Alisol B regulates AMPK/mTOR/SREBPs via directly targeting VDAC1 to alleviate hyperlipidemia.

BACKGROUND: The occurrence of hyperlipidemia is significantly influenced by lipid synthesis, which is regulated by sterol regulatory element binding proteins (SREBPs), thus the development of drugs that inhibit lipid synthesis has become a popular treatment strategy for hyperlipidemia. Alisol B (ALB), a triterpenoid compound extracted from Alisma, has been reported to ameliorate no-nalcoholic steatohepatitis (NASH) and slow obesity. However, the effect of ALB on hyperlipidemia and mechanism are unclear. PURPOSE: To examine the therapeutic impact of ALB on hyperlipidemia whether it inhibits SREBPs to reduce lipid synthesis. STUDY DESIGN: HepG2, HL7702 cells, and C57BL/6J mice were used to explore the effect of ALB on hyperlipidemia and the molecular mechanism in vivo and in vitro. METHODS: Hyperlipidemia models were established using western diet (WD)-fed mice in vivo and oleic acid (OA)-induced hepatocytes in vitro. Western blot, real-time PCR and other biological methods verified that ALB regulated AMPK/mTOR/SREBPs to inhibit lipid synthesis. Cellular thermal shift assay (CETSA), molecular dynamics (MD), and ultrafiltration-LC/MS analysis were used to evaluate the binding of ALB to voltage-dependent anion channel protein-1 (VDAC1). RESULTS: ALB decreased TC, TG, LDL-c, and increased HDL-c in blood, thereby ameliorating liver damage. Gene set enrichment analysis (GSEA) indicated that ALB inhibited the biosynthesis of cholesterol and fatty acids. Consistently, ALB inhibited the protein expression of n-SREBPs and downstream genes. Mechanistically, the impact of ALB on SREBPs was dependent on the regulation of AMPK/mTOR, thereby impeding the transportation of SREBPs from endoplasmic reticulum (ER) to golgi apparatus (GA). Further investigations indicated that the activation of AMPK by ALB was independent on classical upstream CAMKK2 and LKB1. Instead, ALB resulted in a decrease in ATP levels and an increase in the ratios of ADP/ATP and AMP/ATP. CETSA, MD, and ultrafiltration-LC/MS analysis indicated that ALB interacted with VDAC1. Molecular docking revealed that ALB directly bound to VDAC1 by forming hydrogen bonds at the amino acid sites S196 and H184 in the ATP-binding region. Importantly, the thermal stabilization of ALB on VDAC1 was compromised when VDAC1 was mutated at S196 and H184, suggesting that these amino acids played a crucial role in the interaction. CONCLUSION: Our findings reveal that VDAC1 serves as the target of ALB, leading to the inhibition of lipid synthesis, presents potential target and candidate drugs for hyperlipidemia.

Two Undescribed Protostane Triterpenoids from the Rhizome of Alisma plantago-aquatica.

Two undescribed protostane triterpenoids, 11-deoxy-13(17),15-dehydro-alisol B 23-acetate (2) and alisol S (3), together with 21 known ones (1, 4-23), were isolated from the dried rhizome of Alisma plantago-aquatica. Of these compounds, 13(17),15-Dehydro-alisol B 23-acetate (1) and 11-deoxy-13(17),15-dehydro-alisol B 23-acetate (2) are two protostane triterpenoids containing conjugated double bonds in the five-membered ring D that are rarely found from nature resource, while alisol S (3) is a protostane triterpenoid with undescribed tetrahydrofuran moiety linked via C20 -O-C24 at the side chain. Additionally, compound 18 is a new natural product, and cycloartenol triterpenoid 23 is a non protostane triterpenoid firstly isolated from genus Alisma. Their structures were elucidated by extensive spectral analysis of the UV, IR, MS, 1D and 2D NMR, and comparison of the experimental and calculated CD curves.

Study of Herbs Cortex Moutan, Poria cocos, and Alisma orientale and Periodontitis.

INTRODUCTION: The Chinese traditional herbs Cortex Moutan, Poria cocos, and Alisma orientale are considered to have potential to ameliorate periodontitis, although the possible underlying mechanisms remain mostly unknown. Due to the complex formulation of Chinese herbs, it is important to understand the mechanisms of pharmacologic effects of traditional herbs for better application in modern medical treatment. METHODS: Network pharmacology was applied to explore the mechanism of Cortex Moutan, Poria cocos, and Alisma orientale. First we analysed their chemical ingredients using the Traditional Chinese Medicine Systems Pharmacology database and identified 20 active ingredients. Then we analysed the target genes of these 20 active ingredients as well as genes associated with periodontitis and found 74 co-target genes. We further analysed the protein-protein interaction network of these 74 co-target genes using the STRING database and enriched the pathways using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. RESULTS: The top 10 core targets elicited were vascular endothelial growth factor A (VEGFA), interlukin-6 (IL-6), tumour necrosis factor (TNF), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), AKT serine/threonine kinase 1 (AKT1), prostaglandin-endoperoxide synthase 2 (PTGS2), kinase insert domain receptor (KDR), fibroblast growth factor 2 (FGF2), and serpin family E member 1 (SERPINE1). Using these a network of "herbs-ingredients-targetgenes-KEGG pathways." was constructed. CONCLUSIONS: The target and bioprocess network suggested that the pharmacologic effects of Cortex Moutan, Poria cocos, and Alisma orientale may be mainly dependent on their anti-inflammatory potential. Further work is required to eucidate their detailed mechanisms of activity.

New Guaiane-Type Sesquiterpene and Norsesquiterpene from Alisma plantago-aquatica and Their Xanthine Oxidase Inhibitory Activity.

A new sesquiterpene (1) and a new norsesquiterpene (2) belonging guaiane-type skeleton together with six known compounds (3-8) were isolated from the rhizomes of Alisma plantago-aquatica. Their structures were determined by HR-ESI-MS, 1D and 2D NMR spectroscopic methods. Absolute configurations of new compounds were established by experimental and TD-DFT computational ECD spectra. Compounds 1-8 exhibited xanthine oxidase inhibitory activity with their IC50 values in range of 9.4-66.7 µM. The sesquiterpenoids 1-5 displayed the inhibitory activity and hence they could be potential xanthine oxidase inhibitors from A. plantago-aquatica.

Translocation of TiO2 nanoparticles enhances phosphorus uptake by wetland plants: Evidence from Pistia stratiotes and Alisma plantago-aquatica.

Titanium dioxide nanoparticles (nTiO2) and phosphorus (P) are widely present in sewages. To verify the hypothesis and the associated mechanisms that root-to-shoot translocation of nTiO2 can enhance plant P uptake thus P removal during sewage treatment, two wetland plants (Pistia stratiotes and Alisma plantago-aquatica) with different lateral root structures were used to examine the effect of nTiO2 (89.7% anatase and 10.3% rutile) on plant growth and P uptake in a hydroponic system. Inductively coupled plasma-optical emission spectroscopy and transmission electron microscopy-energy dispersive spectroscopy showed that P. stratiotes with well-developed lateral roots translocated 1.4-16 fold higher nTiO2 than A. plantago-aquatica with poorly developed roots, indicating P. stratiotes is efficient in nTiO2 uptake. In addition, nTiO2 root-to-shoot translocation in P. stratiotes increased with increasing nTiO2 concentration, while the opposite occurred in A. plantago-aquatica. Corresponding to the stronger nTiO2 translocation in P. stratiotes, its P uptake efficiency (Imax) and P accumulation were greater than that in A. plantago-aquatica, with Imax being increased by 35.8% and -16.4% and shoot P concentrations being increased by 16.2-64.6% and 11.4%, respectively. The strong positive correlation between Ti and P concentrations in plant tissues (r = 0.72-0.89, P < 0.01) indicated that nTiO2 translocation enhanced P uptake. Moreover, nTiO2-enhanced P uptake promoted plant growth and photosynthetic pigment synthesis. Therefore, wetland plants with well-developed lateral roots like P. stratiotes have potential to be used in P removal from nTiO2-enriched sewages.

Comparative analysis of the structure and function of rhizosphere microbiome of the Chinese medicinal herb Alisma in different regions.

Rhizoma Alismatis, a commonly used traditional Chinese medicine, is the dried tuber of Alisma orientale and Alisma A. plantago-aquatica, mainly cultivated in Fujian and Sichuan provinces (China), respectively. Studies have shown that the rhizosphere microbiome is a key factor determining quality of Chinese medicinal plants. Here we applied metagenomics to investigate the rhizosphere microbiome of Alisma in Fujian and Sichuan, focusing on its structure and function and those genes involved in protostane triterpenes biosynthesis. The dominant phyla were Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria, and Gemmatimonadetes. Compared with Fujian, the rhizosphere of Sichuan has a greater α diversity and stronger microbial interactions but significantly lower relative abundance of archaea. Microbes with disease-suppressing functions were more abundant in Sichuan than Fujian, but vice versa for those with IAA-producing functions. Gemmatimonas, Anaeromyxobacter, and Pseudolabrys were the main contributors to the potential functional difference in two regions. Genes related to protostane triterpenes biosynthesis were enriched in Fujian. Steroidobacter, Pseudolabrys, Nevskia, and Nitrospira may contribute to the accumulation of protostane triterpenes in Alisma. This work fills a knowledge gap of Alisma's rhizosphere microbiome, providing a valuable reference for studying its beneficial microorganisms.

Beneficial Activities of Alisma orientale Extract in a Western Diet-Induced Murine Non-Alcoholic Steatohepatitis and Related Fibrosis Model via Regulation of the Hepatic Adiponectin and Farnesoid X Receptor Pathways.

The hepatic adiponectin and farnesoid X receptor (FXR) signaling pathways play multiple roles in modulating lipid and glucose metabolism, reducing hepatic inflammation and fibrosis, and altering various metabolic targets for the management of non-alcoholic fatty liver disease (NAFLD). Alisma orientale (AO, Ze xie in Chinese and Taeksa in Korean) is an herbal plant whose tubers are enriched with triterpenoids, which have been reported to exhibit various bioactive properties associated with NAFLD. Here, the present study provides a preclinical evaluation of the biological functions and related signaling pathways of AO extract for the treatment of NAFLD in a Western diet (WD)-induced mouse model. The findings showed that AO extract significantly reversed serum markers (liver function, lipid profile, and glucose) and improved histological features in the liver sections of mice fed WD for 52 weeks. In addition, it also reduced hepatic expression of fibrogenic markers in liver tissue and decreased the extent of collagen-positive areas, as well as inhibited F4/80 macrophage aggregation and inflammatory cytokine secretion. The activation of adiponectin and FXR expression in hepatic tissue may be a major mechanistic signaling cascade supporting the promising role of AO in NAFLD pharmacotherapy. Collectively, our results demonstrated that AO extract improves non-alcoholic steatohepatitis (NASH) resolution, particularly with respect to NASH-related fibrosis, along with the regulation of liver enzymes, postprandial hyperglycemia, hyperlipidemia, and weight loss, probably through the modulation of the hepatic adiponectin and FXR pathways.

Natural soluble epoxide hydrolase inhibitors from Alisma orientale and their potential mechanism with soluble epoxide hydrolase.

Inhibition of soluble epoxide hydrolase (sEH) is considered to be an effective treatment for inflammation-related diseases, and small molecules origin from natural products show promising activity against sEH. Two undescribed protostanes, 3ß-hydroxy-25-anhydro-alisol F (1) and 3ß-hydroxy-alisol G (2) were isolated from Alisma orientale and identified as new sEH inhibitors with IC50 values of 10.06 and 30.45 µM, respectively. Potential lead compound 1 was determined as an uncompetitive inhibitor against sEH, which had a Ki value of 5.13 µM. In-depth molecular docking and molecular dynamics simulations revealed that amino acid residue Ser374 plays an important role in the inhibition of 1, which also provides an idea for the development of sEH inhibitors based on protostane-type triterpenoids.

Proteomic insights into protostane triterpene biosynthesis regulatory mechanism after MeJA treatment in Alisma orientale (Sam.) Juz.

Protostane triterpenes in Alisma orientale (Sam.) Juz. have unique structural features with distinct pharmacological activities. Previously we have demonstrated that protostane triterpene biosynthesis could be regulated by methyl jasmonate (MeJA) induction in A. orientale. Here, proteomic investigation reveals the MeJA mediated regulation of protostane triterpene biosynthesis. In our study, 281 differentially abundant proteins were identified from MeJA-treated compared to control groups, while they were mainly associated with triterpene biosynthesis, α-linolenic acid metabolism, carbohydrate metabolism and response to stress/defense. Key enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), squalene epoxidase (SE), oxidosqualene cyclase (OSC) and cytochrome P450s which potentially involved in protostane triterpene biosynthesis were significantly enriched in MeJA-treated group. Basic Helix-loop-helix (bHLH), MYB, and GRAS transcription factors were enhanced after MeJA treatment, and they also improved the expressions of key enzymes in Mevalonate pathway and protostane triterpene. Then, MeJA also could increase the expression of α-galactosidase (α-GAL), thereby promoting carbohydrate decomposition, and providing energy and carbon skeletons for protostane triterpene precursor biosynthesis. As well, exogenous MeJA treatment upregulated 13-lipoxygenase (13-LOX), allene oxide synthase (AOS) and allene oxide cyclase (AOC) involved in α-linolenic acid metabolism, leading to the accumulation of endogenous MeJA and activation of the protostane triterpene biosynthesis transduction. Finally, MeJA upregulated stress/defence-related proteins, as to enhance the defence responses activity of plants. These results were further verified by quantitative real-time PCR analysis of 19 selected genes and content analysis of protostane triterpene. The results provide some new insights into the role of MeJA in protostane triterpene biosynthesis.

Alisma canaliculatum Extract Affects AGS Gastric Cancer Cells by Inducing Apoptosis.

The anti-cancer effects of Alisma canaliculatum extracts (ACE) were identified in AGS gastric cancer cells. Our results showed that ACE inhibited the growth of AGS cells, increased the proportion of sub-G1 phase cells, and depolarized the membrane potential of mitochondria. ACE-induced gastric cancer cell death was associated with Bcl-2, survivin and Bax level changes, and it activated caspase-3 and -9. In addition, it was involved in the activation of MAPKs and increased the reactive oxygen species (ROS). These results suggest that ACE induces apoptosis in AGS gastric cancer cells, and therefore, ACE may have the potential to treat gastric cancer.

Rapid screening for natural lipase inhibitors from Alisma orientale combining high-performance thin-layer chromatography-bioautography with mass spectrometry.

Lipase inhibitors are an attractive class of hypolipidemic compounds, which inhibit the activity of human pancreatic lipase, thereby preventing the absorption of triglycerides in vivo. As a library of promising lead compounds for drug development, traditional Chinese medicine (TCM) has gained growing attention in quick discovery and identification of enzyme inhibitors of natural-origin. The purpose of this work was to discover unknown lipase inhibitors from Alisma orientale by the activity oriented analysis method thin-layer chromatography-bioautography, then use electrospray ionization mass spectrometry technology via the elution based TLC-MS interface to identify their structures. As a result, eleven natural lipase inhibitors from Alisma orientale extracts were identified based on molecular mass and fragment ions obtained by HPTLC-MS, and further confirmed by a series of complementary means including UV spectra, 1H NMR characteristic proton signals and polarity of compounds, eleven lipase inhibitors were tentatively assigned as triterpenoids: alisol B (m/z 495.50 [M + Na]+), alisol B 23-acetate (m/z 537.58 [M + Na]+), 11-deoxy-alisol B (m/z 479.50 [M + Na]+), 11-deoxy-alisol B 23-acetate (m/z 521.50 [M + Na]+), alisol A/epialisol A (m/z 513.50 [M + Na]+), 16-oxo-11-deoxy-alisol A (m/z 511.50 [M + Na]+), 16-oxo-alisol A (527.50 [M + Na] +), alisol C (m/z 509.58 [M + Na]+), alisol C 23-acetate (m/z 551.50 [M + Na]+), alisol M 23-acetate (m/z 567.50 [M + Na]+), and alismanol Q/neoalisol (m/z 493.42 [M + Na]+). The integrated approach is an efficient method for rapid screening lipase inhibitors from complex plant extracts and provides a reasonable and favorable basis for the identification and separation of other enzymatic system and other important compounds with therapeutic values.

Anti-osteoporotic effects of alisol C 23-acetate via osteoclastogenesis inhibition.

Alismatis rhizoma (AR) is the dried rhizome of Alisma orientale (Sam.) Juz. (Alismataceae). This traditional Chinese formula is diuretic, hypoglycemic, and hypolipidemic. Alisol C 23-acetate (AC23A) from AR is anti-inflammatory and ameliorates certain metabolic diseases. However, the mechanism by which AC23A mitigates osteoporosis is unknown. The present study investigated the anti-osteoporotic effects of AC23A in vivo and in vitro. In an ovariectomized (OVX) rat model, AC23A ameliorated OVX-induced organ coefficients and trabecular bone loss. In OVX rats, AC23A treatment lowered serum TRAP5b, CTK, ß-CTX, TNF-α, IL-6, and IL-1ß, raised serum E2, and did not significantly change serum OCN or BALP. AC23A inhibited osteoclast formation in a rat co-culture system without affecting osteoblast activity. RANK (receptor activator of nuclear factor kappaB) signaling channels are vital osteoclastogenesis transcription elements. AC23A inhibited RANK ligand (RANKL)-induced TRAP, c-Fos, MMP9, NFATc1, and CTK expression and JNK phosphorylation. Therefore, AC23A is anti-osteoclastogenic in vitro and in vivo by inhibiting RANKL-induced osteoclast differentiation and function. Moreover, AC23A could help prevent or limit osteoclast-mediated bone diseases by inhibiting osteoclastogenesis.

Changes in Triterpenes in Alismatis rhizoma after Processing Based on Targeted Metabolomics Using UHPLC-QTOF-MS/MS.

Alismatis rhizoma (AR) has been used as an herbal medicine in China for over a thousand years. Crude AR, salt-processed AR (SAR), and bran-processed AR (BAR) are recorded in the Pharmacopoeia of the People's Republic of China. However, the differences of chemical composition between crude AR and its processing products remain limited. In this study, triterpenes were identified from crude AR, SAR, and BAR by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight-mass spectrometer (UHPLC-QTOF-MS/MS). Subsequently, the differences of triterpenes between the crude AR and processed ARs were compared via a targeted metabolomics approach. Finally, a total of 114 triterpenes were identified, of which 83, 100, and 103 triterpenes were found in crude AR, SAR, and BAR, respectively. After salt-processing, there were 17 triterpenes newly generated, 7 triterpenes with trends of increasing, and 37 triterpenes decreased. Meanwhile, 56 triterpenes including 21 newly generated and 35 with significant increases were observed in BAR. This study could be benefit to investigate the processing mechanism of AR, as well as support their clinical applications.

Investigation of the inhibitory effect of protostanes on human carboxylesterase 2 and their interaction: Inhibition kinetics and molecular stimulations.

Carboxylesterase 2 (CES 2), plays a pivotal role in endobiotic homeostasis and xenobiotic metabolism. Protostanes, the major constituents of the genus Alisma, display a series of pharmacological activities. Despite the extensive studies of pharmacological activities, the investigation on inhibitory effects of protostanes against CES 2 is rarely reported. In this study, the inhibitory activities of a library of protostanes (1-25) against human CES 2 were investigated for the first time, using 6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridin-2-yl benzoate (DDAB) as the specific fluorescent probe for human CES 2. Compounds 1, 2, 7, 8, 12, 13, 18, 19, and 25 showed strong inhibitory effects towards CES 2. For the most potent compounds 1, 7, 13, and 25, the inhibition kinetics were further investigated, and these four protostanes were all uncompetitive inhibitors against human CES 2 with the inhibition constant (Ki) values ranging from 0.89 µM to 2.83 µM. In addition, molecular docking and molecular dynamics stimulation were employed to analyze the potential interactions between these protostanes and CES 2, and amino acid residue Gln422 was identified to play a crucial role in the strong inhibition of protostanes towards CES 2.

Alisma genus: Phytochemical constituents, biosynthesis, and biological activities.

The genus Alisma contains 11 species distributed worldwide, of which at least two species (A. orientale [Sam.] Juzep. and A. plantago-aquatica Linn.) have been used as common herbal medicines. Secondary metabolites obtained from the genus Alisma are considered to be the material basis for the various biological functions and medicinal applications. In this review, we mainly focused on the recent investigations of secondary metabolites from plants of the genus Alisma and their biological activities, with the highlighting on the diversity of the chemical structures, the biosynthesis of interesting secondary metabolites, the biological activities, and the relationships between structures and bioactivities.

Hypolipidemic effect of Alisma orientale (Sam.) Juzep on gut microecology and liver transcriptome in diabetic rats.

Alisma orientale (Sam.) Juzep (A. orientale) is a traditional herb that is often used to treat disease including edema and hyperlipidemia. However, the molecular mechanism by which Alisma orientale (Sam.) Juzep exerts its hypolipidemic effects remains unclear. In this study, a diabetic rat model was established by feeding a high-fat and high-sugar diet combined with a low-dose streptozotocin injection (HFS). Then the rats were treated with an A. orientale water extract (AOW), an A. orientale ethanolic extract (AOE) or metform (MET). The gut microflora and liver transcriptome were analyzed by high-throughput next-generation sequencing. Ultra-performance liquid chromatography-triple quadrupole-mass spectrometry was employed to analyze the major compounds in the AOE. The results showed that the serum total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) levels in rats of the AOE group (2.10 g/kg/day, 14 days) were significantly lower than those in the HFS group (p<0.01). Moreover, AOE treatment altered the gut microecology, particularly modulating the relative abundance of gut microflora involved in lipid metabolism compared with the HFS group. Furthermore, compared with the HFS group, the mRNA expression levels of Fam13a, Mapk7, Mpp7, Chac1, Insig1, Mcpt10, Noct, Greb1l, Fabp12 and Hba-a3 were upregulated after the administration of AOE. In contrast, the mRNA expression levels of Lox, Mybl1, Arrdc3, Cyp4a2, Krt20, Vxn, Ggt1, Nr1d1 and S100a9 were downregulated. Moreover, AOE treatment for two weeks markedly promoted the relative abundance of Lachnospiraceae (p = 0.0013). The triterpenoids contents in AOE were alisol A, alisol A 24-acetate, alisol B, alisol B 23-acetate, alisol C 23-acetate, alisol F, alisol F 24-acetate, and alisol G. Our findings above illustrated that the hypolipidemic effect of the triterpenoids of A. orientale is mediated mainly through alteration of the gut microecology and the regulation of genes involved in cholesterol metabolism, especially Insig1.

Alisol A-24-acetate promotes glucose uptake via activation of AMPK in C2C12 myotubes.

BACKGROUND: Alisol A-24-acetate (AA-24-a) is one of the main active triterpenes isolated from the well-known medicinal plant Alisma orientale (Sam.) Juz., which possesses multiple biological activities, including a hypoglycemic effect. Whether AA-24-a is a hypoglycemic-active compound of A. orientale (Sam.) Juz. is unclear. The present study aimed to clarify the effect and potential mechanism of action of AA-24-a on glucose uptake in C2C12 myotubes. METHOD: Effects of AA-24-a on glucose uptake and GLUT4 translocation to the plasma membrane were evaluated. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay. Cell membrane proteins were isolated and glucose transporter 4 (GLUT4) protein was detected by western blotting to examine the translocation of GLUT4 to the plasma membrane. To determine the underlying mechanism, the phosphorylation levels of proteins involved in the insulin and 5'-adenosine monophosphate-activated protein kinase (AMPK) pathways were examined using western blotting. Furthermore, specific inhibitors of key enzymes in AMPK signaling pathway were used to examine the role of these kinases in the AA-24-a-induced glucose uptake and GLUT4 translocation. RESULTS: We found that AA-24-a significantly promoted glucose uptake and GLUT4 translocation in C2C12 myotubes. AA-24-a increased the phosphorylation of AMPK, but had no effect on the insulin-dependent pathway involving insulin receptor substrate 1 (IRS1) and protein kinase B (PKB/AKT). In addition, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and the AKT substrate of 160 kDa (AS160), two proteins that act downstream of AMPK, was upregulated. Compound C, an AMPK inhibitor, blocked AA-24-a-induced AMPK pathway activation and reversed AA-24-a-induced glucose uptake and GLUT4 translocation to the plasma membrane, indicating that AA-24-a promotes glucose metabolism via the AMPK pathway in vitro. STO-609, a calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) inhibitor, also attenuated AA-24-a-induced glucose uptake and GLUT4 translocation. Moreover, STO-609 weakened AA-24-a-induced phosphorylation of AMPK, p38 MAPK and AS160. CONCLUSIONS: These results indicate that AA-24-a isolated from A. orientale (Sam.) Juz. significantly enhances glucose uptake via the CaMKKß-AMPK-p38 MAPK/AS160 pathway.

Protostane-type triterpenoids as natural soluble epoxide hydrolase inhibitors: Inhibition potentials and molecular dynamics.

The inhibition of soluble epoxide hydrolase (sEH) is a promising therapeutic approach to treat inflammation and other disorders. In our present investigation on searching for sEH inhibitors from traditional Chinese medicines, we found that Alisma orientale displayed inhibition of sEH. We constructed a small library of protostane-type triterpenoids (1-25) isolated from A. orientale, and screened their inhibitory activities. Alismanin B (1), 11-deoxy-25-anhydro alisol E (4), 11-deoxy alisol B (5), and 25-O-ethyl alisol A (15) displayed concentration-dependently inhibitory activities against sEH with IC50 values from 3.40 ± 0.57 µM to 9.57 ± 0.88 µM. 11-Deoxy-25-anhydro alisol E (4) and 11-deoxy alisol B (5) were defined as mixed-type competitive inhibitors with Ki values of 12.6 and 3.48 µM, respectively, based on the result of inhibition kinetics. The potential interaction mechanism of 11-deoxy alisol B (5) with sEH was analyzed by molecular docking and molecular dynamics, revealing that amino acid residues Trp336 and Tyr466 were vital for its inhibitory activity.

A biochemometrics strategy for tracing diuretic components of crude and processed Alisma orientale based on quantitative determination and pharmacological evaluation.

We proposed a biochemometrics strategy for tracing diuretic components of herbs based on quantitative determination and pharmacological evaluation. First, a sensitive and robust liquid chromatography coupled with tandem mass spectrometry approach was established for simultaneous quantification of six major triterpenoids in crude and salt-processed Alisma orientale. The separation of triterpenoids was achieved on a BEH C18 column with a mobile phase consisting of acetonitrile and water spiked with 0.1% formic acid. Six major triterpenoids were detected by multiple reaction monitoring in the negative ion mode. Glycyrrhetinic acid was used as the internal standard. The approach showed good linearity. Intra- and inter-day precisions were all within 2.9%. The recovery rates of each triterpenoid ranged from 97.9% to 103.2%. The approach was then successfully employed for quantitative analysis of six triterpenoids in ten batches of crude and salt-processed A. orientale. Second, the diuretic effects of crude and salt-processed A. orientale were evaluated in mice. Third, principal component analysis and canonical correlation analysis were used to uncover the relationship between the contents of six major triterpenoids and the diuretic effect of different crude and salt-processed samples. Alisol B, alisol F, and alisol A have a close positive correlation with the diuretic effect.

Highly potent non-steroidal FXR agonists protostane-type triterpenoids: Structure-activity relationship and mechanism.

Farnesoid X receptor (FXR) is a key regulator in charge of bile acid synthesis, transport, and metabolism. Activation of FXR represses bile acid synthesis and increases its excretion and transport, consequently protecting the liver functions. Thus, FXR is considered as a critical therapeutic target of cholestasis and nonalcoholic steatohepatitis. Herein, we isolated and identified fourteen new protostane-type triterpenoids (1-14) and four known analogues (15-18) from Alisma orientale, and finally constructed a small library of protostane-type triterpenoids (1-70) to investigate their structure-activity relationship with FXR, further leading to obtain compound 15 with potent agonistic activity against FXR (EC50 = 90 nM). Extensive in vitro investigation confirmed high efficacy of compound 15 against FXR in living cell, and revealed its underlying mechanism for FXR activation (amino acid residues Arg331 and Ser332) by molecular docking and site-directed mutagenesis technology.