Alisol B 23-acetate Research Articles

Alisol B 23-Acetate Inhibits IgE/Ag-Mediated Mast Cell Activation and Allergic Reaction.

Alisol B 23-acetate (AB23A), a natural triterpenoid, has been reported to exert hepatoprotective and antitumor activities. Aiming to investigate the anti-inflammatory activity, this study examined the effect of AB23A on mast cells and allergic reaction. AB23A inhibited the degranulation of mast cells stimulated by immunoglobulin E/antigen (IgE/Ag), and also decreased the synthesis of leukotriene C4 (LTC4), production of interlukin-6 (IL-6), and expression of cyclooxygenase-2 (COX-2) in a concentration-dependent manner with no significant cytotoxicity in bone marrow-derived mast cells (BMMCs). AB23A inhibited spleen tyrosine kinase (Syk) and the downstream signaling molecules including phospholipase Cγ (PLCγ), serine-threonine protein kinase/inhibitor of nuclear factor kappa-B kinase/nuclear factor kappa-B (Akt/IKK/NF-κB), and mitogen-activated protein kinases/cytosolic phospholipase A2 (MAPK/cPLA2). Furthermore, AB23A blocked mobilization of Ca2+. Similar results were obtained in other mast cell lines Rat basophilic leukemia (RBL)-2H3 cells and a human mast cell line (HMC-1). In addition, AB23A attenuated allergic responses in an acute allergy animal model, passive cutaneous anaphylaxis (PCA). Taken together, this study suggests that AB23A inhibits the activation of mast cells and ameliorates allergic reaction, and may become a lead compound for the treatment of mast cell-mediated allergic diseases.

Alisol B-23-acetate, a tetracyclic triterpenoid isolated from Alisma orientale, induces apoptosis in human lung cancer cells via the mitochondrial pathway

Alisol B-23-acetate (AB23A), a tetracyclic triterpenoid isolated from the rhizome of Alisma orientale, has been reported to exert anti-proliferative activities in human colon, ovarian and gastric cancer cells. However, the anti-cancer effect of this compound on human lung cancer cells has not yet been thoroughly elucidated. In the present study, we investigated the effects of AB23A on the cell viability and apoptosis in human lung cancer A549 and NCI-H292 cells. The results indicated that AB23A inhibited the growth of A549 and NCI-H292 cells in dose- and time-dependent manner, however, there was only weak cytotoxicity on normal bronchial epithelial cells. The induction of apoptosis by AB23A was demonstrated by DAPI and annexin-V-FITC/PI staining. Further investigation revealed that AB23A decreased mitochondrial membrane potential (MMP) and up regulated reactive oxygen species (ROS) level. Meanwhile, the increased Bax/Bcl-2 ratio, activated caspase-3, caspase-9 and PARP were observed. In addition, AB23A increased the release of cytochrome c from mitochondria and the translocation of apoptotic inducing factor (AIF) into nuclei. Taken together, these results indicated that AB23A induced apoptosis by activating the intrinsic pathway, and suggested that AB23A can be used as a potential modulating agent in lung cancer.

Pharmacokinetics and tissue distribution of five major triterpenoids after oral administration of Rhizoma Alismatis extract to rats using ultra high-performance liquid chromatography–tandem mass spectrometry

Rhizoma Alismatis (RA) was wildly used for treatment of dysuria, pyelonephritis, hyperlipidemia, enteritis diarrhea, diabetes, inflammation, and cancer. Triterpenoids are the major active components of RA, and its extract is mainly composed of alisol A (ALA), alisol B (ALB), alisol C 23-acetate (ALC-23A), alisol A 24-acetate (ALA-24A), and alisol B 23-acetate (ALB-23A). In this study, a simple, reliable, and sensitive ultra high-performance liquid chromatography with triple quadrupole mass spectrometry (UHPLC–MS/MS) method was created and validated for the quantification of the five major triterpenoids in rat plasma and various tissues biosamples (including intestine, stomach, liver, kidney, fat, muscle, brain, heart, lung, spleen, and testes). The plasma and tissues biosamples were pretreated by direct precipitation deproteinization method with acetonitrile. 17α-Hydroxyprogesterone was used as internal standard (IS). The chromatography was performed on a Phenomenex C8 column (30×2.00mm, 1.8μm) at room temperature with gradient elution. Compounds were quantified by selected multi-reactions monitoring (SRM) scanning with positive electric spray ionization mode. The linearity of detection for each triterpene was respectively from 1 to 1000ng/mL for ALC-23A and ALA, from 4 to 4000ng/mL for ALA-24A, from 10 to 10,000ng/mL for ALB, and from 2 to 2000ng/mL for ALB-23B (r>0.99) with low quantification limits of 1–10ng/mL for all analytes. All of the other validation parameters were also in an acceptable range. The validated UHPLC–MS/MS method subsequently applied for the pharmacokinetic and tissue distribution studies of RA extract. After orally given 100mg/kg of RA extract, ALA was the most exposed component, followed by ALB and ALA-24A. Whereas significant gender difference was observed for ALB, ALA, and ALA-24A between female and male rats. The AUC(0-∞) of ALA, ALB, and ALA-24A in female rats were approximately 2–5 fold larger than that in male rats. These triterpenoids also displayed approximately 1.5–2 times longer half-life (t1/2) in female rats. Appearant Km, Vmax and Clint of ALA, ALB, and ALA-24A were calculated by substrate depletion approach, rat P450 CYP3A2 plays an important role in the metabolism of ALA, ALB, and ALA-24A, which is an important factor leading to the different exprosures of ALA, ALB, and ALA-24A between the male rats and the female rats. Furthermore, results from tissue distribution in male rats showed that the main tissue depots of five triterpenoids were the stomach/intestine, followed by the liver, brain, and fat. However, ALA was still measured in the kidney after a long elimination time. ALB and ALB-23B exhibited lower elimination rate in the testis. These results provide a fundamental support for further pharmacological development and clinical safety application of RA.

Alisol B 23-acetate induces autophagic-dependent apoptosis in human colon cancer cells via ROS generation and JNK activation.

Alisol B 23-acetate (AB23A), a natural triterpenoid from the rhizome of Alisma orientale, a Chinese medicinal herb, has multiple physiological activities including anticancer. However, its effect on human colon cancer and the underlying mechanism are not clear. Here, we reported for the first time that AB23A induced cell cycle G1 phase arrest and apoptotic cell death in colon cancer cells. Autophagy also occurred in AB23A-treated HCT116 cells as evidenced by the accumulation of microtubule-associated protein 1 light chain 3 form II (LC3-II) and degradation of SQSTM1/p62. An autophagy inhibitor, 3-methyladenine (3-MA) was found to attenuate AB23A-mediated autophagy, apoptosis, and cell death, indicating that AB23A-induced apoptotic response was dependent on the induction of autophagy. In addition, the treatment of HCT116 cells with AB23A resulted in the generation of reactive oxygen species (ROS) and phosphorylation of c-Jun N-terminal kinase (JNK). A ROS scavenger, N-acetylcysteine (NAC) and a JNK-specific inhibitor, SP600125 attenuated AB23A-induced autophagy and apoptotic cell death. Moreover, NAC was able to eliminate AB23A-induced JNK phosphorylation. This finding provides a novel mechanism of action of AB23A in colon cancer HCT116 cells that AB23A induces autophagic-dependent apoptotic cell death in colon cancer cells, at least in part, though the accumulation of intracellular ROS and subsequent activation of JNK.

Alisol B 23-acetate from the rhizomes of Alisma orientale is a natural agonist of the human pregnane X receptor

BackgroundPregnane X receptor (PXR) is a key regulator of the induction of drug metabolizing enzymes. PXR has been studied for its importance in drug-drug or herb-drug interactions, and it is also a molecular target for the treatment of inflammatory and metabolic diseases. PurposeThis study aims to determine new natural PXR-ligands from traditional plant medicines. MethodsThe PXR activation activity was measured by a mammalian one hybrid assay of PXR. Identification of the active compound from Alisma rhizome (the rhizomes of Alisma orientale) was carried out by bioassay-guided fractionation method. The transcriptional activity of the liver-enriched nuclear receptors was measured by the luciferase reporter assay. The interaction between the SRC-1 and PXR was measured by a mammalian 2-hybrid assay. The expression of endogenous CYP3A4 mRNA in both cultured hPXR-overexpressing hepatoma cells and human primary hepatocytes were measured by quantitative RT-PCR method. ResultsThe extract of Alisma rhizome showed the most potent activation activity by screening of a library of medicinal plant extracts. Alisol B 23-acetate (ABA) was identified to be the active compound of Alisma rhizome. ABA caused a concentration-dependent increase on the PXR-dependent transactivation of a luciferase reporter gene, but did not affect the ligand binding activity of the liver-enriched nuclear receptors, such as CAR, LXR, FXR, PPARα, PPARδ and PPARγ, emphasizing that ABA is a potent and specific agonist of PXR. With ABA treatment, the direct interaction between the ligand-binding domain of PXR and the receptor interaction domain of SRC1 was observed. ABA also induced the expression of endogenous CYP3A4 mRNA in both cultured hPXR-overexpressing hepatoma cells and human primary hepatocytes. ConclusionSince the rhizomes of Alisma orientale are used for a wide range of ailments in traditional Chinese medicine and Japanese Kampo medicine, this study could possibly extend into the clinical usage of these medicines via the mechanism of PXR activation.

Alisol B 23-acetate protects against non-alcoholic steatohepatitis in mice via farnesoid X receptor activation.

Alisol B 23-acetate (AB23A) is a natural triterpenoid isolated from the traditional Chinese medicine rhizoma alismatis, which exhibits a number of pharmacological activities, including anti-hepatitis virus, anti-cancer and antibacterial effects. In this study we examined whether AB23A protected against non-alcoholic steatohepatitis (NASH) in mice, and the mechanisms underlying the protective effects. NASH was induced in mice fed a methionine and choline-deficient (MCD) diet for 4 weeks. The mice were simultaneously treated with AB23A (15, 30, and 60 mg·kg-1·d-1, ig) for 4 weeks. On the last day, blood samples and livers were collected. Serum liver functional enzymes, inflammatoru markers were assessed. The livers were histologically examined using H&E, Oil Red O, Masson's trichrome and Sirius Red staining. Mouse primary hepatocytes were used for in vitro experiments. The mechanisms underlying AB23A protection were analyzed using siRNA, qRT-PCR, and Western blot assays. AB23A treatment significantly and dose-dependently decreased the elevated levels of serum ALT and AST in MCD diet-fed mice. Furthermore, AB23A treatment significantly reduced hepatic triglyceride accumulation, inflammatory cell infiltration and hepatic fibrosis in the mice. AB23A-induced decreases in serum and hepatic lipids were related to decreased hepatic lipogenesis through decreasing hepatic levels of SREBP-1c, FAS, ACC1 and SCD1 and increased lipid metabolism via inducing PPARα, CPT1α, ACADS and LPL. The reduction in inflammatory cell infiltration corresponded to deceased serum levels of mKC and MCP-1 and decreased hepatic gene expression of MCP-1 and VCAM-1. The reduction in hepatic fibrosis was correlated with decreased hepatic gene expression of fibrosis markers. The protective effects of AB23A were FXR-dependent, because treatment with the FXR agonist CDCA mimicked AB23A-induced hepato-protection in the mice, whereas co-administration of FXR antagonist guggulsterone abrogated AB23A-induced hepato-protection. In mouse primary hepatocytes, FXR gene silencing abrogated AB23A-induced changes in gene expression of Apo C-II, CPT1α, ACADS and LPL. AB23A produces protective effects against NASH in mice via FXR activation.

Effects of alisol B 23-acetate on ovarian cancer cells: G1 phase cell cycle arrest, apoptosis, migration and invasion inhibition

BackgroundOvarian cancer is the first leading cause of death among gynecologic malignancies worldwide. Discovery of new chemotherapeutic drugs is still imperative for the improvement of the survival rate. PurposeThis study aims to investigate the anti-cancer potential of alisol B 23-acetate (AB23), a protostane-type triterpene isolated from the Alismatis Rhizoma, in the parental and paclitaxel-resistant ovarian cancer cells. MethodsMTT assay was performed to evaluate cell viability after treatment with AB23, along with flow cytometry for apoptosis and cell cycle analysis. Western blotting was conducted to determine the relative protein level. Wound healing and transwell assays were performed to investigate the effect of AB23 on cell migration and invasion. ResultsAB23 obviously inhibited proliferation of the three ovarian cancer cell lines, down-regulated the protein levels of CDK4, CDK6, and cyclin D1, and blocked the cell cycle progressions in G1 phase. Meanwhile, AB23 induced accumulation of the sub-G1 phase in the three cell lines in a concentration dependent manner. The protein levels of cleaved poly ADP-ribose polymerase (PARP) and the ratio of Bax/Bcl-2 were up-regulated after treatment with AB23. Further study showed that AB23 induced endoplasmic reticulum stress through IRE1 signaling pathway and silencing of IRE1α partially enhanced AB23-induced apoptosis. Wound healing and transwell assays showed that AB23 could also suppress the migration and invasion of HEY cells. Moreover, it down-regulated the protein levels of matrix metalloproteinases MMP-2 and MMP-9. ConclusionAB23 possessed anti-proliferation, anti-migration and anti-invasion activities as a single agent on ovarian cancer cells.

Protective Effects of Alisol B 23-Acetate Via Farnesoid X Receptor-Mediated Regulation of Transporters and Enzymes in Estrogen-Induced Cholestatic Liver Injury in Mice

To investigate protective effects of alisol B 23-acetate (AB23A) against hepatotoxity and cholestasis induced by 17α-ethinylestradiol (EE) in association with farnesoid X receptor (FXR) activation in vivo and in vitro. The cholestatic liver injury model was established by subcutaneous injections of EE in C57BL/6 mice. Serum biomarkers, bile flow assay and H&E staining were used to identify the amelioration of cholestasis after AB23A treatment. Mice primary hepatocytes culture, gene silencing experiment, real-time PCR and Western blot assay were used to elucidate the mechanisms underlying AB23A hepatoprotection. AB23A treatment protected against liver injury induced by EE through increasing hepatic efflux and reducing uptake of bile acid via an induction in efflux transporters (Bsep and Mrp2) and an inhibition in hepatic uptake transporter (Ntcp) expression. AB23A also reduced bile acid synthesis through repressing Cyp7a1 and Cyp8b1, and increased bile acid metabolism through an induction in gene expression of Sult2a1. We further demonstrated that the changes in transporters and enzymes, as well as ameliorative liver histology in AB23A-treated mice were abrogated by FXR antagonist guggulsterone in vivo and were abrogated after FXR was silenced in vitro. AB23A produces protective effects against EE-induced cholestasis, due to FXR-mediated gene regulation.

Alisol B 23-acetate protects against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes involved in bile acid homeostasis

Intrahepatic cholestasis is a clinical syndrome with systemic and intrahepatic accumulation of excessive toxic bile acids that ultimately cause hepatobiliary injury. Appropriate regulation of bile acids in hepatocytes is critically important for protection against liver injury. In the present study, we characterized the protective effect of alisol B 23-acetate (AB23A), a natural triterpenoid, on alpha-naphthylisothiocyanate (ANIT)-induced liver injury and intrahepatic cholestasis in mice and further elucidated the mechanisms in vivo and in vitro. AB23A treatment dose-dependently protected against liver injury induced by ANIT through reducing hepatic uptake and increasing efflux of bile acid via down-regulation of hepatic uptake transporters (Ntcp) and up-regulation of efflux transporter (Bsep, Mrp2 and Mdr2) expression. Furthermore, AB23A reduced bile acid synthesis through repressing Cyp7a1 and Cyp8b1, increased bile acid conjugation through inducing Bal, Baat and bile acid metabolism through an induction in gene expression of Sult2a1. We further demonstrate the involvement of farnesoid X receptor (FXR) in the hepatoprotective effect of AB23A. The changes in transporters and enzymes, as well as ameliorative liver histology in AB23A-treated mice were abrogated by FXR antagonist guggulsterone in vivo. In vitro evidences also directly demonstrated the effect of AB23A on FXR activation in a dose-dependent manner using luciferase reporter assay in HepG2 cells. In conclusion, AB23A produces protective effect against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes.

Alisol B 23-acetate promotes liver regeneration in mice after partial hepatectomy via activating farnesoid X receptor

Liver resection has become a common treatment for liver tumors and hepatocellular carcinoma over the past decades. However, after surgery, the remnant livers in some patients fail to regenerate. Therefore, there is an urgent medical need to develop drugs that can promote liver regeneration. The purpose of the current study is to investigate the promotive effect of alisol B 23-acetate (AB23A) on liver regeneration in mice following partial hepatectomy (PH), and further elucidate the involvement of farnesoid X receptor (FXR) in the liver regeneration-promotive effect using in vivo and in vitro experiments. The results showed that AB23A dose-dependently promoted hepatocyte proliferation via upregulating hepatocyte proliferation-related protein forkhead box M1b (FoxM1b), Cyclin D1 and Cyclin B1 expression, and attenuated liver injury via an inhibition in Cyp7a1 and an induction in efflux transporters Bsep expression resulting in reduced hepatic bile acid levels. These changes in the genes, as well as accelerated liver regeneration in AB23A-treated mice were abrogated by FXR antagonist guggulsterone in vivo. In vitro evidences also directly showed the regulation of these genes by AB23A was abrogated when FXR was silenced. Luciferase reporter assay in HepG2 cells and molecular docking further demonstrated the effect of AB23A on FXR activation in vitro. In conclusions, AB23A produces promotive effect on liver regeneration, due to FXR-mediated regulation of genes involved in hepatocyte proliferation and hepato-protection. AB23A has the potential to be a novel therapeutic option for facilitating efficient liver regeneration in patients subjected to liver resection.

Reversal of P-glycoprotein-mediated multidrug resistance by Alisol B 23-acetate

Herbal drugs were screened for their activity in reversing multidrug resistance (MDR) in P-glycoprotein (P-gp) over-expressing cancer cells. Through bio-assay guided fractionation an active compound was isolated from Rhizoma Alismatis, the underground part of Alisma orientale and the chemical structure of the isolate compound was confirmed by HPLC, LC-MS and NMR as Alisol B 23-acetate (ABA). ABA restored the sensitivity of MDR cell lines HepG2-DR and K562-DR to anti-tumor agents that have different modes of action but are all P-gp substrates. It restored the activity of vinblastine, a P-gp substrate, in causing G 2/M arrest in MDR cells. In a dose-dependent manner, ABA increased doxorubicin accumulation and slowed down the efflux of rhodamin-123 from MDR cells. ABA inhibited the photoaffinity labeling of P-gp by [ 125 I ]iodoarylazidoprazosin and stimulated the ATPase activity of P-gp in a concentration-dependent manner, suggesting that it could be a transporter substrate for P-gp. In addition, ABA was also a partial non-competitive inhibitor of P-gp when verapamil was used as a substrate. Our results suggest that ABA may be a potential MDR reversal agent and could serve as a lead compound in the development of novel drugs.