants further investigations as a new anti-cancer therapeutics derived from natural product genistein.

A systematic review of pharmacokinetic studies on herbal drug Fuzi: Implications for Fuzi as personalized medicine.

BACKGROUND: Fuzi, which is the processed lateral roots of Aconitum carmichaeli Debx. (Ranunculaceae), is a traditional herbal medicine that is well known for its excellent pharmacological effects and acute toxicity. Aconitum alkaloids are responsible for its pharmacological activity and toxicity. Although a large number of studies on Fuzi have been reported, no comprehensive review on its pharmacokinetics has yet been published. PURPOSE: This paper seeks to present a comprehensive review regarding the phytochemistry, pharmacokinetic features and toxicity of Fuzi. The regulation of drug-metabolizing enzymes (DMEs) and efflux transporters (ETs) by Fuzi is also concluded. Additionally, the use of Fuzi as a personalized medicine based on the bioavailability barrier (BB), which mainly comprises DMEs and ETs, is discussed. METHODS: All available information on Fuzi was collected by searching for key words in PubMed, ScienceDirect, CNKI, Google Scholar, Baidu Scholar, and Web of Science. RESULTS: Aconitum alkaloids, which mainly include diester-diterpene alkaloids (DDAs), monoester-diterpene alkaloids (MDAs) and unesterified-diterpene alkaloids (UDAs), could be detected after Fuzi ingestion in vivo. The Aconitum alkaloids are rapidly absorbed in the intestine and extensively distributed in the body. DMEs, especially CYP3A4/5, are responsible for various types of metabolic reactions of the Aconitum alkaloids. ETs, including P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP), are involved in the efflux of the DDAs and MDAs. The kidney is the most important organ involved in the excretion of the Aconitum alkaloids. DDAs are the main toxic compounds present in Fuzi, and their acute toxicity is mainly due to their effects on the voltage-dependent sodium channels. Furthermore, Fuzi can substantially regulate DMEs and ETs. CONCLUSIONS: The toxicity of DDAs is acute. However, further investigations are necessary to determine the exact toxicological mechanisms. The significant impact of Fuzi on DMEs and ETs suggests that the co-administration of Fuzi with drugs that are substrates of DMEs and/or ETs may cause herb-drug interactions (HDIs). The BB network controlled exposure to the Aconitum alkaloids in vivo. Polymorphisms of DMEs and ETs in different individuals contribute to the differences in the efficacy and toxicity of Fuzi ingestion. In the future, the use of Fuzi as personalized medicine based on the BB network is necessary and practical to achieve ideal therapeutic efficacy with minimal toxicity.

Aconitum alkaloids, the major components of Aconitum species, affect expression of multidrug resistance-associated protein 2 and breast cancer resistance protein by activating the Nrf2-mediated signalling pathway.

BACKGROUND: Aconitum alkaloids from Aconitum species are often used to treat arthritis and rheumatic diseases but have the drawback of high toxicity. Identifying their pharmacokinetic behaviour is important for the safe clinical application of Aconitum species. Efflux transporters (ETs), including P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP), have important functions in regulating the pharmacokinetic behaviours of drugs and in herb-herb or herb-drug interactions (HDIs). The Aconitum alkaloids regulate P-gp expression and function, but their effects on MRP2 and BCRP expression remain unknown. PURPOSE: To determine the effects of three Aconitum alkaloids, aconitine (AC), benzoylaconine (BAC), and aconine, on MRP2 and BCRP. METHODS: The levels of the protein and mRNA expression of MRP2 and BCRP in vivo and in vitro were measured via Western blotting and real-time PCR, respectively. Fluorescence signals of MRP2 and BCRP were detected via confocal fluorescence microscopy. A reporter assay using HepG2-C8 cells, which were generated by transfecting plasmids containing the antioxidant response element (ARE)-luciferin gene into HepG2 cells, was used to examine the ARE-luciferin activity. The transport activities of MRP2 and BCRP were tested via flow cytometry using substrate probes. RESULTS: The Aconitum alkaloids significantly up-regulated MRP2 and BCRP expression, accompanied by a marked increase in nuclear factor E2-related factor-2 (Nrf2) expression in the jejunum, ileum, and colon of FVB mice, in the order AC < BAC < aconine. In the in vitro model, the Aconitum alkaloids increased MRP2 and BCRP expression in Caco-2 and LS174T cells, in the order AC < BAC < aconine. Additionally, these alkaloids promoted the translocation of Nrf2 from the cytoplasm to the nucleus and significantly increased ARE-luciferin activity in HepG2-C8 cells. Luteolin, a potent inhibitor of Nrf2, markedly prevented MRP2 and BCRP expression from being induced by the three Aconitum alkaloids. The efflux activity of MRP2 was also significantly increased in cells receiving the same treatment. CONCLUSIONS: The tested Aconitum alkaloids significantly increased the expression of MRP2 and BCRP by activating the Nrf2-mediated signalling pathway and enhanced the efflux activity of MRP2. The potential for herb-herb interactions or HDIs exists when Aconitum species are co-administered with substrate drugs that are transported via MRP2 and BCRP. Therefore, the Aconitum alkaloids may be used as quality indicators for the herbs of Aconitum species.

Development of a hydrophilic interaction chromatography-UPLC assay to determine trigonelline in rat plasma and its application in a pharmacokinetic study.

AIM: Trigonelline (Tr) is the second most abundant alkaloid in coffee beans. This study developed an assay combining hydrophilic interaction chromatography with ultra performance liquid chromatography (HILIC-UPLC) for the quantification of Tr in rat plasma to determine its pharmacokinetic behavior. METHODS: After the administration of Tr by gavage as well as intravenous injection and that of methanol extract of coffee beans (MECB) orally, blood samples from the experimental rats were analyzed using the HILIC-UPLC assay. Pharmacokinetic parameters were determined using the standard non-compartmental method and calculated using Practical Pharmacokinetic Program Version 87/97. RESULTS: The HILIC-UPLC assay was validated with the linear range of 0.12-100 µg·mL(-1) and a lower limit of quantitation of 0.12 µg·mL(-1). Its accuracy, precision, recovery, and stability were within acceptable limits. The AUC(0-∞) (where AUC is the area under the plasma concentration-time curve) values were determined to be (4 066.83 ± 1 244.41) and (3 544.29 ± 908.80) min·µg·mL(-1) after Tr was orally and intravenously administered, respectively. It was (4 566.75 ± 1 435.64) min·µg·mL(-1) after MECB was orally administered. The absolute bioavailability of Tr alone reached 57.37%, whereas that of Tr in MECB was 64.42%. The relative bioavailability of the alkaloid was 112.29%. CONCLUSIONS: The HILIC-UPLC assay for Tr determination is simple and accurate, and also exhibits good reproducibility. The bioavailability of stand-alone Tr and that of Tr in MECB were both good. Tr alone and that in MECB orally administered did not exhibit any significant difference.