Triterpene Oleanolic Acid Research Articles

ANTIBACTERIAL ACTIVITY OF LIANTIBACTERIAL ACTIVITY OF LIANOID AND ARBOREAL SPECIES OF SCHEFFLERA FROM SOUTHERN INDIAANOID AND ARBOREAL SPECIES OF SCHEFFLERA FROM SOUTHERN INDIA

Objective: The genus Schefflera belongs to the family Araliaceae. The secondary metabolites of Schefflera include triterpene glycosides, oleanolic acid, and benzyl glycosides with proven biological activities. The objective of this study was to determine the antibacterial potentials of lianoid and arboreal Schefflera spp., from southern India.
 Methods: Schefflera venulosa, Schefflera stellata, and Schefflera racemosa were collected from the natural forests of Kodagu and Mysore regions of southern Karnataka. The plant parts such as stem bark, leaves, and inflorescence were dried, powdered and known quantity was subjected to Soxhlet extraction based on the solvent polarity. The solvent extracts from each of the species were subjected to preliminary antibacterial screening against five test bacterial strains, namely, Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Enterobacter aerogenes, and Klebsiella pneumoniae by agar disc diffusion method and the inhibition zones were measured. The minimum inhibitory concentrations (MIC’s) were calculated for the positive extracts and represented.
 Results: Of the 30 solvent extracts tested, six solvent extracts showed antibacterial activity. The ethanol and ethyl acetate solvent extracts of all three plants showed positive results for antibacterial activity. The zone of inhibition against the bacterial test pathogens ranged from 6.00±0.00 to 15.00±0.00 mm against B. subtilis, E. aerogenes, S. pyogenes, and K. pneumoniae. The highest zones of inhibition were observed for S. racemosa leaf ethanol extract against B. subtilis, E. aerogenes, and S. pyogenes (10.00 mm to 15.00±0.00 mm). The MIC values of the positive extracts were 5.0 mg/ml in the leaf, flower ethanol, and stem bark ethyl acetate extracts of S. venulosa, leaf ethyl acetate and ethanol extracts of S. stellata and the leaf ethanol extract from S. racemosa, respectively.
 Conclusion: Schefflera spp. from southern India possesses antibacterial potentials, which can be exploited pharmaceutically for potential health benefits against bacterial infections.

Phytosterols and triterpenes from Morinda lucida Benth. exhibit binding tendency against class I HDAC and HDAC7 isoforms.

The important role of histone deacetylases (HDACs) in the development of cancer has been demonstrated by various studies. Thus targeting HDACs with inhibitors is a major focus in anticancer drug research. Although few synthetic HDAC inhibitors (HDIs) have been approved for cancer treatment, they have significant undesirable side effects. Therefore emphases have been placed on natural HDIs as substitutes for the synthetic ones. In a bid to identify more HDIs, this study evaluated the binding tendency of compounds derived from Morinda lucida Benth.towards selected HDACs for the discovery of potent HDIs as potential candidates for anticancer therapeutics, based on the report of anticancer potentials of Morinda lucida-derived extracts and compounds. Givinostat and 49 Morinda-lucida derived compounds were docked against selected HDAC isoforms using AutodockVina, while binding interactions were viewed with Discovery Studio Visualizer, BIOVIA, 2016. Druglikeness and Absorption-Distribution-Metabolism-Excretion (ADME) parameters of the top 7 compounds were evaluated using the Swiss online ADME web tool. The results revealed that out of the 49 compounds, 3 phytosterols (campesterol, cycloartenol, and stigmasterol) and 2 triterpenes (oleanolic acid and ursolic acid) exhibited high HDAC inhibitory activity compared to givinostat. These 5 compounds also fulfill oral drugability of Lipinski rule of five. Morinda lucida-derived phytosterols and triterpenes show high binding tendency towards the selected HDACs and exhibited good drugability characteristics and are therefore good candidates for further studies in the search for therapies against abnormalities linked with over-activity of HDACs.

Phytosterols and triterpenes from Morinda lucida Benth (Rubiaceae) as potential inhibitors of anti-apoptotic BCL-XL, BCL-2, and MCL-1: an in-silico study

Deregulation of the normal cellular apoptotic function is a fundamental element in the etiology of most cancers and the anti-apoptotic B cell lymphoma 2 (BCL‑2) protein family is known to play crucial role in the regulation of this function. Overexpression of this protein family has been implicated in some cancers, such that agents that could inhibit their over-activity are now being explored for anticancer drug development. A number of studies have revealed the anticancer potential of Morinda lucida-derived extracts and compounds. In search of more inhibitors of this anti-apoptotic protein family from plant resources, 47 compounds, identified in Morinda lucida Benth (Rubiaceae) were screened for their inhibitory activities against BCL-XL, BCL-2, and MCL-1 by molecular docking using BINDSURF, while binding interactions of the top compounds were viewed with PyMOL. Druglikeness and Absorption–Distribution–Metabolism–Excretion (ADME) parameters of the top 6 compounds from docking study were evaluated using SuperPred webserver. Results revealed that out of the 47 compounds, 2 triterpenes (ursolic acid and oleanolic acid) and 4 phytosterols (cycloartenol, campesterol, stigmasterol, and β-sitosterol) have higher binding affinities for the selected BCL-2 proteins, compared to known standard inhibitors; these compounds also fulfill oral drugability of Lipinski rule of five. Therefore, since these Morinda lucida-derived phytosterols and triterpenes show high binding affinity toward the selected anti-apoptotic proteins and exhibited good drugability characteristics, they qualify for further study on drug development against cancers characterized by overexpression of this family of protein.

Preparative separation of structural isomeric pentacyclic triterpene oleanolic acid and ursolic acid from natural products by pH-zone-refining countercurrent chromatography.

In this work, pH-zone-refining countercurrent chromatography was investigated in the preparative separation of two bioactive components, oleanolic acid and ursolic acid, from three different natural products, Aralia chinensis, apple peels and Eriobotrya japonica Thunb. Oleanolic acid and ursolic acid are structurally isomeric pentacyclic triterpene acids that are widely distributed in many natural products. However, it was difficult to separate these components with high purity by conventional methods. A biphasic solvent system composed of n-hexane–dichloromethane–methanol–water (7 : 3 : 2 : 8, v/v) was selected, in which an optimized concentration of 10 mmol L−1 trifluoroacetic acid was added in the upper phase as the retainer and 10 mmol L−1 ammonia (with 25–28% NH3) was added in the aqueous phase as the eluter. Consequently, 38.56 mg of oleanolic acid with 99.01% purity was separated from 100 mg of the crude extract of Aralia chinensis, while 65.6 mg of a mixture of ursolic acid (90.98%) and oleanolic acid (6.51%) and 46.6 mg of a mixture of ursolic acid (74.35%) and oleanolic acid (23.61%) were separated from 100 mg of the crude extract of apple peels and 100 mg of the crude extract of Eriobotrya japonica Thunb., respectively, by pH-zone-refining countercurrent chromatography using the above selected biphasic solvent system. The results showed that pH-zone-refining countercurrent chromatography is an efficient method for the preparative separation of pentacyclic triterpene acids from natural products.

Biological Evaluation and Docking Studies of Synthetic Oleanane-type Triterpenoids.

Saponins are potential wide-spectrum antitumor drugs, and copper(I) catalyzed azide–alkyne 1,3-dipolar cycloaddition is a suitable approach to synthesizing saponin-like compounds by regioselective glycosylation of the C2/C3 hydroxyl and C28 carboxylic groups of triterpene aglycones maslinic acid (MA) and oleanolic acid (OA). Biological studies on the T-84 human colon carcinoma cell line support the role of the hydroxyl groups at C2/C3, the influence of the aglycone, and the bulky nature of the substituents in C28. OA bearing a α-d-mannose moiety at C28 (compound 18) focused our interest because the estimated inhibitory concentration 50 was similar to that reported for ginsenoside Rh2 against colon cancer cells and it inhibits the G1–S phase transition affecting the cell viability and apoptosis. Considering that triterpenoids from natural sources have been identified as inhibitors of nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling, docking studies were conducted to evaluate whether NF-κB may be a potential target. Results are consistent with the biological study and predict a similar binding mode of MA and compound 18 to the p52 subunit from NF-κB but not for OA. The fact that the binding site is shared by the NF-κB inhibitor 6,6-dimethyl-2-(phenylimino)-6,7-dihydrobenzo[d][1,3]oxathiol-4(5H)-one supports the result and points to NF-κB as a potential target of both MA and compound 18.

Survival of European Ash Seedlings Treated with Phosphite after Infection with the Hymenoscyphus fraxineus and Phytophthora Species

The European Fraxinus species are threatened by the alien invasive pathogen Hymenoscyphus fraxineus, which was introduced into Poland in the 1990s and has spread throughout the European continent, causing a large-scale decline of ash. There are no effective treatments to protect ash trees against ash dieback, which is caused by this pathogen, showing high variations in susceptibility at the individual level. Earlier studies have shown that the application of phosphites could improve the health of treated seedlings after artificial inoculation with H. fraxineus. Three-year-old F. excelsior seedlings were inoculated with the following pathogens: a H. fraxineus, Phytophthora species mixture (P. plurivora, P. megasperma, and P. taxon hungarica), in combination with two pathogens and mock-inoculated as the control, and then either watered or treated with ammonium phosphite (Actifos). Results showed significant differences in the survival of seedlings and symptoms of disease development among the treatments. Chlorophyll-a fluorescence parameters indicated a decrease in photosynthetic efficiency in infected plants, suggesting that they were under strong biotic stress, but none of the parameters could be used as a reliable bioindicator for ash decline disease. The application of Actifos enhanced the production of triterpenes (ursolic and oleanolic acid), and decreased the production of phenols (tyrosol) and sterols (β-sitosterol) in seedlings infected with H. fraxineus. Treatment with Actifos caused seedlings to enhance their response to pathogen(s) attack and increase their survival probability.

Concurrent analysis of bioactive triterpenes oleanolic acid and β-amyrin in antioxidant active fractions of Hibiscus calyphyllus, Hibiscus deflersii and Hibiscus micranthus grown in Saudi Arabia by applying validated HPTLC method

In this study, we developed a validated HPTLC method for concurrent analysis of two natural antioxidant triterpenes, oleanolic acid (OA) and β-amyrin (BA) in the biologically active fractions (petroleum ether, toluene, chloroform, ethyl acetate and n-butanol) of aerial parts of three Hibiscus species (H. calyphyllus, H. deflersii and H. micranthus). The chromatography was conducted on normal HPTLC (ready to use glass-plate coated with silica gel 60 F254) plate with chloroform and methanol (97:3, V/V) used as mobile phase. The derivatization of the developed plate was done with p-anisaldehyde and scanned at λmax = 575 nm. Well resolved and intense peaks of OA and BA were obtained at Rf = 0.36 and 0.57, respectively. The linear regression equation/correlation coefficient (r2) for OA and BA were Y = 6.65x + 553.35/0.994 and Y = 9.177x + 637.23/0.998, respectively in the linearity range of 100–1200 ng/spot indicated good linear relationship. The low values of %RSD for intra-day/inter-day precision of OA (1.45–1.61/1.38–1.59) and BA (1.52–1.57/1.50–1.53) suggested that the method was precise. The recovery/RSD (%) values for OA and BA were found to be 99.21–99.62/1.39–1.95 and 98.75–99.70/1.56–1.80, respectively assures the reasonably good accuracy of the proposed method. Fifteen samples were analyzed to check the content of OA and BA by using the developed HPTLC methods. The content of OA in different samples were found to be 3.87 (HmP) > 1.212 (HcP) > 0.673 (HdC) > 0.493 (HdP) > 0.168 (HdE) > 0.059 (HcC) > 0.015 (HcE) > 0.008 (HmT) µg/mg of the dried weight of extract. However the content of BA was found as: 2.293 (HmP) > 1.852 (HdT) > 0.345 (HdC) > 0.172 (HmT) > 0.041 (HdE) > 0.008 (HcC) µg/mg of the dried weight of extract. Some Hibiscus species fractions exhibited good antioxidant potential like: HcE (IC50 = 17.6 ± 1.8) > HdB (IC50 = 32.16 ± 0.9) > HmP (IC50 = 80.4 ± 4.5) > HmT (IC50 = 99.7 ± 8.2) when compared with ascorbic acid (IC50 = 14.2 ± 0.5), while other fractions exhibited only mild antioxidant capability. The developed HPTLC method can be further exploited for analysis of these markers in the quality assessment of raw material as well as herbal formulations available in the market.

Pentacyclic triterpenes as α-glucosidase and α-amylase inhibitors: Structure-activity relationships and the synergism with acarbose

In this paper, the inhibition of α-amylase and α-glucosidase by nine pentacyclic triterpenes was determined. For α-amylase inhibitory activity, the IC50 values of ursolic acid, corosolic acid, and oleanolic acid were 22.6±2.4μM, 31.2±3.4μM, and 94.1±6.7μM, respectively. For α-glucosidase inhibition, the IC50 values of ursolic acid, corosolic acid, betulinic acid, and oleanolic acid were 12.1±1.0μM, 17.2±0.9μM, 14.9±1.9μM, and 35.6±2.6μM, respectively. The combination of corosolic acid and oleanolic acid with acarbose showed synergistic inhibition against α-amylase. The combination of the tested triterpenes with acarbose mainly exhibited additive inhibition against α-glucosidase. Kinetic studies revealed that corosolic acid and oleanolic acid showed non-competitive inhibition and acarbose showed mixed-type inhibition against α-amylase. The results provide valuable implications for the triterpenes (ursolic acid, corosolic acid, and oleanolic acid) alone or in combination with acarbose as a therapeutic agent for the treatment of diabetes mellitus.

Discovery of Farnesoid X Receptor Antagonists Based on a Library of Oleanolic Acid 3-O-Esters through Diverse Substituent Design and Molecular Docking Methods.

The pentacyclic triterpene oleanolic acid (OA, 1) with known farnesoid X receptor (FXR) modulatory activity was modified at its C-3 position to find new FXR-interacting agents. A diverse substitution library of OA derivatives was constructed in silico through a 2D fingerprint similarity cluster strategy. With further docking analysis, four top-scored OA 3-O-ester derivatives were selected for synthesis. The bioassay results indicated that all four compounds 3 inhibited chenodeoxycholic acid (CDCA)-induced FXR transactivation in a concentration-dependent mode. Among them 3b and 3d are more active than the parent compound OA. A molecular simulation study was performed to attempt to explain the structure-activity relationship (SAR) and the antagonistic action. To the best of our knowledge, this is the first report on semi-synthetic pentacyclic triterpenoids with FXR-modulatory activities.

The NUTRAOLEOUM Study, a randomized controlled trial, for achieving nutritional added value for olive oils.

BackgroundVirgin olive oil, a recognized healthy food, cannot be consumed in great quantities. We aim to assess in humans whether an optimized virgin olive oil with high phenolic content (OVOO, 429 mg/Kg) and a functional one (FOO), both rich in phenolic compounds (429 mg/Kg) and triterpenic acids (389 mg/kg), could provide health benefits additional to those supplied a by a standard virgin olive oil (VOO).Methods/designA randomized, double-blind, crossover, controlled study will be conducted. Healthy volunteers (aged 20 to 50) will be randomized into one of three groups of daily raw olive oil consumption: VOO, OVOO, and FOO (30 mL/d). Olive oils will be administered over 3-week periods preceded by 2-week washout ones. The main outcomes will be markers of lipid and DNA oxidation, inflammation, and vascular damage. A bioavailability and dose-response study will be nested within this sustained- consumption one. It will be made up of 18 volunteers and be performed at two stages after a single dose of each olive oil. Endothelial function and nitric oxide will be assessed at baseline and at 4 h and 6 h after olive oil single dose ingestion.DiscussionFor the first time the NUTRAOLEUM Study will provide first level evidence on the health benefits in vivo in humans of olive oil triterpenes (oleanolic and maslinic acid) in addition to their bioavailability and disposition.Trial registrationThe Trial has been registered in ClinicalTrials.gov ID: NCT02520739.

SENYAWA INHIBITOR α-GLUKOSIDASE DAN ANTIOKSIDAN DARI KUMIS KUCING DENGAN PENDEKATAN METABOLOMIK BERBASIS FTIR

Plant is well known as an excellent source for bioactive compounds. Metabolomics was reported as a potential tool to accelerate plant acitive compounds identification. In this research, FTIR-based metabolomics method was used to identify active compounds with α-glucosidase inhibitory and antioxidant activity in aerial parts of Orthosiphon stamineus (OS) extract and its fractions. Chemical profile of OS methanolic extracts and hexane, chloroform, butanol, and water fractions were analyzed using infrared spectroscopy. OS extracts and fractions showed inhibitory activity against α-glucosidase enzymes with IC50 value 154.07±30.60-465.83±85.34 µg/mL and antioxidant activity with IC50 value 7.41±0.02-19.35±0.09 µg/mL. Butanol fraction was the fraction with the highest α-glucosidase inhibitory activity and moderate antioxidant activity with IC50 value between 154.07±30.60 µg/mL and 10.84±0.54 µg/mL, respectively. The correlation between the biological activity and chemical composition data were analyzed using Orthogonal Projections to Latent Structures (OPLS). Based on the VIP (variable influence on projection), the coefficient value of the respective OPLS models, and IR database of compounds previously identified in OS, it was suggested that methoxy flavonoid (sinensitin and 5,6,7,3’-tetramethoxy-4’-hydroxy-8-C-preny-lflavone), diterpenes (orthosiphols, orthoarisins, neoorthosiphols, staminols, and staminolactones) and triterpenes (ursolic acid, oleanolic acid, betulinic acid, hydroxybetulinic acid, maslinic acid, α-amyrin and β-amyrin) were identified as responsible compounds for the α-glucosidase inhibitory activity. Meanwhile phenolic (rosmarinic acid), methoxy flavonoid (eupatorin, sinensetin, 5-hydroxy-6,7,3’,4’-tetramethoxyflavone, salvigenin, 6-hydroxy-5,7,3’-trimethoxyflavone and 5,6,7,3’-tetramethoxy-4’-hydroxy-8-C-prenylflavone), diterpenes (orthosiphols, orthoarisins, neoortho-siphols, staminols, and staminolactones) and triterpenes (ursolic acid, oleanolic acid, betulinic acid, hydroxybetulinic acid, maslinic acid, α-amyrin and β-amyrin) were identified as responsible compounds for the antioxidant activity.

Human neutrophil elastase inhibition studied by capillary electrophoresis with laser induced fluorescence detection and microscale thermophoresis

Capillary electrophoresis-laser induced fluorescence (CZE-LIF) and microscale thermophoresis (MST) were used for the first time to study the inhibition of human neutrophil elastase (HNE). We recently studied HNE kinetics (Km and Vmax) by developing an in-capillary CZE-LIF assay based on transverse diffusion of laminar flow profiles (TDLFP) for reactant mixing. In this work, the former assay was adapted to monitor HNE inhibition. Two natural well known HNE inhibitors from the triterpene family, ursolic acid and oleanolic acid, were tested to validate the developed assay. Since the solubility of pentacyclic triterpenes in aqueous media where the enzymatic reaction will take place is limited, the effect of DMSO and ethanol on HNE was studied using microscale thermophoresis (MST). An agglomeration of the enzyme was revealed when preparing the inhibitor in 5% (v/v) DMSO. This phenomenon did not occur in the presence of ethanol. Therefore, ethanol was used as inhibitor solvent, at a limited percentage of 20% (v/v). In these conditions and after optimization of the TDLFP approach, the repeatability (RSD on migration times and peak-areas inferior to 2.2%) of the CZE-LIF assay and the sensitivity (LOQ of few nM) were found to be satisfactory for conducting inhibition assays. IC50 values for ursolic and oleanolic acid were successfully determined. They were respectively equal to 5.62±0.10μM (r2=0.9807; n=3) and to 8.21±0.23μM (r2=0.9887; n=3). Excellent agreement was found between the results obtained by CE and those reported in literature which validates the developed method. Particularly, the CE-based assay is able to rank HNE inhibitors relative to each other.Furthermore, MST technique was used for evaluating HNE interaction with the ursolic acid. Up to 16 capillaries were automatically processed to obtain in one titration experiment the dissociation constant for the HNE-ursolic acid complex. Ki was found to be 2.72±0.66μM (n=3) which is in excellent agreement with the value determined by CE enzyme inhibition studies (Ki=2.81μM) confirming the reliability of the developed CE assay and the competitive inhibition mode of ursolic acid.

Tumour cell derived effects on monocyte/macrophage polarization and function and modulatory potential of Viscum album lipophilic extract in vitro

Macrophages are highly versatile cells that play an important role in tumour microenvironment. Tumour associated macrophages (TAMs) have been linked to both, good or bad prognosis of several cancer types depending on their number, composition and polarization. Viscum album lipophilic extract (VALE) contains several pentacyclic triterpenes known to modulate the activity of monocytes and other immune cells and to exhibit anticancer properties. In our in vitro study, we investigated the effect of tumour cell lines on macrophage polarization and monocyte chemotactic transmigration and examined the modulatory potential of VALE and its predominant triterpene oleanolic acid (OA). Human peripheral blood monocytes were differentiated into monocyte derived macrophages (MDM) using M-CSF and polarized into M1 by IFN-γ and LPS and into M2 macrophages by IL-4 and IL-13 or by co-culture with two different tumour cell lines. Polarized macrophages were subsequently treated with VALE or OA. Phenotypic markers and cytokines were assessed by flow cytometry and immunoanalysis. Migration of human peripheral blood monocytes induced by monocyte chemotactic protein-1 (MCP-1) or supernatants of different tumour cell lines under the influence of VALE or OA was measured in a chemotaxis transmigration assay. In vitro polarized M1 and M2 type macrophages revealed specific phenotypic patterns and tumour cell co-cultured MDM displayed ambiguous phenotypes with M1 as well as M2 associated markers. VALE and OA showed modest influence on cell surface marker profile and cytokine expression of tumour cell co-cultured macrophages. All tumour cell supernatants markedly enhanced the migratory activity of monocytes. VALE and OA significantly inhibited MCP-1 induced monocyte transmigration, whereas monocyte migration initiated by tumour cell derived supernatants was not affected. In our study we reconfirmed that co-culture with different tumour cell lines can result in a mixed macrophage phenotype with M1 as well as M2 patterns, a finding that is important for a better understanding of tumour microenvironment functions. Moreover, we demonstrated that VALE shows slight immunomodulatory effects on tumour cell co-cultured macrophages and modulates monocyte chemotactic transmigration in vitro, indicating promising possibilities of triterpenes from Viscum album L. to contribute in a multimodal concept of anti-cancer therapy in future. Our data contribute to an understanding of monocyte function and macrophage polarization in vitro and of the possibility to influence their behaviour by triterpene containing mistletoe extracts.

Structural basis for 18-β-glycyrrhetinic acid as a novel non-GSH analog glyoxalase I inhibitor.

Glyoxalase I (GLOI), a glutathione (GSH)-dependent enzyme, is overexpressed in tumor cells and related to multi-drug resistance in chemotherapy, making GLOI inhibitors as potential anti-tumor agents. But the most studied GSH analogs exhibit poor pharmacokinetic properties. The aim of this study was to discover novel non-GSH analog GLOI inhibitors and analyze their binding mechanisms. Mouse GLOI (mGLOI) was expressed in BL21 (DE3) pLysS after induction with isopropyl-β-D-1-thiogalactopyranoside and purified using AKTA FPLC system. An in vitro mGLOI enzyme assay was used to screen a small pool of compounds containing carboxyl groups. Crystal structure of the mGLOI-inhibitor complex was determined at 2.3 Å resolution. Molecular docking study was performed using Discovery Studio 2.5 software package. A natural compound 18-β-glycyrrhetinic acid (GA) and its derivative carbenoxolone were identified as potent competitive non-GSH analog mGLOI inhibitors with Ki values of 0.29 μmol/L and 0.93 μmol/L, respectively. Four pentacyclic triterpenes (ursolic acid, oleanolic acid, betulic acid and tripterine) showed weak activities (mGLOI inhibition ratio <25% at 10 μmol/L) and other three (maslinic acid, corosolic acid and madecassic acid) were inactive. The crystal structure of the mGLOI-GA complex showed that the carboxyl group of GA mimicked the γ-glutamyl residue of GSH by hydrogen bonding to the glutamyl sites (residues Arg38B, Asn104B and Arg123A) in the GSH binding site of mGLOI. The extensive van der Waals interactions between GA and the surrounding residues also contributed greatly to the binding of GA and mGLOI. This work demonstrates a carboxyl group to be an important functional feature of non-GSH analog GLOI inhibitors.

Inhibitory Effects of Leaves of Guava (Psidium guajava) on TPA-Induced Inflammation and Tumor Promotion in Two-Stage Carcinogenesis in Mouse Skin

Cancer prevention offers the most cost-effective long-term health strategy. The methanol extract of the leaves of guava(Psidium guajava) exhibits marked antitumor activity in an in vivo two-stage carcinogenesis test in mice using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13- acetate (TPA) as a promoter. From the active fraction of the methanol extract, five triterpene acids, uvaol (1), ursolic acid (2), corosolic acid (3), asiatic aci (4), and oleanolic acid d (5), were isolated and identified. These compounds were evaluated for their inhibitory effects on TPA-induced inflammation (1 µg/ear) in mice, and showed marked anti-inflammatory effects, with a 50% inhibitory dose of 117–657 nmol/ear. The leaves of guava may therefore be effective for cancer prevention.

Changes in Renal Function and Oxidative Status Associated with the Hypotensive Effects of Oleanolic Acid and Related Synthetic Derivatives in Experimental Animals.

PurposeThe triterpene oleanolic acid (OA) is known to possess antihypertensive actions. In the present study we to compared the effects of the triterpene on mean arterial blood pressure (MAP) and kidney function following acute administration in normotensive animals with those of its related oleanane synthetic derivatives (brominated oleanolic acid, Br-OA and oleanolic acid methyl ester, Me-OA). We also used experimental models of hypertension to further explore the effects of sub-chronic oral OA treatment and evaluated influences on oxidative status.MethodsOA was extracted from dried flower buds of Syzygium aromaticum using a previously validated protocol in our laboratory. Me-OA and Br-OA were synthesized according to a method described. Rats were supplemented with lithium chloride (12 mmol L-1) prior to experimentation in order to raise plasma lithium to allow measurements of lithium clearance and fractional excretion (FELi) as indices of proximal tubular Na+ handling. Anaesthetized animals were continuously infused via the right jugular with 0.077M NaCl. MAP was measured via a cannula inserted in the carotid artery, and urine was collected through a cannula inserted in the bladder. After a 3.5 h equilibration, MAP, urine flow, electrolyte excretion rates were determined for 4 h of 1 h control, 1.5 h treatment and 1.5 h recovery periods. OA, Me-OA and Br-OA were added to the infusate during the treatment period. We evaluated sub-chronic effects on MAP and kidney function in normotensive Wistar rats and in two animal models of hypertension, spontaneously hypertensive rats (SHR) and Dahl salt-sensitive (DSS) rats, during 9-week administration of OA (p.o.). Tissue oxidative status was examined in these animals at the end of the study. Increasing evidence suggests that and renal function disturbances and oxidative stress play major roles in the pathogenesis of hypertension.ResultsAcute infusion OA and oleanane derivatives displayed qualitatively similar effects in decreasing MAP and increasing urinary Na+ outputs. The drugs increased the FENa and FELi without influencing GFR indicating that at least part of the overall natriuretic effect involved proximal tubular Na+ reabsorption. Sub-chronic OA administration (p.o.) also elicited hypotensive responses in Wistar, DSS and SHR rats. The MAP lowering effect was more marked in hypertensive animals and were positively correlated with increased urinary Na+ excretion. Compared with respective control rats, OA treatment reduced malondialdehyde (MDA, a marker of lipid peroxidation) and increased activities of antioxidant enzymes; superoxide dismutase and glutathione peroxidase in hepatic, cardiac and renal tissues.ConclusionsOA and oleanane derivatives have similar effects on MAP, kidney function and oxidative stress. The amelioration of oxidative stress and blood pressure lowering effects by OA are more marked in hypertensive animals and correlated with an increased urinary Na+ output.Novelty of the WorkThe results of this study are novel in that they show 1) a correlation between blood pressure reduction and increased urinary Na+ excretion by OA, 2) a more marked MAP reduction in hypertensive animals and 3) a drug-induced decrease in proximal tubule Na+ reabsorption. The results may also be clinically relevant because OA is effective via oral administration.

Tumour cell derived effects on monocyte/macrophage polarization and function and modulatory potential of Viscum album lipophilic extract in vitro.

BackgroundMacrophages are highly versatile cells that play an important role in tumour microenvironment. Tumour associated macrophages (TAMs) have been linked to both, good or bad prognosis of several cancer types depending on their number, composition and polarization. Viscum album lipophilic extract (VALE) contains several pentacyclic triterpenes known to modulate the activity of monocytes and other immune cells and to exhibit anticancer properties. In our in vitro study, we investigated the effect of tumour cell lines on macrophage polarization and monocyte chemotactic transmigration and examined the modulatory potential of VALE and its predominant triterpene oleanolic acid (OA).MethodsHuman peripheral blood monocytes were differentiated into monocyte derived macrophages (MDM) using M-CSF and polarized into M1 by IFN-γ and LPS and into M2 macrophages by IL-4 and IL-13 or by co-culture with two different tumour cell lines. Polarized macrophages were subsequently treated with VALE or OA. Phenotypic markers and cytokines were assessed by flow cytometry and immunoanalysis. Migration of human peripheral blood monocytes induced by monocyte chemotactic protein-1 (MCP-1) or supernatants of different tumour cell lines under the influence of VALE or OA was measured in a chemotaxis transmigration assay.ResultsIn vitro polarized M1 and M2 type macrophages revealed specific phenotypic patterns and tumour cell co-cultured MDM displayed ambiguous phenotypes with M1 as well as M2 associated markers. VALE and OA showed modest influence on cell surface marker profile and cytokine expression of tumour cell co-cultured macrophages. All tumour cell supernatants markedly enhanced the migratory activity of monocytes. VALE and OA significantly inhibited MCP-1 induced monocyte transmigration, whereas monocyte migration initiated by tumour cell derived supernatants was not affected.ConclusionsIn our study we reconfirmed that co-culture with different tumour cell lines can result in a mixed macrophage phenotype with M1 as well as M2 patterns, a finding that is important for a better understanding of tumour microenvironment functions. Moreover, we demonstrated that VALE shows slight immunomodulatory effects on tumour cell co-cultured macrophages and modulates monocyte chemotactic transmigration in vitro, indicating promising possibilities of triterpenes from Viscum album L. to contribute in a multimodal concept of anti-cancer therapy in future. Our data contribute to an understanding of monocyte function and macrophage polarization in vitro and of the possibility to influence their behaviour by triterpene containing mistletoe extracts.

Effects of oleanolic acid on the insulin signaling pathway in skeletal muscle of streptozotocin‐induced diabetic male Sprague‐Dawley rats

The pant-derived triterpene oleanolic acid (OA) has been shown to have antidiabetic effects, but its action on the insulin signaling cascade has not been fully elucidated. The aim of the present study was to investigate the effects of OA on aspects of the phosphatidylinositol 3-kinase/Akt insulin signaling cascade in skeletal muscle of streptozotocin-induced type 1 diabetic male Sprague-Dawley rats. Diabetic and non-diabetic rats were treated with insulin (4 IU/kg), OA (80 mg/kg), and the combination of OA + insulin in acute 60-min and sub-chronic 14-day studies. Single and daily doses were administered in the acute and sub-chronic studies, respectively. In acute studies, phosphorylated (p-) Akt and p-glycogen synthase (GS) expression was evaluated. In sub-chronic studies, GS and glycogen phosphorylase (GP) expression and activity were evaluated, as were glycogen levels. The findings show that OA enhances insulin-stimulated hypoglycemic effects in diabetic rats. In the acute study, OA increased levels of p-Akt and decreased levels of p-GS. In the sub-chronic study, OA increased both GS and GP activity, whereas OA + insulin increased GS and decreased GP activity. Treatment of rats with OA and OA + insulin increased GS expression in the skeletal muscle of diabetic rats and decreased GP expression. Glycogen levels were increased by OA but decreased OA + insulin treatment. Oleanolic acid in synergy with insulin can enhance activation of the insulin signaling pathway. Furthermore, the present study provides evidence of OA activation of insulin signaling enzymes independent of insulin.

Discovery, optimization, and pharmacophore modeling of oleanolic acid and analogues as breast cancer cell migration and invasion inhibitors through targeting Brk/Paxillin/Rac1 axis.

Bioassay-guided fractionation of Terminalia bentzoe L. leaves methanol extract identified the known triterpene oleanolic acid (1) as its major breast cancer cell migration inhibitor. Further chemical optimization afforded five new (9-12 and 15) and seven known (4-8, 13, and 14) semisynthetic analogues. All compounds were tested for their ability to inhibit human breast cancer MDA-MB-231 cells migration, proliferation, and invasion. The results revealed that 3-O-[N-(3'-chlorobenzenesulfonyl)-carbamoyl]-oleanolic acid (11) and 3-O-[N-(5'-fluorobenzenesulfonyl)-carbamoyl]-oleanolic acid (12) were the most active hits at low μM concentration. Western blot analysis indicated the activity of 1, 11, and 12 might be related, at least in part, to the suppression of Brk/Paxillin/Rac1 signaling pathway. Pharmacophore modeling study was conducted to better understand the common structural binding epitopes important for the antimigratory activity. The sulfonyl carbamoyl moiety with an optimal bulkiness electron-deficient phenyl ring is associated with improved activity. This study is the first to discover the antimigratory and anti-invasive activities of oleanolic acid and analogues through targeting the Brk/Paxillin/Rac1 axis.

Solid-phase library synthesis of bi-functional derivatives of oleanolic and maslinic acids and their cytotoxicity on three cancer cell lines.

A wide set of 264 compounds has been semisynthesized with high yields and purities. These compounds have been obtained through easy synthetic processes based on a solid-phase combinatorial methodology. All the members of this library have one central core of a natural pentacyclic triterpene (oleanolic or maslinic acid) and differ by 6 amino acids, coupled with the carboxyl group at C-28 of the triterpenoid skeleton, and by 10 different acyl groups attached to the hydroxyl groups of the A-ring of these molecules. According to the literature on the outstanding and promising pharmacological activities of other similar terpene derivatives, some of these compounds have been tested for their cytotoxic effects on the proliferation of three cancer cell lines: B16-F10, HT29, and Hep G2. In general, we have found that around 70% of the compounds tested show cytotoxicity in all three of the cell lines selected; around 60% of the cytotoxic compounds are more effective than their corresponding precursors, that is, oleanolic (OA) or maslinic (MA) acids; and nearly 50% of the cytotoxic derivatives have IC50 values between 2- to 320-fold lower than their corresponding precursor (OA or MA).