Enhanced Anti-Cancer Potency Using A Combination of Oleanolic Acid and Maslinic Acid to Control Treatment Resistance in Breast Cancer.

The phosphatidylinositol 3-kinase/AKT/mammalian target of Rapamycin (PI3K/AKT/mTOR) pathway is a complex intracellular metabolic pathway leading to cell growth, and tumor proliferation plays an essential role in drug resistance in breast cancer. Therefore, in the present study, the anti-cancer effects of oleanolic acid (OA) and maslinic acid (MA) were investigated to improve the performance of the treatment strategy. We investigated the effect of OA and MA on cell viability using the WST-1 method. The synergistic effect of the combination was analyzed by isobologram analysis. In addition, the effects of the two compounds, individually and in combination, on apoptosis, autophagy, and the cell cycle were investigated in MCF7 cells. In addition, changes in the expression of PI3K/AKT/mTOR genes involved in apoptosis, cell cycle and metabolism were determined by quantitative RT-PCR. MA, OA, and a combination of both caused G0/G1 arrest. Apoptosis also increased in all treated groups. The autophagosomal LC3-II formation was induced 1.74-fold in the MA-treated group and 3.25-fold in the MA-OA-treated group. The combination treatment resulted in increased expression of genes such as GSK3B, PTEN, CDKN1B and FOXO3 and decreased expression of IGF1, PRKCB and AKT3 genes. The results showed that the combination of these two substances showed the highest synergistic effect at the lowest dose and using MA-OA caused cancer cells to undergo apoptosis. The use of combination drugs may reduce the resistance of cancer cells to treatment. However, further studies are needed to clarify cases.

FORMULATION AND EVALUATION OF MASLINIC ACID LOADED TRANSDERMAL PATCHES

Objective: To develop and evaluate Transdermal patch of Maslinic acid for Transdermal drug delivery. The current study is to develop Transdermal drug delivery system. Methods: Suitable method such as Solvent Casting Technique of Film Casting Technique are used for preparation of Transdermal patch. Results: The prepared Transdermal patches were transparent, smooth, uniform and flexible. The method adopted for the preparation of the system was found satisfactory. Conclusion: Various formulations were developed by using hydrophilic and hydrophobic polymers like HPMC E5 and EC respectively in single and combinations by solvent evaporation technique with the incorporation of penetration enhancer such as dimethylsulfoxide and dibutyl phthalate as plasticizer In vitro studies concluded that HPMC E5 patches has better release than that of EC patches, which may be attributed to high water vapour permeability of HPMC patches and hydrophobic nature of EC. An attempt was made to incorporate HPMC E5 and EC to the monolithic system for better release and prolong the duration of release. Formulation F7 containing an equal ratio of HPMC E5: EC (5:5) showed maximum and sustained release of 86.816±0.264 within 24 h. Kinetic models were used to confirm the release mechanism of the formulations. Maslinic acid release from the patches F1 to F7 followed non Fickian diffusion rate controlled mechanism.

Maslinic Acid Attenuates Ischemia/Reperfusion Injury-Induced Myocardial Inflammation and Apoptosis by Regulating HMGB1-TLR4 Axis

Aims: The inflammatory response and apoptosis are the major pathological features of myocardial ischemia/reperfusion injury (MI/RI). Maslinic acid (MA), a natural pentacyclic triterpene with various bioactivities, plays critical roles in the multiple cellular biological processes, but its protective effects on the pathophysiological processes of MI/RI have not been extensively investigated. Our study aimed to determine whether MA treatment alleviate ischemia/reperfusion (I/R)-induced myocardial inflammation and apoptosis both in vitro and in vivo, and further reveal the underlying mechanisms.Methods and results: An MI/RI rat model was successfully established by ligating the left anterior descending coronary artery and H9c2 cells were exposed to hypoxia/reoxygenation (H/R) to mimic I/R injury. In addition, prior to H/R stimulation or myocardial I/R operation, the H9c2 cells or rats were treated with varying concentrations of MA or vehicle for 24 h and two consecutive days, respectively. In this study, our results showed that MA could obviously increase the cell viability and decrease the cardiac enzymes release after H/R in vitro. MA could significantly improve the H/R-induced cardiomyocyte injury and I/R-induced myocardial injury in a dose-dependent manner. Moreover, MA suppressed the expression of inflammatory cytokines (tumor necrosis factor alpha [TNF-α, interleukin-1β [IL-1β and interleukin-6 [IL-6]) and the expressions of apoptosis-related proteins (cleaved caspase-3 and Bax) as well as increased the levels of anti-apoptotic protein Bcl-2 expression both in vitro and in vivo. Mechanistically, MA significantly inhibited nuclear translocation of nuclear factor-κB (NF-κB) p65 after H/R via regulating high mobility group box 1 (HMGB1)/toll-like receptor 4 (TLR4) axis.Conclusion: Taken together, MA treatment may alleviate MI/RI by suppressing both the inflammation and apoptosis in a dose-dependent manner, and the cardioprotective effect of MA may be partly attributable to the inactivation of HMGB1/TLR4/NF-κB pathway, which offers a new therapeutic strategy for MI/RI.

Anti-Inflammatory Activity of Three Triterpene from Hippophae rhamnoides L. in Lipopolysaccharide-Stimulated RAW264.7 Cells

Oleanolic acid (OA), asiatic acid (AA), and maslinic acid (MA) are ubiquitous isomeric triterpene phytochemicals with many pharmacological effects. To improve their application value, we used lipopolysaccharide (LPS) to induce RAW264.7 cells and studied the differences in the anti-inflammatory effects of the triterpenes according to their structural differences. MTT, Griess, and immunofluorescence assays, ELISA, flow cytometry, and Western blotting, were performed. The release of LPS-induced pro-inflammatory mediators, such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), and interleukin (IL-6), was significantly inhibited by OA, AA, and MA at the same concentration, and AA and MA promoted the production of anti-inflammatory factor IL-10. OA, AA, and MA inhibited LPS-induced NF-κB nuclear translocation in RAW264.7 cells. OA and AA inhibited the phosphorylation of ERK1/2, P38, and JNK1/2 in LPS-stimulated RAW264.7 cells. Moreover, OA increased LPS-induced Nrf2 expression and decreased Keap1 expression in RAW264.7 cells. OA, AA, and MA inhibited LPS-stimulated intracellular reactive oxygen species (ROS) production and alleviated mitochondrial membrane potential depletion. Overall, our data suggested that OA, AA, and MA exhibited significant anti-inflammatory effects in vitro. In particular, OA and AA take effects through the MAPKs, NF-κB, and Nrf2 signaling pathways.

First report of isolation of antibacterial ceramides from the leaves of Euclinia longiflora Salisb

Two phytosphingosine-type ceramides (euclinide A (1) and B (2)) were isolated alongside one fatty acid (geddic acid (3)), one fatty acid-1-glyceride ((2 S)-1- O-hentriacontanoyl glycerol (4)), ten triterpenes ( α-amyrin (5), α-amyrin acetate (6), ursolic acid (7), β-amyrin (8), β-amyrin acetate (9), β-amyrin palmitate (10), oleanic acid (11), maslinic acid (12), betulinic acid (13) and cylicodiscic acid (14)) and four sterols ( β -sitosterol (15), stigmasterol (17), and their glucosylated derivatives β-sitosterol-3-O- β-glucopyranoside (16) and stigmasterol-3-O- β-glucopyranoside (18)) from the methanol extract of the leaves of Euclinia longiflora Salisb. using routine chromatographic methods. The structures of the new ceramides and known compounds were determined by analyses of HR-FAB-MS, 1D and 2D NMR (HSQC, HMBC, and NOESY) data and confirmed, where applicable, by comparison with data reported in literature. The methanolic extract and the ceramides were evaluated for their antibacterial activities against different bacterial strains using microdilution method and the MIC values ranged from 6.25 to 50 μg/mL and were considered as moderate activity compared to ciprofloxacin. It is the first report of ceramides in Euclinia longiflora.

Renoprotective Effects of Maslinic Acid on Experimental Renal Fibrosis in Unilateral Ureteral Obstruction Model via Targeting MyD88

Maslinic acid (MA), also named crategolic acid, is a pentacyclic triterpene extracted from fruits and vegetables. Although various beneficial pharmacological effects of MA have been revealed, its effect on renal fibrosis remains unclear. This study was designed to clarify whether MA could attenuate renal fibrosis and determine the putative underlying molecular mechanisms. We demonstrated that MA-treated mice with unilateral ureteral obstruction (UUO) developed a histological injury of low severity and exhibited downregulated expression of fibrotic markers, including α-smooth muscle actin (α-SMA), vimentin, and fibronectin by 38, 44 and 40%, and upregulated expression of E-cadherin by 70% as compared with untreated UUO mice. Moreover, MA treatment restored the expression levels of α-SMA, connective tissue growth factor, and vimentin to 10, 7.8 and 38% of those induced by transforming growth factor (TGF)-β in NRK49F cells. MA decreased expression of Smad2/3 phosphorylation and Smad4 in UUO kidneys and TGF-β treated NRK49F cells (p < 0.05, respectively). Notably, MA specifically interferes with MyD88, an adaptor protein, thereby mitigating Smad4 nuclear expression (p < 0.01 compared to TGF-β treated group) and ameliorating renal fibrotic changes (p < 0.01 for each fibrotic markers compared to TGF-β induced cells). In addition, in the UUO model and lipopolysaccharide-induced NRK49F cells, MA treatment decreased the expression of IL-1β, TGF-α and MCP-1, ICAM-1, associated with the suppression of NF-κB signaling. These findings suggest that MA is a potential agent that can reduce renal interstitial fibrosis, to some extent, via targeting TGF-β/Smad and MyD88 signaling.

Maslinic Acid Inhibits Cervical Intraepithelial Neoplasia by Suppressing Interleukin-6 and Enhancing Apoptosis in a Mouse Model

Background: Cervical intraepithelial neoplasia (CIN) directly precedes cervical cancer, and elevated proinflammatory cytokine interleukins (IL)-6 is implicated in CIN. Objective: As maslinic acid exhibits anti-IL-6 property, the present study sought to determine the effect of maslinic acid on CIN in vitro and in vivo using cell cultures and mouse CIN models, respectively. Methods: The dose-effect of maslinic acid on HeLa cells, a human cervical cancer cell line, was first evaluated, including cytotoxicity, IL-6 secretion, IL-6 receptor (IL-6R) expression, proliferation potential and apoptosis status. A mouse model of CIN was also established, which was then subjected to increasing doses of maslinic acid treatment, followed by assessment of serum IL-6 level, cervical expression of IL-6R, and the proliferation potential and apoptosis of cervical tissues. Results: Maslinic acid dose-dependently inhibited cell growth and proliferation potential, reduced IL-6 secretion, cervical expression of IL-6R and induced apoptosis of HeLa cells in vitro. In the CIN mouse model, serum IL-6 level and cervical expression of IL-6R were elevated, which could be repressed by maslinic acid administration dose-dependently. Additionally, maslinic acid treatment in the CIN mice could also restore the otherwise increased proliferation potential and reduced apoptosis in the cervical tissues. Conclusion: Maslinic acid exhibits potent anti-IL-6 property in the CIN mouse model, and alleviates the disease-related abnormality in proliferation potential and apoptosis state of the cervical tissue cells, demonstrating its usefulness as a promising agent in treating CIN.